POLYMERASE CHAIN-REACTION AMPLIFICATION AND TYPING OF ROTAVIRUS NUCLEIC-ACID FROM STOOL SPECIMENS
POLYMERASE CHAIN-REACTION AMPLIFICATION AND TYPING OF ROTAVIRUS NUCLEIC-ACID FROM STOOL SPECIMENS
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DOI：
10.1128/jcm.28.2.276-282.1990
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发表时间：
1990-02-01
影响因子：
9.4
通讯作者：
FANG, ZY
中科院分区：
文献类型：
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作者：
GOUVEA, V;GLASS, RI;FANG, ZY
通过聚合酶链反应（PCR）直接从粪便标本中扩增轮状病毒编码主要外壳糖蛋白VP7的基因片段。从粪便样品中提取的双链RNA用作逆转录的模板，随后立即在相同的反应混合物中使用Taq聚合酶进行扩增。研究了各种条件以优化扩增基因的产率。MgCl2、二甲基亚砜和模板RNA的浓度至关重要。引物对的选择允许扩增整个区段或特定部分。通过使用来自基因上不同区域的类型特异性引物，我们设计了一种PCR分型方法，其中每个人血清型病毒产生一个特征性片段大小，在琼脂糖凝胶中容易识别。PCR分型方法应用于10个轮状病毒参考株，包括所有6个已知的人类血清型（血清型1，2，3，4，8，和9），和34个粪便标本先前通过酶免疫测定与单克隆抗体的血清分型。分子和血清学方法之间存在绝对相关性。此外，14个粪便标本nonserotypable酶免疫分析与单克隆抗体可以分型的PCR方法。除了直接从粪便中进行轮状病毒检测和分型外，PCR方法还提供了一种快速有效的方法，用于获得大量适合测序、克隆和其他遗传学研究的cDNA，从而无需细胞培养和病毒纯化。
The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR methodprovides a rapid and efficient means fo obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.