Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement
The rapid and accurate detection of foodborne pathogens is essential for ensuring food safety. <i>Escherichia coli</i> O157:H7 (<i>E. coli</i> O157:H7) and <i>Salmonella Typhimurium</i> (<i>S. Typhimurium</i>) are major foodborne pathogenic bacteria th...
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MDPI AG
2025-05-01
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| Series: | Biosensors |
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| author | Yijia Zhang Junkang Pan Qiyi He Zhihao Xu Bruce D. Hammock Dongyang Li |
| author_facet | Yijia Zhang Junkang Pan Qiyi He Zhihao Xu Bruce D. Hammock Dongyang Li |
| author_sort | Yijia Zhang |
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| description | The rapid and accurate detection of foodborne pathogens is essential for ensuring food safety. <i>Escherichia coli</i> O157:H7 (<i>E. coli</i> O157:H7) and <i>Salmonella Typhimurium</i> (<i>S. Typhimurium</i>) are major foodborne pathogenic bacteria that pose significant public health risks, highlighting the need for effective detection methods. In this study, highly sensitive double-antibody sandwich-based enzyme-linked immunosorbent assays (ELISAs) were developed for the rapid detection of <i>E. coli</i> O157:H7 and <i>S. Typhimurium</i>, utilizing a streptavidin-polymerized horseradish peroxidase (SA-PolyHRP)-based signal enhancement system. Systematic optimization was performed on key parameters, including the capture antibody concentration, detection antibody, and blocking agent. Compared to the method using SA-HRP, substitution with SA-PolyHRP significantly improved detection sensitivity, achieving limits of detection (LODs) of 1.4 × 10<sup>4</sup> CFU/mL for <i>E. coli</i> O157:H7 and 6.0 × 10<sup>3</sup> CFU/mL for <i>S. Typhimurium</i>, with sensitivity enhancements of 7.86-fold and 1.83-fold, respectively. Specificity tests confirmed no cross-reactivity with non-target or closely related pathogenic strains. The matrix effect was effectively mitigated through 10-fold and 100-fold dilutions for <i>E. coli</i> O157:H7 and <i>S. Typhimurium</i>, respectively. Both pathogens were successfully detected in beef samples spiked with 5 CFU after 5 h of incubation. This study demonstrates the effectiveness of PolyHRP-based signal enhancement for the highly sensitive and specific detection of foodborne pathogens, offering a promising approach for rapid food safety monitoring and public health protection. |
| format | Article |
| id | doaj-art-68a9f8f7e7f1454792acb8b0db08e76c |
| institution | OA Journals |
| issn | 2079-6374 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj-art-68a9f8f7e7f1454792acb8b0db08e76c2025-08-20T02:33:42ZengMDPI AGBiosensors2079-63742025-05-0115531810.3390/bios15050318Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal EnhancementYijia Zhang0Junkang Pan1Qiyi He2Zhihao Xu3Bruce D. Hammock4Dongyang Li5College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaCollege of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaCollege of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaCollege of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaDepartment of Entomology and Nematology and UCD Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, USACollege of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaThe rapid and accurate detection of foodborne pathogens is essential for ensuring food safety. <i>Escherichia coli</i> O157:H7 (<i>E. coli</i> O157:H7) and <i>Salmonella Typhimurium</i> (<i>S. Typhimurium</i>) are major foodborne pathogenic bacteria that pose significant public health risks, highlighting the need for effective detection methods. In this study, highly sensitive double-antibody sandwich-based enzyme-linked immunosorbent assays (ELISAs) were developed for the rapid detection of <i>E. coli</i> O157:H7 and <i>S. Typhimurium</i>, utilizing a streptavidin-polymerized horseradish peroxidase (SA-PolyHRP)-based signal enhancement system. Systematic optimization was performed on key parameters, including the capture antibody concentration, detection antibody, and blocking agent. Compared to the method using SA-HRP, substitution with SA-PolyHRP significantly improved detection sensitivity, achieving limits of detection (LODs) of 1.4 × 10<sup>4</sup> CFU/mL for <i>E. coli</i> O157:H7 and 6.0 × 10<sup>3</sup> CFU/mL for <i>S. Typhimurium</i>, with sensitivity enhancements of 7.86-fold and 1.83-fold, respectively. Specificity tests confirmed no cross-reactivity with non-target or closely related pathogenic strains. The matrix effect was effectively mitigated through 10-fold and 100-fold dilutions for <i>E. coli</i> O157:H7 and <i>S. Typhimurium</i>, respectively. Both pathogens were successfully detected in beef samples spiked with 5 CFU after 5 h of incubation. This study demonstrates the effectiveness of PolyHRP-based signal enhancement for the highly sensitive and specific detection of foodborne pathogens, offering a promising approach for rapid food safety monitoring and public health protection.https://www.mdpi.com/2079-6374/15/5/318PolyHRPfoodborne pathogenic bacteriaimmunoassaysignal enhancement |
| spellingShingle | Yijia Zhang Junkang Pan Qiyi He Zhihao Xu Bruce D. Hammock Dongyang Li Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement Biosensors PolyHRP foodborne pathogenic bacteria immunoassay signal enhancement |
| title | Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement |
| title_full | Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement |
| title_fullStr | Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement |
| title_full_unstemmed | Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement |
| title_short | Development of Immunoassays for Foodborne Pathogenic Bacteria Detection Using PolyHRP for Signal Enhancement |
| title_sort | development of immunoassays for foodborne pathogenic bacteria detection using polyhrp for signal enhancement |
| topic | PolyHRP foodborne pathogenic bacteria immunoassay signal enhancement |
| url | https://www.mdpi.com/2079-6374/15/5/318 |
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