炭疽杆菌双重荧光定量PCR检测技术的建立和应用

doi:10.11843/j.issn.0366-6964.2019.08.015

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (8): 1658-1665.doi: 10.11843/j.issn.0366-6964.2019.08.015

炭疽杆菌双重荧光定量PCR检测技术的建立和应用

王素华^1*, 帅江冰², 李舟¹, 袁淑辉¹, 吴绍强³, 吕继洲³, 赵治国⁴

1. 温州海关综合技术服务中心, 温州 325027;
2. 杭州海关技术中心, 杭州 310016;
3. 中国检验检疫科学研究院动物检疫所, 北京 100029;
4. 呼和浩特海关技术中心, 呼和浩特 010020

收稿日期:2019-01-03 出版日期:2019-08-23 发布日期:2019-08-19
通讯作者: 王素华,E-mail:29801719@qq.com
作者简介:王素华(1975-),女,山东聊城人,高级兽医师,硕士,主要从事动物及动物产品检疫研究
基金资助:
国家质检总局科技计划项目（2017IK098）；浙江省重大科技专项重点农业项目（2015C02044）

Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis

WANG Suhua^1*, SHUAI Jiangbing², LI Zhou¹, YUAN Shuhui¹, WU Shaoqiang³, Lü Jizhou³, ZHAO Zhiguo⁴

1. Synthesis Technique Service Center of Wenzhou Customs, Wenzhou 325027, China;
2. Technology Center of Hangzhou Customs, Hangzhou 310016, China;
3. Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China;
4. Technology Center of Huhehaote Customs, Hohhot 010020, China

Received:2019-01-03 Online:2019-08-23 Published:2019-08-19
Supported by:

摘要/Abstract

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。

关键词: 炭疽杆菌, BA5345基因, PA基因, 双重荧光定量PCR, 检测方法

Abstract: 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.

Key words: Bacillus anthracis, BA5345 gene, PA gene, duplex real-time PCR, detection method

中图分类号:

S852.616

王素华, 帅江冰, 李舟, 袁淑辉, 吴绍强, 吕继洲, 赵治国. 炭疽杆菌双重荧光定量PCR检测技术的建立和应用[J]. 畜牧兽医学报, 2019, 50(8): 1658-1665.

WANG Suhua, SHUAI Jiangbing, LI Zhou, YUAN Shuhui, WU Shaoqiang, Lü Jizhou, ZHAO Zhiguo. Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(8): 1658-1665.

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炭疽杆菌双重荧光定量PCR检测技术的建立和应用

Establishment and Application of a Duplex Real-time PCR Assay for Detection of Bacillus anthracis

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