FQ-PCR同步检测HTLV-Ⅰ及HTLV-Ⅱ方法的建立及临床应用

Establishment and Clinical Application of Fluorescence Quantitative PCR for Simultaneous Detection of HTLV-Ⅰ and HTLV-Ⅱ

目的:建立可同时检测HTLV-Ⅰ和HTLV-Ⅱ的双重荧光定量PCR(FQ-PCR)方法,并对所建体系进行方法学评价。方法:根据HTLV-Ⅰpol基因和HTLV-Ⅱtax基因的保守序列设计引物及TaqMan-LNA探针,构建重组质粒作为荧光定量PCR的标准品。优化荧光定量PCR反应条件,建立起可同时检测HTLV-Ⅰ和HTLV-Ⅱ的双重荧光定量PCR(FQ-PCR)的方法。结果:结果显示该方法对HTLV-Ⅰ及HTLV-Ⅱ的线性范围分别为108-102 copy/μl和107-10copy/μl;检测灵敏度分别为102copy/μl和10copy/μl。对阳性标本检测均呈阳性,对非目标菌检测均呈阴性。应用该方法对175例临床标本进行考核,有HTLV-Ⅰ阳性6例,HTLV-Ⅱ阳性3例,其中HTLV-Ⅰ和HTLV-Ⅱ双阳性2例。结论:本研究建立的双重FQ-PCR方法特异、快速、灵敏,对HTLV的血液筛查有重要意义。

Objective： To establish the duplex fluorescence quantitative PCR（FQ-PCR） method to detect HTLV-Ⅰ and HTLV-Ⅱ simultaneously,and to evaluate the detecting system in methodology.Methods： Primers and TaqMan-LNA probes were designed according to the conserved sequences of the HTLV-Ⅰ pol gene and the HTLV-Ⅱ tax gene.Engineered plasmids were constructed as the standards of fluorescence quantitative PCR.Conditions and reaction of fluorescence quantitative PCR were optimized to establish the duplex fluorescence quantitative PCR（FQ-PCR） method to detect HTLV-Ⅰ and HTLV-Ⅱ simultaneously.Results： The results showed that this method had a linear range of 10^8-10^2 copy/μl and 10^7-10 copy/μl and a detection sensitivity of 10^2 copy/μl for HTLV-Ⅰ and 10 copy/μl for HTLV-Ⅱ,respectively.Positive results were obtained for all positive samples and negative results were obtained for non-target bacteria detection.This method was applied to 175 clinical samples,and 6 HTLV-Ⅰ positive samples and 3 HTLV-Ⅱ positive samples were identified.Among them 2 samples were positive for both HTLV-Ⅰ and HTLV-Ⅱ.Conclusion： The established duplex FQ-PCR method in present study is specific,fast,and sensitive,which has a significant effect on the blood screening of HTLV.