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

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