荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验替代方法的研究与应用

Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus

【目的】建立一种快速、敏感、特异的检测猪瘟兔化弱毒疫苗(HCLV)的荧光定量PCR方法(FQ-PCR),为猪瘟兔化弱毒疫苗的效力检验提供一替代方法。【方法】在猪瘟兔化弱毒疫苗基因组3′非编码区设计一对针对猪瘟兔化弱毒株的特异性引物和一条MGB探针,并进行反应体系及反应条件优化,以及特异性、灵敏度、稳定性及符合性试验。将此方法用于部分批次疫苗厂疫苗半成品的定量检验,与兔热反应进行比较,寻求两方法的相关性。【结果】试验结果证实,CT值与模板之间呈现良好的相关性,相关系数为0.9998,扩增效率为101.14%;该方法仅扩增HCLV,不扩增HCLV以外的其它病原,显示良好的特异性;具有较高的灵敏度,能检测模板中4.35个cDNA拷贝量,比CSFV-RT-nPCR高1个数量级。将此方法应用于4个厂家17个批次的34份猪瘟兔化弱毒疫苗效力检验,两只兔子均无热反应(不合格)样品共11份,FQ-PCR CT值为21.15—27.30;两只兔子一只呈现定型热,一只无热反应的样品12份,CT值为17.47—23.70;两只兔子都呈现定型热或者一只呈现定型热一只呈现轻热反应(合格品)样品共11份,CT值为17.10—20.8。【结论】建立的HCLV-FQ-PCR方法能特异性的检测疫苗核酸含量,与家兔热反应测定结果存在良好的相关性,可以用于猪瘟疫苗半成品中HCLV定量检测。

[ Objective ] A rapid, sensitive and specific one-step fluorescent quantitative PCR method as a substitute for rabbit fever testing for hog cholera lapinized virus （HCLV） vaccine efficacy was established. [Method ] A pair of primers and a HCLV specific MGB probe were designed on the 3＇UTR region of the HCLV genome for fluorescent quantitative PCR （FQ-PCR）. The method was tested for specificity, sensitivity and conformity after optimization. [Result] The FQ-PCR sensitivity was 4.35 cDNA copies. The correlation coefficient between CT value and cDNA copies was 0.9998. Amplification efficiency was 101.14%. The FQ-PCR method was specific for HCLV and did not show amplifications for CSFV, BVDV, BDV, PRRSV, FMDV and other pathogens. A total of 34 samples from 17 batches of the four vaccine manufacturers were tested after serial dilutions using the rabbit fever test and FQ-PCR. Eleven samples were disqualified for lack of fever in two rabbits with the Ct values falling between 21.15 and 27.30 and viral content of 8.80-102copy/μL-6.52x104copy/μL. Twelve samples induced fever in one rabbit and no fever in the others with the Ct values between 17.47 and 23.70 and viral content of 1,10×10^4copy/μL-8.55-105copy/μL The Ct values of 11 positive samples with both rabbits showing typical fever were from 17.10 to 20.81 with the viral content of 8.27x10^4copy/NL-l.llx106copy/pL. [Conclusion] The FQ-PCR kits established in this study is specific, sensitive and positively correlated with rabbit fever testing, and thus could be used for quantitative examination of semi-finished HCLV vaccine.