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【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 are commonly used lactic acid bacteria in microbial fertilizer production, Their phenotypic characteristics are similar, if the traditional method of identification is time-consuming and laborious, in order to accurately and quickly identify these species, the establishment of species-specific PCR method. 【Method】 The specific primers were designed and screened by Primer-BLAST (Primer-BLAST) and target genes recA and gyrB of GenBank in GenBank. [Results] Lactobacillus, Lactococcus, Pediococcus, Bacillus, Paenibacillus, Brevibacillus and Pseudomonas, Pseudomonas genus (Pseudomonas) were tested in 24 strains of 40 strains of standard strains. Four of the four target species amplified the only target products, respectively, while none of the other species had any target amplification products. Six strains of Lactobacillus isolated from the product were identified by four specific PCR methods. The results were consistent with 16S rDNA sequence analysis and Biolog identification results. 【Conclusion】 The established PCR-specific PCR methods have high intraspecific versatility and interspecific specificity and can be used rapidly and accurately in Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus acidophilus detection and identification, has a good prospect.