特异性检测植物乳杆菌的多重PCR方法

摘要

[Objective]The present study was conducted to establish a specific method for detection of Lactobacillus plantarum without any interference.[Method]Lactobacillus plantarum specific sequences were screened by SMM system,and multiplex PCR detection of different dairy products was conducted based on the designed primer of specific sequences.[Result]The designed seven primers performed good specificity,among which three primers,free from the interference of Lactobacillus pentosus and Lactobacillus plantarum,could produce specific sequences of 101 bp (Primer LP-1 & LP-2),189 bp (Primer LP-5 & LP-6) and 378 bp (Primer LP-9 & LP-10).The limit of detection was 2.2×10 CFU/mL.Using multiplex PCR and agarose gel electrophoresis to test self-made yogurt products with lactic acid bacteria,natural fermented products,and yogurts commodities,Lactobacillus plantarum could be detected to distinguish kindred strains of Lactobacillus plantarum.[Conclusion]Due to its low cost,simple procedures,brief time,high sensitivity and specificity,this method should be used for fast detection of Lactobacillus plantarum in actual production.%[目的]建立特异性检测植物乳杆菌的方法,排除近缘菌的干扰.[方法]利用SMM系统筛选植物乳杆菌种特异序列,根据特异序列设计引物进行不同乳制品的多重PCR检测.[结果]设计的7对引物具有良好的种特异性,其中3对引物可以排除戊糖乳杆菌和副植物乳杆菌的干扰,可扩增获得植物乳杆菌的特异条带101 bp(引物LP-1和LP-2)、189bp(引物LP-5和LP-6)、378 bp(引物LP-9和LP-10).该方法的检测限为2.2×10 CFU/mL.采用多重PCR检测与琼脂糖凝胶电泳图谱分析对自制含有各种乳酸菌酸奶产品、自然发酵产品及市售酸乳产品中植物乳杆菌进行定性检测,可以特异性检测出植物乳杆菌,准确区分植物乳杆菌的近缘菌株.[结论]该多重PCR方法成本低、步骤简单、耗时短、灵敏度高,具有特异性,可作为快速检测植物乳杆菌种的方法在生产中使用.