Establishment of multiplex real-time fluorescent quantitative RT-PCR for rapid detection of Alongshan virus and Songling virus
Objective To establish a multiplex real-time fluorescent quantitative reverse transcription polymerase chain reaction(multiplex real-time qRT-PCR)method for rapid detection of nucleic acids of Alongshan virus(ALSV)and Songling virus(SGLV).Methods Specific primers and TaqMan probes were designed for the conserved regions of the NS 3 gene of ALSV and the S gene of SGLV.A multiplex real-time qRT-PCR method was established for the two viruses,and the specificity,sensitivity,and repeatability of the method were evaluated.Tick specimens were used to verify the method.Results The detection method had no cross-reactivity with six other arboviruses,such as tick-borne encephalitis virus,with a sensitivity up to 1×101 copies/μl,and the coefficient of variation of cycle threshold for repeatability testing was less than 2.00%.Through this method,two groups of ALSV-positive specimens and one group of SGLV-positive specimens were detected from 30 groups of tick specimens collected from Heilongjiang,China in 2019.This method was verified to be 100%consistent with the general PCR method in terms of test results.Conclusion In this study,a highly sensitive and highly specific multiplex real-time qRT-PCR method for rapid detection of ALSV and SGLV has been successfully established.
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