Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development and Evaluation of a Next-Generation Sequencing Panel for the Multiple Detection and Identification of Pathogens in Fermented Foods

  • Dong-Geun Park (Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Seoul National University) ;
  • Eun-Su Ha (Research and Development Center, Sanigen Co., Ltd) ;
  • Byungcheol Kang (Research and Development Center, Sanigen Co., Ltd) ;
  • Iseul Choi (Research and Development Center, Sanigen Co., Ltd) ;
  • Jeong-Eun Kwak (Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Seoul National University) ;
  • Jinho Choi (Research and Development Center, Sanigen Co., Ltd) ;
  • Jeongwoong Park (Research and Development Center, Sanigen Co., Ltd) ;
  • Woojung Lee (Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Seung Hwan Kim (Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Soon Han Kim (Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Ju-Hoon Lee (Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, Center for Food and Bioconvergence, Seoul National University)
  • Received : 2022.11.07
  • Accepted : 2022.11.10
  • Published : 2023.01.28
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Abstract

These days, bacterial detection methods have some limitations in sensitivity, specificity, and multiple detection. To overcome these, novel detection and identification method is necessary to be developed. Recently, NGS panel method has been suggested to screen, detect, and even identify specific foodborne pathogens in one reaction. In this study, new NGS panel primer sets were developed to target 13 specific virulence factor genes from five types of pathogenic Escherichia coli, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium, respectively. Evaluation of the primer sets using singleplex PCR, crosscheck PCR and multiplex PCR revealed high specificity and selectivity without interference of primers or genomic DNAs. Subsequent NGS panel analysis with six artificially contaminated food samples using those primer sets showed that all target genes were multi-detected in one reaction at 108-105 CFU of target strains. However, a few false-positive results were shown at 106-105 CFU. To validate this NGS panel analysis, three sets of qPCR analyses were independently performed with the same contaminated food samples, showing the similar specificity and selectivity for detection and identification. While this NGS panel still has some issues for detection and identification of specific foodborne pathogens, it has much more advantages, especially multiple detection and identification in one reaction, and it could be improved by further optimized NGS panel primer sets and even by application of a new real-time NGS sequencing technology. Therefore, this study suggests the efficiency and usability of NGS panel for rapid determination of origin strain in various foodborne outbreaks in one reaction.

Keywords

Acknowledgement

This work was supported by grants (20161MFDS030 and 21162MFDS027) from the Ministry of Food and Drug Safety, South Korea.

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