Multiplex PCR and a chromogenic DNA macroarray for the detection of Listeria monocytogens, Staphylococcus aureus, Streptococcus agalactiae, Enterobacter sakazakii, Escherichia coli O157:H7, Vibrio parahaemolyticus, Salmonella spp. and Pseudomonas fluorescens in milk and meat samples

• Published in: Journal of microbiological methods 2012, Vol.88 (1), p.110-116
• Main Authors: Chiang, Yu-Cheng, Tsen, Hau-Yang, Chen, Hsin-Yen, Chang, Yu-Hsin, Lin, Chien-Ku, Chen, Chih-Yuan, Pai, Wan-Yu
• Format: Article
• Language: English
• Subjects: agarose; Animals; bacteria; Bacterial Typing Techniques - methods; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cattle; cheeses; Cronobacter sakazakii; diagnostic techniques; DNA; DNA primers; dried milk; Enterobacter sakazakii; Escherichia coli; Escherichia coli O157; fermented milk; Food Contamination - analysis; Fundamental and applied biological sciences. Psychology; gel electrophoresis; Gram-Negative Bacteria - classification; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - isolation & purification; Gram-Positive Bacteria - classification; Gram-Positive Bacteria - genetics; Gram-Positive Bacteria - isolation & purification; Listeria; Macroarray; meat; Meat Products - microbiology; Microbiology; milk; Milk - microbiology; molecular weight; Multiplex PCR; Multiplex Polymerase Chain Reaction - methods; Oligonucleotide Array Sequence Analysis - methods; Pathogenic bacteria; polymerase chain reaction; Pseudomonas fluorescens; Salmonella; sausages; Staphylococcus aureus; Streptococcus agalactiae; Swine; Vibrio parahaemolyticus
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2011.10.021

Food products, such as milk and meat products including cheese, milk powder, fermented milk, sausage, etc. are susceptible to the contamination by pathogenic and deteriorative bacteria. These bacteria include Listeria monocytogens, Staphylococcus aureus, Enterobacter sakazakii, Escherichia coli O157:H7, Salmonella spp., Vibrio parahaemolyticus, Streptococcus agalactiae and Pseudomonas fluorescens, etc. Traditional methods for the detection of these microorganisms are laborious and time consuming. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and PCR primers for the detection of aforementioned microorganisms. By using two sets of multiplex PCR, followed by a chromogenic macroarray system, these organisms in milk or other food products could be simultaneously detected. When the system was used for the inspection of milk or meat homogenate containing 10 0 target cells per milliliter or gram of the sample, all these bacterial species could be identified after an 8 h pre-enrichment step. The system consisting of a multiplex PCR step followed by macroarray allowed us to detect multiple target bacterial species simultaneously without the use of agarose gel electrophoresis. Compared to the commonly used multiplex PCR method, this approach has the additional advantage of detecting more bacterial strains because some bacterial strains generate PCR products with the same molecular sizes which can be differentiated by macroarray but not by electrophoresis. ► A DNA macroarray and mPCR were developed for the detection of major food pathogens. ► The system discriminates pathogens which generate PCR products with the same sizes. ► The whole system is economic and can be easily commercialized into a diagnostic kit.