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

【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,MDV 1基因拷贝数检出限度为32.8拷贝/μL,至少可检出0.006 PFU的CVI988毒株,MDV 3基因拷贝数检出限度为10拷贝/μL,至少可检出0.006 PFU的FC126毒株;重复性好,MDV 1检测方法重复性试验的变异系数小于1%,MDV 3检测方法重复性试验的变异系数小于1.5%。【结论】所建立的实时荧光定量PCR检测方法可分别用于禽用生物制品中外源性MDV1、MDV3毒株的检测。

【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,respectively.About the MDV 1 real-time PCR detection method,the amplification efficiency was 98.8%,the correlation coefficient was 0.992,with the standard curve:Y=-3.351X+38.828(Y=Ct,X=lg(copy number)).About the MDV 3 real-time PCR detection method,the amplification efficiency was 95%,the correlation coefficient was 0.998,and the standard curve:Y=-3.447X+36.496(Y=Ct,X=lg(copy number)).The established detection methods could specially detect MDV 1 or MDV 3 without detecting any other avian virus-associated biological products,virus,the full-length plasmid of UL19 of MDV 2 SB-1 strain,along with production materials for poultry.The sensitivity of MDV 1 real-time PCR detection method was high,with the gene copy number detection limit of 32.8 copies/μL,which could detect at least 0.006 PFU of CVI988 strain.The sensitivity of MDV 3 real-time PCR detection method was high,with the gene copy number detection limit of 10 copies/μL,which could detect at least 0.006 PFU of FC126 strain.The coefficient of variation of the repeatability test was less than 1%in MDV 1 real-time PCR detection method,and less than 1.5%in MDV 3 real-time PCR detection method,respectively.【Conclusion】The established real-time PCR detection methods would be beneficial for detecting exogenous MDV 1 and MDV 3 strains in poultry-derived biological products.