兔病毒性出血症病毒巢式RT-PCR检测方法的建立及应用

Establishment and Application of a Nest PCR Method for Detection of Rabbit Hemorrhagic Disease Virus

为建立一种快速的兔病毒性出血症病毒(rabbit hemorrhagic disease virus,RHDV)病原检测方法,本研究根据Gen-Bank上登录的RHDVVP60基因序列,设计合成内外2对引物,优化PCR反应条件,建立了检测RHDV的巢式RT-PCR方法。该方法对兔轮状病毒、仙台病毒、健康兔肝脏组织的扩增结果均为阴性;该方法第1次扩增的敏感性是10ng,第2次扩增的敏感性是0.1ng,第2次比第1次扩增的敏感性高100倍。建立的巢式RT-PCR方法具有特异性强、敏感性高、重复性好等优点,可以准确快速检测出极低含量的RHDV,将为兔病毒性出血症的病原检测及分子流行病学调查等提供一种快速、简单、高效、特异、灵敏的检测方法。

To establish a rapid rabbit hemorrhagic disease virus（RHDV） pathogen detection methods,this research based on RHDV gene sequence in GenBank,synthesized outer and inner two pairs of primers,and established a nested RT-PCR for RHDV detection.The method used for Lapine rotavirus（LaRV）,Sendai virus（SeV） and healthy rabbit constitution,all of the RT-PCR results were negative;the method for first amplification the sensitivity was 10 ng,the second amplification sensitivity was 0.1 ng,the second amplification was 100 more times sensitive than the first amplification.The established nest RT-PCR method had good specificity and sensitivity,it could detect very low levels of RHDV quickly and accurately,it provided a kind of efficient,rapid,specific and sensitive detection method for pathogen detection and molecular epidemiology of material such as of RHD.