Rapid detection of porcine encephalomyocarditis virus (EMCV) by
isothermal reverse transcription recombinase polymerase amplification
assays
Abstract
Reverse transcription recombinase polymerase amplification assays
combined with the fluorescence detection platform (qRT-RPA) and lateral
flow biosensor (LFB RT-RPA) were developed and validated for rapid
detection of porcine EMCV. The primers and probes were designed based on
the highly conserved region of 3D gene of porcine EMCV. The optimal
reaction condition of qRT-RPA and LFB RT-RPA assay was at 42 °C for 20
min. The assays were highly specific to EMCV and no cross-reactions were
observed with other 7 porcine viruses. With the 10-fold sieral diluted
EMCV genomic RNA as template, the limit of detection of the qRT-RPA
assay was 1.0×10 2 copies and the limit of detection
of LFB RT-RPA was 1.0×10 1 copies. A total of 92
samples collected from EMCV inoculated mice (24), control mice (8) and
pigs showing reproductive failures (60) were examined using developed
RT-RPA and a described qRT-PCR, and the diagnostic agreement between
qRT-RPA (23/92), LFB RT-RPA (25/92) and qRT-PCR (23/92) was 100% and
97.83, respectively. The performances of the developed RT-RPA assays
were comparable to a qRT-PCR, while the RPA assays needed less time and
easy to perform to obtain the positive results and LFB RT-RPA was higher
sensitive than the qRT-PCR. The developed EMCV RT-RPA assays are rapid,
reliable and easy to perform, which provide an attractive and promising
tool for effective detection of EMCV in low-resource settings.