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Development of a fluorescent probe‐based real‐time reverse transcription recombinase‐aided amplification assay for the rapid detection of classical swine fever virus
Author(s) -
Tu Fei,
Yang Xintan,
Xu Shengkui,
Chen Dengjin,
Zhou Lei,
Ge Xinna,
Han Jun,
Zhang Yongning,
Guo Xin,
Yang Hanchun
Publication year - 2021
Publication title -
transboundary and emerging diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.392
H-Index - 63
eISSN - 1865-1682
pISSN - 1865-1674
DOI - 10.1111/tbed.13849
Subject(s) - classical swine fever , reverse transcriptase , virology , virus , biology , recombinase , real time polymerase chain reaction , microbiology and biotechnology , reverse transcription polymerase chain reaction , polymerase chain reaction , gene , gene expression , genetics , recombination
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 (TCID 50 ) 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 R 2 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.