Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples
Reverse transcription multiplex PCR for differentiation between polio- and enteroviruses from clinical and environmental samples
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DOI：
10.1128/jcm.33.6.1442-1447.1995
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发表时间：
1995-06
影响因子：
9.4
通讯作者：
D. Egger;L. Pasamontes;Marianne Ostermayer;K. Bienz
中科院分区：
文献类型：
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作者：
D. Egger;L. Pasamontes;Marianne Ostermayer;K. Bienz
为了快速检测脊髓灰质炎病毒及其与非脊髓灰质炎病毒肠道病毒的区别，我们开发了一种方法，即首先将临床或环境样本接种到细胞培养管中。培养过夜后，用一对能检测所有肠道病毒(T.Hyypiä，P.Auvinen和M.Maaronen，J.Gen.Virol)的引物对培养物进行逆转录多重聚合酶链式反应。)和两对新设计的针对所有36株脊髓灰质炎病毒株的特异性引物对。聚合酶链式反应产物可以通过琼脂糖凝胶中的长度进行明确的鉴定，而脊髓灰质炎病毒株的遗传异质性排除了通过与内部探针反向杂交进行鉴定的可能性。所提出的方法对样品中物质(粪便、污水)的抑制作用高度不敏感。它允许在24小时内检测脊髓灰质炎病毒，并在24小时内将脊髓灰质炎病毒与非脊髓灰质炎病毒肠道病毒区分开来，并同时分离出适合进一步分型的活菌株。
For the rapid detection of polioviruses and their differentiation from nonpoliovirus enteroviruses, we developed a protocol in which clinical or environmental specimens are first inoculated onto cell cultures in tubes. After overnight incubation, the cultures are subjected to reverse transcription multiplex PCR with a primer pair which detects all enteroviruses (T. Hyypiä, P. Auvinen, and M. Maaronen, J. Gen. Virol. 70:3261-3268 1989) and two newly designed primer pairs specific for all 36 poliovirus strains tested. The PCR products can unequivocally be identified by their lengths in agarose gels, whereas the genetic heterogeneity of the poliovirus strains precludes identification by back-hybridization with internal probes. The proposed protocol is highly insensitive to the inhibitory effects of substances in the sample (stool, sewage). It allows for the detection of polioviruses and for polioviruses to be distinguished from nonpoliovirus enteroviruses within 24 h, and it allows for the concomitant isolation of a viable strain suitable for further typing.