Rapid, visual, and equipment-free point-of-care testing for Staphylococcus aureus by direct recombinase polymerase amplification with SYBR Green Ι

Author(s):

Xiaorui Fan,Fangti Han,Binan Zhao,Yan Xu,Xiao Zhao,Xinyi Pu,Yanan Du,Qi Zhang,Xiaoxia Zhang,Wanjing Zhang,Wenjing Wu,Zhiwei Chen,Kai Zhao

Journal:

Acta biochimica et biophysica Sinica

Year:

2021

Abstract:

No abstract available.

PrimerBankID	Target Pathogen	Target Gene
RPB0164	Staphylococcus aureus	\

Isothermal recombinase polymerase amplification-lateral flow detection of SARS-CoV-2, the etiological agent of COVID-19.

Author(s):

Shelite, Thomas R; Uscanga-Palomeque, Ashanti C; Castellanos-Gonzalez, Alejandro; Melby, Peter C; Travi, Bruno L;

Journal:

J VIROL METHODS

Year:

2021

Abstract:

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral cDNA nucleocapsid (N) gene copies/渭L. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/渭L of SARS-CoV-2 N gene containing plasmid. We evaluated 37 nasopharyngeal samples using CDC's N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100 % concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

PrimerBankID	Target Pathogen	Target Gene
RPB0041	MERS-CoV	N gene

Integrating Reverse Transcription Recombinase Polymerase Amplification with CRISPR Technology for the One-Tube Assay of RNA

Author(s):

Wei Feng , Hanyong Peng , Jingyang Xu , Yanming Liu , Kanti Pabbaraju , Graham Tipples, Michael A Joyce , Holly A Saffran , D Lorne Tyrrell , Shawn Babiuk , Hongquan Zhang , X Chris Le

Journal:

Analytical Chemistry

Year:

2021

Abstract:

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.

PrimerBankID	Target Pathogen	Target Gene
RPB0212	SARS-CoV-2	S gene

Visual detection of human metapneumovirus using CRISPR-Cas12a diagnostics

Author(s):

Weidong Qian,Jie Huang,Ting Wang,Xiaoxian He,Guozhang Xu,Yongdong Li

Journal:

Virus research

Year:

2021

Abstract:

Human metapneumovirus (HmPV) is a common and serious virus that causes respiratory tract infection. This study aimed to develop a detection technique by combining reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for clinical diagnosis of HmPV. Herein, four primer pairs targeting partial nucleoprotein (N) gene of HmPV were designed and evaluated. Then, the products amplified by RT-RPA were detected using CRISPR-Cas12a combined with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 35 min or 45 min, and the detection limit was up to 6.97 × 102 copies/mL. And there was no cross reaction with human bocavirus, respiratory syncytial virus, adenovirus and parainfluenza virus. By combining with LF, the detection results were evaluated by naked eyes. Furthermore, 28 clinical samples were applied to examine the performance of RT-RPA-Cas12a system. The detection coincidence rates of RT-RPA-Cas12a-fluorescence and RT-RPA-Cas12a-LF with quantitative RT-PCR were 96.4% and 92.9%, respectively. Together, the new method for detecting HmPV with high sensitivity and specificity based on RT-RPA-Cas12a-fluorescence or LF shows promising potential for clinical diagnosis of HmPV without professional skills or ancillary equipment.

PrimerBankID	Target Pathogen	Target Gene
RPB0094	HMPV	N gene

Detection of Infectious Viruses Using CRISPR-Cas12-Based Assay.

Author(s):

Talwar, Chandana S; Park, Kwang-Hyun; Ahn, Woo-Chan; Kim, Yong-Sam; Kwon, Oh Seok; Yong, Dongeun; Kang, Taejoon; Woo, Euijeon;

Journal:

Biosensors (Basel)

Year:

2021

Abstract:

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has severely influenced public health and economics. For the detection of SARS-CoV-2, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)-based assays have been emerged because of their simplicity, sensitivity, specificity, and wide applicability. Herein, we have developed a CRISPR-Cas12-based assay for the detection of SARS-CoV-2. In the assay, the target amplicons are produced by isothermal reverse transcription recombinase polymerase amplification (RT-RPA) and recognized by a CRISPR-Cas12a/guide RNA (gRNA) complex that is coupled with the collateral cleavage activity of fluorophore-tagged probes, allowing either a fluorescent measurement or naked-eye detection on a lateral flow paper strip. This assay enables the sensitive detection of SARS-CoV-2 at a low concentration of 10 copies per sample. Moreover, the reliability of the method is verified by using nasal swabs and sputum of COVID-19 patients. We also proved that the current assay can be applied to other viruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), with no major changes to the basic scheme of testing. It is anticipated that the CRISPR-Cas12-based assay has the potential to serve as a point-of-care testing (POCT) tool for a wide range of infectious viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0044	MERS-CoV	MERS-CoV E gene
RPB0045	SARS-CoV-2	SARS-CoV S gene
RPB0046	SARS-CoV	SARS-CoV S gene

A Rapid and Sensitive Detection Method for Pseudomonas aeruginosa Using Visualized Recombinase Polymerase Amplification and Lateral Flow Strip Technology

Author(s):

Haitao Yang,Yan Wang,Qiankun Yang,Hui Fan,Lei Wang,Tianmeng Zhang,Zhixing Li,Gang Liu,Panpan Zhao,Huahua Wu,Jingquan Dong,Wei Liang

Journal:

Frontiers in cellular and infection microbiology

Year:

2021

Abstract:

Pseudomonas aeruginosa is a common opportunistic pathogen that causes acute nosocomial necrotizing pneumonia and is the predominant source of chronic lung infections in patients with the genetic disorder cystic fibrosis. Early diagnosis in infected patients and monitoring P. aeruginosa contamination is therefore of great importance in controlling disease spread and development with timely drugs intervention treatment and cut off infection source. Traditional culture-biochemical methods are time consuming and highly dependent on technicians and expensive instruments. To address these challenges, the present study aimed to develop a rapid, sensitive, and specific, on-site detection method for P. aeruginosa based on recombinase polymerase amplification (RPA) combined with lateral flow strip (LFS) technology. The experimental process included screening and modification of primer and probe sets targeting the unique virulence gene elastase B (lasB); specificity detection in 29 strains of P. aeruginosa and 23 closely-related pathogenic bacteria; sensitivity measurements with gradient-diluted P. aeruginosa genomic DNA and probit regression analysis; and clinical application evaluation using 574 patients samples and calculating coincidence rate and kappa index value in comparison with the culture-biochemical method. The P. aeruginosa RPA-LFS assay could complete the amplification process at 37°C constant temperature within 30 min and results could be visualized by the naked eye within 10 min on LFS. The assay displayed high sensitivity with a limit of detection of 3.05 CFU/reaction. It also demonstrated high specificity by showing no cross reaction with other pathogenic bacteria, and rapidness by being completed in less than an hour. Furthermore, when used with clinical samples, the assay had a coincidence rate of 98.26% with the culture-biochemical method and a kappa index value of 0.9433. These data indicate that the RPA-LFS assay represents a major improvement for P. aeruginosa detection, especially in resource-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0125	Pseudomonas aeruginosa	lasB

Development of Rapid and Visual Nucleic Acid Detection Methods towards Four Serotypes of Human Adenovirus Species B Based on RPA-LF Test

Author(s):

Yong Qi,Wei Li,Xiaoling Li,Wanpeng Shen,Jinhai Zhang,Jiameng Li,Ruichen Lv,Nianhong Lu,Liqiang Zong,Susu Zhuang,Qiyuan Gui,Dongming Zhou,Jing Li,Yingjia Xu,Hongbing Shen,Yuexi Li

Journal:

BioMed research international

Year:

2021

Abstract:

Objectives: Human adenoviruses (HAdV) are classified as 7 HAdV species, and some serotypes in species B like HAdV 3, HAdV 7, HAdV 21, and HAdV 55 caused severe symptoms, even fatalities. Patients may be misdiagnosed and inadequately treated without reliable and practical methods for HAdV serotyping. Developing rapid, sensitive, and specific diagnostic methods for HAdV is critical. Methods: Detection methods were established based on a recombinase polymerase amplification (RPA) assay and lateral flow (LF) test. Specific target sequence was screened, targeting which, primers and probes were designed, synthesized, and screened for establishing assay with high amplification efficiency. Primer or probe concentrations and amplification time were optimized. Detection limit, sensitivity, and specificity were evaluated. Results and Conclusions. Simple, sensitive, and specific RPA-LF methods for detection of four serotypes of HAdV together or separately were established, which had detection limits of 10 to 280 copies/reaction comparable to real-time PCR without recognizing other pathogens. The sensitivity and specificity were >92% and >98%, respectively, evaluated by limited clinical samples. The detection can be completed in 25 min without requirement of any instrument except a constant temperature equipment, showing superior detection performance and promising for a wide use in the field and resource-limited area.

