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当前位置: 首页 > 文章 > 炭疽杆菌双重荧光定量PCR检测技术的建立和应用 畜牧兽医学报 2019 (8) 1658-1665
Position: Home > Articles > Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis Chinese Journal of Animal and Veterinary Sciences 2019 (8) 1658-1665

炭疽杆菌双重荧光定量PCR检测技术的建立和应用

作  者：
王素华;帅江冰;李舟;袁淑辉;吴绍强;吕继洲;赵治国
单  位：
杭州海关技术中心;中国检验检疫科学研究院动物检疫所;温州海关综合技术服务中心;呼和浩特海关技术中心
关键词：
炭疽杆菌;BA5345基因;PA基因;双重荧光定量PCR;检测方法
摘  要：
为快速和准确检测炭疽杆菌,本研究以炭疽杆菌染色体上的BA5345基因和pXO1质粒上的PA基因为靶基因设计特异性引物和探针,建立了炭疽杆菌双重荧光定量PCR检测方法,对其反应的特异性、敏感性和重复性进行了分析,并与常规PCR方法一起对临床样品进行检测。结果显示,所建立方法特异性强,与蜡样芽胞杆菌群中其他芽胞杆菌无交叉反应;对BA5345基因和PA基因的最低检测限分别为8.0和7.8拷贝·μL~(-1),标准曲线相关系数大于0.99,组内和组间CV均小于1.5%。对35份污染皮毛样品检测显示BA5345基因和PA基因的检出率分别为80.0%和82.9%,与常规PCR检测方法相比,敏感性更高。本研究为炭疽杆菌的检测和流行病学调查提供了一种快速、准确的检测方法。
译  名：
Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis
作  者：
WANG Suhua;SHUAI Jiangbing;LI Zhou;YUAN Shuhui;WU Shaoqiang;Lü Jizhou;ZHAO Zhiguo;Synthesis Technique Service Center of Wenzhou Customs;Technology Center of Hangzhou Customs;Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine;Technology Center of Huhehaote Customs;
关键词：
Bacillus anthracis;;BA5345 gene;;PA gene;;duplex real-time PCR;;detection method
摘  要：
In order to detect Bacillus anthracis rapidly and accurately, two sets of specific primers and TaqMan probes were designed according to the chromosomal sequence BA5345 gene and virulent plasmid sequence PA gene to establish a duplex real-time PCR assay for detection of Bacillus anthracis. The specificity, sensitivity and repeatability were tested, respectively. Then the duplex real-time PCR was used to detect clinical samples and compared with conventional PCR. Results showed that the Bacillus anthracis could be identified specifically and without any cross reaction with other bacillus of Bacillus cereus group. Besides, the detection limits were 8.0 and 7.8 copies·μL~(-1) for BA5345 gene and PA gene, respectively. The coefficients of variations were less than 1.5% for both intra-assay and inter-assay. Thirty-five samples contaminated by Bacillus anthracis were detected by the established assay, BA5345 positive rate was 80.0% and PA positive rate was 82.9%. The sensitivity of duplex real-time PCR was significantly higher than that of conventional PCR. The results indicate that the detection assay could be applied for rapid and high through-put detection of Bacillus anthracis.

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