Design and evaluation of Bifidobacterium genus-specific primer for quantification

Abstract:

[Objective] The aim of this study was to design a pair of absolute quantitative specific primers for detecting the number of the low content Bifidobacterium in different samples.[Methods] The complete genome sequence of 57 strains of Bifidobacterium was downloaded from NCBI, and the specific primers of Bifidobacterium were designed based on their common single-copy core genes. Primers were screened by PCR and rescreened by specificity. Then ddPCR (Droplet Digital PCR) was used to verify the specificity, sensitivity and practicability of the selected primers.[Results] The specificity of Bif-D-9 primer was best, and 4 strains Bifidobacterium were amplified but 20 strains of non-Bifidobacterium could not be amplified. The diluted DNA was quantitatively by ddPCR, and the amplification results show a linear decreasing trend, which proves that the sensitivity is better. At the same time, Bif-D-9 combined with ddPCR can quantitatively determine the copy number of Bifidobacterium in infant feces is 71 copies/μL, and the maternal feces contained 2.7 copies/μL, which proves the practicality is good.[Conclusion] The primer Bif-D-9 was high specificity, high sensitivity and accuracy of Bifidobacterium, and is suitable for the quantitative study of Bifidobacterium in complex environmental samples.

Key words:Bifidobacterium;genus-specific primer;quantitative;evaluation;droplet digital PCR