ABSTRACT
Objective:To develop a novel method for detection of genomic RNA of 2019-nCoV with reverse-transcription thermophilic helicase-dependent amplification (tHDA) and lateral flow dipsticks.Methods:This study included 143 PCR-negative nucleic acid specimens and 20 PCR- positive nucleic acid specimens collected from patients from January to April 2020 at Institute of Clinical Laboratory, Jinling Hospital. 5 pairs of primers were designed for conserved sequence of both gene N and E of 2019-nCoV, and the amplicons were analyzed by gel electrophoresis to screen out the most efficient primers. High level (5×10 5 copies/ml) and low level (5×10 2 copies/ml) templates were amplified, and lateral flow dipsticks (LFD) were used to detect amplification products. The amplification time and visualization time were optimized to visualize the results, and the optimal tHDA-LFD reaction system was established. Simulated specimens with low, medium, and high concentrations were detected for 15 times, and precision was assessed. The limit of detection was evaluated using tenfold serial dilutions of 2019-nCoV in contrived samples. To evaluate the cross-reactivity, six samples of respiratory viruses, including influenza A virus, influenza B virus, human adenovirus, human respiratory syncytial virus, human parainfluenza virus and coronavirus 229E were tested using this assay. The tHDA-LFD assay was performed to detect 2019-nCoV in 163 clinical specimens stored in our laboratory. The clinical diagnostic efficacy was determined. Results:The established RT-tHDA one-step method combined with LFD was adopted, which could be conducted within 60 min and was reproducible with good precision, showing 100% positive concordance rate. The assay showed no cross reaction with other six human respiratory pathogens. The limit of detection of the RT-tHDA assay was 5×10 2 copies/ml for both N and E genes detected by LFD. The diagnostic efficacy evaluation showed that the sensitivity of the method was 95.00%(19/20), and the specificity was 100.00%(143/143). The positive predicted value of the method was 100.00% (19/19)and the negative predicted value was 99.31%(143/144). Compared with real-time RT-qPCR assay, it showed Kappa value of 0.971 ( P<0.0001). Conclusion:The proposed tHDA-LFD assay is a rapid and visualized method to detect 2019-nCoV.
ABSTRACT
Objective@#To establish and hevaluate a detection method for influenza virus using reverse transcriptase-recombinase polymerase amplification (RT-RPA).@*Methods@#RT-RPA was developed for detection of influenza viruses (type A and B) and subtyping of H1 and H3 using the primers targeted matrix and hemagglutinin (HA) genes of influenza A virus, and non-structural (NS) protein gene of influenza B virus. The specificity and sensitivity of RT-RPA were determined.@*Results@#The RT-RPA for the detection of influenza viruses showed specific amplification products of corresponding target gene, but no amplification products for other respiratory viruses, indicating that the method had good specificity. The detection limits of RT-RPA were 100 copies/μl. RT-RPA combined with SYBR Green I was used for the detection of influenza B virus with the detection limit of 100 copies/μl.@*Conclusions@#The feasibility of detecting influenza virus by RT-RPA was preliminarily confirmed.