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

doi:10.3864/j.issn.0578-1752.2013.01.019

Abstract

Abstract: 【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.

Key words: Lapinized hog cholera virus , efficacy testing , fluorescent quantitative PCR , TaqMan-MGB

CHEN Kai, YAO Hua-Wei, WANG Chang-Jiang, XU Lu, FAN Xue-Zheng, ZHAO Qi-Zu, ZOU Xing-Qi, ZHU Yuan-Yuan, ZHAO Yan, YANG Guang-You, WANG Qin. Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus[J].Scientia Agricultura Sinica, 2013, 46(1): 162-169.

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

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