应用DPO-PCR方法特异性检测志贺氏菌

中国畜牧兽医

应用DPO-PCR方法特异性检测志贺氏菌

徐义刚¹，李丹丹²，刘忠梅¹，吴岩¹，李苏龙¹，魏冬旭¹

（1.黑龙江出入境检验检疫局检验检疫技术中心，黑龙江哈尔滨 150001；2.海南出入境检验检疫局检验检疫技术中心，海南海口 570125）

收稿日期:2013-09-17 出版日期:2014-03-20 发布日期:2014-05-15
作者简介:徐义刚(1978—），男，吉林人，博士后，研究方向：病原微生物诊断与防治技术。
基金资助:
国家质检总局科技计划项目(2012IK157)；质检公益性行业科研专项(201310126)。

Specific Detection of Shigella Using DPO-PCR Method

XU Yi-gang¹，LI Dan-dan²，LIU Zhong-mei¹，WU Yan¹，LI Su-long¹，WEI Dong-xu¹

(1.Technical Center of Heilongjiang Entry-exit Inspection and Quarantine Bureau, Harbin 150001, China;2.Technical Center of Hainan Entry-exit Inspection and Quarantine Bureau, Haikou 570125, China)

Received:2013-09-17 Online:2014-03-20 Published:2014-05-15

摘要/Abstract

摘要： 本研究利用一种高特异性PCR引物设计方法——双启动寡核苷酸引物 (dual-priming oligonucleotide, DPO)，以志贺氏菌属侵袭性质粒抗原（ipaH）基因为靶基因，设计DPO引物，建立了特异性检测志贺氏菌属的DPO-PCR方法。结果显示，所建立的DPO-PCR方法检测灵敏度为1.65×10² CFU/mL；与常规PCR引物相比，DPO引物设计简易，简化了PCR引物设计；DPO引物退火温度范围宽，在50～70 ℃退火温度内均可对靶基因进行高效扩增； DPO引物结构特殊，具有比常规PCR引物更高的特异性，反应中无非特异性扩增产生。实践检测结果显示，利用该方法对133份冻/鲜肉类、果蔬类、鲜牛奶、鸡蛋和熟食样本进行检测，共检出15份志贺氏菌阳性样本，经国标法(GB/T 4789.5—2012)复检，两者检测结果一致，显示出良好的实用性和可靠性，为志贺氏菌快速、准确的检测提供了新手段。

关键词: 志贺氏菌; 侵袭性质粒抗原（ipaH）基因; DPO-PCR方法

Abstract: In this study, a dual-priming oligonucleotide (DPO)-based PCR method for specific detection of Shigella was established using ipaH gene of Shigella as the target gene. The results showed that detection sensitivity of the DPO-PCR method was 1.65×10² CFU/mL. Compared with conventional PCR primers, the DPO primers were easily designed, which simplified the design procedure of PCR primers. DPO primers were not sensitive to annealing temperature, which could efficiently amplify the target gene in the annealing temperature range from 50 to 70 ℃. Moreover, due to the special structure of DPO primers, it had a higher specificity than conventional PCR primers, and none nonspecific amplifications were produced in reaction. In the practical application, tests on 133 samples including frozen/fresh meat, fruits and vegetables, fresh milk, eggs and cooked food by the DPO-PCR method showed that 15 samples were Shigella positive, which were in accordance with the testing results using GB method (GB/T 4789.5—2012), showing good practicality and reliability. The DPO-PCR method provided a new tool for fast and accurate detection of Shigella.

Key words: Shigella; ipaH gene; DPO-PCR method

徐义刚，李丹丹，刘忠梅，吴岩，李苏龙，魏冬旭. 应用DPO-PCR方法特异性检测志贺氏菌[J]. 中国畜牧兽医.

XU Yi-gang，LI Dan-dan，LIU Zhong-mei，WU Yan，LI Su-long，WEI Dong-xu. Specific Detection of Shigella Using DPO-PCR Method[J]. .

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