Research on the quantification method to detect pathogenic bacteria in instant rice noodles by 3-plex droplet digital polymerase chain reaction test

Abstract

ObjectiveThis study aimed to establish a quantitative 3-plex droplet digital polymerase chain reaction （ddPCR） method for simultaneously detecting the copy numbers of Salmonella， Bacillus cereus， and Listeria monocytogenes in instant food.MethodsThree pairs of primers and probes corresponding to three single-copy-genes were selected as target genes. The genes were the essC gene in Bacillus cereus， ttrA/ttrC gene in Salmonella， and invasion-associated endopeptidase gene in Listeria monocytogenes. The specificity of the primers and probes were verified by real-time fluorescence quantitative PCR separately. A 3-plex ddPCR method was constructed to detect the copy numbers of three pathogenic bacteria simultaneously.ResultsThe linear ranges were： 25-22 687 copies/20 µL for Salmonella， 19-15 620 copies/20 µL for Bacillus cereus， and 18-23 373 copies/20 µL for Listeria monocytogenes. The three linear correlation coefficients were r≥0.999. relative standard deviation （RSD）≤12% at six concentrations and repeated thrice， indicating favorable repeatability. The minimum detection limits were six copies/20 µL for Salmonella， three copies/20 µL for Bacillus cereus， and seven copies/20 µL for Listeria monocytogenes. When a simulated sample of contaminated rice noodles was detected by 3-plex ddPCR and the plate counting method， the deviation between these two methods was <9%， indicating a good consistency in the results.ConclusionThe 3-plex ddPCR method for the simultaneous and quantitative detection of Salmonella， Bacillus cereus， and Listeria monocytogenes in instant food was quicker， more sensitive， and more accurate than the plate-counting method.

Keywords

• foodborne pathogens
• salmonella
• bacillus cereus
• listeria monocytogenes
• 3-plex droplet digital pcr
• instant food