PrimerBankID	Target Pathogen	Target Gene
RPB0088	HAdV	\
RPB0089	HAdV	\
RPB0090	HAdV	\
RPB0091	HAdV	\
RPB0092	HAdV	\

Rapid and simultaneous visual screening of SARS-CoV-2 and influenza virufses with customized isothermal amplification integrated lateral flow strip

Author(s):

Yong Sun , Panzhu Qin , Jun He , Weiwei Li , Yonglin Shi , Jianguo Xu , Qian Wu , Qingqing Chen , Weidong Li , Xinxin Wang , Guodong Liu , Wei Chen

Journal:

Biosensors and Bioelectronics

Year:

2022

Abstract:

Due to the similar clinical symptoms of influenza (Flu) and coronavirus disease 2019 (COVID-19), there is a looming infection threat of concurrent Flu viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this work, we introduce a customized isothermal amplification integrated lateral flow strip (LFS) that is capable performing duplex reverse transcription-recombinase polymerase amplification (RT-RPA) and colorimetric LFS in a sequential manner. With customized amplification primer sets targeted to SARS-CoV-2 (opening reading frame 1a/b and nucleoprotein genes) and Flu viruses (Flu A and Flu B), the platform allows the rapid and simultaneous visual screening of SARS-CoV-2 and Flu viruses (Flu A and Flu B) without cross reactivity, false positives, and false negatives. Moreover, it maximally eases the detection, reduces the detection time (1 h), and improves the assay performance to detect as low as 10 copies of the viral RNA. Its clinical application is powerfully demonstrated with 100% accuracy for evaluating 15 SARS-CoV-2-positive clinical samples, 10 Flu viruses-positive clinical samples, and 5 negative clinical samples, which were pre-confirmed by standard qRT-PCR. We envision this portable device can meet the increasing need of online monitoring the serious infectious diseases that substantially affects health care systems worldwide.

PrimerBankID	Target Pathogen	Target Gene
RPB0204	SARS-CoV-2	Orf1ab-gene
RPB0205	SARS-CoV-2	N-gene

A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing

Author(s):

Harriet D Gliddon,Dan Frampton,Vanisha Munsamy,Jude Heaney,Thomas Pataillot-Meakin,Eleni Nastouli,Alexander S Pym,Adrie J C Steyn,Deenan Pillay,Rachel A McKendry

Journal:

Microbiology spectrum

Year:

2021

Abstract:

Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings. IMPORTANCE Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings. This article describes a new workflow that can diagnose drug-resistant TB in a shorter time, with less equipment, and for a lower price than current methods. The amount of TB DNA is first increased without the need for bulky and costly thermocycling equipment. The DNA is then read using a portable sequencer called a MinION, which indicates whether there are tell-tale changes in the DNA that indicate whether the TB strain is drug resistant. Our workflow could play an important role in the future in the fight against the public health challenge that is TB drug resistance.

PrimerBankID	Target Pathogen	Target Gene
RPB0113	Mycobacterium	rpoB
RPB0114	Mycobacterium	katG
RPB0115	Mycobacterium	inhA

Microfluidic Chip with Two-Stage Isothermal Amplification Method for Highly Sensitive Parallel Detection of SARS-CoV-2 and Measles Virus

Author(s):

Qin Huang,Xiaohui Shan,Ranran Cao,Xiangyu Jin,Xue Lin,Qiurong He,Yulei Zhu,Rongxin Fu,Wenli Du,Wenqi Lv,Ying Xia,Guoliang Huang

Journal:

Micromachines

Year:

2021

Abstract:

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 μL and 10.6 μL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

PrimerBankID	Target Pathogen	Target Gene
RPB0110	SARS-CoV-2	\
RPB0111	Measles morbillivirus	\
RPB0211	SARS-CoV-2	\

Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus infection

Author(s):

Zhengtu Li,Mingdie Wang,Teng Xu,Yangqing Zhan,Fengyi Chen,Ye Lin,Shaoqiang Li,Jing Cheng,Feng Ye

Journal:

Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi

Year:

2022

Abstract:

Background: Rapid and reliable diagnostic methods for Aspergillus fumigatus infection are urgently needed. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 13a (Cas13a) has high sensitivity and specificity in the diagnosis of viral infection. However, its potential use in detecting A. fumigatus remains unexplored. A highly sensitive and specific method using the CRISPR/Cas13a system was developed for the reliable and rapid detection of A. fumigatus. Methods: The conserved internal transcribed spacer (ITS) region of A. fumigatus was used to design CRISPR-derived RNA (crRNA) and the corresponding recombinase polymerase amplification (RPA) primer sequence with the T7 promoter for the CRISPR assay. Twenty-five clinical isolates and 43 bronchoalveolar lavage fluid (BALF) remaining from routine examinations of patients with confirmed pulmonary aspergillosis were collected to further validate the CRISPR assay. Results: No amplification signal was observed when genomic DNA from closely clinically related Aspergillus species, such as Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, as well as Monascus purpureus Went and Escherichia coli, was tested by this assay, and the detection limit for A. fumigatus was 3 copies in a single reaction system. Validation experiments using the 25 clinical isolates demonstrated 91.7% specificity for the A. fumigatus section, and the sensitivity was 100% when first-generation sequencing was used as the standard. There was no significant difference between the PCR and CRISPR methods (P = 1.0), and the diagnosis results of the two methods were consistent (Kappa = 0.459, P = 0.003). Conclusion: The study offers a new validated CRISPR/Cas13a technique for A. fumigatus detection, providing a simple, rapid and affordable test that is ready for application in the diagnosis of A. fumigatus infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0084	Aspergillus fumigatus	\

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Author(s):

Fei Hu, Yanfei Liu, Shuhao Zhao, Zengming Zhang, Xichen Li, Niancai Peng, and Zhuangde Jiang

Journal:

Biosensors and Bioelectronics

Year:

2022

Abstract:

The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

PrimerBankID	Target Pathogen	Target Gene
RPB0202	SARS-CoV-2	\

A Recombinase Polymerase Amplification-Coupled Cas12a Mutant-Based Module for Efficient Detection of Streptomycin-Resistant Mutations in Mycobacterium tuberculosis

Author(s):

Peng Liu,Xinjie Wang,Juan Liang,Qian Dong,Jinping Zhang,Dongxin Liu,Shuai Wang,Jing Bi,Wenqi Liu,Zhaoqin Wang,Liang Chen,Lei Liu,Xingxu Huang,Guoliang Zhang

Journal:

Frontiers in microbiology

Year:

2022

Abstract:

Drug-resistant tuberculosis (TB) is a serious public health problem and threat to global TB prevention and control. Streptomycin (STR) is the earliest and classical anti-TB drug, and it is the earliest drug that generated resistance to anti-TB treatment, which limits its use in treating TB and impedes TB control efforts. The rapid, economical, and highly sensitive detection of STR-resistant TB may help reduce disease transmission and morbimortality. CRISPR/CRISPR-associated protein (Cas) is a new-generation pathogen detection method that can detect single-nucleotide polymorphisms with high sensitivity and good specificity. In this study, a Cas12a RR detection system that can recognize more non-traditional protospacer-adjacent motif-targeting sequences was developed based on Cas12a combined with recombinase polymerase amplification technology. This system detects 0.1% of the target substance, and the entire detection process can be completed within 60 min. Its sensitivity and specificity for detecting clinical STR-resistant Mycobacterium tuberculosis were both 100%. Overall, the Cas12 RR detection system provides a novel alternative for the rapid, simple, sensitive, and specific detection of STR-resistant TB, which may contribute to the prompt treatment and prevention of disease transmission in STR-resistant TB.

PrimerBankID	Target Pathogen	Target Gene
RPB0112	Mycobacterium	rpsl gene

iSCAN-V2: A One-Pot RT-RPA-CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection.

