| 摘要: |
| 【目的】建立一种能够实际应用于猪瘟兔化弱毒疫苗中猪瘟病毒含量检测的荧光定量PCR方法。【方法】根据GenBank中登录号为AF091507的猪瘟病毒（CSFV）兔化弱毒株的全长基因组序列，在其5′非编码区设计2对特异性引物（标准品引物、定量引物）和1条探针(qPCR-P)，通过标准品引物进行RT-PCR扩增及T7 RNA聚合酶体外转录构建标准品RNA。对荧光定量PCR方法的各项条件进行优化，建立猪瘟兔化弱毒荧光定量PCR检测方法，并采用该法对成品猪瘟兔化弱毒疫苗中的病毒含量进行检测。【结果】成功构建了体外转录的标准品RNA，建立了检测猪瘟兔化弱毒的荧光定量PCR方法。该方法检测灵敏度可达10拷贝/μL，比RT-nPCR方法的灵敏度高出1个数量级；该法对标准品RNA检测的线性范围为1.0×108～1.0×102拷贝/μL。对1.0×108,1.0×106,1.0×103拷贝/μL 3种稀释度的标准品RNA进行重复性试验，批内变异系数分别为0.54%，0.52%和0.39%；批间变异系数分别为1.47%，1.85%和1.01%，具有良好的可重复性。应用该方法对不同厂家猪瘟兔化弱毒疫苗中的病毒含量进行测定，可知同一厂家脾淋苗中的病毒含量比细胞苗高出1～2个数量级，而不同厂家同一类型疫苗中病毒含量也有差异，其中脾淋苗差异不大，而细胞苗间的差异较大，最高可达27.2倍。【结论】建立的猪瘟兔化弱毒荧光定量PCR方法，具有敏感性高、特异性强、重复性好的特点，为实际工作中对猪瘟兔化弱毒疫苗的质量监控提供了重要的技术支撑。 |
| 关键词: 猪瘟兔化弱毒 荧光定量PCR 体外转录 标准品RNA |
| DOI： |
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| 基金项目:国家“863”重大项目“家畜重要病毒病基因工程疫苗研究和创制”（2006AA10A204） |
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| Development and preliminary application of real-time PCR assay for detection and quantitation of the lapinized Chinese strain of classical swine fever virus |
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| Abstract: |
| 【Objective】The study was to develop a real-time assay for detecting and quantifying the virus loads of the lapinized Chinese strain vaccine against classical swine fever virus（CSFV）.【Method】Two pairs of primes and a specific TaqMan probe were designed on the 5′-untranslated region (UTR) of the CSFV genome whose accession number is AF091507 on Genbank.A standard RNA was constructed through RT-PCR with the Standard’s Primes and then in vitro transcripted.The real-time PCR assay was developed by optimizing the variety of conditions.This assay was applied to quantify the virus loads of several lapinized virus vaccines against CSFV in order to obtain a preliminary evaluation.【Result】The real-time PCR assay for detecting and quantifying the virus loads of the lapinized Chinese strain vaccine against CSFV was developed successfully.The minimum detection limit was 10 copies/μL,10 times higher than that of the conventional RT-nPCR.The linear range was from 1.0×108 copies/μL to 1.0×102 copies/μL.Data of the repetitive tests showed good reproducibilities and the coefficients variations of 1.0×108,1.0×106,1.0×103 copies/μL within and between batches were 0.54%,0.52%,0.39% and 1.47%,1.85%,1.01%,respectively.The results of clinical application showed that the virus load in the spleen vaccine was 10～100 fold higher than that of the cell-based vaccine compared with each manufacturer’s products.However,compared with different manufacturers,the virus loads in spleen vaccine were similar,and that in the cell-based vaccine were discrepant.The maximum discrepancy was up to 27.2 folds.【Conclusion】The real-time PCR assay for detecting and quantifying the CSFV has high detection specificity and sensitivity,and good reproducibilities as well,which provides technical support for clinically monitoring the quality of the lapinized Chinese strain vaccine against CSFV. |
| Key words: lapinized Chinese strain of CSFV real-time PCR in vitro transcription standard RNA |
