鹅细小病毒SYBR GreenⅠ荧光定量PCR检测方法的建立及应用

Establishment and Application of SYBR GreenⅠReal-time Fluorescent Quantitative PCR Detection Method for Goose Parvovirus

为探讨鹅细小病毒(Goose parvovirus,GPV)在鹅胚上皮细胞(goose embryo epithelial cells,GEEC)中的增殖规律,研究采用荧光染料SYBR GreenⅠ渗入法,建立了检测GPV的荧光定量PCR方法,并用该方法分析了GPV在GEEC中的生长情况,绘制了生长曲线。结果显示,鹅细小病毒SYBR GreenⅠ荧光定量PCR检测方法建立的标准曲线具有良好的重复性和特异性,与模板浓度呈现良好的线性关系。在线性浓度范围内,随着模板量的减少,其对应的Ct值相应增大,得到的标准曲线方程为y=-3.3412x+38.494,相关系数R2为0.998。特异性试验结果显示检测新城疫病毒(Newcastle disease virus,NDV)、鸭甲型肝炎病毒(Duck hepatitis A virus,DHAV)、禽腺病毒(Avian adenovirus,FAdV)均为阴性。敏感性试验结果显示最低检测限为100拷贝/反应,而普通PCR的最低检测限为1000拷贝/反应,熔解曲线为单一特征峰型,熔解温度为80℃±0.5℃。病毒生长曲线绘制结果显示该病毒在GEEC细胞中有较好的增殖,72 h前病毒量逐渐增高,病毒量在72 h达到峰值。研究结果为GPV的定量检测提供了一种快速有效的方法。

The objective of this study was to establish a real-time PCR method for detection of the proliferation of goose parvovirus(GPV)in goose embryo epithelial cells(GEEC).The fluorescence dye SYBR GreenⅠinfiltration method was used and the growth of GPV in GEEC was analyzed and the growth curve was drawn.The results showed that the standard curve established by SYBR GreenⅠquantitative real-time PCR assay showed good repeatability and specificity,and it showed a good linear relationship with template concentration.In the linear concentration range,with the decrease of the template amount,the correspondin g Ct value increases correspondingly,and the standard curve equation is y=-3.3412x+38.494,R 2 is 0.998.Sensitivity test showed that the lowest detection limit was 100 copies/reaction,while the lowest detection limit of common PCR was 1000 copies/reaction.The melting curve is a single characteristic peak,and the melting temperature is 80℃+0.5℃.The virus growth curve showed that the virus had a good proliferation in GEEC,and the amount of 60 h increased gradually,indicating that it was rapidly proliferating before 72 h,and the amount of virus reached the highest in 72 h.This result is consistent with that of synchronous TCID 50 assay,and it is more sensitive and accurate.It provides a rapid and effective method for the quantitative detection of goose parvovirus.