Rapid and visual detection of Staphylococcus aureus in milk using a recombinase polymerase amplification-lateral flow assay combined with immunomagnetic separation

Author(s):

Ya-Lei Wang,Xin Zhang,Quan Wang,Peng-Xuan Liu,Wei Tang,Rong Guo,Hai-Yang Zhang,Zhao-Guo Chen,Xian-Gan Han,Wei Jiang

Journal:

Journal of applied microbiology

Year:

2022

Abstract:

Aims: The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk. Methods and results: Under optimum conditions, the average capture efficiency values for S. aureus strains (104 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction. Conclusions: The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min. Significance and impact of the study: The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.

PrimerBankID	Target Pathogen	Target Gene
RPB0163	Staphylococcus aureus	nuc

Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B

Author(s):

Li-Guo Liang,Miao-Jin Zhu,Rui He,Dan-Rong Shi,Rui Luo,Jia Ji,Lin-Fang Cheng,Xiang-Yun Lu,Wei Lu,Fu-Ming Liu,Zhi-Gang Wu,Nan-Ping Wu,Hang Chen,Zhe Chen,Hang-Ping Yao

Journal:

journal of medical virology

Year:

2023

Abstract:

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0021	SARS-CoV-2	N
RPB0022	SARS-CoV-2	E
RPB0023	SARS-CoV-2	ORF1ab
RPB0024	Influenza A virus (H1N1)	M
RPB0025	Influenza A virus (H3N2)	HA
RPB0026	Influenza B virus	PA

Computer vision enabled funnel adapted sensing tube (FAST) for power-free and pipette-free nucleic acid detection

Author(s):

Mengdi Bao,Shuhuan Zhang,Chad Ten Pas,Stephen J Dollery,Ruth V Bushnell,F N U Yuqing,Rui Liu,Guoyu Lu,Gregory J Tobin,Ke Du

Journal:

Lab On A Chip

Year:

2022

Abstract:

A simple, portable, and low-cost microfluidic system-funnel adapted sensing tube (FAST) is developed as an integrated, power-free, and pipette-free biosensor for viral nucleic acids. This FAST chip consists of four reaction chambers separated by carbon fiber rods, and the reagents in each chamber are transferred and mixed by manually removing the rods. Rather than using electrical heaters, only a hand warmer pouch is used for an isothermal recombinase polymerase amplification (RPA) and CRISPR-Cas12a reaction. The signal produced by the RPA-CRISPR reaction is observed by the naked eye using an inexpensive flashlight as a light source. The FAST chip is fabricated using water-soluble polyvinyl alcohol (PVA) as a sacrificial core, which is simple and environmentally friendly. Using a SARS-CoV-2 fragment as a target, a ∼10 fM (6 × 103 copies per μL) detection limit is achieved. To generalize standard optical readout for individuals without training, a linear kernel algorithm is created, showing an accuracy of ∼100% for identifying both positive and negative samples in FAST. This power-free, pipette-free, disposable, and simple device will be a promising tool for nucleic acid diagnostics in either clinics or low-resource settings.

PrimerBankID	Target Pathogen	Target Gene
RPB0031	SARS-CoV-2	S

Nanozyme-strip for rapid and ultrasensitive nucleic acid detection of SARS-CoV-2

Author(s):

Xiangqin Meng, Sijia Zou, Dandan Li , Jiuyang He, Ling Fang, Haojue Wang, Xiyun Yan, Demin Duan, Lizeng Gao

Journal:

Biosensors & Bioelectronics

Year:

2022

Abstract:

