Development of a real-time PCR assay for direct detection and quantification of Bacillus sporothermodurans in ultra-high temperature milk.

Abstract

In our study, a new and highly sensitive real-time PCR Taqman assay was developed for the direct and specific detection of Bacillus sporothermodurans in UHT milk. The target region was selected based on the 16S rRNA gene profiles of 11 B. sporothermodurans from GenBank. A standard curve was created using a reference strain of B. sporothermodurans, DSM 10599. A low limit of detection for B. sporothermodurans in UHT milk (10 cfu/mL) was obtained. Furthermore, a total of 110 UHT milk samples from several supermarkets were directly assessed to detect and quantify B. sporothermodurans using the real-time PCR Taqman developed. The B. sporothermodurans counts obtained were highly correlated with the microbial plate counts in the UHT milk samples. This is the first time that B. sporothermodurans has been quantified directly from UHT milk. This technique could be applicable as a routine tool for preventing the growth of these bacteria by allowing for the rapid screening of raw milk samples in dairy plants. As expected, the probability of bacterial growth in UHT milk packages increased with the B. sporothermodurans counts in the raw milk.