PCR-HRM方法快速鉴定布鲁氏菌的初步应用

目的 本研究旨在建立准确、快速地鉴定布鲁氏菌菌种及生物分型的PCR-HRM(高分辨率熔解曲线)方法。方法 根据目的基因序列,参考文献合成6对基因扩增引物(1对布鲁氏菌属特异引物,5对种间特异引物),应用PCR-HRM方法鉴定布鲁氏菌属6个种19个生物型的标准菌株,并初步应用到临床分离的35株布鲁氏菌中。结果 采用布鲁氏菌属特异引物(Bspp),6个种19个生物型的标准菌株均扩增出同样形状的溶解曲线,与其它对照菌株的曲线不同;采用5对种特异引物,6个种的标准菌株均有特征性PCR-HRM曲线;临床分离的35株布鲁氏菌的PCR-HRM曲线与羊种布鲁氏菌标准菌株的一致。结论 该研究采用的PCR-HRM分析方法,获得了布鲁氏菌属及6个种标准菌株的不同曲线图,可准确鉴定临床分离的羊种布鲁氏菌,可用于临床微生物实验室疑似布鲁氏菌感染的初步鉴定。

Clinical application of PCR and high resolution melting analysis for rapid identification of Brucella isolates

The aim of this study is to develop a rapid and accurately species typing method for Brucella isolates by using High Resolution Melting (HRM) analysis. Six pairs of primers were used according to the reference for the sequence of purpose gene. Nineteen biotypes of six species Brucella standard strains were identified by PCR-HRM analysis and this analysis was used to detect the 35 clinical isolates. Results showed Brucella amplified specific melting curves were different from contrasted strains with primer Bspp. The six species Brucella standard strains have own characteristic curve shape from each others by PCR-HRM analysis with five pairs of primers. Thirty-five clinical isolates of Brucella have entirely consistent with PCR-HRM curve shape with Brucella melitensis standard strains. So, PCR-HRM analysis methods can accurately identify Brucella strains, especially clinical isolated Brucella melitensis, and may be used in clinical microbiology laboratories.