The coronavirus disease 2019 (COVID-19) pandemic has created a huge demand for sensitive and rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard for SARS-CoV-2 detection is reverse transcription-polymerase chain reaction (RT-PCR)-based nucleic acid amplification. However, RT-PCR is time consuming and requires specialists and large instruments that are unattainable for point-of-care testing (POCT). To develop POCT for SARS-CoV-2, we combined recombinase polymerase amplification (RPA) and FeS2 nanozyme strips to achieve facile nucleic acid amplification and subsequent colorimetric signal enhancement based on the high peroxidase-like activity of the FeS2 nanozymes. This method showed a nucleic acid limit of detection (LOD) for SARS-CoV-2 of 200 copies/mL, close to that of RT-PCR. The unique catalytic properties of the FeS2 nanozymes enabled the nanozyme-strip to amplify colorimetric signals via the nontoxic 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Importantly, the detection of clinical samples of human papilloma virus type 16 (HPV-16) showed 100% agreement with previous RT-PCR results, highlighting the versatility and reliability of this method. Our findings suggest that nanozyme-based nucleic acid detection has great potential in the development of POCT diagnosis for COVID-19 and other viral infections.

PrimerBankID	Target Pathogen	Target Gene
RPB0032	SARS-CoV-2	ORF1ab

Aptamer-based colorimetric detection of methicillin-resistant Staphylococcus aureus by using a CRISPR/Cas12a system and recombinase polymerase amplification

Author(s):

Luyu Wei,Zhilong Wang,Jia Wang,Xiaohong Wang,Yiping Chen

Journal:

Analytica chimica acta

Year:

2022

Abstract:

Detection of methicillin-resistant Staphylococcus aureus (MRSA) with superior accuracy, timeliness, and simplicity is highly valuable in clinical diagnosis and food safety. In this study, an aptamer-based colorimetric biosensor was developed to detect MRSA by using a CRISPR/Cas12a system and recombinase polymerase amplification (RPA). The aptamer of silver ion (Ag+) pre-coupled to magnetic nanoparticles was employed not only as the substrate of trans-cleavage in the CRISPR/Cas12a system, but also as the modulator of Ag+-3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction, innovatively integrating the powerful CRISPR/Cas12a system with convenient colorimetry. The utilized aptamer containing consecutive and interrupted cytosine: cytosine mismatched base pairs also served as a signal amplifier because of the one-to-multiple binding of the aptamer to Ag+. Using triple amplification of RPA, multiple-turnover nuclease activity of Cas12a, and cytosine-Ag+-cytosine coordination chemistry, MRSA was detected as low as 8 CFU mL-1. Moreover, its satisfactory accuracy in the analysis of real samples, together with visualization and simplicity, revealed the great potential of the proposed biosensor as a robust antibiotic-resistant bacteria detection platform.

PrimerBankID	Target Pathogen	Target Gene
RPB0162	Staphylococcus aureus	mecA

Duplex real-time fluorescent recombinase polymerase amplification for the rapid and sensitive detection of two resistance genes in drug-resistant Staphylococcus aureus

Author(s):

Zhonglin Shi,Yanan Li,Anzhong Hu,Junsheng Cui,Min Shao,Ling Zhu,Ke Yang,Yong Liu,Guoqing Deng,Cancan Zhu

Journal:

Journal of microbiological methods

Year:

2022

Abstract:

In the clinic, drug-resistant Staphylococcus aureus (S. aureus) is the most common suppurative infection pathogen in humans. It can cause local infections in humans and animals, such as pneumonia, mastitis, and other systemic illnesses. At present, the detection of drug-resistant S. aureus includes traditional isolation by culture and antimicrobial susceptibility tests. However, these methods are complicated in experimental design, specialized in operation and time consuming. Therefore, a rapid and accurate drug-resistant S. aureus detection technology is urgently needed. In this study, we combined duplex pairs of fluorescent probes with recombinase polymerase amplification (RPA) to realize the simultaneous detection of two resistance genes in drug-resistant S. aureus. The method shows low detection limit, detecting 20 copies within 10 min. The analytical specificity of this method was evaluated with several related drug-resistant bacterial strains (Non-resistant S. aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), and the positive signal was only observed with drug-resistant S. aureus. In addition, the clinical suitability of this method was verified by 30 clinical isolates. Compared with qPCR, the coincidence rate of drug resistance genes were 100% (mecA) and 96.7% (ermA), respectively. These results show that the duplex real-time fluorescent RPA assay is a rapid, low detection limit and specific detection of mecA and ermA genes in isolates of drug-resistant S. aureus.

