The Detection of Foodborne Pathogenic Bacteria in Seafood Using a Multiplex Polymerase Chain Reaction System

Abstract

Multiplex polymerase chain reaction (PCR) assays are mainly used to simultaneously detect or identify multiple pathogenic microorganisms. To achieve high specificity for detecting foodborne pathogenic bacteria, specific primers need to be designed for the target strains. In this study, we designed and achieved a multiplex PCR system for detecting eight foodborne pathogenic bacteria using specific genes: toxS for Vibrio parahaemolyticus, virR for Listeria monocytogenes, recN for Cronobacter sakazakii, ipaH for Shigella flexneri, CarA for Pseudomonas putida, rfbE for Escherichia coli, vvhA for Vibrio vulnificus, and gyrB for Vibrio alginolyticus. The sensitivity of the single system in this study was found to be 20, 1.5, 15, 15, 13, 14, 17, and 1.8 pg for V. parahaemolyticus, L. monocytogenes, E. coli O157:H7, C. sakazakii, S. flexneri, P. putida, V. vulnificus, and V. alginolyticus, respectively. The minimum detection limit of the multiplex system reaches pg/μL detection level; in addition, the multiplex system exhibited good specificity and stability. Finally, the assays maintained good specificity and sensitivity of 104 CFU/mL for most of the samples and we used 176 samples of eight aquatic foods, which were artificially contaminated to simulate the detection of real samples. In conclusion, the multiplex PCR method is stable, specific, sensitive, and time-efficient. Moreover, the method is well suited for contamination detection in these eight aquatic foods and can rapidly detect pathogenic microorganisms.

A Comprehensive Review for the Surveillance of Human Pathogenic Microorganisms in Shellfish

“…For example, multiplex PCR assays can simultaneously target two or more bacteria in a single reaction mixture, increasing the efficiency of pathogen detection compared to conventional PCR by reducing the detection time and costs. Li et al [ 155 ], for example, recently designed a multiplex PCR system with good specificity and sensitivity for the detection of a total of eight food-borne bacterial pathogen species, including V. parahaemolyticus , Listeria monocytogenes , Cronobacter sakazakii , Shigella flexneri , Pseudomonas putida , E. coli , V. vulnificus and V. alginolyticus , targeting the toxS , virR , recN , ipaH, rfbE, vvhA and gyrB1 genes, respectively. Nevertheless, this multiplex PCR approach does not quantify targeted amplicons, whereas quantitative PCR (qPCR) is able to extrapolate quantification using the Ct values and calibration curves.…”

Electrochemical Detection of ompA Gene of C. sakazakii Based on Glucose-Oxidase-Mimicking Nanotags of Gold-Nanoparticles-Doped Copper Metal-organic Frameworks

“…The bacterial culture method for C. sakazakii is a traditional method that takes 5 to 7 days to obtain results and cannot meet the needs for rapid detection and screening of bacteria. Some other methods that are classified into protein-based biosensing method [5][6][7] and DNA-based biosensing method [8][9][10][11][12][13][14][15][16] have been promoted and applied in the detection of C. sakazakii. Protein-based immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) [5] and lateral flow immunoassays [6], are based on antibody-antigen interactions, which require a certain concentration of targets in samples, and sometimes cannot be applied in the detection at an early infection stage.…”

“…Protein-based immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) [5] and lateral flow immunoassays [6], are based on antibody-antigen interactions, which require a certain concentration of targets in samples, and sometimes cannot be applied in the detection at an early infection stage. DNA-based methods including polymerase chain reaction (PCR)-based biosensing methods [8,9], fluorescence biosensing Sensors 2023, 23, 4396 2 of 14 methods [10,11], colorimetric biosensing methods [12,13], and electrochemical biosensing methods [14][15][16] have been intensively promoted and investigated for the detection of C. sakazakii. Due to the properties of low cost, ease of fabrication, time-saving, and ease of operation, several kinds of electrochemical biosensors, including amperometric biosensors [14,16] and voltametric biosensors [15], have been fabricated for C. sakazakii detection.…”