中蜂囊状幼虫病毒RT-PCR检测方法的建立  

Development of A RT-PCR Method for Determination of Chinese Sacbrood Virus

作　　者：马鸣潇[1] 李明[1] 袁春颖[2] 李鹏飞[1] 张轶博[1] 苏玉虹[1] 曲祖乙[1] 

机构地区：[1]辽宁医学院畜牧兽医学院,辽宁锦州121001 [2]辽宁省蜜蜂原种场,辽宁兴城125100

出　　处：《中国生物制品学杂志》2010年第4期425-427,共3页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30972200);辽宁省教育厅资助(2009A464)

摘　　要：目的建立中蜂囊状幼虫病毒RT-PCR检测方法。方法参照已发表的中蜂囊状幼虫病毒(CSBV)基因序列,针对其结构蛋白基因设计1对特异性引物,提取感染CSBV的囊状幼虫RNA,反转录为cDNA,以其为模板,进行PCR扩增,对检测方法进行优化,验证该方法的特异性及敏感性,并对31份临床样品进行检测。结果所建立的CSBVRT-PCR检测方法特异性和敏感性良好,以含CSBV结构蛋白基因的重组质粒为模板,最低可检出10pgDNA。临床症状诊断和电镜观察均为阴性的22份样品经RT-PCR检测,有2份扩增出目的条带。结论已建立了中蜂囊状幼虫病毒RT-PCR检测方法,为CSBV检测、流行病学调查及动物模型的建立奠定了基础。Objective To develop a RT-PCR method for determination of Chinese sacbrood virus（CSBV）. Methods A pair of primers specific to structural protein gene of CSBV were designed according to the published CSBV gene sequence, with which the RNA of CSBV was extracted and reversely transcribed to cDNA as a template for PCR amplification. The developed RT-PCR method was optimized, verified for specificity and sensitivity, and used for determination of 31 clinical samples. Results The developed RTPCR method for CSBV showed high specificity and sensitivity, and its minimum detection limit using the recombinant plasmid containing CSBV structural protein gene as a template was 10 pg DNA. Twenty-two samples diagnosed as negative by clinical symptoms and electron microscopy were confirmed by the developed RT-PCR method, from 2 of which the target genes were amplified. Conclusion The developed RT-PCR method laid a foundation of determination, epidemiological investigation and establishment of animal model of CSBV infection.

关 键 词：中蜂囊状幼虫病毒 反转录聚合酶链式反应 特异性 敏感性 

分 类 号：R373.9[医药卫生—病原生物学] Q789[医药卫生—基础医学]