金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR方法的建立与初步应用

发布时间：2018-06-07 17:49

本文选题：金黄色葡萄球菌 + 绿脓杆菌　；参考：《中国比较医学杂志》2017年05期

【摘要】：目的建立金黄色葡萄球菌、绿脓杆菌和肺炎克雷伯杆菌多重PCR检测方法,为实验动物细菌检测提供快速、灵敏、简便的方法。方法根据金黄色葡萄球菌nuc基因、绿脓杆菌Las I基因、肺炎克雷伯杆菌Pho E基因和细菌通用16S rRNA基因设计出特异性引物;经过多重PCR引物浓度和退火温度的优化,特异性和敏感性的检测,建立多重PCR体系。应用该PCR体系对人工感染标本、实验动物粪便样本进行验证检测,与传统检测方法对比。结果多重PCR分别扩增出金黄色葡萄球菌(153 bp)、绿脓杆菌(600 bp)、肺炎克雷伯杆菌(368 bp)和通用(520 bp)的目的条带。多重敏感性为10 pg,特异性检测未从其他细菌中检测出目的条带。应用建立的多重PCR体系检测出不同组合的人工感染标本,从76只粪便检测出绿脓杆菌阳性1例,与传统检测方法结果一致。结论本文建立的多重PCR方法具有特异、灵敏、简便、快速等优点,为实验动物微生物的快速检测提供了可靠的方法。
[Abstract]:Objective to establish a multiplex PCR method for the detection of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae. Methods specific primers were designed according to Staphylococcus aureus nuc gene, Pseudomonas aeruginosa Las I gene, Klebsiella pneumoniae Pho E gene and bacterial general 16s rRNA gene. A multiplex PCR system was established for the detection of specificity and sensitivity. The PCR system was used to verify and detect the samples of artificial infection and animal faeces, and compared with the traditional methods. Results the target bands of Staphylococcus aureus 153 BP, Pseudomonas aeruginosa 600 BP, Klebsiella pneumoniae 368 BP and General purpose 520 BP) were amplified by multiplex PCR. The multiplex sensitivity was 10 PG, and the target bands were not detected by specific detection from other bacteria. The multiplex PCR system was used to detect different combinations of artificially infected specimens. One case of Pseudomonas aeruginosa positive was detected from 76 feces, which was consistent with the traditional method. Conclusion the multiplex PCR method developed in this paper has the advantages of specificity, sensitivity, simplicity and rapidity, which provides a reliable method for the rapid detection of microbes in laboratory animals.
【作者单位】：河北省实验动物重点实验室河北医科大学实验动物学部;河北医科大学第二医院;
【基金】：国家科技支撑计划(2015BAI07B02-05)
【分类号】：R440

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