Development of a culture-independent polymerase chain reaction-based assay for the detection of lactobacilli in stretched cheese

ABSTRACT The purpose of this research was to develop a culture‐independent assay based on polymerase chain reaction (PCR) for the specific detection of lactobacilli present in very low amounts in natural microflora during the processing of traditional pasta filata cheese. The utility of casein micelle solubilization with Triton X‐100 was also evaluated before proceeding to DNA extraction and PCR detection. By using primers related to cell envelope proteinase and gassericin B genes, proteolytic lactobacilli were successfully detected in samples from which they had not previously been isolated by plate counting. The sensitivity of the method is estimated to be 1000 cfu/mL. The speed of the DNA extraction protocol and the sensitivity and reliability of the PCR assay even make it feasible to detect nondominant populations of Lactobacillus species in cheese.