Development of propidium monoazide-recombinase polymerase amplification (PMA-RPA) assay for rapid detection of Streptococcus pyogenes and Streptococcus agalactiae

Author(s):

Jing Chen,Yuanyang Wang,Xiaoqing Liu,Guopei Chen,Xuejian Chen,Jiaping Chen,Zhongdong Liu,Jingwen Gong,Guowu Yang,Quanxue Lan

Journal:

Molecular and cellular probes

Year:

2018

Abstract:

Streptococcus pyogenes (Group A Streptococcus, GAS) and Streptococcus agalactiae (Group B Streptococcus, GBS) are common pathogens that threaten public health. In this study, a double recombinase polymerase (RPA) amplification assay was developed to rapidly detect these pathogens. Specificity tests revealed that the GAS and GBS strains were positive for speB and SIP genes, respectively. In clinical samples, the double assay performed similarly to the traditional biochemical method. The limits of detection were both ≤100 copies per reaction. In tests for simulant-contaminated samples, bacterial-culture media containing 103 CFU/mL original concentrations of S. pyogenes and S. agalactiae were positive in RPA assays after incubating for 4 h. Results can be obtained at 37 °C in 20 min. To determine whether propidium monoazide (PMA) can eliminate the influence of DNA extracted from dead cells, a bacterial suspension was treated with PMA before DNA extraction. Findings of RPA assay showed that DNA extracted from dead cells had no fluorescence signal. Therefore, the PMA-RPA assay is a promising technology for field tests and rapid point-of-care diagnosis.

PrimerBankID	Target Pathogen	Target Gene
RPB0166	Streptococcus pyogenes	speB
RPB0167	Streptococcus pyogenes	SIP
RPB0512	Group A Streptococcus	speB
RPB0513	Group B Streptococcus	SIP

Rapid Detection of Avian Influenza A Virus (H7N9) by Lateral Flow Dipstick Recombinase Polymerase Amplification

Author(s):

Shiwei Ma,Xue Li,Bo Peng,Weihua Wu,Xin Wang,Hui Liu,Lihong Yuan,Shisong Fang,Jiahai Lu

Journal:

Biological & pharmaceutical bulletin

Year:

2018

Abstract:

Avian influenza A (H7N9) virus has caused several epidemics and infection in both human and poultry. With mutation, the H7N9 virus gained its fifth endemic in China. Early diagnosis is crucial for the control of viral spread in poultry and prognosis of infected patients. In this study, we developed and evaluated a lateral flow dipstick recombinase polymerase amplification (LFD-RPA) assay for rapid detection of both hemagglutinin and neuraminidase gene of H7N9. Our H7-LFD-RPA and N9-LFD-RPA assay were able to detect 32 fg H7N9 nucleic acid which is more convenient and rapid than previous methods. Through detecting 50 influenza positive samples, cross-reaction was not found with other subtypes of influenza virus. The 100% analytical specificity and sufficient analytical sensitivity results agreed the real time RT-PCR assay. The results data demonstrated that our method performed well and could be applied to the detection of H7N9 virus. This LFD-RPA assay provides a candidate method for rapid point-of-care diagnosis of H7N9.

PrimerBankID	Target Pathogen	Target Gene
RPB0101	Influenza A virus (H7N9)	HA gene
RPB0102	Influenza A virus (H7N9)	NA gene

Reverse transcription recombinase polymerase amplification with lateral flow dipsticks for detection of influenza A virus and subtyping of H1 and H3

Author(s):

Ning Sun,Weiping Wang,Jie Wang,Xinyue Yao,Fangfang Chen,Xiaojun Li,Yi Yinglei,Bo Chen

Journal:

Molecular and cellular probes

Year:

2018

Abstract:

Three reverse transcription recombinase polymerase amplification assays with lateral flow dipsticks (RT-RPA-LFD) were developed for identification of the matrix and hemagglutinin (HA) genes to detect influenza A virus and distinguish subtypes H1 and H3. Assessment of the assays' specificity showed that there was no cross-reactivity with other targets. Their limits of detection were 123.6 copies per reaction for the matrix gene, 677.1 copies per reaction for the H1 HA gene, and 112.2 copies/reaction for the H3 HA gene. Of 111 samples tested by RT-RPA-LFD assays, 27 were positive for influenza A virus, 14 were positive for H1, and 10 were positive for H3. Compared to the results obtained from real-time RT-PCR assays, the sensitivity of RT-RPA-LFD assays was 75%, 93.33% and 71.43% for the matrix, H1, and H3, with 100% specificity. The sensitivity of RT-RPA-LFD assays is lower than that of real-time RT-PCR, comparable or better than that of conventional RT-PCR, and much better than that of RIDTs. In conclusion, these assays offer an efficient and reliable tool for identification and subtyping of influenza A virus (subtype H1 and H3) in the resource-limited setting.

