应用双重实时荧光定量PCR方法检测鸡马立克氏病血清1型病毒

本研究建立了鸡马立克氏病血清1型病毒（MDV1）绝对定量检测方法。研究中选择MDV1特有的Meq基因的一段保守序列作为检测对象，将其克隆到质粒载体中，作为阳性标准品；同时将管家基因．鸡卵铁转蛋白（Ovo）特异性基因片段克隆到质粒载体上作为内参照的标准品。经荧光定量PCR（FQ-PCR）法扩增获得MDV1的FQ-PCR两条标准曲线，建立了MDV1双重FQ-PCR检测方法。应用该方法绝对定量检测了实验攻毒鸡及吉林省某地发病鸡只的羽髓、淋巴细胞等组织样本中单位细胞病毒拷贝数，并与琼脂扩散（AGP）、常规PCR等检测方法进行比较。结果表明，不论实验攻毒鸡还是自然发病鸡，羽髓中病毒富含量均高于其它组织，每百万宿主细胞内病毒含量为10^7～10^8拷贝；FQ-PCR检测MD发病鸡只的阳性率高于AGP，达100％；该方法的灵敏度比常规PCR检测高10～100倍，在单位细胞内可灵敏地检测到2．78个拷贝的病毒。该方法可以在不同的样品中有效的绝对定量检测MDVl。

Application of duplex fluorescent quantitative polymerase-chain reaction for detecting Marek's disease virus serotype 1

The development of the method for absolute quantification of Marek's disease virus serotype 1 (MDV1) was described in this paper. The plasmid DNAs bearing a unique DNA fragment of Meq gene from MDV1 and a fragment of the chicken ovotransferrin gene were used to quantify virus and host genomes respectively. A series of duplex fluorescent quantitative PCRs (FQ-PCR) were carried out to produce the standard curves. Absolute quantification of MDV1 was performed successfully on samples from feather tips, lymphocytes and other tissues collected from experimentally infected chickens and naturally infected chickens in Jilin province. The results indicated that the detected MDV1 in feather tip was the most abundant in all of samples (107-108 copies per million cells). The FQ-PCR had a higher positive rate (100 %) compared with AGP and regular PCR methods, and its sensitivity was 10-100 times than that of regular PCR, and the lower detection limit of the MDV1 assay was 2.78 copies. This method is useful in the absolute quantification of MDV1 viruses in different samples.