Establishment and application of a TTV detection method based on real-time PCR technology
Objective:To develop a TTV detection technique with higher sensitivity and specificity,providing crucial tech-nical support for uncovering the role of TTV in process of various diseases.Methods:For more accurate and sensitive TTV detec-tion,this study analyzed all published subtypes of TTV genome sequences,on the basis of which a real-time PCR assay targeting the UTR was established and compared with the more widely used PCR assays reported in the references.Results:The method established in this study showed good linear relationship within the range of 1×107 to 1×101 copies/μL standard sample concentra-tions,with a correlation coefficient of 1.000,a slope of-3.446,and a detection limit of 1×101 copies/μL.The repeatability test results demonstrated an intra-assay coefficient of variation of 7.22%,indicating strong repeatability and stability of the method.Compared with four sets of primers widely used in previous studies,our established real-time PCR detection method showed sig-nificantly higher sensitivity(P<0.01)in detecting 30 clinical samples.Sanger sequencing results confirmed that all 30 positive samples detected by our method were TTV-positive,with a detection specificity of 100%.Conclusion:This study adopts a real-time PCR detection method based on the TaqMan probe,which has high sensitivity and wide coverage of genotype range,espe-cially for quantitative detection in cases of low TTV viral load.It provides important technical support for the pathogenicity of the TTV virus and its application as an immune marker.
Torque teno virusGenome amplification and sequencingReal-time PCR detection
万方数据
维普
