特异性检测植物乳杆菌的多重PCR方法

A specific method for detection of Lactobacillus plantarum by multiplex PCR

【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101bp(引物LP-1和LP-2)、189bp(引物LP-5和LP-6)、378bp(引物LP-9和LP-10)。该方法的检测限为2.2×10CFU/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。

[Objective]The present study was conducted to establish a specific method for detection of Lactobacillus plantarum without any interference. [Method ]Laetobacillus plantamm specific sequenees were screened by SMM system, and muhiplex 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, fi＇ee from the interference of Lactobaeillus pentosus and LactobacilIus plantamm, 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 2.2x10 CFU/mL . Using multiplex PCR and agarose gel electrophoresis to test self-made yogurt products with lactic acid bacteria, natural ferment- ed products, and yogurts commodities, Laetobacillus plantarum could be detected to distinguish kindred strains of Lacto- bacillus 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.