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Alicyclobacillus  acidoterrestris  is a major concern in fruit juice industry due to its spoilage potential of acidic fruit juice. Quantifying the expression levels of functional genes by real-time quantitative polymerase chain reaction (RT-qPCR) is necessary to elucidate the response mechanisms of  A. acidoterrestris  to acid stress. However, appropriate reference genes (RGs) for data normalization are required to obtain reliable RT-qPCR results. In this study, eight novel candidate RGs were screened based on transcriptome datasets of  A. acidoterrestris  under acid stress. The expression stability of eight new RGs and commonly used RG 16s rRNA was assessed using geNorm, NormFinder, and BestKeeper algorithms. Moreover, the comprehensive analysis using the RefFinder program and the validation using target gene  ctsR  showed that  dnaG  and  dnaN  were the optimal multiple RGs for normalization at pH 4.0;  ytvI ,  dnaG , and 16s rRNA at pH 3.5;  icd  and  dnaG  at pH 3.0; and  ytv I,  dnaG , and  spoVE  at pH 2.5. This study revealed for the first time that  A. acidoterrestris  had different suitable RGs under different acid conditions, with implications for further deciphering the acid response mechanisms of this spoilage-causing bacterium.

Transcriptome-Based Selection and Validation of Reference Genes for Gene Expression Analysis of Alicyclobacillus acidoterrestris Under Acid Stress