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Detection of spore forming Paenibacillus macerans in raw milk
Atinuke M Olajide1, Gisèle LaPointe1
1CRIFS, Department of Food Science, University of Guelph, Ontario N1G 2W1, Canada.
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.
Insights
A new real-time PCR assay (PMassay) effectively detects Paenibacillus macerans in raw milk, even with background microbial interference. This method offers sensitive and specific detection crucial for monitoring milk quality.
Area of Science:
- Food Microbiology
- Molecular Biology
- Dairy Science
Background:
- Paenibacillus macerans contributes to milk spoilage during prolonged storage.
- The native milk microbiota complicates accurate enumeration of Paenibacillus species in raw milk.
Purpose of the Study:
- To develop and validate a SYBR Green real-time PCR assay for specific detection of Paenibacillus macerans (PMassay) in raw milk.
- To compare the specificity and sensitivity of the PMassay against a total Paenibacillus detection assay (TPassay).
Main Methods:
- Development of a qualitative SYBR Green real-time PCR assay targeting the groEL gene for P. macerans detection.
- Testing assay specificity against dairy-related spore-forming isolates and evaluating performance with varying levels of background DNA.
- Enrichment of raw milk samples using Reinforced Clostridial Medium with D-cycloserine (RCM-D) under anaerobic conditions followed by PCR analysis.
Main Results:
- The PMassay demonstrated high specificity, with non-P. macerans isolates yielding a Ct > 30.
- The assay maintained sensitivity (detection limit of 2 log CFU/ml) even with up to 95% background DNA substitution.
- Paenibacillus was detected in 10 out of 16 raw milk samples after 7 days of enrichment, with P. macerans quantified between 0.3 and 4.6 log CFU/ml.
Conclusions:
- The developed PMassay provides a sensitive and specific tool for detecting Paenibacillus macerans in raw milk.
- The assay is robust against background microbial DNA, making it suitable for commercial applications in dairy quality control.
