摘要:
根据猪PRRSV的ORF7基因序列,设计并合成了1对引物,以PRRSV RNA为模板,筛选最佳反应条件,建立了检测PRRSV的RT-PCR方法。应用该方法对病毒细胞培养物进行基因扩增,均获得了分子量为443bp的特异性目的片段,而对PCV2、PRV、CSFV及Marc-145细胞进行同条件检测,结果均为阴性;PRRSV扩增产物测序结果与文献报道的PRRSV其他毒株序列同源性达92%~98%。敏感性试验表明,该体系可检测到2.39T CID50的PRRSV;对2004~2005年送检的81份临床样品进行检测,阳性率为23.4%。上述研究结果表明,建立的RT-PCR特异性好、敏感性高,可用于猪繁殖与呼吸综合征的诊断和流行病学调查。
Abstract:
Based on the ORF7 gene sequence of the porcine reproductive and respiratory syndrome virus(PRRSV),a pair of primers were designed and synthesized.By means of the template of RNA of PRRSV,the RT-PCR technique for detecting PRRSV was established.The 443 bp specific fragments were obtained subsequently from our laboratory’s virocyte culture.Negative test results were found on the Porcine Circovirus Type 2,Pseudorabies Virus,Classical Swine Fever Virus,and Marc-145 cells.The amplified sequence of PCR product was 92%-98% in agreement with those published data on PRRSV strains.The sensitivity of RT-PCR reached 2.39TCID50.In 2004-2005,23.4% of the 81 clinical specimens tested to be positive.The results showed that the newly developed RT-PCR method had high specificity and sensitivity,and that it could be applied for PRRSV diagnosis and epidemiological investigation.
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