Rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method
Rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method
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DOI：
10.1002/jmv.20223
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发表时间：
2004-12-01
影响因子：
12.7
通讯作者：
Asano, Y
中科院分区：
文献类型：
--
作者：
Okamoto, S;Yoshikawa, T;Asano, Y
评价水痘-带状疱疹病毒（VZV）环介导等温扩增（LAMP）快速诊断病毒感染的可靠性。VZV特异性引物只扩增VZV DNA；在与其他病毒DNA模板的反应中未观察到LAMP产物。该方法的特异性通过琼脂糖凝胶电泳和浊度测定两种独立的测定方法得到证实。琼脂糖凝胶电泳测定VZV LAMP的灵敏度为500拷贝/管。然而，使用浊度测定法检测，灵敏度为1,000拷贝/管。在这些初步的验证研究之后，评估了VZV LAMP在临床标本中检测病毒DNA的可靠性。从水疱性皮肤疹患者收集的32份拭子样本进行了VZV DNA检测。通过先前建立的VZV实时PCR技术，在样品编号10-32中确认了VZV。VZV LAMP产品采用样品13 ~ 32的浊度检测（灵敏度；87.0%，特异性；阳性预测值100%；阴性预测值100%；75%）。虽然在三个样品中可以检测到低水平的VZV DNA，但结果不同（样品编号10-12），但在这些样品中未检测到VZV LAMP产物，表明该试验的检测限较高。在接下来的实验中考察了VZV LAMP法对DNA提取步骤的要求。浊度测定在所有20个阳性拭子样本（样品编号13-32）中检测到VZV LAMP产品，无论DNA提取如何。(C) 2004 Wiley-Liss, Inc。
The reliability of varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) was evaluated for rapid diagnosis of viral infection. VZV-specific primers only amplified VZV DNA; no LAMP products were observed in reactions performed with other viral DNA templates. The specificity of this method was confirmed by two independent determinations, agarose gel electrophoresis and a turbidity assay. The sensitivity of VZV LAMP, determined by agarose gel electrophoresis, were 500 copies/tube. Detection using the turbidity assay, however, gave a sensitivity of 1,000 copies/tube. After these initial validation studies, reliability of VZV LAMP was evaluated for the detection of viral DNA in clinical specimens. Thirty-two swab samples collected from patients with vesicular skin eruptions were tested for VZV DNA. VZV was confirmed in sample numbers 10-32 by VZV real-time PCR, a previously established technique. VZV LAMP products were detected using turbidity from samples 13 to 32 (sensitivity; 87.0%, specificity; 100%, positive predictive value; 100%, negative predictive value; 75%). Although low levels of VZV DNA could be detected in the three samples exhibiting divergent results (samples numbers 10-12), no VZV LAMP product was detected in these samples, indicating a higher detection limit for this assay. Requirement of a DNA extraction step in the VZV LAMP method was examined in next experiment. The turbidity assay detected a VZV LAMP product in all of the 20 positive swab samples (samples numbers 13-32), regardless of DNA extraction. (C) 2004 Wiley-Liss, Inc.