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

环介等温扩增法(LAMP)扩增DNA具有高特异性、高效率、高速度的特点。然而，与实时荧光聚合酶链式反应相比，LAMP不足以定量检测靶DNA。在本研究中，我们用Q探针建立了HSV-1和HSV-2的LAMP定量检测方法，该探针通过特定位置的染料与鸟嘌呤碱基之间的电子转移而猝灭。在单链反应中，定量灯在15min内扩增出HSV DNA。建立的HSV-1和HSV-2定量检测灯灵敏度高，重复性好。然而，不可能确定多重扩增的最佳条件。此外，还评价了一种新的分子筛查GCV耐药HHV-6B的方法。新的分子方法的结果与直接测序法的结果一致。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，我们建立了实时荧光聚合酶链式反应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.