牦牛奶酪中乳酸菌的随机扩增多态性研究

Randomly Amplified Polymorphic DNA (RAPD) Analysis of Lactic Acid Bacteria from Yak Milk Cheese

目的:探讨随机扩增多态性(RAPD)技术在牦牛奶酪中乳酸菌基因型研究方面的应用价值。方法:采用M13引物,对退火温度、Mg2+浓度、引物用量、Taq酶用量、模板量进行单因素梯度试验,建立最佳反应条件,然后对分离自牦牛奶酪的39株乳酸菌进行扩增,用NTsys 2.10e软件分析扩增图谱,计算遗传相似系数,进行聚类分析,并与16S rRNA测序得到的菌种鉴定结果进行对比。结果:39株乳酸菌相互间的遗传相似系数在0.47～0.98之间,当相似系数为0.78时,菌株被分成10个组,除X23、Y36和Y37之外,其余菌株均按照不同的菌种聚类,与16S rRNA测序结果基本一致,聚类有较强的地域相关性。结论:RAPD技术可用于牦牛奶酪中乳酸菌的基因型研究,可用于菌种鉴定和遗传亲缘关系分析。

Objective： To explore the application value of randomly amplified polymorphic DNA （RAPD）-PCR for the genotype evaluation of lactic acid bacteria isolated from yak milk cheeses. Methods： In order to establish a reliable RAPD protocol with primer M13, factors such as annealing temperature, Mg2＋ concentration, primer amount, Taq polymerase and DNA template for affecting the quality and reproducibility of RAPD profiles were investigated. The optimal PCR procedure and reaction reagent concentration were employed for RAPD amplification of 39 isolates from yak milk cheese on the basis of previous identification of 16S rRNA sequence analysis. The electrophoretic profiles were analyzed using NTsys2.10e software, genetic similarity coefficients were calculated and a cluster dendrogram was plotted. Results： Genetic similarity coefficients of 39 strains ranged from 0.47 to 0.98. The cluster dendrogram showed that 39 isolates were classified into 10 groups using the similarity coefficient of 0.78 as a cut-off point. The discrimination of groups was consistent with the assignment of the species based on 16S rRNA sequence analysis, except for strains X23, Y36 and Y37. Conclusion： RAPD technique is useful in taxonomy of lactic acid bacteria isolated from yak milk cheese. The accuracy of species identification can be improved by the combination of RAPD with other kinds of genotypic methods.