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

• DOI: 10.3389/fmicb.2023.1179934
• Publication Date: 2023-07-14T01:32:10Z
• Authors: 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
• Keywords: Primer (cosmetics); Amplicon; Yersinia enterocolitica

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