Gouvea V, Allen J R, Glass R I, Fang Z Y, Bremont M, Cohen J, McCrae M A, Saif L J, Sinarachatanant P, Caul E O
Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 30333.
J Clin Microbiol. 1991 Mar;29(3):519-23. doi: 10.1128/jcm.29.3.519-523.1991.
We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.
我们采用聚合酶链反应（PCR）来检测不可培养的B组和C组轮状病毒，并引入了一种简单便捷的技术从粪便标本中纯化病毒RNA。粪便提取物中存在的双链RNA通过吸附到羟基磷灰石上进行纯化，并用作逆转录和聚合酶扩增的模板。选择针对B组（基因8）和C组（基因6）轮状病毒的引物对，以扩增在溴化乙锭染色的琼脂糖凝胶中易于识别的cDNA拷贝的组特征大小。这些引物对分别用于单独的PCR检测中，或与针对A组轮状病毒（基因9）的引物对合并用于联合PCR检测，以同时检测所有三个轮状病毒组。该方法非常灵敏，用于鉴定粪便标本中B组和C组轮状病毒的人源和猪源毒株。使用内部组特异性引物进行的第二次PCR扩增进一步提高了检测的灵敏度，并确认了第一次扩增中获得的诊断结果。