Author(s):

Aman, Rashid; Marsic, Tin; Sivakrishna Rao, Gundra; Mahas, Ahmed; Ali, Zahir; Alsanea, Madain; Al-Qahtani, Ahmed; Alhamlan, Fatimah; Mahfouz, Magdy;

Journal:

Front Bioeng Biotechnol

Year:

2022

Abstract:

Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0070	SARS-CoV-2	N gene

Rapid Detection and Differentiation of Legionella pneumophila and Non -Legionella pneumophila Species by Using Recombinase Polymerase Amplification Combined With EuNPs-Based Lateral Flow Immunochromatography

Author(s):

Jungang Du,Biao Ma,Jiali Li,Yaping Wang,Tianyu Dou,Shujuan Xu,Mingzhou Zhang

Journal:

Frontiers in chemistry

Year:

2022

Abstract:

Legionella, a waterborne pathogen, is the main cause of Legionnaires' disease. Therefore, timely and accurate detection and differentiation of Legionella pneumophila and non-Legionella pneumophila species is crucial. In this study, we develop an easy and rapid recombinase polymerase amplification assay combined with EuNPs-based lateral flow immunochromatography (EuNPs-LFIC-RPA) to specifically distinguish Legionella pneumophila and non-Legionella pneumophila. We designed primers based on the mip gene of Legionella pneumophila and the 5S rRNA gene of non-Legionella pneumophila. The recombinase polymerase amplification reaction could go to completion in 10 min at 37°C, and the amplification products could be detected within 5 min with EuNPs-LFIC strips. Using a florescent test strip reader, the quantitative results were achieved by reading the colored signal intensities on the strips. The sensitivity was 1.6 × 101 CFU/ml, and a linear standard linear curve plotted from the test strip reader had a correlation coefficient for the determination of Legionella pneumophila (R 2 = 0.9516). Completed concordance for the presence or absence of Legionella pneumophila by EuNPs-LFIC-RPA and qPCR was 97.32% (κ = 0.79, 95% CI), according to an analysis of practical water samples (n = 112). In short, this work shows the feasibility of EuNPs-LFIC-RPA for efficient and rapid monitoring of Legionella pneumophila and non-Legionella pneumophila in water samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0106	Legionella pneumophila	mip gene
RPB0107	Legionella pneumophila	5S rRNA gene

Enhanced Isothermal Amplification for Ultrafast Sensing of SARS-CoV-2 in Microdroplets

Author(s):

Mengyun Zhou , Chuan Fan , Lirong Wang , Tailin Xu , Xueji Zhang

Journal:

Analytical Chemistry

Year:

2022

Abstract:

Rapid and high-throughput screening is critical to control the COVID-19 pandemic. Recombinase polymerase amplification (RPA) with highly accessible and sensitive nucleic acid amplification has been widely used for point-of-care infection diagnosis. Here, we report an integrated microdroplet array platform composed of an ultrasonic unit and minipillar array to enhance the RPA for ultrafast, high-sensitivity, and high-throughput detection of SARS-CoV-2. On such a platform, the independent microvolume reactions on individual minipillars greatly decrease the consumption of reagents. The microstreaming driven by ultrasound creates on-demand contactless microagitation in the microdroplets and promotes the interaction between RPA components, thus greatly accelerating the amplification. In the presence of microstreaming, the detection time is 6-12 min, which is 38.8-59.3% shorter than that of controls without microstreaming, and the end-point fluorescence intensity also increased 1.3-1.7 times. Furthermore, the microagitation-enhanced RPA also exhibits a lower detection limit (0.42 copy/μL) for SARS-CoV-2 in comparison to the controls. This integrated microdroplet array detection platform is expected to meet the needs for high-throughput nucleic acid testing (NAT) to improve the containment of viral transmission during the epidemic, as well as provide a potential platform for the timely detection of other pathogens or viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0203	SARS-CoV-2	SARS-Cov-2 N gene

Integrated Trinity Test With RPA-CRISPR/Cas12a-Fluorescence for Real-Time Detection of Respiratory Syncytial Virus A or B

Author(s):

Ling Gong,Xiaowen Wang,Zhu Li,Guichuan Huang,Wei Zhang,Jin Nie,Chunyan Wu,Daishun Liu

Journal:

Frontiers in microbiology

Year:

2022

Abstract:

Respiratory syncytial virus (RSV) is a common virus that causes respiratory infection, especially severe respiratory infection in infants and young children, the elderly people over 65 years old, and people with weak immunity. Currently, RSV infection has no effective vaccine and antiviral treatment. The number of deaths due to RSV infection increases every year. Moreover, RSV A infection occurs in a large number and has severe clinical symptoms and complications than RSV B infection. Therefore, the development of a simple, rapid, and inexpensive detection method with high amplification efficiency, high sensitivity, and specificity is very important for the diagnosis of RSV A or RSV B infection, which can help in the early clinical medication and prevent the progress of the disease. Therefore, we developed an integrated trinity test with an RPA-CRISPR/Cas12a-fluorescence (termed IT-RAISE) assay system to detect RSV A or RSV B. The characteristic of the IT-RAISE system is that after target recognition, the reporter single-stranded DNA (ssDNA) is cleaved by Cas12a that is activated by different crRNAs to detect the generated fluorescent signal. This method is simple and helps in adding all reagents rapidly. It is a high-sensitive method that can detect 1.38 × 101 copies/μl of the target sequences, and it can distinguish RSV A or RSV B infection within 37 min. In addition, clinical specimens were detected for IT-RAISE system. It was found that the sensitivity and specificity of RSV A were 73.08 and 90%, respectively, and those of RSV B were 42.86 and 93.33%, respectively. The cost of ONE specimen for IT-RAISE system was approximately $ 2.6 (excluding rapid RNA extraction and reverse transcription costs). IT-RAISE system has good clinical application prospects for detecting RSV A or RSV B infection; it is a simple, rapid, and inexpensive method with high amplification efficiency, high sensitivity, and high specificity. The IT-RAISE system might also detect other viral or bacterial infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0126	RSV A	\
RPB0127	RSV B	\

Rapid One-Tube RPA-CRISPR/Cas12 Detection Platform for Methicillin-Resistant Staphylococcus aureus

Author(s):

Yanan Li,Zhonglin Shi,Anzhong Hu,Junsheng Cui,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu,Ling Zhu

Journal:

Diagnostics (Basel, Switzerland)

Year:

2022

Abstract:

Methicillin-resistant Staphylococcus aureus (MRSA) is a severe health threat causing high-level morbidity and mortality in health care environments and in community settings. Though existing diagnostic methods, including PCR and culture-based methods, are routinely used in clinical practice, they are not appropriate for rapid point-of-care testing (POCT). Recently, since the development of the CRISPR/Cas technology, new possibilities for rapid point-of-care detection have emerged. In this study, we developed a rapid, accurate, and contamination-free platform for MRSA detection by integrating recombinase polymerase amplification (RPA) with the Cas12 system into one tube. Using this approach, visual MRSA detection could be achieved in 20 min. Based on the one-tube RPA-CRISPR/Cas12a platform, the assay results are visualized by lateral flow test strips (LFS) and fluorescent-based methods, including real-time and end-point fluorescence. This platform allows specific MRSA detection with a sensitivity of 10 copies for the fluorescence method and a range of 10-100 copies for the LFS. The results of 23 samples from clinical MRSA isolates showed that the coincidence rate was 100% and 95.7% of the fluorescence method and LFS, respectively, compared to qPCR. In conclusion, the one-tube RPA-CRISPR/Cas12a platform is an effective method for MRSA detection with significant potential in future practical POCT applications.

PrimerBankID	Target Pathogen	Target Gene
RPB0158	Staphylococcus aureus	mecA gene

Visualized Genotyping from "Sample to Results" Within 25 Minutes by Coupling Recombinase Polymerase Amplification (RPA) With Allele-Specific Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

Author(s):

Likun Zhang , Xueping Ma , Danni Liu , Jingwen Shan , Yanan Chu , Jieyu Zhang , Xiemin Qi , Xiaohui Huang , Bingjie Zou, Guohua Zhou

Journal:

J Biomed Nanotechnol .J Biomed Nanotechnol J Biomed Nanotechnol

Year:

2022

Abstract:

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0206	SARS-CoV-2	CYP2C19*2
RPB0207	SARS-CoV-2	CYP2C19*3
RPB0208	SARS-CoV-2	A570D
RPB0209	SARS-CoV-2	D80A
RPB0210	SARS-CoV-2	T473K

Establishment and Clinical Application of a RPA-LFS Assay for Detection of Capsulated and Non-Capsulated Haemophilus influenzae

Author(s):

Yan Wang,Aibo Liu,Mei Fu,Jingjing Guo,Lei Wang,Xiaohua Zuo,Fenfen Ma

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

A recombinase polymerase amplification-lateral flow strip assay was established for detection of the outer membrane protein P6 (omp6) and the capsule encoding gene bexA of Haemophilus influenzae and the detection limit, sensitivity, and specificity were determined. Specific primers and probes were designed based on the published nucleotide sequences of omp6 and bexA. The minimum detection limit was determined with standard strains and the practical applicability of the RPA-LFS assay was assessed by detection of 209 clinical samples. The results confirmed that the RPA-LFS assay was both specific and sensitive for the detection of capsulated and non-capsulated H. influenzae with a detection limit of 1 CFU/µL. The detection rate of the 209 clinical samples was 97.1%, while the detection rate of capsulated H. influenzae was 63.2%. The detection results were consistent with the traditional culture method and dual polymerase chain reaction (PCR), confirming the applicability of the RPA-LFS assay.

