A Culture-Independent Method for Enumeration of Viable Load of Lactobacillus acidophilus NCFM by using Real-Time PCR

Abstract

AbstractThe study was performed to develop a non-culture method to quantify viable loads of Lactobacillus acidophilus NCFM using RNA-based molecular technique. The ‘growth curve’ and ‘cycle threshold curve’ were developed respectively by plate counting and using cycle threshold (CT) values. ‘Standard curve’ was constructed using cells per milliliter and relative CT values. A maximum viable count (1.5 ± 0.15) × 1010 cells/mL with a minimum CT value 20.18 ± 0.56 was achieved following 18 h of growth. The two parameters were inversely proportional to each other over the exponential growth. The ‘standard curve’ corresponded to equation y = 2E + 28e−2.034x (y = cells/mL, x = CT value; R2 = 0.993), and no sample showed significant difference between ‘plate count’ and relative ‘PCR count’ following the validation process. Industrial adaptation of this method in dairy processing could potentially contribute to a faster enumeration of viable L. acidophilus NCFM compared to plate counting.