中蜂囊状幼虫病毒SYBR Green Ⅰ实时荧光定量PCR方法的建立与应用

Establishment and application of SYBR Green Ⅰreal-time fluorescent quantitative PCR method for Chinese sacbrood virus

通过RT-PCR方法在云南中华蜜蜂体内检测中蜂囊状幼虫病毒(CSBV),该病毒主要以AmSBV-YN(GenBank ID:KX819276.1)病毒株的形式存在。为了精确鉴定、快速检测中蜂囊状幼虫病毒,根据AmSBV-YN的CDS保守区域,设计合成特异性引物并建立相对应的SYBR GreenⅠ实时荧光定量PCR方法。结果显示,该方法的标准曲线循环域值(C_(t))与标准品模板浓度在2.34×10^(2)~2.34×10^(9)copies/μL间的线性关系良好,相关系数R~2为0.994,斜率为-3.118。该方法与蜜蜂残翅病毒、黑蜂王台病毒、蜜蜂以色列急性麻痹病毒不发生交叉反应,表明该方法特异性强;重复性评价结果显示,批内与批间的变异系数均小于3%。所建立的荧光定量PCR方法对中华蜜蜂样品的检测结果显示,阳性率达90.0%,高于常规PCR方法的检测阳性率73.3%,说明该方法具有良好的适用性。结果表明,该方法可应用于对中蜂囊状幼虫病毒的快速检测,为中蜂囊状幼虫病的及时防控提供了有力的技术支持。

RT-PCR was used to detect Chinese sacbrood virus(CSBV) in Yunnan honeybee(Apis cerana cerana).The virus mainly existed as AmSBV-YN(Gen Bank ID:KX819276.1) virus strain.In order to accurately identify and quickly detect CSBV,a SYBR GreenⅠreal-time fluorescent quantitative PCR method was established using specific primers and probes according to the CDS conservative region of AmSBV-YN.The results showed that there was a good linear relationship between the cyclic domain of standard curve(C_t) and the standard template concentration in the range of 2.34×10^(2)to 2.34×10^(9)copies/μL,the correlation coefficient R~2was 0.994 and the slope was-3.118.This method has strong specificity and does not cross react with deformed wing virus,black queen cell virus,Israeli acute paralysis virus.Repeatability evaluation results showed that the coefficients of variation within and between batches were less than 3%.The established AmSBV-YN fluorescence quantitative PCR method for the detection of A.cerana cerana samples showed that the positive rate was 90.0%,which was higher than 73.3%of the conventional PCR method,indicating that the method has good applicability.The results indicated that the method can be applied to the rapid detection of CSBV and provide strong technical support for the timely prevention and control of CSBV.