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