鸡白痢沙门菌套式PCR检测方法的建立及初步应用

发布时间：2018-04-11 15:37

  本文选题：鸡白痢沙门菌 + 套式PCR　； 参考：《动物医学进展》2017年08期

【摘要】：为了建立一种快速、高效和简便的鸡白痢沙门菌套式PCR检测方法,根据沙门菌侵袭蛋白基因invA的高度保守区域,利用Primmer 5.0软件设计2对特异性的套式引物,以提取的沙门菌DNA为模板克隆invA基因,将其连入T载体并计算拷贝数作为套式PCR的模板,在对反应条件进行优化的基础上,建立最佳的套式PCR反应条件,确定其上、下游引物用量分别为0.3μL,套式PCR的第1次退火温度为57.4℃,第2次为53.5℃。应用该方法对已构建好的不同浓度的标准阳性质粒作为模板进行检测,其灵敏度为102拷贝数/20μL,远高于常规PCR的106拷贝数/20μL。用建立的套式PCR方法对从新疆石河子某鸡场采集的25样品进行检测,与传统的诊断方法比较符合率达95.00%。建立的鸡白痢沙门菌套式PCR检测方法具有快速、可行、特异性强、准确率高等特点,为鸡沙门菌病的早期诊断和研究提供了技术支撑。
[Abstract]:In order to establish a rapid, efficient and simple nested PCR method for detection of Salmonella pullorum, two pairs of specific nested primers were designed by using Primmer 5.0 software according to the highly conserved region of invA gene of Salmonella pullorum invasion protein.The extracted Salmonella DNA was used as template to clone the invA gene, and was inserted into T vector and the copy number was calculated as template of nested PCR. Based on the optimization of reaction conditions, the optimal reaction conditions of nested PCR were established, and the optimal reaction conditions were determined.The amount of downstream primer was 0.3 渭 L, the first annealing temperature of nested PCR was 57.4 鈩，

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