不同毒力番鸭呼肠孤病毒双重PCR鉴别方法的建立

Establishment of Duplex PCR for Identification of Muscovy Duck Reovirus with Different Virulence

自2001年我国出现番鸭感染呼肠孤病毒(MDRV)报道以来,本课题组从病鸭体内分离到2株在临床上致病力差别很大的病毒毒株,并分别命名为YJL株和YB株。本研究根据这2株病毒基因特点,分别针对YB株P10蛋白基因和YJL株P17蛋白基因设计2对特异性引物,通过条件优化建立了鉴别2个毒株的双重PCR。结果显示:该方法可同时扩增出YB 288 bp、YJL 441 bp长度的特异性片段,而对其他番鸭病病原无扩增结果;该方法最佳反应条件为95℃5 min,94℃1 min,55℃45 s,72℃1 min,30个循环,72℃延伸10 min;该方法最低能检出10 pg YB和YJL株RNA模板;使用单重PCR和多重PCR方法对60份临床病料检测验证,两者符合率为100%。结果表明该方法特异性、敏感性和重复性良好,具有快速、准确的特点,可用于这2种不同毒力毒株的鉴别。

Since Muscovy duck reovirus （MDRV）infection was discovered in China in 2001,two MDRV strains with quite different pathogenicity, which were named as YJL and YB strain respectively, have been isolated from diseased ducks by our group. In this study,two pairs of specific primers were designed according to coding genes of YB P10 and YJL P17 respectively, based on the genomie differences of YB and YJL strain and through optimization of PCR conditions, a detection method of duplex PCR was established, which could be used for identification of the two strains. The results showed that：the expected fragments of 288 bp of YB strain and 441 bp of YJL strain could be simultaneously amplified by this duplex PCR,while amplification results of other muscovy duck pathogens were negative;The optimum reaction conditions for duplex PCR were as follows： 95 % 5 rain, 94 % 1 min, 55 ℃ 45 s, 72 ℃ 1 min, 30 cycles,72 0（2 10 min; The template amount used in this detection could be as low as 10 pg; Detection of 60 copies of clinical samples by single PCR and duplex PCR showed a coincidence rate of 100%. With a good specificity, sensitivity and repeatability, this method could be used for identification of two kinds of virulence of MDRV fast and accurately.