PEDV和TGEV双重TaqMan荧光定量一步法RT-PCR检测方法的建立及应用

发布时间：2018-11-15 09:08

【摘要】：本文通过对猪腹泻病毒(Porcine epidemic diarrhea virus,PEDV)和猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)基因组生物信息学分析,分别在两种病毒基因保守区设计特异性探针和Real-Time PCR引物,并建立了检测PEDV和TGEV的双重实时荧光定量RT-PCR方法。结果表明,本研究建立的方法对其他常见病毒无交叉检出,具有较强的特异性;应用本方法对PEDV和TGEV检测灵敏度均可达101个拷贝。应用本法组装PEDV和TGEV双重实时荧光定量PCR试剂盒,该试剂盒批次内、批次间的变异系数CV(coefficient of variation,CV)分别低于0.58%和3.7%。应用该试剂盒对97份病例进行检测,阳性率提高了14.93%。本研究建立的一步法荧光定量RT-PCR方法具有快速、特异性好、灵敏度高、定量且重复性和稳定性好等优点,在PEDV和TGEV感染的快速鉴别诊断,以及流行病学调查方面都有广阔的应用前景。
[Abstract]:Based on the analysis of genomic bioinformatics of porcine diarrhea virus (Porcine epidemic diarrhea virus,PEDV) and porcine transmissible gastroenteritis virus (Transmissible gastroenteritis virus,TGEV), specific probes and Real-Time PCR primers were designed in the conserved region of the two viruses. A dual real-time fluorescence quantitative RT-PCR method for the detection of PEDV and TGEV was established. The results showed that the method was specific to other common viruses, and the sensitivity of this method to PEDV and TGEV could reach 101 copies. PEDV and TGEV dual real-time fluorescent quantitative PCR kits were assembled by this method. The coefficient of variation (CV (coefficient of variation,CV) between batches was lower than 0.58% and 3.7% respectively. The positive rate of 97 cases was increased by 14.93%. The one-step fluorescence quantitative RT-PCR method developed in this study has the advantages of fast, specificity, high sensitivity, quantitative, reproducibility and stability. It can be used in the rapid differential diagnosis of PEDV and TGEV infection. And the epidemiology investigation has the broad application prospect.
【作者单位】：广东海大畜牧兽医研究院有限公司;
【基金】：广东省科技计划项目——猪伪狂犬突变株的鉴定及新型、高效猪伪狂犬疫苗开发与产业化(2015B020203006)
【分类号】：S852.651

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