PrimerBankID	Target Pathogen	Target Gene
RPB0098	Influenza A virus	matrix gene
RPB0099	Influenza A virus	H1
RPB0100	Influenza A virus	H3

Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay

Author(s):

Michael Frimpong,Hubert Senanu Ahor,Ahmed Abd El Wahed,Bernadette Agbavor,Francisca Naana Sarpong,Kenneth Laing,Mark Wansbrough-Jones,Richard Odame Phillips

Journal:

PLoS neglected tropical diseases

Year:

2019

Abstract:

Background: Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. Methodology and principal findings: A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95). Conclusion: The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.

PrimerBankID	Target Pathogen	Target Gene
RPB0118	Mycobacterium ulcerans	IS2404

Development of a reverse transcription recombinase polymerase amplification assay for rapid detection of human respiratory syncytial virus.

Author(s):

Xi, Yun; Xu, Chang-Zhi; Xie, Zhi-Zhi; Zhu, Dong-Lin; Dong, Jie-Ming; Xiao, Gang;

Journal:

MOL CELL PROBE

Year:

2019

Abstract:

Respiratory syncytial virus (RSV) is one of the most important causative agents that causing respiratory tract infection in children and associated with high morbidity and mortality. A diagnostic method would be a robust tool for identification of RSV infection, especially in the resource-limited settings. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technique which has been widely employed to detect human/animal pathogens. In present study, a probe-based reverse transcription RPA (RT-RPA) assay was established for the detection of RSV. The primers and probe were designed based on the sequences of the conserved nucleocapsid (N) gene. The minimal detection limit of the RT-RPA assay for the detection of RSV B was 19 copies of RNA molecules at 95% probability, whereas the detection limit for RSV A was 104 copies molecule. The assay was RSV-specific since it had no non-specific reactions with other common human pathogens. The clinical performance of the RT-RPA assay was validated using 188 nasopharyngeal aspirates (NPAs). The nucleic acid extraction of the samples was performed by use of the magnetic bead-based kit which didn't require the heavy and expensive centrifuge. The coincidence rates between RT-RPA and qRT-PCR for the clinical samples was 96%, indicating the RT-RPA assay had good diagnostic performance on clinical samples. The real-time RT-RPA assay combined with the manual genome extraction method make it potential to detect clinical samples in field, providing a possible solution for RSV diagnosis in remote rural areas in developing countries.

PrimerBankID	Target Pathogen	Target Gene
RPB0050	RSV A/B	N gene
RPB0129	RSV	(N) gene

Utilization of recombinase polymerase amplification method combined with lateral flow dipstick for visual detection of respiratory syncytial virus.

Author(s):

Xu, Yu-Zhong; Fang, Du-Zhi; Chen, Fang-Fang; Zhao, Qin-Fei; Cai, Chao-Ming; Cheng, Ming-Gang;

Journal:

MOL CELL PROBE

Year:

2020

Abstract:

Respiratory syncytial virus (RSV) is a major causative agent of respiratory tract infection necessitating hospitalization in children. A rapid diagnostic method would facilitate early detection of RSV infection and timely implementation of special treatment. Here, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with lateral flow dipstick (LFD) was evaluated for rapid visual detection of RSV. The primers were designed to target the conserved L gene. The RT-RPA-LFD assay could simultaneously detect RSV subtype A and B with the same detection limit of 10 copies of a given RNA molecule. Moreover, the assay showed no cross-reactivity with other common human pathogens. The performance of the RT-RPA-LFD assay was evaluated by testing 136 nasopharyngeal aspirates (NPAs). The agreement of the detection results between RT-RPA-LFD and qRT-PCR was 100% (34 positive and 102 negative cases). In summary, the developed RT-RPA-LFD assay had good performance in detecting RSV in clinical specimens, thus providing a novel alternative solution for the detection of RSV under field conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0049	RSV A/B	UTR gene
RPB0128	RSV	conserved L gene

