Suzuki Ryota, Yoshikawa Tetsushi, Ihira Masaru, Enomoto Yoshihiko, Inagaki Shoji, Matsumoto Koichi, Kato Koji, Kudo Kazuko, Kojima Seiji, Asano Yoshizo
Division of Pharmacy, Fujita Health University Hospital, Toyoake, Aichi, Japan.
J Virol Methods. 2006 Mar;132(1-2):216-21. doi: 10.1016/j.jviromet.2005.09.008. Epub 2005 Nov 11.
Cytomegalovirus (CMV) loop-mediated isothermal amplification (LAMP) was performed on DNA extracted from CMV (AD-169)-, herpes simplex virus (HSV) 1 (KOS)-, HSV-2 (186)-, varicella-zoster virus (Oka-vaccine)-, human herpesvirus (HHV)-6 A (U1102)-, HHV-6 B (Z29)-, and HHV-7 (RK)-infected cells. Although amplified CMV demonstrated typical ladder patterns, no LAMP product was detected in reactions performed with other viral DNAs. The sensitivity of the CMV LAMP was 500 copies/tube, as determined by either agarose gel electrophoresis or turbidity assay. To determine whether CMV LAMP could be used for quantitative analysis of viral DNA, threshold times, defined as the time (in seconds) to reach the threshold level (0.1), were measured by amplification of serial dilutions of the plasmid DNA. The standard curve exhibited a correlation coefficient of 0.944, a slope of -208.1, and a y-intercept of 3261.4. Following these initial validation experiments, we analyzed 180 samples collected serially from 20 pediatric hematopoietic stem cell transplant recipients. Detection of CMV DNA in whole blood (WB) was tested by CMV LAMP and real-time polymerase chain reaction (PCR). When >500 copies/tube (>5000 copies/200 microl of WB) was defined as positive for CMV infection, the sensitivity, specificity, positive predictive value, and negative predictive values of the CMV LAMP were 80.0, 98.9, 66.7, and 99.4%, respectively.
对从巨细胞病毒（CMV，AD - 169株）、单纯疱疹病毒1型（HSV - 1，KOS株）、HSV - 2（186株）、水痘 - 带状疱疹病毒（Oka疫苗株）、人类疱疹病毒（HHV） - 6 A（U1102株）、HHV - 6 B（Z29株）和HHV - 7（RK株）感染的细胞中提取的DNA进行了巨细胞病毒环介导等温扩增（LAMP）。尽管扩增的CMV呈现出典型的梯状条带模式，但在用其他病毒DNA进行的反应中未检测到LAMP产物。通过琼脂糖凝胶电泳或浊度测定法确定，CMV LAMP的灵敏度为500拷贝/管。为了确定CMV LAMP是否可用于病毒DNA的定量分析，通过对质粒DNA系列稀释液进行扩增，测量了达到阈值水平（0.1）所需的时间（以秒为单位），即阈值时间。标准曲线的相关系数为0.944，斜率为 - 208.1，截距为3261.4。在这些初步验证实验之后，我们分析了从20名儿科造血干细胞移植受者连续采集的180份样本。通过CMV LAMP和实时聚合酶链反应（PCR）检测全血（WB）中的CMV DNA。当将>500拷贝/管（>5000拷贝/200微升WB）定义为CMV感染阳性时，CMV LAMP的灵敏度、特异性、阳性预测值和阴性预测值分别为80.0%、98.9%、66.7%和99.4%。
