Detection of spore forming Paenibacillus macerans in raw milk

Abstract

Paenibacillus macerans can cause spoilage of milk during extended storage. However, the natural milk microbiota interferes with the enumeration of Paenibacillus species in raw milk. In this study, a qualitative SYBR Green real-time PCR assay based on the groEL gene was developed for detecting P. macerans (PMassay) in raw milk and compared with one designed for total Paenibacillus detection (TPassay). The specificity of the PMassay was confirmed against a panel of dairy-related spore forming isolates. In the presence of background DNA substituted up to 95%, P. macerans DNA could still be detected by the PMassay although interference occurred as non-target DNA substitution increased. The PMassay was sensitive (detection limit of 2 log CFU/ml in milk) and specific as non-P. macerans isolates gave a Ct > 30. After enrichment of raw milk for 7 days at 37 °C in Reinforced Clostridial Medium with D-cycloserine (RCM-D) under anaerobiosis, Paenibacillus was detected in 10 of the 16 raw milk samples tested. Enrichment in RCM-D yielded about 0.5 to 5.8 log CFU/ml total Paenibacillus and 0.3 to 4.6 log CFU/ml P. macerans in the samples. The assay could be useful in commercial settings, allowing a sensitive detection of P. macerans.