应用特异PCR快速鉴定微生物肥料中4种乳酸菌

Rapid identification of four lactic acid bacteria using specific PCR in microbial fertilizers

【目的】植物乳杆菌(Lactobacillus plantarum)、鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(L.acidophilus)和德氏乳杆菌(L.delbrueckii)是微生物肥料生产中常用的乳酸菌,它们表型特征相似,若采用传统方法鉴定则费时费力,为准确、快速地鉴定这些种,建立种特异PCR方法。【方法】利用NCBI中Primer-BLAST(引物设计和特异性检验工具),以GenBank数据库中上述菌种的recA和gyrB为靶基因,设计和筛选种特异性引物从而建立相应特异PCR鉴定方法。【结果】经过乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、片球菌属(Pediococcus)、芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、短芽孢杆菌属(Brevibacillus)和假单胞菌属(Pseudomonas)7个属24个种共40株标准菌株的实验验证,4个目标种分别扩增出唯一的目的产物,而其他种均无目的扩增产物。采用建立的4种特异PCR方法对产品中分离的16株乳杆菌进行鉴定,结果与16S rDNA序列分析、Biolog鉴定结果一致。【结论】建立的特异PCR鉴定方法均具有较高的种内通用性和种间特异性,可快速、准确的用于微生物制剂中植物乳杆菌、德氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌的检测和鉴定,具有较好的应用前景。

[Objective] Lactobacillus plantarum, L. rhamnosus, L. acidophilus and L. delbrueckii, which were widely used in microbial fertilizers, were indistinguishable from each other just by phenotypic characteristics. Rapid identification of the lactic acid bacteria by specific PCR was necessary for detection and ecological evaluation of microbial fertilizers. [Methods] In this study, based on recA and gyrB genes, four pairs of species-specific primers were designed, and the corresponding specific PCR methods were established. [Results] Forty reference strains belonging to seven genera, such as Lactobacillus, Lactococcus, Pediococcu, Bacillus, Paenibacillus, Brevibacillus and Pseudomonas, were tested. The results showed that, the target single band was consistently amplified from only the 4 strains of lactic acid bacteria. Meanwhile, sixteen strains of lactic acid bacteria from microbial fertilizer products were identified by this specific PCR, the results were consistent with that of 16S rDNA sequence analysis and Biolog. [Conclusion] The methods of specific PCR were species-specific and effective, and could be used in the rapid identification of L. plantarum, L. rhamnosus, L. acidophilus and L. delbrueckii. The results will provide technical supports for the detection and ecological evaluation of microbial fertilizer.