猪瘟兔化弱毒疫苗中病毒含量实时荧光定量RT-PCR检测方法的建立及初步应用

Abstract

Abstract: To establish a rapid, sensitive and specific method for evaluation of viral load of hog cholera lapinized virus (HCLV) vaccine, a Real-time RT-PCR assay was established using two pairs of primers and an internal TaqMan probe from the 5' non-structural region of HCLV genome. The sensitivity of the assay was 1.20×10⁵ copies/mL. The assay was specific and showed no application reaction with porcine reproductive and respiratory syndrome live vaccines, porcine epidemic encephalitis B live vaccines, swine paratyphoid and pseudorabies live vaccines. The coefficient of variation (CV) of 3 different vaccine samples of HCLV was 0.29% to 0.39% in intra-assay and 0.32% to 0.61% in inter-assay, respectively. Using this method to detect 7 different vaccines of HCLV from six different plants, the results showed that there were significant differences in viral loads among the selected HCLV vaccines. In conclusion, this Real-time RT-PCR was a highly sensitive, specific and stable method which could be applied to preliminarily evaluate the viral loads of HCLV vaccines.

Key words: hog cholera lapinized virus (HCLV); Real-time RT-PCR; viral loads

CLC Number:

S852.4

FAN Bin, XU Wen-hua, HAN Jian-qiang, MA Zhi-liang, HU Qi, XIN Ai-guo, ZHANG Yi-fang. Development and Preliminary Application of Real-time RT-PCR Assay for Detecting Quantitation of Hog Cholera Lapinized Virus in Vaccine[J]. , 2014, 41(12): 56-61.

References

1 仇华吉,童光志,沈荣显. 猪瘟兔化弱毒疫苗——半个世纪的回顾[J]. 中国农业科学,2005,38(8):1675～1685.
2 王伟,邹月红,李雪松,等. 猪瘟石门系强毒株SYBR Green-Ⅰ染料法实时荧光定量PCR方法的建立[J]. 中国畜牧兽医,2011,38(3):104～107.
3 王琴. 猪瘟流行现状及中国猪瘟净化策略[J]. 中国猪业,2012,7(10):45～47.
4 邓力,张彦明,李维维,等. 猪瘟兔化弱毒实时荧光定量PCR检测方法的建立及初步应用[J]. 西北农林科技大学学报(自然科学版),2011,39(2):1～8.
5 史子学,徐兴然,涂长春. 用实时荧光定量RT-PCR方法检测猪瘟病毒[J]. 中国预防兽医学报,2007,29(6):467～470.
6 宁宜宝,吴文福. 我国猪瘟流行新特点与疫苗免疫研究[J]. 中国兽药杂志,2011,45(8):33～37.
7 刘大伟,张小飞,胡来根,等. 猪瘟兔化弱毒疫苗株TaqMan实时荧光定量RT-PCR检测方法的建立及初步应用[J]. 中国预防兽医学报,2010,32(6):446～450.
8 李军,潘艳,禤雄标,等. 猪瘟病毒实时荧光定量RT-PCR检测方法的建立和初步应用[J]. 中国畜牧兽医,2011,38(3):116～119.
9 陈玉栋,张楚瑜,邹俊煊,等. 建立快速定量检测猪瘟兔化弱毒苗的实时荧光定量PCR技术[J]. 中国病毒学,2003,18(2):26～30.
10 陈锴,姚华伟,王长江,等. 实时荧光定量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[J]. 中国农业科学,2013,46(1):162～169.
11 葛叶,张兴娟,朱庆虎,等. 实时荧光定量RT-PCR方法与兔体定型热试验对于检测猪瘟兔化弱毒疫苗的平行关系[J]. 中国预防兽医学报,2011,33(9):699～703.
12 Hoffmann B, Beer M, Schelp C, et al. Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever[J]. J Virol Methods, 2005, 130(1～2):36～44.
13 Risatti G, Holinka L, Lu Z, et al. Diagnostic evaluation of a real-time reverse transcriptase PCR assay for detection of classical swine fever virus[J]. J Clin Microbiol, 2005, 43(1):468～471.
14 Zhang X J, Han Q Y, Sun Y, et al. Development of a triplex TaqMan real-time RT-PCR assay for differential detection of wild-type and HCLV vaccine strains of classical swine fever virus and bovine viral diarrhea virus 1[J]. Res Vet Sci, 2012, 92(3):512～518.

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Development and Preliminary Application of Real-time RT-PCR Assay for Detecting Quantitation of Hog Cholera Lapinized Virus in Vaccine

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