PCR-HRM方法分析16S rRNA基因进行细菌鉴定的可行性研究

Feasibility Study on Analysis of 16S rRNA Gene for Bacterial Identification by PCR-HRM Method

目的建立一种基于16S rRNA基因进行聚合酶链式-高分辨率熔解曲线(PCR-HRM)分析方法,用于快速鉴定临床常见感染细菌。方法收集中国人民解放军北部战区总医院2017年8月~2018年10月期间临床分离的167株常见细菌,选取16S rRNA三个高度保守区域(V1,V3,V6)基因序列用于引物设计,在含有饱和荧光染料的体系中进行PCR-HRM分析以区分不同细菌种属。将167株实验菌分为葡萄球菌、链球菌、非发酵阴性杆菌以及其它常见临床分离菌四组进行实验,分别进行基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和PCR-HRM分析并对结果进行对比,不一致的鉴定结果以测序为金标准。同时进行PCR-HRM方法鉴定临床常见病原菌的灵敏度、重复性和盲法验证试验。结果 PCR-HRM方法与MALDI-TOF MS对167株临床常见分离菌鉴定正确率分别为98.2%和97.0%,这两种方法无明显统计学差异(χ^2=1.97,P=0.727>0.05)。PCR-HRM方法可检测2 pg/μl的模板浓度;将6株革兰阴性菌分成一式三份同时进行PCR-HRM重复性试验,同种实验菌的三份熔解曲线图完全聚类;其盲法验证实验准确率100%。结论该方法通过使用3对16S rRNA引物构造多重差异曲线可对临床常见细菌进行分类到属级甚至种级,且灵敏度高、特异度强,可以作为鉴定临床常见感染菌的新选择。

Objective A polymerase chain reaction-high resolution melting curve analysis(PCR-HRM) method based on 16s rRNA gene was developed for rapid identification of common clinical infected bacteria.Methods 167 common bacteria isolated clinically from August 2017 to October 2018 in the General Hospital of Northern Theater Command of the Chinese People’s Liberation Army,and three highly conserved regions(V1,V3,V6) of GenBank were selected for primer design.PCR-HRM analys in systems containing saturated fluorescent dyes to distinguish between different bacterial species.167 strains were divided into four groups:Staphylococcus,Streptococcus,non-fermentative negative bacilli and other common clinical isolates.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) and PCR-HRM analysis were performed and the results were compared.The inconsistent results were determined by sequencing as the gold standard.The sensitivity,specificity and reproducibility of HRMA method for identification of common clinical pathogens were evaluated.Results The correct rate of identification between PCR-HRM and MALDI-TOF MS was 98.2% and 97.0%,respectively.There was no significant difference between the two methods(χ^2=1.97,P=0.727>0.05).The template concentration of 2 pg/μl could be detected by PCR-HRM method,and 6 Gram-negative bacteria were divided into three strains and the PCR-HRM repeatability test was carried out at the same time,and the three fusion curves of the same tested bacteria were completely clustered,and the accuracy of the experiment was 100% by blind method.Conclusion By using 3 pairs of 16s rRNA primers to construct multiple differential curves,common clinical bacteria can be classified to genus or even species,with high sensitivity and specificity,which can be used as a new choice for identification of common clinical infective bacteria.