A new sterility test for cow's milk using alanine racemase gene as the index

Oligonucleotide primers designed from consensus sequences of alanine racemase genes were used for a sterility test of cow's milk by polymerase chain reaction (PCR). Commercial cow's milk in two 250 ml packages was separately centrifuged at 5000 x g for 10 min, bacterial cells in each precipitate were cultivated at 30 and 55 degrees C for 5 h in Luria-Bertani medium, and the cells from each culture were mixed and used for the PCR after being treated with 0.1 N NaOH at 60 degrees C for 10 min. When we performed the PCR using DNAs from various bacteria and eukaryotes as the templates, a unique PCR product of about 390 bp was amplified only from the bacteria. The sensitivity of the PCR method was such that an initial inoculum of 1 CFU of Bacillus stearothermophilus, Escherichia coli, and Pseudomonas fluorescens per 250 ml of cow's milk could be detected. When we analyzed 14 types of commercial cow's milk, all samples which were positive by the standard sterility test at 30 or 55 degrees C were also found positive by the PCR method.