建立快速定量检测猪瘟兔化弱毒苗的荧光定量PCR技术

Development of a Fluorogenic Quantitative PCR Assay for Rapid Quantification of Hog cholera lapinized virus

在猪瘟病毒兔化弱毒疫苗株的5挿潜嗦肭杓埔欢砸锖鸵惶跤馓秸?利用荧光定量PCR原理,结合LightCycler检测系统,首次建立了定量检测猪瘟兔化弱毒苗方法。结果表明,该方法的灵敏度为102拷贝数,线性范围为107-102,达6个数量级;标准样品的变异系数为2.3%-5.1%(n=10),疫苗样品组内实验变异系数为0.85%-2.8%(n=5)、组间实验为2.5%-7.3%(n=5),对同一样品分5次RNA提取和逆转录,其变异系数为5.0%;对9份疫苗样品进行了检测,与兔体定型热反应方法相比较,有很好的相关性;整个检测过程仅需4h。该法可望取代传统的兔体定型热反应用于疫苗生产过程中的效价测定及指导疫苗的配制,也为猪瘟病毒分子生物学研究提供了一种新的、简捷有效的工具。

A rapid and reproducible method was first established for assessment of Hog cholera lapinized virus (HCLV) loads in Classical Swine Fever vaccine using Flurogenic quantitative PCR (FQ-PCR) combines LightCycler sequence detection system. The method contains a pair of primers and an internal daul-labled fluorogenic probe spanning the part of 5?noncoding region (5扤CR) of HCLV, the use of such a probe combined with the 5?3?nuclease activity of Taq polymerase allows direct quantification of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. The sensitivity of the assay was 102 copies per reaction. The assay is linear within 6-log dynamic rang. The coefficient of variation (CV) of the standard of Ct value is 2.3% -5.1% (n =10); The CV of vaccine sample is 0.85%-2.8% in intra-assay and 2.5%-7.3% in inter-assay (n =5), respectively; The CV of the same sample in different RNA isolation and reverse transcription is 5.0 % (n=5). Nine vaccines were quantified by this method and give similar but more accurate results compared to the conventional rabbit fever reaction. The entire assay, including RNA isolation, reverse transcription, and quantification, could be completed within 4 hours. In conclusion, the high sensitivity, simplicity, and reproducibility of the HCLV RNA quantification which allows the screening of large numbers of samples, combined with its wide dynamic rang, makes this method especially suitable for evaluating the viral loads and guiding how to confect the vaccine, it also provides a novel and simple research tool for CSFV.