SYBR GreenⅠ实时荧光PCR法检测禽活疫苗中禽网状内皮组织增生症病毒

Detection of Avian Reticuloendotheliosis Virus of Avian Live Vaccine by SYBR Green Real-time PCR

关键词:  网状内皮组织增生症病毒  实时荧光PCR  禽活疫苗

Key words:  REV  Real-time PCR  Avian Live Vaccine

摘要:

为快速、准确检测禽活疫苗中的禽网状内皮组织增生症病毒，本研究根据REVp30基因上的一段保守序列，利用Primer Premier 5.0软件设计合成了１对特异性引物，建立了基于SYBR Green I 模式的实时荧光PCR 方法（Real-time PCR），并以常规PCR产物为标准品绘制了标准曲线，对方法的特异性、敏感性、重复性、与经典方法的符合率进行了评价。结果表明：扩增产物片段大小为222bp，与预期片段大小相符，测序结果证实为REV靶序列；标准曲线具有良好的线性关系，相关系数为1.0，扩增效率为94.2%，溶解温度Tm=86.0±0.50℃，无引物二聚体；该方法只从REV阳性样本检出扩增信号，其他病原无扩增信号，特异性好；最低可检测26.97拷贝数的阳性标准品，比常规PCR敏感1000倍；组内变异系数、组间变异系数分别为0.79%～5.36%，1.61%～5.25%。运用建立的实时荧光PCR方法对17批禽用活疫苗进行检测，并与间接免疫荧光法（IFA）进行同步比较试验，结果两者符合率为100%。且实时荧光PCR法更快捷、更方便，结果判定更为直观可用于疫苗中REV污染情况的监测。

Abstract:

IIn order todetect avian reticuloendotheliosis virus(REV) of avian live vaccine quickly and accurately，a pair of specific primer was designed and synthesized usingPrimer Premier 5.0 according to conservative sequence of REV p30 gene in this study. The real-time PCR method based on the SYBR Green I pattern（Real-time PCR）was developed. The standard curve was constructed by using the product of conventional PCR,the specificity, sensitivity, reproducibility and the coincidence rate to IFA were evaluated. The result showed that the length of the PCR product was 222bp as expected and the amplified sequence was approved to be identical with the REV target sequence. The standard curve had a good linear relationship , the correlation coefficient was 1.0 and the amplification efficiency was 94.2%. Melting peak appeared at 86.8±0.5℃,without primer-dimer. This method only detected amplified signals from REV positive sample ,so it was identified to have good specificity. The standard curve covered a linear range of 2.697×109～2.697×103 copies. The minimum copies of the positive sample detected was 26.97 , 1000 times more sensitive than the conventional PCR. The Coefficient of Variation(CVs) of intra assay and inter assay were in the range of 4.80%~5.72% and 0.75%~3.87%, respectively. The real-time PCR was used to detect 17 batches of Avian live vaccine, comparing with indirecti- ng immunofluorescence(IFA).The results showed that the coincidence rate of these two methods was 100%. This real time PCR method has been proven to be a useful tool for the detection of REV in avian live vaccine because it was more rapid, more convenient and more intuitive.