7种食源性致病菌GeXP多重PCR检测方法的建立及其应用

doi:10.11843/j.issn.0366-6964.2020.10.022

摘要/Abstract

摘要： 本研究旨在建立一种能够同时鉴别包括大肠杆菌O157：H7、沙门菌、空肠弯曲菌、单核细胞增生李斯特菌、霍乱弧菌、副溶血弧菌和金黄色葡萄球菌7种常见食源性致病菌的GeXP多重PCR检测方法。根据这些致病菌在GenBank上公布的保守基因序列，设计合成了7对特异性GeXP引物。用单一或混合细菌样品的DNA模板优化反应条件，设置对照组，构建重组质粒，随机组合不同浓度的样品，验证所建立的GeXP方法的特异性、敏感性和准确性。最后用该方法检测120份临床样品，进一步验证所建立的GeXP检测方法的准确性和可靠性。结果显示，单一或混合模板的GeXP检测均能特异性出现相应清晰峰值，可在10³拷贝·μL^-1水平上同时特异地检测出7种细菌病原体，不同浓度模板混合时，本试验所建立的方法依然可检测出对应病原体。检测120份临床样品，GeXP多重PCR阳性率为2.50%（3/120）~15.83%（19/120），普通PCR阳性率为2.50%（3/120）~15.00%（18/120），GeXP多重PCR多检出8份阳性，表明GeXP方法更为敏感与准确。本研究建立的同时鉴别7种食源性致病菌的GeXP多重PCR检测方法，具有高通量、特异性强和敏感性高的特点，为食源性常见致病菌感染或混合感染提供了快速分子诊断方法。

关键词: 食源性致病菌, GeXP, 多重PCR, 特异性嵌合引物

Abstract: This experiment was developed to simultaneously detect the seven common foodborne pathogenic bacterias include E. coli O157:H7, Salmonella, V. cholera, L. monocytogenes, C. jejuni, V. Parahemolyticus and S. aureus. Seven pairs of specific primers were designed according to the conserved sequences of the genes from each pathogen available in the GenBank database. Single and mixed pathogen DNA templates were used to evaluate the specificity of the GeXP-multiplex assay. Control group was set up, recombinant plasmids were constructed, and samples of different DNA concentrations were randomly combined to verify the sensitivity, specificity, accuracy and anti-interference of the established GeXP method. To certify the accuracy and reliability of the GeXP assay, it was evaluated using 120 clinical specimens that were compared with the single PCR. The obtained results showed that the corresponding specific fragments of genes were amplified by the single and the multiplex GeXP PCR assay. The detection limit of GeXP was 10³ copies·μL^-1 when all of seven bacterial pathogens were detected. The results of the interference assay showed the presence of specific amplification peaks when different templates. The detection rate of the GeXP multiplex PCR method was 2.50% (3/120)-15.83% (19/120) while the conventional PCR was 2.50% (3/120)-15.00% (18/120), and GeXP multiple PCR detected 8 more positive cases, which means that, the GeXP was more sensitive and accurate in the detection of the clinical samples. In conclusion, this GeXP-based multiplex PCR is a high-throughput, specific and sensitive test to detect seven common foodborne pathogenic bacterias. This assay provides a method in rapid molecular diagnosis for mix clinical seven common foodborne pathogenic bacterias.

Key words: foodborne pathogenic bacterias, genome lab gene expression profiler, multiplex PCR, separation identification

中图分类号:

张艳芳, 谢芝勋, 曾婷婷, 刘加波, 谢丽基, 邓显文, 谢志勤, 罗思思, 黄娇玲, 王盛. 7种食源性致病菌GeXP多重PCR检测方法的建立及其应用[J]. 畜牧兽医学报, 2020, 51(10): 2536-2546.

ZHANG Yanfang, XIE Zhixun, ZENG Tingting, LIU Jiabo, XIE Liji, DENG Xianwen, XIE Zhiqin, LUO Sisi, HUANG Jiaoling, WANG Sheng. Development and Application of a GeXP-multiplex PCR Assay for Detection of Seven Foodborne Pathogenic Bacterias[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2536-2546.

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