布鲁氏菌和贝纳氏柯克斯氏体双重PCR检测方法的建立与初步应用

doi:10.13523/j.cb.20140213

中国生物工程杂志

2014, Vol. 34

Issue (2): 78-83 DOI: 10.13523/j.cb.20140213

技术与方法

布鲁氏菌和贝纳氏柯克斯氏体双重PCR检测方法的建立与初步应用

彭武丽^1,2, 罗展荣³, 季新成², 员丽娟², 史茜², 于学辉²

1. 石河子大学生命科学学院石河子 832000;
2. 新疆出入境检验检疫局乌鲁木齐 830063;
3. 中国人民解放军69016部队乌鲁木齐 830001

Establishment and Preliminary Application of a Duplex PCR Assay for Detecting Brucella and Coxiella burnetii

PENG Wu-li^1,2, LUO Zhan-rong³, JI Xin-cheng², YUN Li-juan², SHI Qian², YU Xue-hui²

1. College of Life Science of Shihezi University, Shihezi 832000, China;
2. Xinjiang Exit-import Inspection and Quarantine Bureau, Urumqi 830063, China;
3. 69016 Armed Forces of Chinese People's Liberation Army, Urumqi 830001, China

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摘要： 为建立同步检测布鲁氏菌和贝纳氏柯克斯氏体的双重PCR方法，根据GenBank上公布的布鲁氏菌Bp26基因和贝纳氏柯克斯氏体IS1111a基因，利用 Primer premier 5.0软件各设计一对特异性引物，分别扩增出219 bp和130 bp的目的片段，通过优化PCR反应体系和扩增条件，建立起了能够同步快速检测布鲁氏菌和贝纳氏柯克斯氏体的双重PCR方法，该方法具有较好的特异性、可重复性和较高的灵敏性。利用该双重PCR方法对流产牛抗凝全血、血清、流产胎儿及奶液等172份临床样品进行检测，共检测到布鲁氏菌感染阳性样品53份，贝纳氏柯克斯氏体感染阳性样品10份，混合感染阳性样品2份，阳性检出率分别为30.8% 、5.8% 、1.2%。初步临床应用结果表明，该方法可用来安全、同步、快速、灵敏地对布鲁氏菌和贝纳氏柯克斯氏体进行检测。

关键词： 布鲁氏菌病; Q热; 感染; 双重PCR; 共扩增

Abstract: To establish a duplex PCR assay for simultaneously detecting Brucella and Coxiella burnetii, two genus-specific genes sequences according to the Bp26 of Brucella spp. and IS1111a of Coxiella burnetii were obtained in GenBank and two specific primers pairs were designed respectively. After optimizing the PCR reactions and amplification conditions, the duplex assay was finally established. This assay had better specificity, repeatability and higher sensitivity. Using this assay to detect 172 samples including bloods,serums, fetuses and milk of abortion cattle in clinic,53 brucellosis positive samples and 10 Q fever positive samples were detected out, the positive rates were separately 30.8% and 5.8%, 2 co-infection positive samples were detected out also and the positive rate was 1.2%. The above preliminary clinical results indicated this assay can be used for safely, simultaneously, rapidly and sensibly detecting Brucella and Coxiella burnetii.

Key words: Brucellosis Q fever Infection Duplex PCR Co-amplification

收稿日期: 2013-10-24 出版日期: 2014-02-25

ZTFLH:

Q819

基金资助: 国家质量监督检验检疫总局科研项目（20141K239，2011IK017）资助项目

通讯作者: 季新成 E-mail: jixincheng@126.com

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引用本文:

彭武丽, 罗展荣, 季新成, 员丽娟, 史茜, 于学辉. 布鲁氏菌和贝纳氏柯克斯氏体双重PCR检测方法的建立与初步应用[J]. 中国生物工程杂志, 2014, 34(2): 78-83.

PENG Wu-li, LUO Zhan-rong, JI Xin-cheng, YUN Li-juan, SHI Qian, YU Xue-hui. Establishment and Preliminary Application of a Duplex PCR Assay for Detecting Brucella and Coxiella burnetii. China Biotechnology, 2014, 34(2): 78-83.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140213 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I2/78

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