PrimerBankID	Target Pathogen	Target Gene
RPB0096	Haemophilus influenzae	omp6
RPB0097	Haemophilus influenzae	bexA

Clinical Validation of a Rapid Variant-Proof RT-RPA Assay for the Detection of SARS-CoV-2

Author(s):

Dounia Cherkaoui, Judith Heaney, Da Huang,Matthew Byott, Benjamin S. Miller,Eleni Nastouli, and Rachel A. McKendry

Journal:

Diagnostics (Basel).

Year:

2022

Abstract:

The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.

PrimerBankID	Target Pathogen	Target Gene
RPB0200	SARS-CoV-2	E gene
RPB0201	SARS-CoV-2	RdRP gene

Glycerol Additive Boosts 100-fold Sensitivity Enhancement for One-Pot RPA-CRISPR/Cas12a Assay

Author(s):

Mei Lin , Huahua Yue , Tian Tian , Erhu Xiong , Debin Zhu , Yongzhong Jiang , Xiaoming Zhou

Journal:

Analytical Chemistry

Year:

2022

Abstract:

CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.

PrimerBankID	Target Pathogen	Target Gene
RPB0198	SARS-CoV-2	Orf1ab-gene
RPB0199	SARS-CoV-2	N-gene

Development and Clinical Application of a Recombinase Polymerase Amplification-Lateral Flow Strip Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii

Author(s):

Lei Wang,Dunpo Sun,Li Chen,Ping Zhou,Kun Wang,Fang Wang,Xingqi Lei,Yan Wang,Yingzhi Lu,Guanhong Huang,Xuzhu Gao

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Acinetobacter baumannii is a worldwide, primary cause of respiratory tract infections, septicemia, urinary apparatus infections, and secondary meningitis. It can be fatal. Rapid and accurate detection methods are needed to control the spread of carbapenem-resistant A. baumannii (CRAB). Current molecular diagnostic methods are limited and not suitable for on-site detection. In this study, an isothermal detection method using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) was developed to target the blaOXA-51 and blaOXA-23 genes of A. baumannii. The reaction was completed in about 40 min at 37°C. This method can also effectively distinguish A. baumannii and CRAB. The limit of detection of 100-101 CFU/reaction was equal to that of other detection methods. The detection accuracy was equal to that of the qPCR method with the use of clinical samples. The RPA-LFS assay is portable, rapid, and accurate and could replace existing detection methods for on-site detection of A. baumannii and CRAB.

PrimerBankID	Target Pathogen	Target Gene
RPB0080	Acinetobacter baumannii	bla OXA-51
RPB0081	Acinetobacter baumannii	bla OXA-23

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2

Author(s):

Gaihua Cao, Danqun Huo, Xiaolong Chen, Xianfeng Wang, Shiying Zhou, Shixian Zhao, Xiaogang Luo, Changjun Hou

Journal:

Talanta

Year:

2022

Abstract:

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min. Most important of all, we developed an automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) with a 3D-printed microfluidic chip for sensitively detecting SARS-CoV-2, which addressed aerosol contamination issue and provided a more accurate and high-throughput detection during the on-site detection process. The detection limits of S gene and Orf1ab gene were as low as 0.68 fM and 4.16 fM. Furthermore, we used the lateral flow strip to realize visualization and point of care testing (POCT) of SARS-CoV-2. Therefore, profit from the efficient amplification of RPA and the high specificity of CRISPR/Cas13a, APHF-analyzer and the lateral flow strip to simultaneous detection of S gene and Orf1ab gene would be applied as a promising tool in the field of SARS-CoV-2 detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0037	SARS-CoV-2	S
RPB0038	SARS-CoV-2	ORF1ab

Rapid Detection of Klebsiella pneumoniae Carrying Virulence Gene rmpA2 by Recombinase Polymerase Amplification Combined With Lateral Flow Strips

Author(s):

Na Li,Lei Wang,Fang Wang,Huimin Chen,Shuan Tao,Qing Zhu,Liping Liu,Wei Liang,Fang Ma

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Highly virulent Klebsiella pneumoniae often causes invasive infections with high morbidity and mortality rates, posing an immense clinical challenge. Rapid and accurate detection of pathogenic bacteria is of great significance for treatment and preventive control. Conventional detection by polymerase chain reaction (PCR) is limited by a dependence on laboratory equipment and professional staff. Recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS) can rapidly amplify and visualize target genes in a short period of time. The aim of this study was to develop an RPA-LFS technique for detection of the K. pneumoniae virulence gene rmpA2. Primers were designed against conserved sequences specific to the virulence gene, and primer probe design was optimized by introducing base substitution to obtain a specific and sensitive primer-probe combination for clinical detection. We tested 65 actual samples collected from clinics to evaluate the performance of the newly established RPA-LFS system in comparison with conventional PCR methods and qPCR methods. The RPA-LFS assay was performed at for 25 min a constant temperature of 37°C, and results could be observed without instrumentation. The system could specifically identify highly virulent K. pneumoniae carrying the virulence gene rmpA2 with a minimum detection limit of 10-1 ng/μL and 10 copies/μL. For the 65 clinical samples tested, The RPA-LFS assay results were in complete agreement with the qPCR results and PCR results. The RPA-LFS assay provides a rapid, accurate, and simple method for identification of highly virulent K. pneumoniae carrying rmpA2.

PrimerBankID	Target Pathogen	Target Gene
RPB0103	Klebsiella pneumoniae	rmpA2

Rapid, Simple, and Highly Specific Detection of Streptococcus pneumoniae With Visualized Recombinase Polymerase Amplification

Author(s):

Fang Wang,Yan Wang,Xia Liu,Lei Wang,Kun Wang,Chenglai Xu,Guanhong Huang,Xuzhu Gao

Journal:

Frontiers in cellular and infection microbiology

Year:

2022

Abstract:

Streptococcus pneumoniae is a major pathogen that causes microbiological illness in humans. The introduction of polyvalent vaccines has resulted in a significant decrease in pneumococcal-related mortality. However, pneumococcal infections continue to be a leading cause of death in children under the age of 5 and adults over the age of 65 worldwide. A speedy and highly sensitive diagnostic tool is necessary for routine adoption to adequately manage patients and control the spread of infection. In this study, we investigated a new nucleic acid amplification technique, isothermal recombinase polymerase amplification (RPA), which amplifies DNA at 37°C under isothermal conditions with high specificity, efficiency, and rapidity. Using the autolysin gene lytA as the molecular diagnostic target, an RPA primer-probe combination was designed and optimized for the detection of S. pneumoniae. This RPA reaction produced amplification products labeled with specific chemical markers, to be detected with gold-nanoparticle-based lateral flow strips (LFS), reducing the reliance on equipment and trained personnel. The high specificity of the RPA-LFS technique was demonstrated with the specific detection of 22 strains of S. pneumoniae but not 25 closely related pathogenic bacteria. The assay showed good sensitivity, and detected S. pneumoniae down to 3.32 colony-forming units/μL. When used on clinical samples, the assay provided accurate and consistent results compared with PCR. The compliance with the culture-biochemistry method was 98.18% and the kappa index was 0.977. These results reveal that the RPA-LFS test significantly improved S. pneumoniae identification, particularly in resource-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0165	Streptococcus pneumoniae	lytA

Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics

Author(s):

Menglu Hu, Zhiqiang Qiu, Zirong Bi, Tian Tian, Yongzhong Jiang, and Xiaoming Zhou

Journal:

Proceedings of the National Academy of Sciences of the United States of America

Year:

2022

Abstract:

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.

