MHV、MPV和Reo-3三重TaqMan定量PCR检测方法的建立OA

Objective To establish a highly specific and sensitive triplex TaqMan quantitative PCR method for the detection of mouse hepatitis virus(MHV),mouse parvovirus(MPV),and reovirus type 3(Reo-3)in laboratory mice.Methods Recombinant plasmid standards were constructed using conserved genomic regions of the three target viruses.Specific primers and probes were designed,and a triplex TaqMan quantitative PCR(qPCR)system was optimized through preliminary experiments.Sensitivity was evaluated using 10-fold serial dilutions(101-107 copies/μL).Intra-and inter-assay repeatability were assessed via reproducibility experiments.Results A strong linear correlation(R 2>0.99)was observed between copy number and Ct value at template concentrations of 102-107 copies/μL.Intra-assay and inter-assay coefficients of variation(CV)were both below 5%.No cross-reactivity was observed with mouse cytomegalovirus(MCMV),vesicular stomatitis virus(VSV),Sendai virus(SeV),or pneumonia virus of mice(PVM).Conclusion The triplex TaqMan quantitative PCR method established in this study has high sensitivity,good repeatability,and strong specificity.It enables the rapid,specific,and sensitive detection of MHV,MPV,and Reo-3,allows the simultaneous detection of multiple pathogens in a single tube,and can be applied to the detection of clinical samples from laboratory animals and to epidemiological investigations,thereby providing effective scientific and technological support for the prevention and control of viral infection and the interruption of transmission.