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

Author(s):

Behrmann, Ole; Bachmann, Iris; Spiegel, Martin; Schramm, Marina; Abd El Wahed, Ahmed; Dobler, Gerhard; Dame, Gregory; Hufert, Frank T;

Journal:

CLIN CHEM

Year:

2020

Abstract:

BACKGROUND:The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS:We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS:The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS:With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

PrimerBankID	Target Pathogen	Target Gene
RPB0079	SARS-CoV-2	N gene

An Isothermal Method for Sensitive Detection of Mycobacterium tuberculosis Complex Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a Cis and Trans Cleavage

Author(s):

Haipo Xu,Xiaolong Zhang,Zhixiong Cai,Xiuqing Dong,Geng Chen,Zhenli Li,Liman Qiu,Lei He,Bin Liang,Xiaolong Liu,Jingfeng Liu

Journal:

The Journal of molecular diagnostics : JMD

Year:

2020

Abstract:

Tuberculosis is one of the most serious infectious diseases, resulting in death worldwide. Traditional detection methods are not enough to meet the clinical requirements of rapid diagnosis, high specificity, and high sensitivity. Fast, sensitive, and accurate detection of Mycobacterium tuberculosis (MTB) is urgently needed to treat and control tuberculosis disease. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas12a) exhibit strong nonspecific degradation ability of exogenous single-strand nucleic acids (trans cleavage) after specific recognition of target sequence. We purified Cas12a protein and selected a proper guide RNA based on conserved sequences of MTB from designed guide RNA library. Then, we proposed a novel detection method based on recombinase polymerase amplification and CRISPR/Cas12a nuclease system for specific and sensitive detection of MTB DNA. The assay, based on fluorescence detection, showed 4.48 fmol/L of limit of detection and good linear correlation of concentration with fluorescence value (R2 = 0.9775). It also showed good performance in distinguishing other bacteria. Furthermore, its clinical performance was evaluated by 193 samples and showed sensitivity of 99.29% (139/140) and specificity of 100% (53/53) at 99% CI, compared with culture method. Taken together, the CRISPR/Cas12a system showed good specificity, excellent sensitivity, and excellent accuracy for MTB detection, and it meets requirements of MTB detection in clinical samples and has great potential for clinical translation.

PrimerBankID	Target Pathogen	Target Gene
RPB0117	Mycobacterium	IS1081

Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples

Author(s):

Jinqiang Hu,Yi Wang,Huimin Ding,Chunpeng Jiang,Yao Geng,Xincheng Sun,Jianzhou Jing,Hui Gao,Zhangcun Wang,Caiwen Dong

Journal:

International journal of food microbiology

Year:

2020

Abstract:

Currently, rapid, sensitive, and convenient visual detection methods for Staphylococcus aureus (S. aureus) are scarce. In this study, a novel detection method based on recombinase polymerase amplification (RPA) and polymer flocculation sedimentation (PFS) was developed. Twelve effective primer combinations derived from four forward primers F1, F2, F3, F4, and three reverse primers R1, R2, R3 targeting the nuc gene of S. aureus were designed and screened by a polymerase chain reaction and RPA methods. RPA reaction conditions, including temperature, time, and volume as well as PEG8000 and NaCl concentrations range, were optimized. Moreover, the specificity and sensitivity of the RPA-PFS assay were further analyzed. Finally, the potential use of the RPA-PFS assay was evaluated using artificially S. aureus contaminated food samples, including pork, beef, shrimp, fish, cheese, cabbage, leftover rice, egg, milk, and orange juice. Results showed that the SA5 (F2/R2) combination was the optimal primer candidate. The optimal temperature range, the shortest time and the minimal volume of RPA reaction were 40-42 °C, 10 min and 10 μL, respectively and the optimal PEG8000/NaCl concentrations were 0.2 g/mL and 2.5 M, respectively, for the adsorption between magnetic beads and RPA products. The RPA-PFS method could detect as little as 13 fg genomic DNA of S. aureus and was also specific for five target S. aureus as well as twenty-seven non-target foodborne bacteria. The limit of detection of RPA-PFS for S. aureus in artificially contaminated food samples was 38 CFU/mL (g). Besides, RPA-PFS has directly been judged by the naked eye and has totally taken less than 20 min. In short, the assay RPA-PFS developed in this study is a rapid, sensitive, and specific visual detection method for S. aureus.

