纳豆芽孢杆菌和嗜酸乳杆菌融合子的多重PCR鉴定

摘要

A multiplex polymerase chain reaction (mPCR) assay for the rapid identification of Lactobacillus acidophilus-Bacillus natto fusant was developed in this paper.The primer pair P1/P2 was designed based on the spo0A gene encoding the stage 0 sporulation protein A of Bacillus subtilis,as well as the primer pair P3/P4 according to the conserved sequence of the β-galactosidase gene of 13 lactic acid bacterial (LAB) strains.Using the genomic DNA of the parental strains as template,PCR systems for each primer pair were optimized under the predetermined annealing temperature.The specificity analysis showed that P1/P2 could amplify the spo0A fragment (308 bp) but all seven LAB strains gave negative results;P3/P4 could amplify the target fragment (576 bp) from all LAB and five identified positive fusants but not from Bacillus subtilis.Results of multiplex PCR demonstrated that the optimal PCR system for P1/P2 could only amplify its own 308 bp target DNA,while in the optimal system for P3/P4 both target DNA fragments could be obtained.The results of identification of 50 fusant strains by multiplex PCR showed 100％ consistency with that of traditional PCR and phenotype identification.The mPCR system contained 1 ng/μL template DNA,0.4 μmol/L primer (each),0.25 mmol/L dNTP,0.06 U/μL Taq polymerase,and the PCR conditions were as follows:5 min predenaturation at 94 ℃,30 cycles of denaturation at 94 ℃ 30 s,annealing at 53 ℃℃ for 30 s,and extension at 72 ℃ for 60 s.This method proved to be a rapid,convenient method with high specificity for the efficient identification of spore-forming bacteria,LAB and their fusant.%为建立纳豆芽孢杆菌和嗜酸乳杆菌融合子快速鉴定的多重聚合酶链式反应(polymerase chain reaction,PCR)方法,根据芽孢杆菌的芽孢形成早期因子基因spo0A和13株乳酸菌β-半乳糖苷酶基因的保守序列分别设计引物对P1/P2和P3/P4,以亲本菌株DNA为模板,优化每对引物在适宜退火温度条件下的PCR体系,在最优体系下的特异性分析结果表明P1/P2能特异性扩增出spo0A基因片段(308 bp),而7株乳酸菌全部呈阴性;P3/P4可扩增出所有受试乳酸菌和经表型鉴定确定的阳性融合子中的目标基因片段(576 bp),而对芽孢杆菌无扩增.多重PCR结果表明在P 1/P2的最优体系中只有308 bp片段的扩增,而在P3/P4的最优体系中可实现2个片段的共扩增,对融合后获得的50个菌株的鉴定结果与单重PCR和表型鉴定结果100％吻合.该体系中模板DNA 1 ng/μL,每条引物0.4 μmol/L,脱氧核糖核昔三磷酸0.25 mmol/L,Taq酶0.06 U/μL;PCR时94℃预变性5 mina每个循环(共30个)94℃30 s、53℃30 s、72℃60 s,产物末端72℃延伸7 min.该方法简便、快速、特异性好,适用于芽孢杆菌、乳酸菌以及二者融合子的快速鉴定.