PrimerBankID	Target Pathogen	Target Gene
RPB0196	SARS-CoV-2	O-gene
RPB0197	SARS-CoV-2	N-gene

Rapid detection of methicillin-resistant Staphylococcus aureus in positive blood-cultures by recombinase polymerase amplification combined with lateral flow strip

Author(s):

Arpasiri Srisrattakarn,Pimchanok Panpru,Patcharaporn Tippayawat,Aroonwadee Chanawong,Ratree Tavichakorntrakool,Jureerut Daduang,Lumyai Wonglakorn,Aroonlug Lulitanond

Journal:

PloS one

Year:

2022

Abstract:

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), is an important bacterium that causes community and healthcare-related infections throughout the world. However, the current conventional detection methods are time-consuming. We therefore developed and evaluated a recombinase polymerase amplification-lateral flow strip (RPA-LF) approach for detection of MRSA in positive blood-culture samples. Sixty positive blood-cultures from a hospital were tested directly without DNA extraction and purification before the amplification reaction. RPA primers and probes were designed for nuc (encoding thermonuclease) and mecA (encoding penicillin-binding protein 2a) genes to diagnose S. aureus and its methicillin-resistance status. The RPA reaction occurred under isothermal conditions (45°C) within 20 min and a result was provided by the LF strip in a further 5 min at room temperature. The evaluation of RPA-LF using blood-culture samples showed 93.3% (14/15) sensitivity for identifying S. aureus, and no cross-amplification was seen [100% (45/45) specificity]. For detection of methicillin resistance, the RPA-LF test provided 100% (16/16) sensitivity and 97.7% (43/44) specificity. The RPA-LF is rapid, highly sensitive, robust and easy to use. It can be used for direct detection of MRSA with no requirement for special equipment.

PrimerBankID	Target Pathogen	Target Gene
RPB0156	Staphylococcus aureus	nuc
RPB0157	Staphylococcus aureus	mecA

Multiple ligation-Assisted recombinase polymerase amplification for highly sensitive and selective colorimetric detection of SARS-CoV-2

Author(s):

Tasnima Alam Asa, Pradeep Kumar, Jaehyeon Lee, Young Jun Seo

Journal:

Talanta

Year:

2023

Abstract:

In this paper we present a new method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), targeting a specific region "N gene." Under isothermal reaction conditions, we integrated ligation (Lig; high selectivity) and recombinase polymerase amplification (RPA; high sensitivity) processes, obtaining a robust method of detection. For point-of-care testing, we incorporated our laboratory-produced pyrophosphate ion (PPi)-sensing probe (PK-probe) for colorimetric analysis of the reaction. The total detection system was efficient and effective at diagnosing this RNA virus-mediated disease rapidly (30 min). In a full-genome SARS-CoV-2 study, our PK-probe/Lig-RPA system functioned with a limit of detection of 1160 copies/ml, with a single-mismatch level of selectively, and it was highly selective even in the presence of bacterial genomes commonly found in the human mouth and nose. This robust, straightforward, selective, efficient, and ultrasensitive colorimetric detection method, with potential for point-of-care analysis, should also be effective in detecting a diverse range of other RNA-based diseases.

PrimerBankID	Target Pathogen	Target Gene
RPB0019	SARS-CoV-2	N

Multiplexed lateral flow assay integrated with orthogonal CRISPR-Cas system for SARS-CoV-2 detection

Author(s):

Gaoxing Su,Min Zhu,Diyuan Li,Mengting Xu,Yuedong Zhu,Yan Zhang,Hongyan Zhu,Feng Li,Yanyan Yu

Journal:

Sensors and Actuators:B. Chemical

Year:

2022

Abstract:

The development of field-deployable detection platform amenable for multiplexed genes testing will significantly improve the efficiency and reliability during point-of-care testing (POCT) applications. In this regard, an orthogonal CRISPR-Cas-mediated multiplexed lateral flow assay (designated as OC-MLFA) is proposed for SARS-CoV-2 genome detection. Taking the advantage of activation and cleavage preferences between Cas12a and Cas13a, orthogonal (two-independent-channel signal readout) CRISPR-Cas system is investigated. Lateral flow strips with two target lines are designed to accommodate the orthogonal CRISPR system. The interference between Cas12a and Cas13a channels can be effectively eliminated via the elaborate nucleic acids and lateral flow strips design. The high preamplification efficiency from reverse transcription recombinase polymerase amplification (RT-RPA) and Cas enzyme mediated trans-cleavage process bring the sensitivity of our OC-MLFA method to 10 copies per test (30 μL). Nasopharyngeal swab clinical samples with different cycle threshold (Ct) values according to the RT-PCR method were analyzed with the proposed OC-MLFA, during which 76 out of 76 detection accuracy was obtained. Featured with the multiplexed genes detection simultaneously in one reaction and colorimetric readout through single strip, the OC-MLFA we proposed herein ensures great accuracy and efficiency, which endows promising field-deployable POCT application feasibility.

PrimerBankID	Target Pathogen	Target Gene
RPB0033	SARS-CoV-2	ORF1ab
RPB0034	SARS-CoV-2	N

Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation

Author(s):

Ya-Lei Wang,Xin Zhang,Quan Wang,Peng-Xuan Liu,Wei Tang,Rong Guo,Hai-Yang Zhang,Zhao-Guo Chen,Xian-Gan Han,Wei Jiang

Journal:

Journal of applied microbiology

Year:

2022

Abstract:

Aims: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. Methods and results: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. Conclusions: The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. Significance and impact of the study: The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

PrimerBankID	Target Pathogen	Target Gene
RPB0163	Staphylococcus aureus	nuc

Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B

Author(s):

Li-Guo Liang,Miao-Jin Zhu,Rui He,Dan-Rong Shi,Rui Luo,Jia Ji,Lin-Fang Cheng,Xiang-Yun Lu,Wei Lu,Fu-Ming Liu,Zhi-Gang Wu,Nan-Ping Wu,Hang Chen,Zhe Chen,Hang-Ping Yao

Journal:

journal of medical virology

Year:

2023

Abstract:

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0021	SARS-CoV-2	N
RPB0022	SARS-CoV-2	E
RPB0023	SARS-CoV-2	ORF1ab
RPB0024	Influenza A virus (H1N1)	M
RPB0025	Influenza A virus (H3N2)	HA
RPB0026	Influenza B virus	PA

Computer vision enabled funnel adapted sensing tube (FAST) for power-free and pipette-free nucleic acid detection

Author(s):

Mengdi Bao,Shuhuan Zhang,Chad Ten Pas,Stephen J Dollery,Ruth V Bushnell,F N U Yuqing,Rui Liu,Guoyu Lu,Gregory J Tobin,Ke Du

Journal:

Lab On A Chip

Year:

2022

Abstract:

A simple, portable, and low-cost microfluidic system-funnel adapted sensing tube (FAST) is developed as an integrated, power-free, and pipette-free biosensor for viral nucleic acids. This FAST chip consists of four reaction chambers separated by carbon fiber rods, and the reagents in each chamber are transferred and mixed by manually removing the rods. Rather than using electrical heaters, only a hand warmer pouch is used for an isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a reaction. The signal produced by the RPA-CRISPR reaction is observed by the naked eye using an inexpensive flashlight as a light source. The FAST chip is fabricated using water-soluble polyvinyl alcohol (PVA) as a sacrificial core, which is simple and environmentally friendly. Using a SARS-CoV-2 fragment as a target, a ∼10 fM (6 × 103 copies per μL) detection limit is achieved. To generalize standard optical readout for individuals without training, a linear kernel algorithm is created, showing an accuracy of ∼100% for identifying both positive and negative samples in FAST. This power-free, pipette-free, disposable, and simple device will be a promising tool for nucleic acid diagnostics in either clinics or low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0031	SARS-CoV-2	S

Nanozyme-strip for rapid and ultrasensitive nucleic acid detection of SARS-CoV-2

Author(s):

Xiangqin Meng, Sijia Zou, Dandan Li , Jiuyang He, Ling Fang, Haojue Wang, Xiyun Yan, Demin Duan, Lizeng Gao

Journal:

Biosensors & Bioelectronics

Year:

2022

Abstract:

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0032	SARS-CoV-2	ORF1ab

Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system and recombinase polymerase amplification

Author(s):

Luyu Wei,Zhilong Wang,Jia Wang,Xiaohong Wang,Yiping Chen

Journal:

Analytica chimica acta

Year:

2022

Abstract:

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with superior accuracy, timeliness, and simplicity is highly valuable in clinical diagnosis and food safety. In this study, an aptamer-based colorimetric biosensor was developed to detect MRSA by using a CRISPR/Cas12a system and recombinase polymerase amplification (RPA). The aptamer of silver ion (Ag+) pre-coupled to magnetic nanoparticles was employed not only as the substrate of trans-cleavage in the CRISPR/Cas12a system, but also as the modulator of Ag+-3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction, innovatively integrating the powerful CRISPR/Cas12a system with convenient colorimetry. The utilized aptamer containing consecutive and interrupted cytosine: cytosine mismatched base pairs also served as a signal amplifier because of the one-to-multiple binding of the aptamer to Ag+. Using triple amplification of RPA, multiple-turnover nuclease activity of Cas12a, and cytosine-Ag+-cytosine coordination chemistry, MRSA was detected as low as 8 CFU mL-1. Moreover, its satisfactory accuracy in the analysis of real samples, together with visualization and simplicity, revealed the great potential of the proposed biosensor as a robust antibiotic-resistant bacteria detection platform.

