Development of quantitative multiplex DNA amplification method by loop-mediated isothermal amplification (LAMP) using Qprobe.

环介导等温扩增方法（LAMP）以高特异性、效率和速度扩增DNA。然而，与实时PCR相比，LAMP不足以定量靶DNA。本研究建立了一种以Q探针为探针的HSV-1和HSV-2的LAMP定量检测方法。在单重反应中，定量的LAMPs在15 min内扩增HSV DNA。HSV-1和HSV-2的LAMP定量检测灵敏度高，重复性好。然而，不可能确定多重扩增的最佳条件。此外，我们还建立了一种新的分子生物学方法--Q探针法，用于筛选HHV-6 B的抗GCV株。新的分子生物学方法的结果与直接测序的结果一致。序列特异性DNA-RNA嵌合探针（循环探针）也是检测SNP的合适方法。LAMP和循环探针的结合可能是一种很好的定量分析工具。作为第一步，我们建立了联合收割机实时荧光定量PCR与循环探针相结合的VZV野毒株和Oka疫苗株的定量检测方法。

The loop-mediate isothermal amplification method (LAMP) amplifies DNA with high specificity efficiency, and speed. However, the LAMP is insufficient for quantification of target DNA in comparison to the real-time PCR. In this study, we developed quantitative LAMP assay for HSV-1 and HSV-2 by Qprobe, which is quenched via electron transfer between the dye and guanine base at particular position. In singleplex reaction, the quantitative LAMPs amplified HSV DNA within 15min. The quantitative LAMP for HSV-1 and HSV-2 had high sensitivity and reproducible. However, it was impossible to determine an optimal condition for multiplex amplification. In addition, a new molecular method by using Qprobe was evaluated for screening of GCV resistant HHV-6B. The results of the new molecular method were consistent with those of the direct sequence. The sequence-specific DNA-RNA chimeric probe (cycling probe) is also appropriate method for the detection of SNP. The combination of LAMP and cycling probe might be good tool for quantitative analysis. As the first step, we developed quantitative assay combine real-time PCR with cycling probe to discriminate wild type and Oka-vaccine strain of VZV.