Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

Lactobacillus paracasei is a mesophilic lactic acid bacterium technologically active in food fermentation. Culture-independent methods have rapidly been recognized as a valuable alternative to culture-dependent methods for lactic acid bacteria enumeration. In the present work, the efficacy of different protocols to extract DNA from yoghurt were compared, real-time PCR (qPCR) targeting tuf gene for L. paracasei enumeration was evaluated, and qPCR and plate counts of L. paracasei in yoghurt samples were compared. Total DNA concentrations from commercial yoghurts were higher using DNAzol method 2 than using the other tested methods. Standard curves presented suitable mean efficiency values of 91 % (pure L. paracasei strain CTT 7501), 95 % (pure L. paracasei strain FNU), and 103 % (yoghurt with L. paracasei strain FNU). Limit of detection is 3 log DNA copy number, corresponding to 2.78 log CFU, a suitable range of CFU enumeration for probiotic bacteria in yoghurt samples, considering that they should be present in large amounts. The L. paracasei (CFU) enumerated by qPCR were compared to culturable L. paracasei enumerated by plate counts at 7, 14, 21, and 28 days of yoghurt manufacture. Differences between qPCR and plate counts were observed only 28 days after yoghurt preparation, counts were similar at 7, 14, and 21 days. In conclusion, this qPCR assay is a useful and rapid tool to enumerate L. paracasei in yoghurt, although it does not distinguish dead and viable cells.