PrimerBankID	Target Pathogen	Target Gene
RPB0162	Staphylococcus aureus	mecA

Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus

Author(s):

Zhonglin Shi,Yanan Li,Anzhong Hu,Junsheng Cui,Min Shao,Ling Zhu,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu

Journal:

Journal of microbiological methods

Year:

2022

Abstract:

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus.

PrimerBankID	Target Pathogen	Target Gene
RPB0160	Staphylococcus aureus	mecA
RPB0161	Staphylococcus aureus	ermA

A New Auto-RPA-Fluorescence Detection Platform for SARS-CoV-2

Author(s):

Jing Tian, Biao Chen, Bin Zhang, Tantan Li, Zhiqiang Liang, Yujin Guo, Huping Jiao, Fenghong Liang, Longquan Xiang, Fanzhong Lin, Ruiwen Ren, Qingbin Liu

Journal:

Laboratory Medicine

Year:

2023

Abstract:

Objective: The outbreak of COVID-19 caused by SARS-CoV-2 has led to a serious worldwide pandemic. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)-based methods were recommended for routine detection of SARS-CoV-2 RNA. Because the reaction time and analytical sensitivity of qRT-PCR limits the diagnosis of SARS-CoV-2, development of a quick process of SARS-CoV-2 detection technology with high analytical sensitivity remains urgent. Methods: We combined isothermal amplification and fluorescence detection technology to develop a new auto-recombinase polymerase amplification (RPA)-fluorescence platform that could be used in the diagnosis of SARS-CoV-2. Results: By optimization of primers and probes, the RPA platform could detect SARS-CoV-2 nucleotides within 15 min. The limits of detection and specificity of the auto-RPA-fluorescence platform were 5 copies/µL and 100%, respectively. The accuracy of detection of the auto-RPA-fluorescence platform in the 16 positive samples was 100%. Conclusion: The RPA platform is a potential technology for the diagnosis of SARS-CoV-2 infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0017	SARS-CoV-2	N
RPB0018	SARS-CoV-2	S

Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

Author(s):

Aline Dorendorf,Iris Bachmann,Martin Spiegel,Ahmed Abd El Wahed,Gregory Dame,Frank Hufert

Journal:

Journal of clinical virology plus

Year:

2022

Abstract:

Background: Human coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children. Objectives: For fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. Study design: The analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed. Results: Our HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively. Conclusions: We were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.

PrimerBankID	Target Pathogen	Target Gene
RPB0093	HCoV-NL63	N-gene
RPB0171	HCoV-NL63	N-gene

An ultrasensitive and rapid "sample-to-answer" microsystem for on-site monitoring of SARS-CoV-2 in aerosols using "in situ" tetra-primer recombinase polymerase amplification

Author(s):

Shanglin Li, Bao Li, Xinyue Li, Ce Liu, Xiao Qi, Yin Gu, Baobao Lin, Lingli Sun, Lan Chen, Bingqian Han, Jiazhen Guo, Yanyi Huang, Shuangsheng Wu, Lili Ren, Jianbin Wang, Jingwei Bai, Jianxin Ma, Maosheng Yao, Peng Liu

Journal:

Biosensors & Bioelectronics

Year:

2022

Abstract:

Airborne transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for aerosol monitoring of SARS-CoV-2 to prevent sporadic outbreaks of COVID-19. The inadequate sensitivity of conventional methods and the lack of an on-site detection system limited the practical SARS-CoV-2 monitoring of aerosols in public spaces. We have developed a novel SARS-CoV-2-in-aerosol monitoring system (SIAMs) which consists of multiple portable cyclone samplers for collecting aerosols from several venues and a sensitive "sample-to-answer" microsystem employing an integrated cartridge for the analysis of SARS-CoV-2 in aerosols (iCASA) near the sampling site. By seamlessly combining viral RNA extraction based on a chitosan-modified quartz filter and "in situ" tetra-primer recombinase polymerase amplification (tpRPA) into an integrated microfluidic cartridge, iCASA can provide an ultra-high sensitivity of 20 copies/mL, which is nearly one order of magnitude greater than that of the commercial kit, and a short turnaround time of 25 min. By testing various clinical samples of nasopharyngeal swabs, saliva, and exhaled breath condensates obtained from 23 COVID-19 patients, we demonstrate that the positive rate of our system was 3.3 times higher than those of the conventional method. Combining with multiple portable cyclone samplers, we detected 52.2% (12/23) of the aerosol samples, six times higher than that of the commercial kit, collected from the isolation wards of COVID-19 patients, demonstrating the excellent performance of our system for SARS-CoV-2-in-aerosol monitoring. We envision the broad application of our microsystem in aerosol monitoring for fighting the COVID-19 pandemic.

PrimerBankID	Target Pathogen	Target Gene
RPB0036	SARS-CoV-2	ORF1ab

Recombinase Polymerase Amplification Combined with Fluorescence Immunochromatography Assay for On-Site and Ultrasensitive Detection of SARS-CoV-2

Author(s):

Guangyu Wang , Xingsheng Yang, Hao Dong, Zhijie Tu , Yong Zhou, Zhen Rong , Shengqi Wang

Journal:

Pathogens

Year:

2022

Abstract:

This study established a portable and ultrasensitive detection method based on recombinase polymerase amplification (RPA) combined with high-sensitivity multilayer quantum dot (MQD)-based immunochromatographic assay (ICA) to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RPA-MQD-based ICA method is reported for the first time and has the following advantages: (i) RPA is free from the constraints of instruments and can be promoted in point-of-care testing (POCT) scenarios, (ii) fluorescence ICA enhances the portability of detection operation so that the entire operation time is controlled within 1 h, and (iii) compared with common colorimetric-based RPA-ICA, the proposed assay used MQD to provide strong and quantifiable fluorescence signal, thus enhancing the detection sensitivity. With this strategy, the proposed RPA-MQD-based ICA can amplify and detect the SARS-CoV-2 nucleic acid on-site with a sensitivity of 2 copies/reaction, which is comparable to the sensitivity of commercial reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Moreover, the designed primers did not cross-react with other common respiratory viruses, including adenovirus, influenza virus A, and influenza virus B, suggesting high specificity. Thus, the established portable method can sensitively detect SARS-CoV-2 nucleic acid without relying on equipment, having good application prospects in SARS-CoV-2 detection scenarios under non-lab conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0035	SARS-CoV-2	S

A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance

Author(s):

Maturada Patchsung,Aimorn Homchan,Kanokpol Aphicho,Surased Suraritdechachai,Thanyapat Wanitchanon,Archiraya Pattama,Khomkrit Sappakhaw,Piyachat Meesawat,Thanakrit Wongsatit,Artittaya Athipanyasilp,Krittapas Jantarug,Niracha Athipanyasilp,Juthamas Buahom,Supapat Visanpattanasin,Nootaree Niljianskul,Pimchai Chaiyen,Ruchanok Tinikul,Nuanjun Wichukchinda,Surakameth Mahasirimongkol,Rujipas Sirijatuphat,Nasikarn Angkasekwinai,Michael A Crone,Paul S Freemont,Julia Joung,Alim Ladha,Omar Abudayyeh,Jonathan Gootenberg,Feng Zhang,Claire Chewapreecha,Sittinan Chanarat,Navin Horthongkham,Danaya Pakotiprapha,Chayasith Uttamapinant

Journal:

The CRISPR Journal

Year:

2023

Abstract:

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0425	SARS-CoV-2	N
RPB0426	SARS-CoV-2	S gene

Sensitive and rapid RT-RPA-Cas12a-mediated detection method capable of human rhinovirus A and/or C species by targeting VP4

