Open AccessA Dual Filtration-Based Multiplex PCR Method for Simultaneous Detection of Viable Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on Fresh-Cut Cantaloupe
Author(s) -
Kai Feng,
Wenzhong Hu,
Aili Jiang,
- Sarengaowa,
You-Hai Xu,
Yi Zou,
Liu Yang,
Xin Wang
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0166874
Subject(s) - listeria monocytogenes , propidium monoazide , staphylococcus aureus , microbiology and biotechnology , escherichia coli , biology , multiplex , multiplex polymerase chain reaction , filtration (mathematics) , bacteria , food science , polymerase chain reaction , bioinformatics , biochemistry , statistics , genetics , mathematics , gene
Fresh-cut cantaloupe is particularly susceptible to contamination with pathogenic bacteria, such as Escherichia coli O157:H7, Listeria monocytogenes , and Staphylococcus aureus . Therefore, development of rapid, yet accurate detection techniques is necessary to ensure food safety. In this study, a multiplex PCR system and propidium monoazide (PMA) concentration were optimized to detect all viable pathogens in a single tube. A dual filtration system utilized a filtration membrane with different pore sizes to enrich pathogens found on fresh-cut cantaloupe. The results revealed that an optimized multiplex PCR system has the ability to effectively detect three pathogens in the same tube. The viable pathogens were simultaneously detected for PMA concentrations above 10 μg/ml. The combination of a nylon membrane (15 μm) and a micro pore filtration membrane (0.22 μm) formed the dual filtration system used to enrich pathogens. The achieved sensitivity of PMA-mPCR based on this dual filtration system was 2.6 × 10 3 cfu/g for L . monocytogenes , 4.3 × 10 cfu/g for E . coli O157:H7, and 3.1 × 10 2 cfu/g for S . aureus . Fresh-cut cantaloupe was inoculated with the three target pathogens using concentrations of 10 3 , 10 2 , 10, and 1 cfu/g. After 6-h of enrichment culture, assay sensitivity increased to 1 cfu/g for each of these pathogens. Thus, this technique represents an efficient and rapid detection tool for implementation on fresh-cut cantaloupe.