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Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized milk.
MedLine Citation:
PMID:  22720921     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
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 (20mL) 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 20mL 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 4mL 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.
Authors:
T Soejima; J Minami; K Iwatsuki
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of dairy science     Volume:  95     ISSN:  1525-3198     ISO Abbreviation:  J. Dairy Sci.     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-06-22     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2985126R     Medline TA:  J Dairy Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  3634-42     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Affiliation:
Morinaga Milk Industry Co. Ltd., 5-1-83, Higashihara, Zama, Kanagawa 252-8583, Japan.
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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