One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a.

Author(s):

Sun, Yangyang; Yu, Lei; Liu, Chengxi; Ye, Shanting; Chen, Wei; Li, Dechang; Huang, Weiren;

Journal:

19 J Transl Med

Year:

2021

Abstract:

BACKGROUND:COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.METHODS:We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.RESULTS:The OR-DETECTR detection process can be completed in one tube, which takes approximately 50min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/ul input (RNA standard) and 1 copy/ul input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/ul input.CONCLUSIONS:The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

PrimerBankID	Target Pathogen	Target Gene
RPB0061	SARS-CoV-2	RdRp
RPB0062	SARS-CoV-2	RdRp and Ngene

One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

Author(s):

Jia Xie,Xiaohan Yang,Lei Duan,Keyi Chen,Pan Liu,Wenli Zhan,Changbin Zhang,Hongyu Zhao,Mengru Wei,Yuan Tang,Mingyong Luo

Journal:

Frontiers in microbiology

Year:

2021

Abstract:

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0085	Coxsackievirus A6	VP1 gene

Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene.

Author(s):

Huang, Pei; Jin, Hongli; Zhao, Yongkun; Li, Entao; Yan, Feihu; Chi, Hang; Wang, Qi; Han, Qiuxue; Mo, Ruo; Song, Yumeng; Bi, Jinhao; Jiao, Cuicui; Li, Wujian; He, Hongbin; Wang, Hongmei; Ma, Aimin; Feng, Na; Wang, Jianzhong; Wang, Tiecheng; Yang, Songtao; Gao, Yuwei; Xia, Xianzhu; Wang, Hualei;

Journal:

PLoS Negl Trop Dis

Year:

2021

Abstract:

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2脳101 copies/渭l viral RNA for the UpE assay and 1.2 copies/渭l viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.

PrimerBankID	Target Pathogen	Target Gene
RPB0047	MERS-CoV	MERS-CoV UpE gene
RPB0048	MERS-CoV	MERS-CoV N gene
RPB0108	MERS-CoV	UpE
RPB0109	MERS-CoV	N gene

Sensitive electrochemical biosensor combined with isothermal amplification for point-of-care COVID-19 tests

Author(s):

Hyo Eun Kim, Ariadna Schuck, See Hi Lee, Yunjong Lee, Minhee Kang, and Yong-Sang Kim

Journal:

Biosensors and Bioelectronics

Year:

2021

Abstract:

We report an electrochemical biosensor combined with recombinase polymerase amplification (RPA) for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2. The electrochemical biosensor based on a multi-microelectrode array allows the detection of multiple target genes by differential pulse voltammetry. The RPA reaction involves hybridization of the RPA amplicon with thiol-modified primers immobilized on the working electrodes, which leads to a reduction of current density as amplicons accumulate. The assay results in shorter “sample-to-answer” times than conventional PCR without expensive thermo-cycling equipment. The limits of detection are about 0.972 fg/μL (RdRP gene) and 3.925 fg/μL (N gene), which are slightly lower than or comparable to that of RPA assay results obtained by gel electrophoresis without post-amplification purification. The combination of electrochemical biosensors and the RPA assay is a rapid, sensitive, and convenient platform that can be potentially used as a point-of-care test for the diagnosis of COVID-19.

PrimerBankID	Target Pathogen	Target Gene
RPB0217	SARS-CoV-2	N gene
RPB0218	SARS-CoV-2	RdRP gene

Combined recombinase polymerase amplification/rkDNA-graphene oxide probing system for detection of SARS-CoV-2.

Author(s):

Choi, Moon Hyeok; Lee, Jaehyeon; Seo, Young Jun;

Journal:

ANAL CHIM ACTA

Year:

2021

Abstract:

The development of rapid, highly sensitive, and selective methods for the diagnosis of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should help to prevent the spread of this pandemic virus. In this study, we combined recombinase polymerase amplification (RPA), as a means of isothermal DNA amplification, with an rkDNA-graphene oxide (GO) probe system to allow the rapid detection of SARS-CoV-2 with high sensitivity and selectivity. We used in situ enzymatic synthesis to prepare an rkDNA probe that was complementary to an RPA-amplified sequence of the target N-gene of SARS-CoV-2. The fluorescence of this rkDNA was perfectly quenched in the presence of GO. When the quenched rkDNA-GO system was added to the RPA-amplified sequence of the target SARS-CoV-2, the fluorescence recovered dramatically. The combined RPA/rkDNA-GO system exhibited extremely high selectivity (discrimination factor: 17.2) and sensitivity (LOD = 6.0 aM) for the detection of SARS-CoV-2. The total processing time was only 1.6 h. This combined RPA/rkDNA-GO system appears to be a very efficient and simple method for the point-of-care detection of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0058	SARS-CoV-2	N gene

A microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay for rapid COVID-19 detection.

