Kou Xiao-Xi, Wu Qing-Ping, Fan Hong-Ying
Guangdong Instisute of Microbiology, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangzhou 510070, China.
Wei Sheng Wu Xue Bao. 2006 Apr;46(2):328-30.
Rotavirus is one of the major cause of the viral gastroenteritis throughout the world. A nucleic acid sequence-based amplification(NASBA) technique for the detection of rotavirus in faecal specimens was developed and compared to the RT-PCR technique. Primers were designed according to the high conserved region of VP7 gene. Amplicons were detected by denaturating agarose gel, and then demonstrated by Northern hybridization with digoxigenin-labeled oligonucleotide probe. The anticipative specific amplification product is 392bp,and no nonspecific products appear even the concentration of nontarget nucleic acid as high as 1 microg/microL. A detection limit of 50 pg target RNA/mL is obtained when the optimal amplification time of 3h used.The NASBA assay will be a favourable alternative to RT-PCR for the investigation of rotavirus outbreaks as a routine diagnostic test in the near future because it is shown to be highly sensitive, specific and do not require specialized equipment.
轮状病毒是全球病毒性肠胃炎的主要病因之一。开发了一种基于核酸序列扩增（NASBA）的技术用于检测粪便标本中的轮状病毒，并与逆转录聚合酶链反应（RT-PCR）技术进行比较。根据VP7基因的高度保守区域设计引物。扩增产物通过变性琼脂糖凝胶检测，然后用洋地黄毒苷标记的寡核苷酸探针进行Northern杂交验证。预期的特异性扩增产物为392bp，即使非靶核酸浓度高达1μg/μL也无非特异性产物出现。当使用3小时的最佳扩增时间时，检测限为50pg靶RNA/mL。NASBA检测法因其高度灵敏、特异且不需要专门设备，在不久的将来作为常规诊断试验用于调查轮状病毒爆发时，将是RT-PCR的一个良好替代方法。
