Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis
Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis
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DOI：
10.1016/j.jcv.2010.12.009
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发表时间：
2011-04-01
影响因子：
8.8
通讯作者：
Houpt, Eric
中科院分区：
文献类型：
--
作者：
Liu, Jie;Kibiki, Gibson;Houpt, Eric
背景：几种病毒可以引起腹泻病，这是世界范围内发病率和死亡率的主要原因。现有的诊断方法包括ELISA和核酸扩增，通常单独进行。目的：（1）建立一种同时检测肠道主要病毒病原体的多重检测方法。(2)定量的病毒载量归一化与extrinsic control.Study设计：一个简单的协议相结合的一步多重逆转录-聚合酶链反应（RT-PCR）与微球为基础的荧光检测诺如病毒GI和GII，轮状病毒，星状病毒，sapovirus，腺病毒。外源性对照，噬菌体MS 2，掺入到每个粪便样品之前，核酸提取样品之间的核酸提取和amplification.Results的效率归一化：荧光结果是定量的，几乎一样灵敏的相应的单重真实的时间RT-PCR（qRT-PCR）分析样品。在对坦桑尼亚腹泻住院患者的229份粪便样本进行检测后，该检测方法对所有分析物的灵敏度和特异性均在88%至100%之间。MS 2样品之间的荧光强度的差异表明变量提取效率，并用于更好地完善每个speciments.Conclusions的病毒载量：这一步核酸为基础的检测，使快速，灵敏和特异性的检测胃肠炎的主要病毒原因。该测定产生的定量结果可为临床研究提供信息，特别是在混合感染的情况下。(C)2011爱思唯尔有限公司版权所有。
Background: Several viruses can cause diarrheal disease, a leading cause of morbidity and mortality worldwide. Existing diagnostic methods include ELISA and nucleic acid amplification, usually performed individually.Objectives: (1) To develop a multiplexed assay for simultaneous detection of major enteric viral pathogens. (2) Quantitation of viral load by normalizing with an extrinsic control.Study design: A simple protocol combining a one-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) with microsphere-based fluorescence detection was developed for norovirus GI and GII, rotavirus, astrovirus, sapovirus, and adenovirus. An extrinsic control, bacteriophage MS2, was spiked into each fecal sample before nucleic acid extraction to normalize between samples for the efficiency of nucleic acid extraction and amplification.Results: The fluorescent results were quantitative and nearly as sensitive as the corresponding singleplex real time RT-PCR (qRT-PCR) assay on analytic samples. Upon testing 229 fecal samples from inpatients with diarrhea in Tanzania the assay yielded between 88% and 100% sensitivity and specificity for all analytes. The difference in fluorescence intensities of MS2 between samples indicated variable extraction efficiency and was used to better refine the viral load of each specimen.Conclusions: This one-step nucleic acid-based assay enables rapid, sensitive and specific detection of the major viral causes of gastroenteritis. The quantitation yielded by the assay is informative for clinical research particularly in the context of mixed infections. (C) 2011 Elsevier B.V. All rights reserved.