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Evaluation of a facile method of template DNA preparation for PCR-bascd detection and typing of lactic acid bacteria

机译：简便的模板DNA制备方法的评估，以PCR为基础的乳酸菌检测和分型

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摘要

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, Laaococcus, 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 Cram-positive and Cram-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.
机译：我们研究的目的是开发一种方便可靠的生成模板DNA的方法，用于常规基于PCR的乳酸菌（LAB）检测和分型。使用尿素，SDS和NaOH的组合从乳杆菌，乳球菌，球菌和Leuconostoc中提取的模板DNA在PCR中用属特异性引物产生了预期大小的扩增子。除了LAB外，还可以采用拟议的方法从许多Cram阳性和Cram阴性细菌菌株中生成PCR兼容的模板DNA。通过提出的方法从各种乳酸杆菌标准菌株制备的DNA模板。在rep-PCR中也用BOXA1R引物产生了区分指纹。该研究的重要发现是，使用尿素-SDS-NaOH方法制备的模板DNA和市售DNA分离试剂盒，在rep-PCR中获得了LAB菌株的可比谱带。当通过UPGAMA方法进行聚类分析时，在81.8-96.7范围内获得的高骰子系数值进一步证明了这一点。通过检测发酵样品中LAB属的特定扩增子，验证了该DNA提取方法在基于PCR的发酵食品（如大麦，艾德巴特和盐发酵黄瓜）中直接检测LAB的应用潜力。当先前通过PCR在盐发酵黄瓜中检测到29种细菌致癌LAB菌株（Bac〜+）时，所提出的模板DNA提取方法的适用性得到进一步证实[Singh，AK，Ramesh，A.，2008.优势乳酸和拮抗乳酸的继承发酵黄瓜中的细菌：基于PCR的方法的见解。餐饮。微生物。 [25,278-287]在基于BOX元素的rep-PCR中产生了区分指纹，并与参考LAB菌株形成了簇。

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