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

环介导等温扩增法（LAMP）具有高特异性、高效率和快速扩增DNA的特点。然而，与实时PCR相比，LAMP在目标DNA的定量方面是不够的。在本研究中，我们建立了HSV-1和HSV-2的定量LAMP检测方法，该方法通过特定位置的染料和鸟嘌呤碱基之间的电子转移来猝灭。在单复合反应中，定量lamp在15min内扩增HSV DNA。该方法对HSV-1和HSV-2具有较高的灵敏度和重复性。然而，不可能确定一个最佳条件的多重放大。此外，我们还评价了一种利用Qprobe分子筛选抗GCV的HHV-6B的新方法。新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与循环探针的结合可能是定量分析的好工具。首先，建立了实时PCR和循环探针相结合的VZV野生型和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.