Park Dong-Geun, Kwon Joon-Gi, Ha Eun-Su, Kang Byungcheol, Choi Iseul, Kwak Jeong-Eun, Choi Jinho, Lee Woojung, Kim Seung Hwan, Kim Soon Han, Park Jeongwoong, Lee Ju-Hoon
Department of Food and Animal Biotechnology, Seoul National University, Seoul, Republic of Korea.
Department of Agricultural Biotechnology, Seoul National University, Seoul, Republic of Korea.
Front Microbiol. 2023 Jul 13;14:1179934. doi: 10.3389/fmicb.2023.1179934. eCollection 2023.
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 (, , , , , and ) 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 10 to 10 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 10 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.
检测和确定食源性病原体爆发的源头具有挑战性。新一代测序（NGS）面板方法通过在一次反应中实现对各种细菌的高效筛选和鉴定，提供了一种潜在的解决方案。在本研究中，开发并优化了针对六种目标病原体（、、、、、和）的18个特定毒力因子基因的新型NGS面板引物组。通过单重PCR对引物组的特异性和选择性进行了验证，确认了预期的扩增子大小。交叉检查和多重PCR表明引物组或致病DNA混合物中没有干扰。对加标水样的NGS面板分析在一次反应中检测到了所有18个目标基因，每个目标病原体的病原体浓度范围为10至10菌落形成单位（CFU）。值得注意的是，来自毒力因子基因的总序列读数与每个目标病原体的CFU呈正相关。然而，该方法在10 CFU的低病原体浓度下表现出相对较低的灵敏度和偶尔的假阳性结果。为了验证检测和鉴定结果，对相同的加标水样独立进行了两组定量实时PCR（qPCR）分析，与NGS面板分析相比，产生了几乎相同的效率和特异性。比较统计分析和Spearman相关性分析通过显示NGS面板序列读数与qPCR循环阈值（Ct）值之间的负相关，进一步支持了结果的相似性。为了增强NGS面板分析以实现更好的检测，优化引物组和实时NGS测序技术至关重要。尽管如此，本研究为将NGS面板分析应用于多种食源性病原体检测提供了有价值的见解，强调了其在确保食品安全方面的潜力。
