肠道中乳酸杆菌属特异性定量引物的筛选及验证

Screening and evaluation of genus-specific primer for quantification of Lactobacillus in gut

摘要:

[目的]乳酸杆菌与人和动物的健康有密切关系，它的存在及含量变化可以作为评价宿主健康的指标之一。在乳酸杆菌定量研究中，特异性引物往往是定量成功的关键。然而，已有引物质量参差不齐，难以保证其特异性。本文旨在通过理论与试验的方法快速筛选出用于定量的乳酸杆菌属特异性引物，同时为今后引物筛选和设计提供理论基础。[方法]查阅文献、挑选出12对基于16S rRNA基因序列设计的乳酸杆菌属引物，通过MEGA 6.0软件确定引物相对位置，计算引物匹配率，以引物相对位置和匹配率为依据重新组合引物，获得理论特异性乳酸杆菌属引物，再通过琼脂糖凝胶电泳和QX200 Droplet Digital PCR系统对新组合引物的特异性进行检验。[结果]通过理论与试验相结合的方法确定了一对特异性较好的乳酸杆菌属定量引物Lab1，它的扩增产物大小约300 bp。ddPCR系统检验结果发现其特异性和灵敏性较好，还可以有效定量粪便中的乳酸杆菌。[结论]引物设计理论结合特异性试验这种方法可以快速有效地筛选出特异性较好的引物，同时为今后引物筛选和设计提供理论基础。

关键词:乳酸杆菌属;特异性;引物筛选;微滴式数字PCR;验证

Abstract:

[Objective] Lactobacillus is closely related to human or animal health, and its presence and content changes can be used as health indicators. Usually, specific primers are the key to successful quantification. Redesigning primers is time-consuming and hard to guarantee its specificity. This study was to screen genus-specific primers for Lactobacillus by theoretical and experimental methods rapidly and effectively, and also to provide a theoretical basis for screening and designing of primers in the future.[Methods] We selected 12 pairs of primers based on the 16S rRNA gene sequence from published literatures and evaluated them using primer design software. Based on the evaluation results, we recombined the primers to obtain the theoretically specific Lactobacillus primers, and test the specificity of the new combination of Lactobacillus primers using the gel electrophoresis and the QX200 droplet digital PCR system. [Results] We screened out a pair of Lactobacillus genus-specific primer named Lab-F1/Lab-R1 by primer-designing software and tests, and its product size was about 300 bp. The ddPCR tests showed that the specificity and sensitivity of the Lab-F1/Lab-R1 were better, and it also could effectively quantify Lactobacillus in fecal samples. [Conclusion] This method can quickly and efficiently screen out the specific primers with better specificity and provide a theoretical basis for the future screening and designing of primers.

Key words:Lactobacillus;specificity;primer screening;droplet digital PCR;evaluation