Author(s):

Weidong Qian,Xuefei Wang,Jie Huang,Jian Liu,Si Chen,Ting Wang,Dandan Zhang,Yongdong Li

Journal:

Virus research

Year:

2022

Abstract:

Human rhinovirus (HRV), the main etiologic agent of the common cold, is responsible for significant morbidity, medical costs, and the loss of productivity in the workplace and school. To prevent the spread of HRV, accurate, low-cost and rapid diagnostics of HRV is crucial for identifying those at-risk for the illness associated with HRV, with the most frequently detected species, including HRV species A (HRV-A) and C (HRV-C). Here, a novel HRV-A and/or HRV-C molecular diagnostic assay that integrates reverse-transcription recombinase polymerase amplification assay (RT-RPA) amplification with CRISPR/Cas12a detection, with the result readout using a fluorescence detector or lateral flow strip (LFS). The established assay could be completed within 50 min without complex instruments and skilled technicians. The limit of detection of the RT-RPA-Cas12a-mediated real-time fluorescence or LFS assay could reach 0.1 copy/μl, and 0.5 copy/μl for the end-point fluorescence assay with a UV light illuminator readout, respectively. Meanwhile, the assay demonstrates excellent specificity without cross-reactivity to non-target viruses. Furthermore, they were appraised using 80 clinical samples, and RT-RPA-Cas12a-mediated fluorescence or LFS assay displayed high-accuracy with positive and negative predictive agreement of 96.7%, 95% and 100%, respectively. Taken together, the RT-RPA-Cas12a-mediated assay is a rapid, sensitive, and specific detection tool for routine and on-site detection method for HRV-A and/or HRV-C infections, and shows great promise for use in resource-poor or constrained settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0095	HRV	VP4

Rapid and Sensitive Genotyping of SARS-CoV-2 Key Mutation L452R with an RPA- Pf Ago Method

Author(s):

Chenjie Zhao , Lihong Yang , Xue Zhang, Yixin Tang, Yue Wang, Xiaofu Shao, Song Gao, Xin Liu, Pei Wang

Journal:

Analytical Chemistry

Year:

2022

Abstract:

In the two years of COVID-19 pandemic, the SARS-CoV-2 variants have caused waves of infections one after another, and the pandemic is not ending. The key mutations on the S protein enable the variants with enhanced viral infectivity, immune evasion, and/or antibody neutralization resistance, bringing difficulties to epidemic prevention and control. In support of precise epidemic control and precision medicine of the virus, a fast and simple genotyping method for the key mutations of SARS-CoV-2 variants needs to be developed. By utilizing the specific recognition and cleavage property of the nuclease Argonaute from Pyrococcus furiosus (PfAgo), we developed a recombinase polymerase amplification (RPA) and PfAgo combined method for a rapid and sensitive genotyping of SARS-CoV-2 key mutation L452R. With a delicate design of the strategy, careful screening of the RPA primers and PfAgo gDNA, and optimization of the reaction, the method achieves a high sensitivity of a single copy per reaction, which is validated with the pseudovirus. This is the highest sensitivity that can be achieved theoretically and the highest sensitivity as compared to the available SARS-CoV-2 genotyping assays. Using RPA, the procedure of the method is finished within 1.5 h and only needs a minimum laboratorial support, suggesting that the method can be easily applied locally or on-site. The RPA-PfAgo method established in this study provides a strong support to the precise epidemic control and precision medicine of SARS-CoV-2 variants and can be readily developed for the simultaneous genotyping of multiple SARS-CoV-2 mutations.

PrimerBankID	Target Pathogen	Target Gene
RPB0030	SARS-CoV-2	S（containing the L452R mutation site）

Specific lateral flow detection of isothermal nucleic acid amplicons for accurate point-of-care testing

Author(s):

Ting Zheng, Xianming Li, Yanjun Si, Minjin Wang, Yuzhen Zhou, Yusheng Yang, Na Liang, Binwu Ying, Peng Wu

Journal:

Biosensors & Bioelectronics

Year:

2023

Abstract:

For point-of-care testing (POCT), coupling isothermal nucleic acid amplification schemes (e.g., recombinase polymerase amplification, RPA) with lateral flow assay (LFA) readout is an ideal platform, since such integration offers both high sensitivity and deployability. However, isothermal schemes typically suffers from non-specific amplification, which is difficult to be differentiated by LFA and thus results in false-positives. Here, we proposed an accurate POCT platform by specific recognition of target amplicons with peptide nucleic acid (PNA, assisted by T7 Exonuclease), which could be directly plugged into the existing RPA kits and commercial LFA test strips. With SARS-CoV-2 as the model, the proposed method (RPA-TeaPNA-LFA) efficiently eliminated the false-positives, exhibiting a lowest detection concentration of 6.7 copies/μL of RNA and 90 copies/μL of virus. Using dual-gene (orf1ab and N genes of SARS-CoV-2) as the targets, RPA-TeaPNA-LFA offered a high specificity (100%) and sensitivity (RT-PCR Ct < 31, 100%; Ct < 40, 71.4%), and is valuable for on-site screening or self-testing during isolation. In addition, the dual test lines in the test strips were successfully explored for simultaneous detection of SARS-CoV-2 and H1N1, showing great potential in response to future pathogen-based pandemics.

PrimerBankID	Target Pathogen	Target Gene
RPB0027	SARS-CoV-2	ORF1ab
RPB0028	SARS-CoV-2	N
RPB0029	Influenza A virus (H1N1)	M1
RPB0410	SARS-CoV-2	ORF1ab gene
RPB0411	Influenza A virus (H1N1)	N gene

Rapid and Visual Detection of SARS-CoV-2 RNA Based on Reverse Transcription-Recombinase Polymerase Amplification with Closed Vertical Flow Visualization Strip Assay

Author(s):

Song, Yumeng; Huang, Pei; Yu, Mengtao; Li, Yuanguo; Jin, Hongli; Qiu, Jiazhang; Li, Yuanyuan; Gao, Yuwei; Zhang, Haili; Wang, Hualei;

Journal:

Microbiol Spectr

Year:

2023

Abstract:

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was initially identified in 2019, after which it spread rapidly throughout the world. With the progression of the epidemic, new variants of SARS-CoV-2 with faster transmission speeds and higher infectivity have constantly emerged. The proportions of people asymptomatically infected or reinfected after vaccination have increased correspondingly, making the prevention and control of COVID-19 extremely difficult. There is therefore an urgent need for rapid, convenient, and inexpensive detection methods. In this paper, we established a nucleic acid visualization assay targeting the SARS-CoV-2 nucleoprotein (N) gene by combining reverse transcription-recombinase polymerase amplification with closed vertical flow visualization strip (RT-RPA-VF). This method had high sensitivity, comparable to that of reverse transcription-quantitative PCR (RT-qPCR), and the concordance between RT-RPA-VF and RT-qPCR methods was 100%. This detection method is highly specific and is not compatible with bat coronavirus HKU4, human coronaviruses 229E, OC43, and HKU1-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), or other respiratory pathogens. However, multiple SARS-CoV-2 variants are detectable within 25鈥塵in at 42掳C using this visual method, including RNA transcripts of the Wuhan-Hu-1 strain at levels as low as 1 copy/渭L, the Delta strain at 1 copy/渭L, and the Omicron strain at 0.77 copies/渭L. The RT-RPA-VF method is a simple operation for the rapid diagnosis of COVID-19 that is safe and free from aerosol contamination and could be an affordable and attractive choice for governments seeking to promote their emergency preparedness and better their responses to the continuing COVID-19 epidemic. In addition, this method also has great potential for early monitoring and warning of the epidemic situation at on-site-nursing points. IMPORTANCE The global COVID-19 epidemic, ongoing since the initial outbreak in 2019, has caused panic and huge economic losses worldwide. Due to the continuous emergence of new variants, COVID-19 has been responsible for a higher proportion of asymptomatic patients than the previously identified SARS and MERS, which makes early diagnosis and prevention more difficult. In this manuscript, we describe a rapid, sensitive, and specific detection tool, RT-RPA-VF. This tool provides a new alternative for the detection of SARS-CoV-2 variants in a range as low as 1 to 0.77 copies/渭L RNA transcripts. RT-RPA-VF has great potential to ease the pressure of medical diagnosis and the accurate identification of patients with suspected COVID-19 at point-of-care.

