Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater

Dong-Geun Park; Joon-Gi Kwon; Eun-Su Ha; Byungcheol Kang; Iseul Choi; Jeong-Eun Kwak; Jinho Choi; Woojung Lee; Seung Hwan Kim; Soon Han Kim; Jeongwoong Park; Ju-Hoon Lee

doi:10.3389/fmicb.2023.1179934

Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater

Front Microbiol. 2023 Jul 13:14:1179934. doi: 10.3389/fmicb.2023.1179934. eCollection 2023.

Authors

Dong-Geun Park^{1

2

3

4}, Joon-Gi Kwon^{1

2

3

4}, Eun-Su Ha⁵, Byungcheol Kang⁵, Iseul Choi⁵, Jeong-Eun Kwak^{1

2

3

4}, Jinho Choi⁵, Woojung Lee⁶, Seung Hwan Kim⁶, Soon Han Kim⁶, Jeongwoong Park⁵, Ju-Hoon Lee^{1

2

3

4}

Affiliations

¹ Department of Food and Animal Biotechnology, Seoul National University, Seoul, Republic of Korea.
² Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
³ Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
⁴ Center for Food and Bioconvergence, Seoul National University, Seoul, Republic of Korea.
⁵ Research and Development Center, Sanigen Co., Ltd, Anyang, Republic of Korea.
⁶ Division of Food Microbiology, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju, Republic of Korea.

Abstract

Detecting and identifying the origins of foodborne pathogen outbreaks is a challenging. The Next-Generation Sequencing (NGS) panel method offers a potential solution by enabling efficient screening and identification of various bacteria in one reaction. In this study, new NGS panel primer sets that target 18 specific virulence factor genes from six target pathogens (Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) were developed and optimized. The primer sets were validated for specificity and selectivity through singleplex PCR, confirming the expected amplicon size. Crosscheck and multiplex PCR showed no interference in the primer set or pathogenic DNA mixture. The NGS panel analysis of spiked water samples detected all 18 target genes in a single reaction, with pathogen concentrations ranging from 10⁸ to 10⁵ colony-forming units (CFUs) per target pathogen. Notably, the total sequence read counts from the virulence factor genes showed a positive association with the CFUs per target pathogen. However, the method exhibited relatively low sensitivity and occasional false positive results at low pathogen concentrations of 10⁵ CFUs. To validate the detection and identification results, two sets of quantitative real-time PCR (qPCR) analyses were independently performed on the same spiked water samples, yielding almost the same efficiency and specificity compared to the NGS panel analysis. Comparative statistical analysis and Spearman correlation analysis further supported the similarity of the results by showing a negative association between the NGS panel sequence read counts and qPCR cycle threshold (Ct) values. To enhance NGS panel analysis for better detection, optimization of primer sets and real-time NGS sequencing technology are essential. Nonetheless, this study provides valuable insights into applying NGS panel analysis for multiple foodborne pathogen detection, emphasizing its potential in ensuring food safety.

Keywords: NGS panel; foodborne pathogen; multiple detection; next-generation sequencing; real-time PCR.