PrimerBankID	Target Pathogen	Target Gene
RPB0159	Staphylococcus aureus	nuc gene

Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis.

Author(s):

Huang, Zhen; Tian, Di; Liu, Yang; Lin, Zhen; Lyon, Christopher J; Lai, Weihua; Fusco, Dahlene; Drouin, Arnaud; Yin, Xiaoming; Hu, Tony; Ning, Bo;

Journal:

BIOSENS BIOELECTRON

Year:

2020

Abstract:

Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

PrimerBankID	Target Pathogen	Target Gene
RPB0077	SARS-CoV-2	ORF1ab and N gene
RPB0078	SARS-CoV-2	N gene

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification

Author(s):

Ole Behrmann,Matthias Hügle,Franz Eckardt,Iris Bachmann,Cecilia Heller,Marina Schramm,Carrie Turner,Frank T Hufert,Gregory Dame

Journal:

Micromachines

Year:

2020

Abstract:

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-β-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

PrimerBankID	Target Pathogen	Target Gene
RPB0104	Klebsiella pneumoniae	khe
RPB0105	Klebsiella pneumoniae	blaNDM-1

Rapid detection of Mycobacterium tuberculosis based on antigen 85B via real-time recombinase polymerase amplification

Author(s):

Y Xu,P Wu,H Zhang,J Li

Journal:

Letters in applied microbiology

Year:

2021

Abstract:

Tuberculosis (TB), as a common infectious disease, still remains a severe challenge to public health. Due to the unsatisfied clinical needs of currently available diagnostic vehicles, it is desired to establish a new approach for universally detecting Mycobacterium tuberculosis. Herein, we designed a real-time recombinase polymerase amplification (RPA) technology for identifying M. tuberculosis within 20 min at 39°C via custom-designed oligonucleotide primers and probe, which could specifically target antigen 85B (Ag85B). Particularly, the primers F4-R4 produced the fastest fluorescence signal with the probe among four pairs of designed primers in the RPA assays. The optimal primers/probe combination could effectively identify M. tuberculosis with the detection limit of 4·0 copies per μl, as it could not show a positive signal for the genomic DNA from other mycobacteria or pathogens. The Ag85B-based RPA could determine the genomic DNA extracted from M. tuberculosis with high reliability (100%, 22/22). More importantly, when testing clinical sputum samples, the real-time RPA displayed an admirable sensitivity (90%, 95% CI: 80·0-96·0%) and specificity (98%, 95% CI: 89·0-100·0%) compared to traditional smear microscopy, which was similar to the assay of Xpert MTB/RIF. This real-time RPA based Ag85B provides a promising strategy for the rapid and universal diagnosis of TB.

PrimerBankID	Target Pathogen	Target Gene
RPB0116	Mycobacterium	Ag85B

Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA.

Author(s):

Kim, Youngeun; Yaseen, Adam B; Kishi, Jocelyn Y; Hong, Fan; Saka, Sinem K; Sheng, Kuanwei; Gopalkrishnan, Nikhil; Schaus, Thomas E; Yin, Peng;

Journal:

medRxiv.

Year:

2020

Abstract:

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 渭L reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

PrimerBankID	Target Pathogen	Target Gene
RPB0076	SARS-CoV-2	\

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii

Author(s):

Shuang Liu,Guangtao Huang,Yali Gong,Xiaojun Jin,Yudan Meng,Yizhi Peng,Junning Zhao,Xiaolu Li,Qin Li

Journal:

Burns & trauma

Year:

2020

Abstract:

Background:Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. Methods: In this study, a 15-μL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla OXA-51 gene by PCR, qPCR and 15 μL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 μL-RPA data for detecting the carbapenem-resistance gene bla OXA-23 were comparatively assessed. Results: The detection limit of the bla OXA-51 gene by 15 μL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 μL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 μL RPA data for detecting bla OXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. Conclusion: We demonstrated that the new 15 μL RPA assay for detecting bla OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

PrimerBankID	Target Pathogen	Target Gene
RPB0082	Acinetobacter baumannii	blaOXA-51
RPB0083	Acinetobacter baumannii	bla OXA-23

An enhanced isothermal amplification assay for viral detection.

