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	\