基于鞭毛蛋白基因的坚强芽孢杆菌特异性套式PCR和荧光定量PCR方法的建立

Establishment of Specific Detection Methods by Nested PCR and qPCR for Bacillus firmus Based on the hag Gene

关键词:  坚强芽孢杆菌  鞭毛蛋白  hag基因  套式PCR  荧光定量PCR

Key words:  Bacillus firmus  Flagellin  hag gene  Nested PCR  Fluorescent quantitative PCR

摘要:

细菌鞭毛蛋白编码基因hag具两端的保守序列及中间可变区域，在细菌的分类和鉴定中可作为分子标记。本研究克隆了坚强芽孢杆菌鞭毛蛋白编码基因hag部分序列，根据所得核酸序列设计套式引物Bfho和Bfhi，进行菌株的套式PCR特异性检测。此外，采用内引物Bfhi建立坚强芽孢杆菌荧光定量PCR特异性检测方法并确定该方法的检测限，对15个模拟样品进行检测并对阳性组中坚强芽孢杆菌的含量进行定量分析。结果显示，克隆得到的坚强芽孢杆菌hag基因长为1213 bp，经比对，与枯草芽孢杆菌hag基因相似性为13%−15%。荧光定量PCR方法对坚强芽孢杆菌菌株PC004和PC024的检测限分别为17.3×103和19.7×103 CFU/ml。模拟样品检测结果显示，15个样品中检测出7个阳性，并同时定量各样品中坚强芽孢杆菌的含量。本研究建立的坚强芽孢杆菌检测方法特异性强、操作时间短、灵敏度高，可为该菌的实际检测提供技术支持。

Abstract:

Hag, a Flagellin coding gene, has conserved domains on the ends and a central domain that is highly variable in the length and the sequence, and hence has been used as a molecular marker in bacterial classification and identification. In this study, the hag gene was amplified from Bacillus firmus with the primer set Bhag, which was designed based on the conserved regions of the hag gene of B. subtilis and other bacteria. Two primer sets, Bfho and Bfhi, were designed based on the obtained sequence of hag of B. firmus and were used in the nested PCR for the species-specific detection of B. firmus. Furthermore we developed another specific detection method using fluorescent quantification PCR based on the inner primer set Bfhi, and tested the detection limit. Some simulated samples were tested with this FQ-PCR method and in the positive samples the concentrations of B. firmus were determined with the same method. The sequence of hag cloned from B. firmus had 1213 bp and was only 13%15% similar to that of B. subtilis according to NCBI BLAST, and most differences existed in the variable region. The detection limits of the FQ-PCR method were 17.3×103 CFU/ml and 19.7×103 CFU/ml for B. firmus strains PC004 and PC024 respectively. In 15 simulated samples, 7 were detected positive with the FQ-PCR method, and the concentrations of B. firmus in the samples were determined as well. The detection methods developed in our study have technical advantages such as high specificity, time-saving, and high sensitivity, and thus may become valuable tools in the practical application.