PrimerBankID	Target Pathogen	Target Gene
RPB0160	Staphylococcus aureus	mecA
RPB0161	Staphylococcus aureus	ermA

A New Auto-RPA-Fluorescence Detection Platform for SARS-CoV-2

Author(s):

Jing Tian, Biao Chen, Bin Zhang, Tantan Li, Zhiqiang Liang, Yujin Guo, Huping Jiao, Fenghong Liang, Longquan Xiang, Fanzhong Lin, Ruiwen Ren, Qingbin Liu

Journal:

Laboratory Medicine

Year:

2023

Abstract:

Objective: The outbreak of COVID-19 caused by SARS-CoV-2 has led to a serious worldwide pandemic. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)-based methods were recommended for routine detection of SARS-CoV-2 RNA. Because the reaction time and analytical sensitivity of qRT-PCR limits the diagnosis of SARS-CoV-2, development of a quick process of SARS-CoV-2 detection technology with high analytical sensitivity remains urgent. Methods: We combined isothermal amplification and fluorescence detection technology to develop a new auto-recombinase polymerase amplification (RPA)-fluorescence platform that could be used in the diagnosis of SARS-CoV-2. Results: By optimization of primers and probes, the RPA platform could detect SARS-CoV-2 nucleotides within 15 min. The limits of detection and specificity of the auto-RPA-fluorescence platform were 5 copies/µL and 100%, respectively. The accuracy of detection of the auto-RPA-fluorescence platform in the 16 positive samples was 100%. Conclusion: The RPA platform is a potential technology for the diagnosis of SARS-CoV-2 infection.

PrimerBankID	Target Pathogen	Target Gene
RPB0017	SARS-CoV-2	N
RPB0018	SARS-CoV-2	S

Rapid detection of human coronavirus NL63 by isothermal reverse transcription recombinase polymerase amplification

Author(s):

Aline Dorendorf,Iris Bachmann,Martin Spiegel,Ahmed Abd El Wahed,Gregory Dame,Frank Hufert

Journal:

Journal of clinical virology plus

Year:

2022

Abstract:

Background: Human coronaviruses are one of the leading causes for respiratory tract infections and for frequent primary care consultation. The human coronavirus NL63 (HCoV..µNL63) is one representative of the seasonal coronaviruses and capable of infecting the upper and lower respiratory tract and causative agent for croup in children. Objectives: For fast detection of HCoV-NL63, we developed an isothermal reverse transcription recombinase polymerase amplification (RT-RPA) assay. Study design: The analytical sensitivities of the RT-RPA assay were identified for in vitro transcribed ribonucleic acid (RNA) and for genomic viral RNA from cell culture supernatant. Moreover, specificity was tested with nucleic acids from other human coronaviruses and a variety of clinically relevant respiratory viruses. Finally, a clinical nasopharyngeal swab sample with spiked genomic viral HCoV-NL63 RNA was analyzed. Results: Our HCoV-NL63 RT-RPA assay is highly specific and has an analytical sensitivity of 13 RNA molecules/reaction for in vitro transcribed RNA. For genomic viral RNA from cell culture supernatant spiked into a clinical nasopharyngeal swab sample the assay...s analytical sensitivity is 170 RNA molecules/reaction. The assay shows amplification of the lowest detectable target copy number after 8 minutes and 7 minutes, respectively. Conclusions: We were able to design a sensitive and specific RT-RPA assay for the detection of HCoV-NL63. Additionally, the assay is characterized by short duration, isothermal amplification, and simple instrumentation.

