Ⅰ群4型禽腺病毒特异性SYBR Green Ⅰ荧光定量PCR检测方法的建立与应用

Establishment and Application of SYBR GreenⅠ Fluorescent Quantitative PCR for Detecting GroupⅠ Fowl Adenovirus Serotype 4

为建立Ⅰ群4型禽腺病毒特异性SYBR GreenⅠ荧光定量PCR检测方法,参考Gen Bank中已发布的禽腺病毒的六邻体基因(Hexon)序列设计两对引物,通过PCR扩增出Hexon基因的954 bp目的片段,克隆至p MD-19T载体,提取阳性质粒测定浓度,10倍系列稀释作为荧光定量PCR的标准品模板,制备标准曲线。结果显示,荧光定量PCR熔解曲线峰值单一,产蛋下降综合症病毒(EDSV)、传染性喉气管炎病毒(ILTV)、禽白血病病毒(ALV)等外源病毒均无扩增曲线出现,对标准品模板最低检出值为1.2×101拷贝/μL。对7份Ⅰ群4型禽腺病毒的细胞培养物的检出率为100%,表明建立的实时荧光定量PCR检测方法准确可行。

In order to establish a SYBR Green Ⅰ real-time fluorescence quantitative PCR for detection of group Ⅰ fowl adenovirus serotype 4, two pairs of primers were designed with reference to Hexon gene of published fowl adenovirus sequence in GenBank. The 954 bp fragment of Hexon gene was amplified by PCR and cloned into pMD-19T vector. Positive plasmids were extracted and serially diluted 10 times to prepare for a standard curve for quantitative PCR. The results showed that the melting curve had one specific peak and there was no amplification detected from exogenous virus such as egg drop syndrome virus （EDSV）, infectious laryngotraeheitis virus （ILTV）and avian leukosis virus （ALV）, and the lowest detection limit was 1.2×10^4 copies/μL for the standard template. The cell cultures of 7 samples of group I fowl adenovirus serotype 4 was detected in laboratory and the detection rate was 100%, which indicated that the established real-time fluorescence quantitative PCR method was accurate and feasible.