呼肠孤病毒Ⅲ型（Re03）RT—PCR检测方法的建立

目的建立呼肠孤病毒Ⅲ型（Re03）RT—PCR检测方法，应用于实验动物及人用动物源性材料及生物制品外源Re03的检测。方法根据已发表的呼肠孤病毒Ⅲ型（Re03）M1基因序列，设计合成引物。提取Re03细胞毒RNA，以其为模板，进行PCR扩增。优化反应条件，进行特异性、敏感性、重复性试验。结果建立的Re03RT—PCR检测方法特异、敏感、稳定。以Re03RNA逆转录产物为模板，所能检测RNA最小模板浓度为0．42pg／μL，可检测病毒最小滴度为10^-9／mL。结论建立的呼肠孤病毒Ⅲ型（Re03）RT—PCR检测方法可用于实验动物及人用动物源性材料及生物制品外源Re03的检测。

Development of A RT-PCR Method for Determination of Mammalian Orthoreovirus 3

Objective To develop a RT-PCR method for determination of Mammalian Orthoreovirus 3 （ Reo3 ） in laboratory animal and the biological materials or the biological products for human use from the animal. Methods Two pair of primers specific to the sequences of M1 gene were designed according to the published Mammalian Orthoreovirus 3. With which the RNA of Reo3 was extracted and reversely transcribed to eDNA as a template for PCR amplification. The developed RT-PCR method was optimized, verified for specificity and sensitivity. Results The developed RT-PCR method is good in ensitivity, specificity and stability, and its minimum detection limit using the recombinant plasmid containing Reo3 gene as atemplate was 0.42pg/μL,and the lowest detection virus titer is 10^-9/mL. Conclusion The developed RT-PCR method can be used in detecting the Mammalian Orthoreovirus 3 （Reo3） in laboratory animaland the biological materials or the biological products for human use from the animal.