Evaluation of a facile method of template DNA preparation for PCR-based detection and typing of lactic acid bacteria.
Evaluation of a facile method of template DNA preparation for PCR-based detection and typing of lactic acid bacteria.
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DOI：
10.1016/j.fm.2009.03.006
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发表时间：
2009-08
影响因子：
5.3
通讯作者：
A. Singh;A. Ramesh
中科院分区：
文献类型：
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作者：
A. Singh;A. Ramesh
本研究的目的是建立一种简便可靠的模板DNA生成方法，用于乳酸菌的常规PCR检测和分型。使用尿素、SDS和NaOH的组合从乳杆菌属、乳球菌属、片球菌属和明串珠菌属提取模板DNA，在使用属特异性引物的PCR中产生预期大小的扩增子。除了LAB之外，所提出的方法还可以用于从许多革兰氏阳性和革兰氏阴性细菌菌株中产生PCR相容的模板DNA。用该方法制备的乳酸杆菌标准菌株DNA模板，在重复PCR中与BOXA1R引物反应，也能产生区分性指纹。研究的一个重要发现是，使用尿素-SDS-NaOH法制备的模板DNA和市售的DNA分离试剂盒，在rep-PCR中获得了可比较的LAB菌株带型。UPGAMA方法聚类分析得到的骰子系数在81.8 - 96.7之间，进一步证明了这一点。通过检测发酵样品中乳酸菌属的特异性扩增子，验证了该DNA提取方法用于发酵食品样品中乳酸菌的PCR直接检测的应用潜力。当先前通过PCR在盐发酵黄瓜中检测到29种细菌素原性LAB菌株（Bac+）时，进一步证实了所提出的模板DNA提取方法的适用性[Singh，A.K.，Ramesh，A.，2008.发酵黄瓜中优势和拮抗乳酸菌的演替：基于PCR方法的见解。食物Microbiol. 25，278 - 287]在基于BOX元件的rep-PCR中产生了区分指纹，并与参考LAB菌株形成了簇。
The objective of our investigation was to develop a convenient and reliable method of generating template DNA for routine PCR-based detection and typing of lactic acid bacteria (LAB). Template DNA extracted from Lactobacillus, Lactococcus, Pediococcus and Leuconostoc using a combination of urea, SDS and NaOH yielded amplicons of expected size in PCR with genus-specific primers. Apart from LAB, the proposed method could also be adopted to generate PCR-compatible template DNA from a number of Gram-positive and Gram-negative bacterial strains. DNA template prepared by the proposed method from various standard strains of Lactobacillus sp. also generated discriminating fingerprints with BOXA1R primer in rep-PCR. A significant finding of the investigation was that a comparable banding profile of LAB strains was obtained in rep-PCR using template DNA prepared by urea–SDS–NaOH method and a commercially available DNA isolation kit. This was further evidenced by high dice coefficient values obtained in the range of 81.8–96.7 when cluster analysis was performed by UPGAMA method. The application potential of this DNA extraction method for PCR-based direct detection of LAB in fermented food samples such as dahi, idli batter and salt-fermented cucumber was validated by detecting specific amplicons of LAB genera in the fermented samples. The applicability of the proposed template DNA extraction method was further substantiated when 29 bacteriocinogenic LAB strains (Bac+) previously detected in salt-fermented cucumber by PCR [Singh, A.K., Ramesh, A., 2008. Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: Insights from a PCR-based approach. Food. Microbiol. 25, 278–287] generated differentiating fingerprints in BOX element based rep-PCR and formed clusters with reference LAB strains.