猪德尔塔冠状病毒RT-PCR检测方法的建立及应用

发布时间：2018-05-19 03:34

本文选题：猪德尔塔冠状病毒 + RT-PCR　；参考：《中国兽医科学》2017年03期

【摘要】：为建立一种能够快速、准确检测猪德尔塔冠状病毒(PD Co V)的方法,根据G en Bank上已发表的PDCo V的M基因序列,设计并合成1对特异性引物,扩增的目的基因片段的大小为359 bp。通过优化反应条件,建立了检测PDCo V的RT-PCR方法,并对其敏感性、特异性和重复性进行了检测。结果显示,仅PDCo V阳性模板可扩增得到约359 bp的目的条带,而猪繁殖与呼吸综合征病毒、猪瘟病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、轮状病毒、牛病毒性腹泻病毒、猪细小病毒、猪圆环病毒2型、伪狂犬病病毒、猪链球菌2型、大肠杆菌的扩增结果均为阴性。敏感性试验结果显示,该方法能检测到的最低核酸质量浓度为1 ng/L。应用该方法对110份临床疑似发病的腹泻猪样品进行检测,结果检出13份PDCo V阳性,阳性检出率为11.8%。应用Blast对测序结果进行比较分析,结果表明检测的阳性样品与其他PDCo V序列的同源性为92.2%~100%。M基因的系统进化分析表明,阳性样品与PDCo V中国分离株的亲缘关系较近。以上结果表明,本研究成功建立了PDCo V的RT-PCR方法,该方法具有良好的特异性、敏感性和重复性,可用于PDCo V的临床诊断及流行病学监测。
[Abstract]:In order to establish a rapid and accurate detection method for porcine Delta coronavirus (PRV) PD Co V, a pair of specific primers were designed and synthesized according to the M gene sequence of PDCo V published on G en Bank. The amplified target gene fragment size was 359bp. By optimizing the reaction conditions, a RT-PCR method for the detection of PDCo V was established, and its sensitivity, specificity and repeatability were determined. The results showed that only PDCo V positive template could amplify about 359bp, while porcine reproductive and respiratory syndrome virus, swine fever virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, rotavirus, bovine viral diarrhea virus were obtained. The amplification results of porcine parvovirus, porcine circovirus 2, pseudorabies virus, Streptococcus suis type 2 and Escherichia coli were all negative. The sensitivity test results show that the minimum mass concentration of nucleic acid detected by this method is 1 ng / L. This method was used to detect PDCo V in 110 suspected diarrhea pigs. The positive rate of PDCo V was 11. 8%. Blast was used to compare the sequencing results. The results showed that the homology between the positive samples and other PDCo V sequences was 92.2and the phylogenetic analysis of 100.M gene showed that the relationship between the positive samples and the Chinese isolates of PDCo V was close. The results show that the RT-PCR method of PDCo V has been successfully established, which has good specificity, sensitivity and reproducibility, and can be used in the clinical diagnosis and epidemiological monitoring of PDCo V.
【作者单位】：天津市畜牧兽医研究所;天津市畜禽健康养殖技术工程中心;
【基金】：天津市农业科技转化与推广项目(201301030)
【分类号】：S852.651

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