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Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized
T Soejima1, J Minami, K Iwatsuki
1Morinaga Milk Industry Co. Ltd., 5-1-83, Higashihara, Zama, Kanagawa 252-8583, Japan. t_soezim@morinagamilk.co.jp
Abstract:
Pasteurized milk is a complex food and contains numerous PCR inhibitors and can often contain high levels of dead Enterobacteriaceae cells, depending on the condition of food sanitation. Usually, propidium monoazide (PMA) or ethidium monoazide PCR techniques decrease the number of dead bacteria by up to 3.5 log to the associated dead bacteria with no treatment. However, this difference could be insufficient to completely inhibit DNA amplification in the PCR from 10(6) cells of dead Enterobacteriaceae bacteria/mL, potentially contaminated in pasteurized milk. Actually, such potentially high levels of dead Enterobacteriaceae cells in milk has prevented milk researchers from applying PMA- or ethidium monoazide PCR to the assay of viable Enterobacteriaceae cells in milk. We, therefore, developed a rapid PMA real-time PCR whose minimum levels of detection were 1.5 log cfu/PCR for Cronobacter muytjensii and Escherichia coli, and 2.5 log cfu/PCR for Salmonella enteritidis without DNA purification in milk matrices. The PMA real-time PCR allowed us to specifically detect viable Enterobacteriaceae cells (5-10 cfu/mL) in pasteurized milk (20 mL) within 7.5h of total testing time, following the hygienic guidelines for pasteurized milk in the United States and European Union. The long DNA amplification (mainly 2,451 bp) of the 16S-23S rRNA gene was completely suppressed in highly contaminated dead Enterobacteriaceae cells (7.5 log cfu of Cronobacter muytjensii) in 20 mL of pasteurized milk by 23-μM PMA treatment. Although the contamination of the PCR reaction with 5% milk usually causes great inhibition, our method led to the successful elongation of PCR from viable Enterobacteriaceae cells still in the pasteurized milk matrices finally corresponding to 2 to 4 mL of milk PCR inhibitors without a DNA purification step. To comply with current customer demands for chilled pasteurized milk at the most excellent possible quality, our new technique could enable laboratory persons in a factory to conduct rapid milk coliform testing before shipping from a factory.
Insights
This study developed a rapid propidium monoazide (PMA) real-time PCR method to accurately detect viable Enterobacteriaceae in pasteurized milk. The new technique overcomes PCR inhibition from dead bacteria, enabling faster quality control for safe milk.
Area of Science:
- Food Microbiology
- Molecular Biology
- Analytical Chemistry
Background:
- Pasteurized milk can contain PCR inhibitors and high levels of dead Enterobacteriaceae, hindering accurate viability testing.
- Existing propidium monoazide (PMA) or ethidium monoazide PCR methods are insufficient to overcome inhibition from high concentrations of dead bacteria in milk.
- This limitation has prevented the reliable application of these PCR techniques for assessing viable Enterobacteriaceae in milk matrices.
Purpose of the Study:
- To develop a rapid PMA real-time PCR method for specific detection of viable Enterobacteriaceae in pasteurized milk.
- To overcome the challenges posed by PCR inhibitors and dead bacterial cells present in milk.
- To enable faster and more accurate quality control testing of pasteurized milk before shipment.
Main Methods:
- Development of a rapid PMA real-time PCR assay without a DNA purification step.
- Optimization of PMA treatment (23-μM) to suppress DNA amplification from dead Enterobacteriaceae cells.
- Testing the method's detection limits for Cronobacter muytjensii, Escherichia coli, and Salmonella enteritidis in milk.
Main Results:
- The developed PMA real-time PCR achieved minimum detection levels of 1.5 log cfu/PCR for C. muytjensii and E. coli, and 2.5 log cfu/PCR for S. enteritidis.
- Specific detection of viable Enterobacteriaceae (5-10 cfu/mL) in 20 mL of pasteurized milk within 7.5 hours.
- Complete suppression of DNA amplification from high levels of dead C. muytjensii (7.5 log cfu) in 20 mL of milk.
- Successful PCR amplification from viable Enterobacteriaceae in the presence of milk inhibitors, equivalent to 2-4 mL of milk, without DNA purification.
Conclusions:
- The rapid PMA real-time PCR method effectively detects viable Enterobacteriaceae in pasteurized milk, even with high levels of dead cells and PCR inhibitors.
- This technique adheres to US and EU hygienic guidelines for pasteurized milk quality control.
- The method offers a rapid, reliable solution for factory laboratories to perform coliform testing, ensuring high-quality chilled pasteurized milk.
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