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

Author(s):

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

Journal:

The CRISPR Journal

Year:

2023

Abstract:

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

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

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

Author(s):

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

Journal:

Virus research

Year:

2022

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0095	HRV	VP4

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

Author(s):

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

Journal:

Analytical Chemistry

Year:

2022

Abstract:

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

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

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

Author(s):

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

Journal:

Biosensors & Bioelectronics

Year:

2023

Abstract:

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

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

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

Author(s):

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

Journal:

Microbiol Spectr

Year:

2023

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0040	MERS-CoV	N gene

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

Author(s):

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

Journal:

Frontiers in Cellular and Infection Microbiology

Year:

2023

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0389	Influenza A virus	M gene

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

Author(s):

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

Journal:

Frontiers in Bioengineering and Biotechnology

Year:

2023

Abstract:

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

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

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

Author(s):

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

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

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

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

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

Author(s):

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

Journal:

Sens Actuators B Chem

Year:

2023

Abstract:

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

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

Reverse transcription recombinase-aided amplification assay for avian influenza virus

Author(s):

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

Journal:

Virus Genes

Year:

2023

Abstract:

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

PrimerBankID	Target Pathogen	Target Gene
RPB0388	Influenza A virus	M gene