Identification of new reference genes for colony counting by reverse-transcription quantitative PCR in Bifidobacterium animalis

Bifidobacterium animalis, one of the predominant bacteria in the intestines of humans and other mam-mals, is widely added to dairy products. We employed RNA sequencing to analyze gene expression variance on a genome-wide scale and found stable reference genes (RG) in B. animalis. A total of 1,665 genes were identified by analyzing the data from the transcriptome under 4 different conditions, and 13 probable candidate RG with variation coefficient values <0.1 were validated using reverse-transcription quantitative PCR (RT-qP-CR). The amplification efficiency of candidate RG were ranging from 94.16% to 126.25%. We integrated the analysis results of BestKeeper, geNorm, NormFinder, and RefFinder algorithms and revealed that rplD and atpA comprehensive ranked 1.68 and 2.82, respectively, which were more stable than traditional RG. Compared with plate count (1.58 x 10(6) cfu/mL), the concentra-tions of B. animalis AR668 by RT-qPCR using rplD, atpA, and 16S rRNA as RG were 2.27 x 10(6), 2.24 x 10(6), and 6.66 x 10(6) cfu/mL, respectively, after 10 h of fermentation in fermented skim milk. It suggested that rplD and atpA as RG can be accurate for colony counting of B. animalis. Our study provides the founda-tion for more accurate analysis of colony counting by RT-qPCR of B. animalis in dairy foods.

Automated summary

In this study, gene expression variance in B. animalis was analyzed using RNA sequencing, and stable reference genes were identified. Through integration of multiple analysis algorithms, rplD and atpA were determined as stable reference genes. Using these two genes for colony counting, accurate results were obtained in fermented skim milk. This study provides a foundation for accurate analysis of colony counting of B. animalis in dairy foods.