一种基于real-time PCR技术的TTV检测方法的建立及应用OACSTPCD

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.