PrimerBankID	Target Pathogen	Target Gene
RPB0040	MERS-CoV	N gene

Rapid detection of influenza A viruses using a real-time reverse transcription recombinase-aided amplification assay

Author(s):

Huan Cui,Cheng Zhang,Fei Tu,Kui Zhao,Yunyi Kong,Jie Pu,Lei Zhang,Zhaoliang Chen,Yuanyuan Sun,Yujie Wei,Chuncai Liang,Juxiang Liu,Jun Liu,Zhendong Guo

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2023

Abstract:

Introduction: Influenza A viruses (IAVs) are important pathogens of respiratory infections, causing not only seasonal influenza but also influenza pandemics and posing a global threat to public health. IAVs infection spreads rapidly, widely, and across species, causing huge losses, especially zoonotic IAVs infections that are more harmful. Fast and sensitive detection of IAVs is critical for controlling the spread of this disease. Methods: Here, a real-time reverse transcription recombinase-aided amplification (real-time RT-RAA) assay targeting conserved positions in the matrix protein gene (M gene) of IAVs, is successfully established to detect IAVs. The assay can be completed within 20 min at 42°C. Results: The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability, which was comparable to the sensitivity of the RT-qPCR assay. The specificity assay showed that the real-time RT-RAA assay was specific to IAVs, and there was no cross-reactivity with other important viruses. In addition, 100%concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 120 clinical specimens. Discussion: The results suggested that the real-time RT-RAA assay we developed was a specific, sensitive and reliable diagnostic tool for the rapid detection of IAVs.

PrimerBankID	Target Pathogen	Target Gene
RPB0389	Influenza A virus	M gene

Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins13a combined with magnetic beads, chemiluminescence and reverse transcription-recombinase aided amplification for detection of avian influenza a (H7N9) virus

Author(s):

Hongpan Xu,Lijun Peng,Jie Wu,Adeel Khan,Yifan Sun,Han Shen,Zhiyang Li

Journal:

Frontiers in Bioengineering and Biotechnology

Year:

2023

Abstract:

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins (CRISPR-Cas) have promising prospects in the field of nucleic acid molecular diagnostics. However, Clustered Regularly Interspaced Short Palindromic Repeats-based fluorescence detection technology is mainly hindered by proteins with conjugated double bonds and autofluorescence, resulting in high fluorescence background, low sensitivity and incompatible reaction systems, which are not conducive to automatic clinical testing. Chemiluminescence (CL) detection technology has been applied mainly owing to its greatly high sensitivity, as well as low background and rapid response. Therefore, we developed a rapid, ultrasensitive and economical detection system based on Clustered Regularly Interspaced Short Palindromic Repeats-Clustered Regularly Interspaced Short Palindromic Repeats-Associated Proteins 13a combined with magnetic beads (MBs) and chemiluminescence (CL) (Cas13a-MB-CL) to detect Influenza A (H7N9), an acute respiratory tract infectious disease. The carboxyl functionalized magnetic beads (MBs-COOH) were covalently coupled with aminated RNA probe while the other end of the RNA probe was modified with biotin. Alkaline phosphatase labeled streptavidin (SA-ALP) binds with biotin to form magnetic beads composites. In presence of target RNA, the collateral cleavage activity of Cas13a was activated to degrade the RNA probes on MBs and released Alkaline phosphatase from the composites. The composites were then magnetically separated followed by addition of ALP substrate Disodium 2-chloro-5-{4-methoxyspiro [1,2-dioxetane-3,2'-(5'-chloro) tricyclo (3.3.1.13,7) decan]-4-yl}-1-phenyl phosphate (CDP-star), to generate the chemiluminescence signal. The activity of Associated Proteins 13a and presence of target RNA was quantified by measuring the chemiluminescence intensity. The proposed method accomplished the detection of H7N9 within 30 min at 25°C. When combined with Reverse Transcription- Recombinase Aides Amplification (RT-RAA), the low detection limit limit of detection was as low as 19.7 fM (3S/N). Our proposed MB-Associated Proteins 13a-chemiluminescence was further evaluated to test H7N9 clinical samples, showing superior sensitivity and specificity.

PrimerBankID	Target Pathogen	Target Gene
RPB0403	Influenza A virus (H7N9)	HA
RPB0404	Influenza A virus (H7N9)	NA

A versatile integrated tube for rapid and visual SARS-CoV-2 detection

Author(s):

Jingsong Xu,Xi Wang,Shuang Yang,Lei He,Yuting Wang,Jiajun Li,Qian Liu,Min Li,Hua Wang

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

The coronavirus disease 2019 (COVID-19) caused by novel severe acute respiratory coronavirus 2 (SARS-CoV-2) has been rapidly spreading worldwide. Rapid and widespread testing is essential to promote early intervention and curb the ongoing COVID-19 pandemic. Current gold standard reverse transcription-polymerase chain reaction (RT-PCR) for detecting SARS-CoV-2 is restricted to professional laboratories and well-trained personnel, thus, limiting its widespread use in resource-limited conditions. To overcome these challenges, we developed a rapid and convenient assay using a versatile integrated tube for the rapid and visual detection of SARS-CoV-2. The reaction conditions of the method were optimized using SARS-CoV-2 RNA standards and the sensitivity and specificity were further determined. Finally, it was verified on clinical specimens. The assay was completed within 40 min, and the result was visible by the naked eye. The limits of detection (LODs) for the target ORF1ab and N genes were 50 copies/μl. No cross-reactivity was observed with the RNA standard samples of four respiratory viruses or clinical samples of common respiratory viral infections. Ninety SARS-CoV-2 positive and 30 SARS-CoV-2 negative patient specimens were analyzed. We compared these results to both prior and concurrent RT-PCR evaluations. As a result, the overall sensitivity and specificity for detection SARS-CoV-2 were 94.5 and 100.0%, respectively. Conclusion: The integrated tube assay has the potential to provide a simple, specific, sensitive, one-pot, and single-step assay for SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0441	SARS-CoV-2	ORF1ab gene
RPB0442	SARS-CoV-2	N gene

RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern

Author(s):

Guiyue Tang,Zilong Zhang,Wei Tan,Fei Long,Jingxian Sun,Yingying Li,Siwei Zou,Yujiao Yang,Kezhu Cai,Shenwei Li,Zhiyi Wang,Jiakun Liu,Guobing Mao,Yingxin Ma,Guo-Ping Zhao,Zhen-Gan Tian,Wei Zhao

Journal:

Sens Actuators B Chem

Year:

2023

Abstract:

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and ΔH69-V70 were discriminated by RRCd, demonstrating 100% specificity in clinical samples with C t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

PrimerBankID	Target Pathogen	Target Gene
RPB0413	SARS-CoV-2	S gene
RPB0414	SARS-CoV-2	N gene
RPB0415	SARS-CoV-2	E gene
RPB0416	MERS-CoV	N gene
RPB0417	Influenza A virus (H1N1)	H1N1 HA1 N gene
RPB0418	Influenza A virus (H1N1)	H1N1 HN1 N gene

Reverse transcription recombinase-aided amplification assay for avian influenza virus

Author(s):

Suchun Wang,Qingye Zhuang,Nan Jiang,Fuyou Zhang,Qiong Chen,Ran Zhao,Yang Li,Xiaohui Yu,Jinping Li,Guangyu Hou,Liping Yuan,Fuliang Sun,Zihao Pan,Kaicheng Wang

Journal:

Virus Genes

Year:

2023

Abstract:

Avian influenza virus (AIV) infection can lead to severe economic losses in the poultry industry and causes a serious risk for humans. A rapid and simple test for suspected viral infection cases is crucial. In this study, a reverse transcription recombinase-aided amplification assay (RT-RAA) for the rapid detection of all AIV subtypes was developed. The reaction temperature of the assays is at 39 °C and the detection process can be completed in less than 20 min. The specificity results of the assay showed that this method had no cross-reaction with other main respiratory viruses that affect birds, including Newcastle disease virus (NDV) and infectious bronchitis virus (IBV). The analytical sensitivity at a 95% confidence interval was 102 RNA copies per reaction. In comparison with a published assay for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), the κ value of the RT-RAA assay in 384 avian clinical samples was 0.942 (p < 0.001). The sensitivity and specificity of the RT-RAA assay for avian clinical sample detection was determined as 97.59% (95% CI 93.55-99.23%) and 96.79% (95% CI 93.22-98.59%), respectively. The RT-RAA assay for AIV in this study provided an effective and practicable tool for AIV molecular detection.

PrimerBankID	Target Pathogen	Target Gene
RPB0388	Influenza A virus	M gene