Author(s):

Qian, Jason; Boswell, Sarah A; Chidley, Christopher; Lu, Zhi-Xiang; Pettit, Mary E; Gaudio, Benjamin L; Fajnzylber, Jesse M; Ingram, Ryan T; Ward, Rebecca H; Li, Jonathan Z; Springer, Michael;

Journal:

Nat Commun

Year:

2020

Abstract:

Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45鈥塵in from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.

PrimerBankID	Target Pathogen	Target Gene
RPB0074	SARS-CoV-2	S gene
RPB0075	SARS-CoV-2	N gene

Simultaneous Dual-Gene Diagnosis of SARS-CoV-2 Based on CRISPR/Cas9-Mediated Lateral Flow Assay

Author(s):

Xiong, Erhu; Jiang, Ling; Tian, Tian; Hu, Menglu; Yue, Huahua; Huang, Mengqi; Lin, Wei; Jiang, Yongzhong; Zhu, Debin; Zhou, Xiaoming;

Journal:

ANGEW CHEM INT EDIT

Year:

2020

Abstract:

Few methods for the detection of SARS-CoV-2 currently have the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Developed here is a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (Orf1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions

PrimerBankID	Target Pathogen	Target Gene
RPB0059	SARS-CoV-2	E gene
RPB0060	SARS-CoV-2	Orf1ab gene

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

Author(s):

Xiong, Dan; Dai, Wenjun; Gong, Jiaojiao; Li, Guande; Liu, Nansong; Wu, Wei; Pan, Jiaqiang; Chen, Chen; Jiao, Yingzhen; Deng, Huina; Ye, Junwei; Zhang, Xuanxuan; Huang, Huiling; Li, Qianyun; Xue, Liang; Zhang, Xiuming; Tang, Guanghui; 

Journal:

PLOS BIOL.

Year:

2020

Abstract:

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

PrimerBankID	Target Pathogen	Target Gene
RPB0072	SARS-CoV-2	ORF1ab
RPB0073	SARS-CoV-2	N gene

Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Author(s):

Lau, Yee Ling; Ismail, Ilyiana Binti; Mustapa, Nur Izati Binti; Lai, Meng Yee; Tuan Soh, Tuan Suhaila; Haji Hassan, Afifah; Peariasamy, Kalaiarasu M; Lee, Yee Leng; Abdul Kahar, Maria Kahar Bador; Chong, Jennifer; Goh, Pik Pin;

Journal:

PLoS One

Year:

2021

Abstract:

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/渭L RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

PrimerBankID	Target Pathogen	Target Gene
RPB0071	SARS-CoV-2	N gene

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

Author(s):

El Wahed, Ahmed Abd; Patel, Pranav; Maier, Melanie; Pietsch, Corinna; Rüster, Dana; Böhlken-Fascher, Susanne; Kissenkötter, Jonas; Behrmann, Ole; Frimpong, Michael; Diagne, Moussa Moïse; Faye, Martin; Dia, Ndongo; Shalaby, Mohamed A; Amer, Haitham; Elgamal, Mahmoud; Zaki, Ali; Ismail, Ghada; Kaiser, Marco; Corman, Victor M; Niedrig, Matthias; Landt, Olfert; Faye, Ousmane; Sall, Amadou A; Hufert, Frank T; Truyen, Uwe; Liebert, Uwe G; Weidmann, Manfred; 

Journal:

ANAL CHEM

Year:

2021

Abstract:

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0063	SARS-CoV-2	RdRp gene
RPB0064	SARS-CoV-2	E gene
RPB0065	SARS-CoV-2	N gene

Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection.

Author(s):

Zhang, Wei S; Pan, Jianbin; Li, Feng; Zhu, Min; Xu, Mengting; Zhu, Hongyan; Yu, Yanyan; Su, Gaoxing;

Journal:

ANAL CHEM

Year:

2021

Abstract:

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.

PrimerBankID	Target Pathogen	Target Gene
RPB0066	SARS-CoV-2	ORF1ab gene
RPB0067	SARS-CoV-2	N gene