title: A Dual Filtration-Based Multiplex PCR Method for Simultaneous Detection of Viable Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on Fresh-Cut Cantaloupe.
abstract: 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 × 103 cfu/g for L. monocytogenes, 4.3 × 10 cfu/g for E. coli O157:H7, and 3.1 × 102 cfu/g for S. aureus. Fresh-cut cantaloupe was inoculated with the three target pathogens using concentrations of 103, 102, 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.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China.|College of Life Science, Dalian Nationalities University, Dalian, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Feng
authors.affiliation: College of Life Science, Dalian Nationalities University, Dalian, China.
authors.forename: Wenzhong
authors.initials: W
authors.lastname: Hu
authors.affiliation: College of Life Science, Dalian Nationalities University, Dalian, China.
authors.forename: Aili
authors.initials: A
authors.lastname: Jiang
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China.|College of Life Science, Dalian Nationalities University, Dalian, China.
authors.lastname: Sarengaowa
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Xu
authors.affiliation: College of Life Science, Dalian Nationalities University, Dalian, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zou
authors.affiliation: College of Life Science, Dalian Nationalities University, Dalian, China.
authors.forename: Liu
authors.initials: L
authors.lastname: Yang
authors.affiliation: College of Life Science, Dalian Nationalities University, Dalian, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
mesh_terms: D001386:Azides / Q000737:chemistry; D015373:Bacterial Typing Techniques; D028463:Cucumis melo / Q000382:microbiology; D004269:DNA, Bacterial / Q000235:genetics*; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005374:Filtration / Q000295:instrumentation / Q000379:methods; D005506:Food Contamination; D005516:Food Microbiology; D006801:Humans; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011419:Propidium / Q000031:analogs & derivatives / Q000737:chemistry; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001386:Azides; D004269:DNA, Bacterial; C533957:propidium monoazide; D011419:Propidium