利用SYBR Green real-time PCR方法监控鸡外周血淋巴细胞中马立克病毒的载量

Monitoring Mareks disease virus load in peripheral blood leukocytes of chicken u sing SYBR Green real-time PCR

建立基于SYBR GreenⅠ的real-time FQ-PCR方法检测并定量鸡外周血淋巴细胞(peripheral blood leukocytes,PBLs)中马立克病毒(Mareks disease virus,MDV)的载量(以meq基因为定量标准),并将此技术的敏感性与标准PCR方法进行对比。应用PCR技术从MDV-1攻毒后的霞烟鸡PBLs中扩增MDVmeq基因的部分片段,纯化上述基因的DNA扩增产物,然后克隆到pGM-T载体,重组质粒通过PCR和EcoRⅠ酶切及测序分析进行确认;将浓度梯度的meq基因的重组标准品质粒DNA进行real-time FQ-PCR,建立其相应的标准曲线。同样以质粒DNA为模板,real-time FQ-PCR方法的最低检出率为10拷贝,而标准PCR的最低检出率则为32拷贝。结果表明,在检测MDV时,real-time FQ-PCR敏感度比标准PCR要高。

The aim of this study was to develop a SYBR Green real-time polymerase chain reaction assay to detect and quantify Marek＇s disease virus （MDV） loads in peripheral blood leukocytes （PBLs） targeting meq gene of the virus and compare the sensitivity of it with that of a standard polymerase chain reaction for the monitoring of Marek＇s disease virus. Total DNA was isolated from the samples of PBLs of birds after the challenge with MDV-1 and meq gene was amplified by PCR from total DNA. Then the purified PCR products of rneq were cloned into vector pGM-T. The positive clones were confirmed by the digestion with restriction endonuclease EcoR Ⅰ and amplifi-cation of PCR and sequences analysis of the inserted fragments. The standard curves of meq were established through SYBR Green real-time PCR assay of the recombinant plasmid DNA specimens which were diluted into a series of standard concentrations. The SYBR Green real-time PCR assay had a minimum detection limit of 10 copies of meq gene when plasmid DNA was used as the tem-plate. The standard PCR assay had a minimum detection limit of 32 copies of meq gene when plas-mid DNA was used as the template. SYBR Green real-time PCR assay showed a high sensitivity in detection of Marek＇s disease virus compared with assays using standard PCR methods.