Zeng S Q, Halkosalo A, Salminen M, Szakal E D, Puustinen L, Vesikari T
Vaccine Research Center, University of Tampere, Medical School, Vaccine Research Center, Biokatu 10, FIN-33520 Tampere, Finland.
J Virol Methods. 2008 Nov;153(2):238-40. doi: 10.1016/j.jviromet.2008.08.004. Epub 2008 Sep 17.
The standard diagnosis of rotavirus gastroenteritis is based on the demonstration of rotavirus antigen in stools using an enzyme immunoassay (EIA). In this study, a one-step quantitative RT-PCR (Q-PCR) was used for sensitive detection of rotavirus in diarrheal stools. The primers and TaqMan probe for the Q-PCR were selected from a highly conserved region of the non-structural protein 3 (NSP3) of rotavirus. After validation, the test was applied to study rotavirus EIA positive (N=25) and EIA negative (N=143) stool specimens from cases of acute gastroenteritis of all degrees of severity in a prospective follow-up cohort of infants from 2 months to 2 years of age. Q-PCR detected all 25 EIA positive rotavirus antigens and seven additional cases that were rotavirus EIA negative, i.e. 28% more rotavirus positive cases than identified by EIA. It is concluded that Q-PCR using primers targeted at NSP3 is a rapid and sensitive method for diagnosing acute rotavirus gastroenteritis.
轮状病毒肠胃炎的标准诊断是基于使用酶免疫测定法（EIA）在粪便中检测到轮状病毒抗原。在本研究中，采用一步定量逆转录聚合酶链反应（Q-PCR）对腹泻粪便中的轮状病毒进行灵敏检测。用于Q-PCR的引物和TaqMan探针是从轮状病毒非结构蛋白3（NSP3）的高度保守区域中选取的。经过验证后，该检测方法应用于研究前瞻性随访队列中2个月至2岁婴儿所有严重程度的急性肠胃炎病例的粪便样本，其中轮状病毒EIA检测呈阳性的有25例（N = 25），呈阴性的有143例（N = 143）。Q-PCR检测出所有25例EIA阳性的轮状病毒抗原，另外还检测出7例EIA阴性的病例，即比EIA检测出的轮状病毒阳性病例多28%。结论是，使用针对NSP3的引物进行Q-PCR是诊断急性轮状病毒肠胃炎的一种快速且灵敏的方法。
