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首页> 外文期刊>Journal of Clinical Microbiology >Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination
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Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

机译：同时基因分型和人类T淋病病毒1、2和3型前病毒载量测定的多重实时PCR分析方法的开发和验证

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The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 105 to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r2 = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.
机译：人类T淋巴病毒（HTLV）的前病毒载量仍然是疾病进展的最佳替代指标。已经开发出实时PCR技术来检测和定量世界性HTLV 1a型（HTLV-1a）和HTLV-2。由于观察到亚型和基因型多样性的增长水平，我们开发了用于同时检测，基因分型和定量HTLV-1、2和3的前病毒载量的多重定量PCR。我们的测定使用 tax 类型特异性引物和双标记探针，动态范围为10 5 至10个HTLV拷贝。分析了163个样本，其中每个HTLV基因型内的所有不同亚型都可以检测到。将我们多重测定的前病毒载量测定性能与另一家研究所开发的基于SYBR绿色检测的先前发表的HTLV-1单重定量PCR的性能进行了比较。线性回归分析显示，用这两种方法测得的前病毒负荷值之间具有统计学显着性（ P <0.0001）和强（ r 2 = 0.87）相关性独特的实时PCR分析。总之，我们的新颖测定法可在单个扩增反应中提供准确的分子诊断和基因分型，以及确定HTLV感染个体的前病毒载量。此外，当当前可用的血清学检测还不够时，我们的分子检测可以提供另一种选择。

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