鸡传染性贫血病毒SYBR Green Ⅰ法荧光定量PCR检测方法的建立及其在疫苗污染检测中的应用

DEVELOPMENT AND APPLICATION OF SYBR GREEN Ⅰ QUANTITATIVE PCR FOR THE DETECTION OF CHICKEN INFECTIOUS ANEMIA VIRUS CONTAMINATION IN ATTENUATED VACCINES

为了更快速而灵敏地检测禽弱毒疫苗中可能存在的鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)污染,根据CIAV基因组保守区域VP2部分基因设计和合成了1对特异性引物,通过优化反应条件建立了快速检测CIAV的SYBR GreenⅠ荧光定量PCR方法。结果显示,建立的检测方法灵敏度可达6.36 copies/μL,是常规PCR灵敏度的1000倍。该方法与禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)、禽白血病病毒(Avian leukemia virus,ALV)、鸡马立克氏病病毒(Mareks disease virus,MDV)等均无交叉反应,特异性良好;批内和批间重复试验显示变异系数均小于2.5%,呈现良好的可重复性。在模拟试验中,建立的SYBR Green I荧光定量PCR能够检测到1000羽份弱毒疫苗中1个EID50的低剂量污染,应用该方法从送检的14种(批)商品化禽用弱毒疫苗中检测到2份为CIAV阳性,阳性样品按照经典的SPF鸡检查法进行检测也为CIAV阳性。因此,本研究建立的SYBR GreenⅠ荧光定量PCR检测方法灵敏度高,特异性强,重复性好,适合用于疫苗中CIAV污染的检验。

To detect the contamination of Chicken infectious anemia virus （CIAV） in attenuated poultry vaccines, one pair of specific primers were designed and synthesized within conserved region VP2 partial gene sequence of CIAV for development and optimization of a SYBR Green I real-time quantitative PCR assay. The sensitivity limit of the SYBR Green I quantitative PCR was 6.36 copies/μl, which was 1000 times higher than that of conventional PCR assay. Under experimental condition, the detection limit was I EID50/1000 plumes. The SYBR Green I quantitative PCR had good specificity to CIAV and had no cross-reaction with other REV, ALV and MDV genes. The coefficients of variations of both intra-assay and inter-assay were less than 2.5%, suggesting good repeatability. Two out of 14 commercial poultry vaccine samples were tested as CIAV positive confirmed in classical SPF chicken experiment. These results suggest specificity, sensitivity and repeatability and can be used in diagnostic attenuated vaccines. by using the SYBR Green I quantitative PCR method, which were that the SYBR Green I quantitative PCR developed here has good laboratories for detection of a low dosage CIAV contamination in