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

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

Analysis of metabolites of red seabream (Pagrus major) from different geographical origins by capillary electrophoresis time-of-flight mass spectrometry.

Red seabream (Pagrus major), a migratory fish, is characterized by high protein levels in the muscle. South Korean and Japanese red seabreams have a general distribution pattern; however, distinguishing them based on their geographical origin is difficult. In this study, we used capillary electrophoresis time-of-flight mass spectrometry (CE-TOF/MS) to analyze the red seabream muscle metabolome to investigate how can distinguish the origin of the fish. The metabolites were extracted using 50% acetonitrile in water. Chromatographic separation was successfully used to classify the metabolite profiles of Japanese and South Korean red seabream. Principal component analysis and hierarchical cluster analysis showed good ability to categorize the samples according to their origin. Amino acids showed the greatest quantitative difference in South Korean and Japanese muscle samples. Specifically, the L-alanine, L-glutamic acid, L-isoleucine, dimethylglycine, and L-valine levels in Japanese red seabream samples were significantly higher than those in South Korean samples. In contrast, the levels of trimethylamine N-oxide and inosine monophosphate in South Korean muscle samples were significantly higher than those in Japanese red muscle samples. The monitored metabolite profiles suggest that South Korean and Japanese red seabreams can be identified on the basis of amino acid levels

Analyzing the Metabolomic Profile of Yellowtail (<i>Seriola quinquerdiata</i>) by Capillary Electrophoresis–Time of Flight Mass Spectrometry to Determine Geographical Origin

Country-of-origin violations have occurred in which some merchants have fraudulently sold cheap Japanese yellowtail (Seriola quinqueradiata) by presenting them as domestic Korean products. There are many methods for determining the origins of marine organisms, such as molecular genetic methods and isotope analysis. However, this study aimed to develop a method for determining the origins of aquatic products using metabolite analysis technology. Ten yellowtail each from Korea and Japan were analyzed by capillary electrophoresis–time of flight/mass spectrometry (CETOF/MS). Hierarchical cluster analysis (HCA) and principal component analysis (PCA) results showed highly differing aspects between the Korean and Japanese samples. In the tricarboxylic acid (TCA) cycle, citric, malic, oxaloglutaric, and fumaric acids exhibited significant differences between Korean and Japanese yellowtail. Sixteen of the twenty essential amino acids analyzed as metabolites also differed significantly. All amino acids were involved in protein digestion, absorption, and metabolism. All 16 amino acid contents were higher in Japanese yellowtail than in Korean yellowtail, except for glutamine. The fasting period was found to be the biggest factor contributing to the difference in amino acid contents, in addition to environmental factors (including feeding habits). These significant differences indicated that metabolomics could be used to determine geographical origin

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A formulated red ginseng extract inhibits autophagic flux and sensitizes to doxorubicin-induced cell death

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)–monomeric red fluorescent protein (mRFP)–LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP–mRFP–LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC. Keywords: autophagic flux, cell death, ginseng extrac