Author(s):

Liu, Dan; Shen, Haicong; Zhang, Yuqian; Shen, Danyu; Zhu, Mingyang; Song, Yanling; Zhu, Zhi; Yang, Chaoyong;

Journal:

LAB CHIP

Year:

2019

Abstract:

The COVID-19 pandemic, caused by SARS-CoV-2, currently poses an urgent global medical crisis for which there remains a lack of affordable point-of-care testing (POCT). In particular, resource-limited areas need simple and easily disseminated testing solutions to manage the outbreak. In this work, a microfluidic-integrated lateral flow recombinase polymerase amplification (MI-IF-RPA) assay was developed for rapid and sensitive detection of SARS-CoV-2, which integrates the reverse transcription recombinase polymerase amplification (RT-RPA) and a universal lateral flow (LF) dipstick detection system into a single microfluidic chip. The single-chamber RT-RPA reaction components are mixed with running buffer, and then delivered to the LF detection strips for biotin- and FAM-labelled amplified analyte sequences, which can provide easily interpreted positive or negative results. Testing requires only a simple nucleic acid extraction and loading, then incubation to obtain results, approximately 30 minutes in total. SARS-CoV-2 armored RNA particles were used to validate the MI-IF-RPA system, which showed a limit of detection of 1 copy per 渭L, or 30 copies per sample. Chip performance was further evaluated using clinically diagnosed cases of COVID-19 and revealed a sensitivity of 97% and specificity of 100%, highly comparable to current reverse transcription-polymerase chain reaction (RT-PCR)-based diagnostic assays. This MI-IF-RPA assay is portable and comprises affordable materials, enabling mass production and decreased risk of contamination. Without the need for specialized instrumentation and training, MI-IF-RPA assay can be used as a complement to RT-PCR for low-cost COVID-19 screening in resource-limited areas.

PrimerBankID	Target Pathogen	Target Gene
RPB0057	SARS-CoV-2	N gene

Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings.

Author(s):

Cherkaoui, Dounia; Huang, Da; Miller, Benjamin S; Turbé, Valérian; McKendry, Rachel A;

Journal:

BIOSENS BIOELECTRON

Year:

2021

Abstract:

The COVID-19 pandemic is challenging diagnostic testing capacity worldwide. The mass testing needed to limit the spread of the virus requires new molecular diagnostic tests to dramatically widen access at the point-of-care in resource-limited settings. Isothermal molecular assays have emerged as a promising technology, given the faster turn-around time and minimal equipment compared to gold standard laboratory PCR methods. However, unlike PCR, they do not typically target multiple SARS-CoV-2 genes, risking sensitivity and specificity. Moreover, they often require multiple steps thus adding complexity and delays. Here we develop a multiplexed, 1-2 step, fast (20-30 min) SARS-CoV-2 molecular test using reverse transcription recombinase polymerase amplification to simultaneously detect two conserved targets - the E and RdRP genes. The agile multi-gene platform offers two complementary detection methods: real-time fluorescence or dipstick. The analytical sensitivity of the fluorescence test was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick method was 130 (95% CI: 82-500) RNA copies per reaction. High specificity was found against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing in decentralised settings, with minimal or equipment-free incubation methods and a user-friendly prototype smartphone application. This rapid, simple, ultrasensitive and multiplexed molecular test offers valuable advantages over gold standard tests and in future could be configurated to detect emerging variants of concern.

PrimerBankID	Target Pathogen	Target Gene
RPB0042	MERS-CoV	E gene
RPB0043	MERS-CoV	RdRP gene

Autonomous Lab-on-Paper for Multiplexed, CRISPR-based Diagnostics of SARS-CoV-2

Author(s):

Kun Yin , Xiong Ding , Ziyue Li , Maroun M Sfeir , Enrique Ballesteros , Changchun Liu

Journal:

Lab on a Chip

Year:

2021

Abstract:

The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0215	SARS-CoV-2	N gene
RPB0216	SARS-CoV-2	S gene

A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV-2.

Author(s):

Wang, Pei; Ma, Chao; Zhang, Xue; Chen, Lizhan; Yi, Longyu; Liu, Xin; Lu, Qunwei; Cao, Yang; Gao, Song;

Journal:

Front Cell Infect Microbiol

Year:

2021

Abstract:

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV-2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV-2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

PrimerBankID	Target Pathogen	Target Gene
RPB0068	SARS-CoV-2	N gene
RPB0069	SARS-CoV-2	ORF1ab gene

Optimization of reaction condition of recombinase polymerase amplification to detect SARS-CoV-2 DNA and RNA using a statistical method

Author(s):

Kevin Maafu Juma, Teisuke Takita, Kenji Ito, Masaya Yamagata,Shihomi Akagi, Emi Arikawa, Kenji Kojima, Manish Biyani,Shinsuke Fujiwara, Yukiko Nakura, Itaru Yanagihara, and Kiyoshi Yasukawa

Journal:

Biochemical and Biophysical Research Communications

Year:

2021

Abstract:

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.

PrimerBankID	Target Pathogen	Target Gene
RPB0213	SARS-CoV-2	nucleocapsid phosphoprotein gene
RPB0214	SARS-CoV-2	ORF8 protein gene

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	\