外源性马立克氏病病毒荧光定量PCR检测方法的建立  

Establishment of Real-Time PCR Method for Detection of Extraneous Marek’s Disease Virus

作　　者：苏佳 赵炜 刘丹[1] 王嘉[1] 白洪旭 吴华伟[1] 薛青红[1] 陈晓春[1] SU Jia;ZHAO Wei;LIU Dan;WANG Jia;BAI HongXu;WU HuaWei;XUE QingHong;CHEN XiaoChun(China Institute of Veterinary Drug Control,Beijing 100081)

机构地区：[1]中国兽医药品监察所,北京100081

出　　处：《中国农业科学》2023年第20期4125-4136,共12页Scientia Agricultura Sinica

基　　金：兽药行业公益性重点项目(GY202105)。

摘　　要：【目的】为解决现有兽用生物制品外源性马立克氏病病毒(Marek’s disease virus,MDV)检验方法灵敏度低、检测时间长、鉴别性不强的问题,研究分别建立MDV血清1型(MDV serotype 1,MDV 1)和MDV血清3型(MDV serotype 3,MDV 3)毒株的实时荧光定量PCR检测方法,用于禽用生物制品纯净性控制。【方法】从NCBI下载MDV 1、MDV血清2型(MDV serotype 1,MDV 2)和MDV 3各毒株UL19序列,进行核苷酸和氨基酸序列比对分析;分别针对MDV 1 CVI988毒株、MDV 3 FC126毒株的UL19设计特异性引物和相应的Taqman探针,建立实时荧光定量PCR检测方法;分别构建相应的重组质粒作为阳性标准品,建立标准曲线,并评价所建立方法基因拷贝数检测的灵敏度;分别对其他禽用病毒类生物制品、毒种、MDV 2 SB-1毒株UL19的全长质粒及生产原材料(SPF鸡胚尿囊液、胚体、尿囊膜、鸡胚成纤维细胞)进行检测,评价检测方法的特异性;分别对600、60、6、0.6、0.06、0.006、0.0006 PFU的CVI988毒株和FC126毒株进行检测,评价所建立方法检测活病毒粒子的灵敏度;分别以不同稀释度的阳性标准品质粒为模板,进行3次重复性检测,计算变异系数,分析所建立检测方法的可重复性。【结果】MDV UL19在同一血清型内核苷酸和推导的氨基酸同源性达99.99%,具有很高的保守性,在不同血清型间核苷酸同源性只有约75%,推导的氨基酸同源性只有约85%;分别建立了MDV 1和MDV 3两种实时荧光定量PCR检测方法;MDV 1检测方法标准曲线的扩增效率E=98.8%,相关系数R^(2)=0.992,标准曲线方程Y=-3.351X+38.828(Y=Ct,X=lg(拷贝数)),MDV 3检测方法标准曲线的扩增效率E=95.0%,相关系数R^(2)=0.998,标准曲线方程Y=-3.447X+36.496(Y=Ct,X=lg(拷贝数));所建立的两种检测方法特异性好,MDV 1或MDV 3毒株扩增曲线良好,其他禽用病毒类生物制品、毒种、MDV 2 SB-1毒株UL19的全长质粒及生产原材料未出现特异性扩增曲线;灵敏度高,M【Objective】In order to solve the problems of low sensitivity,long detection time,and poor discrimination of existing exogenous Marek’s disease virus(MDV)testing methods,this study was designed to establish two real-time PCR detection methods for the identification of MDV serotype 1(MDV 1)and MDV serotype 3(MDV 3)strains,which could be used for purity control of poultry-derived biological products.【Method】The UL19 sequences of MDV 1,MDV serotype 2(MDV 2)and MDV 3 strains were downloaded from the NCBI database and were used for nucleotide and amino acid homology comparison.A pair of specific primers and corresponding Taqman probe was designed from the known sequence of conserved UL19 of MDV 1 CVI988 strain and MDV 3 FC126 strain,respectively,and two real-time PCR detection methods were established.The corresponding recombinant plasmids were constructed and used as positive standards to make standard curves,and the sensitivity of gene copy number of the methods were evaluated.Other avian virus-associated biological products,virus,the full-length plasmid of UL19 of MDV 2 SB-1 strain and the raw materials for production(SPF chicken embryo allantoic fluid,embryonic body,allantoic membrane,chicken embryo fibroblasts)were detected to evaluate the specificity of the established methods.600,60,6,0.6,0.06,0.006 and 0.0006 PFU of CVI988 or FC126 strains were detected,respectively,and the sensitivity of the established two methods for detecting live virions was evaluated.Three repeatability tests were performed using corresponding recombinant plasmids of different dilutions,and the correlation coefficient were calculated to analyze the reproducibility of the two established detection methods.【Result】The nucleotide and the derived amino acid homology of MDV UL19 in the same serotype was highly conserved with 99.99%,and the nucleotide homology between different serotypes was only about 75%,while the derived amino acid homology was only about 85%.MDV 1 and MDV 3 real-time PCR detection methods were established,resp

关 键 词：马立克氏病病毒 实时荧光定量PCR 外源病毒 检测 

分 类 号：S852.65[农业科学—基础兽医学]