Multiple-centre clinical evaluation of an ultrafast single-tube assay for SARS-CoV-2 RNA.

Background The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed Methods Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively Results The limit of detection was 1 copies/μl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples Conclusion This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas

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Reverse Transcription Recombinase-Aided Amplification Assay With Lateral Flow Dipstick Assay for Rapid Detection of 2019 Novel Coronavirus.

Comparing the significance of the utilization of next generation and third generation sequencing technologies in microbial metagenomics.

Classical swine fever (CSF), which is caused by the CSF virus (CSFV), remains one of the most economically important diseases of the global swine industry. Rapid and reliable detection of CSFV is critical for controlling CSF. In this study, a novel fluorescent probe-based real-time reverse transcription recombinase-aided amplification (rRT-RAA) assay, targeting a highly conserved position within the 5' non-translated region (5'NTR) among all CSFV genotypes, was developed for the detection of CSFV. The assay is highly specific to CSFV and does not cross react with other important viruses. Sensitivity analysis revealed that the assay could detect two 50% tissue culture infectious dose (TCID50 ) of CSFV RNA per reaction at 95% probability, which is comparable to that of a documentary reverse transcription quantitative PCR (RT-qPCR) assay for CSFV. The rRT-RAA assay exhibited good reproducibility, with intra- and inter-assay coefficient of variation values of <8.0%. Of the 135 samples (including 102 clinical tissue samples and 33 different cell culture isolates of CSFV), 50 and 52 samples were tested positive for CSFV by rRT-RAA and RT-qPCR, respectively. The coincidence rate between the two assays was 98.5% (133/135). Further linear regression analysis showed a significant correlation between the rRT-RAA and RT-qPCR assays with an R2 value of 0.8682. Interestingly, the amplification products of the rRT-RAA assay could be directly observed with naked eyes under a portable blue light imager, making it possible for an on-site testing. Our results indicate that the rRT-RAA assay is a robust diagnostic tool for the rapid detection of CSFV.

Development of a fluorescent probe-based real-time reverse transcription recombinase-aided amplification assay for the rapid detection of classical swine fever virus

Background The implementation of fast, sensitive and specific diagnostic tools such as those for point-of-care testing (POCT) can assure public health security and food safety against foodborne infectious disease outbreaks. Norovirus is the most common pathogen causing foodborne outbreaks and the most frequent cause of acute gastroenteritis in humans. This frequency in outbreaks highlights the importance of the development and application of new portable diagnostic concepts and sensitive, multiplex, accurate, real-time (SMART) technologies considering the recommendations of Public Health Authorities accentuating personal and food hygiene measures to limit the spread of the virus. The development of POCT using handheld devices has significantly increased in recent years to detect norovirus due to the undeniable advantages of these methods such as rapidness, ultra-sensitivity and simplicity for norovirus diagnosis, in comparison to the ‘gold standard’ quantitative polymerase chain reaction (qPCR). Scope and approach Recent progress in the development of POCT devices and changing detection priorities in the food industry highlights the importance of efficient POCT methods selection, therefore, this review sought to outline the comparison of emerging POCT methods to be used for the analysis of food, water and clinical samples including molecular methods, biosensing technologies, immunodiagnostics, microarrays, and other prospective methods whereby implementation could aid the investigation of outbreaks of norovirus. Key findings In this study, advantages and disadvantages of POCT methods are illustrated and the prospective identification of POCT methods are compared and discussed. In summary, the concluding remarks illustrate the future trends and directions for POCT methods which can be applied to detect norovirus and other viruses.

Current state-of-the-art diagnostics for Norovirus detection: Model approaches for point-of-care analysis

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.

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Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

Hepatitis B virus (HBV) is a widespread blood-borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma, particularly in south-east Asian and African countries where HBV is highly endemic and the budget and resources are limited. Therefore, simple, rapid, and portable field detection methods are crucial to efficiently control HBV infection. In this study, using heat-treated DNA, we developed two-field applicable detection assays for HBV based on recombinase-aided amplification (RAA). One was an internal controlled duplex RAA assay using a portable real-time fluorescence detection device, another was an instrument-free visual observation assay using lateral flow dipsticks. The entire experimental time was greatly shortened to less than 40 minutes at 39.0°C. The sensitivities, specificities, and clinical performance of both assays were evaluated. Compared with quantitative polymerase chain reaction assay as a reference, our results demonstrated that the two RAA-based assay obtained 97.18% and 95.77% of sensitivity, respectively, and the specificity was 100%, by testing a total of 157 serum samples with HBsAg positive. We conclude that the advantages of rapidity, simplicity, portability, and visualization of proposed two assays make them great potentials in point-of-care testing of HBV infection by untrained people in resource-limited situations.

Field applicable detection of hepatitis B virus using internal controlled duplex recombinase‐aided amplification assay and lateral flow dipstick assay

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses timely makes it a powerful tool for public health emergency response. Third-generation sequencing (TGS) offers advantages in speed and length of detection over second-generation sequencing (SGS). Here, we presented the end-to-end workflows for both Oxford Nanopore MinION and Pacbio Sequel on a viral disease emergency event, along with Ion Torrent PGM as a reference. A specific pipeline for comparative analysis on viral genomes recovered by each platform was assembled, given the high errors of base-calling for TGS platforms. All the three platforms successfully identified and recovered at least 85% Norovirus GII genomes. Oxford Nanopore MinION spent the least sample-to-answer turnaround time with relatively low but enough accuracy for taxonomy classification. Pacbio Sequel recovered the most accurate viral genome, while spending the longest time. Overall, Nanopore metagenomics can rapidly characterize viruses, and Pacbio Sequel can accurately recover viruses. This study provides a framework for designing the appropriate experiments that are likely to lead to accurate and rapid virus emergency response.

Comparison of third-generation sequencing approaches to identify viral pathogens under public health emergency conditions.

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Applicability of duplex real time and lateral flow strip reverse-transcription recombinase aided amplification assays for the detection of Enterovirus 71 and Coxsackievirus A16

Field applicable detection of hepatitis B virus using internal controlled duplex recombinase‐aided amplification assay and lateral flow dipstick assay

Comparison of third-generation sequencing approaches to identify viral pathogens under public health emergency conditions.