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

【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。

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

【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-6.52×104copy/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 1.10×104copy/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 8.27×104copy/μL-1.11×106copy/μL.【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.