Universal diagnostic RT-PCR protocol for arboviruses
Universal diagnostic RT-PCR protocol for arboviruses
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DOI：
10.1016/s0166-0934(98)00003-2
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发表时间：
1998-05-01
影响因子：
3.1
通讯作者：
Kuno, G
中科院分区：
文献类型：
--
作者：
Kuno, G
A.对选定数量的PCR方案进行评估，以确定它们是否可以作为检测和鉴定所有虫媒病毒的通用方案。在这项研究中，四个参数，影响RT-PCR的效率（RNA提取方法，逆转录酶的选择，DNA聚合酶的选择和热循环程序）进行了评估。这些参数的最佳组合采用硅胶膜旋转柱，RAV-2逆转录酶，Tth DNA聚合酶，和一个简单的修改公布的热循环程序的使用。用该方法设计的50多对引物对5个虫媒病毒科的病毒RNA进行了扩增，结果令人满意。对于每个测试的引物对，通过该通用方案获得的灵敏度和特异性与通过原始方案获得的灵敏度和特异性相当;并且对于一些引物，观察到灵敏度提高。还发现，对商业RT-PCR试剂盒的建议方案进行简单修改可以产生几乎相同的结果，并作为另一种通用方案。使用通用的诊断逆转录-聚合酶链反应（RT-PCR）方案，可以更有效地对临床或生物标本进行针对属于许多家族的大量RNA病毒的同时筛查，以在因鉴别诊断困难而复杂的情况下进行病原学确定。此外，这样的通用协议有助于降低基于PCR的诊断操作的成本，并使机构内或协作机构之间的基于PCR的诊断的质量标准化。一个合理的策略是分两个阶段进行诊断，在第一阶段使用广泛的组反应性引物，以缩小可能的病原体的范围，并在第二阶段使用病毒特异性引物进行鉴定。然而，在采用这种策略之前，必须提供针对大量虫媒病毒的更具有组反应性的引物，而目前还没有这样的引物。此外，对于策略的第二阶段，需要为每种病毒选择最佳的一对或多对引物。(C)1998年由Elsevier Science B. V.出版，版权所有。
A. selected number of PCR protocols were evaluated to determine if they could serve as a universal protocol for detecting and identifying all arboviruses. In this study, four parameters that affect the efficacy of RT-PCR (RNA extraction method, choice of reverse transcriptase, choice of DNA polymerase and thermocycling program) were evaluated in combination. The most optimal combination of those parameters employed use of silica gel membrane spin column, RAV-2 reverse transcriptase, Tth DNA polymerase, and a simple modification of a published thermocycling program. By this modified protocol, viral RNA could be amplified satisfactorily with more than 50 pairs of primers designed for diagnosis of arboviruses representing five families. The sensitivity and specificity obtained by this universal protocol were comparable to those obtained by the original protocol for each primer pair tested; and for some primers, improved sensitivity was observed. It was also found that a simple modification of a suggested protocol of a commercial RT-PCR kit could produce nearly identical results and serve as another universal protocol. With the use of a universal diagnostic reverse transcriptase-polymerase chain reaction (RT-PCR) protocol, simultaneous screening of clinical or biological specimens against a large number of RNA viruses belonging to many families can be performed more efficiently for etiologic determination in the situations complicated by the difficulty of differential diagnosis. Furthermore, such a universal protocol facilitates reducing the cost of PCR-based diagnostic operation and standardizing the qualities of PCR-based diagnosis within an institution or among collaborating institutions. A logical strategy is to conduct diagnosis in two stages by using broadly group-reactive primers in the first stage to narrow the range of possible etiologic agents and using virus-specific primers in the second stage for identification. Before such a strategy is employed, however, more group-reactive primers for a large number of arboviruses, for which no such primers currently exist, must be made available. Furthermore, the best pair or pairs of primers need to be selected for each virus for the second stage of the strategy. (C) 1998 Published by Elsevier Science B.V. All rights reserved.