细菌16S rRNA基因实时荧光定量及分型方法的建立

Establishment of quantifying and typing analysis of 16S rRNA gene by real-time PCR

摘要:

目的：细菌培养、组织学、免疫学及普通PCR方法等在病原菌的检测方面都存在一定的不足，不能满足临床的需要，探索快速可靠的败血症和化脓性脑膜炎等细菌性感染疾病诊断的新方法是目前亟待解决的关键问题。方法：分析细菌16S rRNA 基因序列，在高度保守区自行设计通用引物和探针，以及革兰阳性和阴性分型探针，对12种标准菌株、23种临床培养分离株、乙型肝炎病毒、新型隐球菌及白色念珠菌、人基因组DNA等进行了荧光定量检测, 分析三种探针检测结果的相关性。结果：细菌16S rRNA基因荧光定量PCR具有较好的敏感性和特异性，最低能检测到10个拷贝的16S rRNA基因，即相当于2个细菌，与病毒、真菌及人基因组DNA等无交叉阳性反应；12种标准菌株、23种临床培养分离株均进行三种探针的荧光定量检测：通用探针均为阳性，18株革兰阳性菌株通过革兰阳性探针（G+Probe探针）检测为阳性，17株革兰阴性菌株通过革兰阴性探针（G-Probe探针）检测为阳性，反之均为阴性，吻合率100％。结论：建立了用通用引物和分型双荧光探针的细菌荧光定量PCR定量、分型方法。其检测方便，快速、敏感性和特异性高，符合率好，同时进行定量和分型，在病原菌检测方面具有推广及应用价值

Abstract:

OBJECTIVE: To explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia. METHODS: A pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed. RESULTS: The determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gram-positive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains. CONCLUSIONS: The FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.