PrimerBankID	Target Pathogen	Target Gene
RPB0093	HCoV-NL63	N-gene
RPB0171	HCoV-NL63	N-gene

An ultrasensitive and rapid "sample-to-answer" microsystem for on-site monitoring of SARS-CoV-2 in aerosols using "in situ" tetra-primer recombinase polymerase amplification

Author(s):

Shanglin Li, Bao Li, Xinyue Li, Ce Liu, Xiao Qi, Yin Gu, Baobao Lin, Lingli Sun, Lan Chen, Bingqian Han, Jiazhen Guo, Yanyi Huang, Shuangsheng Wu, Lili Ren, Jianbin Wang, Jingwei Bai, Jianxin Ma, Maosheng Yao, Peng Liu

Journal:

Biosensors & Bioelectronics

Year:

2022

Abstract:

Airborne transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for aerosol monitoring of SARS-CoV-2 to prevent sporadic outbreaks of COVID-19. The inadequate sensitivity of conventional methods and the lack of an on-site detection system limited the practical SARS-CoV-2 monitoring of aerosols in public spaces. We have developed a novel SARS-CoV-2-in-aerosol monitoring system (SIAMs) which consists of multiple portable cyclone samplers for collecting aerosols from several venues and a sensitive "sample-to-answer" microsystem employing an integrated cartridge for the analysis of SARS-CoV-2 in aerosols (iCASA) near the sampling site. By seamlessly combining viral RNA extraction based on a chitosan-modified quartz filter and "in situ" tetra-primer recombinase polymerase amplification (tpRPA) into an integrated microfluidic cartridge, iCASA can provide an ultra-high sensitivity of 20 copies/mL, which is nearly one order of magnitude greater than that of the commercial kit, and a short turnaround time of 25 min. By testing various clinical samples of nasopharyngeal swabs, saliva, and exhaled breath condensates obtained from 23 COVID-19 patients, we demonstrate that the positive rate of our system was 3.3 times higher than those of the conventional method. Combining with multiple portable cyclone samplers, we detected 52.2% (12/23) of the aerosol samples, six times higher than that of the commercial kit, collected from the isolation wards of COVID-19 patients, demonstrating the excellent performance of our system for SARS-CoV-2-in-aerosol monitoring. We envision the broad application of our microsystem in aerosol monitoring for fighting the COVID-19 pandemic.

PrimerBankID	Target Pathogen	Target Gene
RPB0036	SARS-CoV-2	ORF1ab

Recombinase Polymerase Amplification Combined with Fluorescence Immunochromatography Assay for On-Site and Ultrasensitive Detection of SARS-CoV-2

Author(s):

Guangyu Wang , Xingsheng Yang, Hao Dong, Zhijie Tu , Yong Zhou, Zhen Rong , Shengqi Wang

Journal:

Pathogens

Year:

2022

Abstract:

This study established a portable and ultrasensitive detection method based on recombinase polymerase amplification (RPA) combined with high-sensitivity multilayer quantum dot (MQD)-based immunochromatographic assay (ICA) to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RPA-MQD-based ICA method is reported for the first time and has the following advantages: (i) RPA is free from the constraints of instruments and can be promoted in point-of-care testing (POCT) scenarios, (ii) fluorescence ICA enhances the portability of detection operation so that the entire operation time is controlled within 1 h, and (iii) compared with common colorimetric-based RPA-ICA, the proposed assay used MQD to provide strong and quantifiable fluorescence signal, thus enhancing the detection sensitivity. With this strategy, the proposed RPA-MQD-based ICA can amplify and detect the SARS-CoV-2 nucleic acid on-site with a sensitivity of 2 copies/reaction, which is comparable to the sensitivity of commercial reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Moreover, the designed primers did not cross-react with other common respiratory viruses, including adenovirus, influenza virus A, and influenza virus B, suggesting high specificity. Thus, the established portable method can sensitively detect SARS-CoV-2 nucleic acid without relying on equipment, having good application prospects in SARS-CoV-2 detection scenarios under non-lab conditions.

PrimerBankID	Target Pathogen	Target Gene
RPB0035	SARS-CoV-2	S

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