Abstract:
Objective To establish a multiplex quantitative polymerase chain reaction（qPCR）assay for the detections of six common foodborne pathogens.
Methods Specific primers and multiplex qPCR probes were designed to target six common foodborne pathogens: Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus, Norovirus, Escherichia coli O157:H7, and Staphylococcus aureus. The primers, probes, and reaction conditions were optimized for the establishment of the multiplex qPCR assay. The assay was evaluated through standard curve construction, limit of detection （LOD） analysis, specificity test, interference test and simulation sample testing to assess its applicability.
Results A multiplex quantitative PCR assay targeting six pathogens was established based on two reaction systems. Each system detects three pathogens, with LOD of 10 copies/µL for both systems. The correlation coefficient （R2） for both systems was greater than 0.99, and the P-value was less than 0.001. Amplification curves were observed for the six target pathogens, while no amplification was seen for non-target pathogens, indicating high specificity of the assay. Additionally, no interference or inhibition was observed when multiple pathogens at different concentrations were tested simultaneously. Testing with simulated samples showed that the multiplex qPCR assay could specifically detect all the six target pathogens.
Conclusion This study established a sensitive and specific multiplex qPCR assay, which can be used for the rapid detection of six common foodborne pathogens.
下载: