蜜蜂畸翅病毒SYBR GreenⅠ荧光定量RT-PCR检测方法的建立

为建立蜜蜂畸翅病毒SYBR GreenⅠ荧光定量RT-PCR检测方法,通过构建蜜蜂畸翅病毒(DWV)RDRP基因部分序列的标准质粒作为模板,进行了标准曲线的建立,并对所建立方法进行了特异性和灵敏度测试。结果表明:所建立的荧光定量RT-PCR扩增效率E=100%,R2=0.999,最低检出量为100个拷贝;该方法仅能检出DWV病毒,而不能检测出蜜蜂黑王台病毒、囊状幼虫病毒,具有很好的特异性。表明已成功建立了特异灵敏的DWV实时荧光定量RT-PCR检测方法。采用所建方法对昆明部分蜂场进行了DWV感染状况调查,结果表明昆明地区东方蜜蜂带毒率为54%,提示DWV病毒在昆明地区东方蜜蜂中存在流行。

A real time quantitative RT-PCR assay for the detection of Deformed Wing Virus in honeybee

Area l-time RT-PCR assay, based on SYBR Green （SG） chemistry, were developed, for the detection of a important honeybee viruse： Deformed Wing Virus （DWV）, the reaction were optimized to yield the highest sensitivity and specificity. Viral detection and identification were confirmed by melting curve analysis and sequencing of the PCR products. The R2 was 0.999 and the effciency of the PCR reaction was 100%. The detection limit for the DWV RT-PCR was determined by the lowest dilution that showed specifc amplification for the positive control, which was 102 copies. The technique was used to evaluate Kunming feld samples and establish the distribution of the viruse. Sacbrood virus was present at 54% frequencies.