Detection technology for food pathogenic bacteria based on multiplex real-time fluorescence quantitative PCR
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Zhichun Cao
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Zhonghou Cao
Abstract
Aiming at the low detection efficiency and long-time consumption of pathogenic bacteria in bulk ready-to-eat foods, this paper designs a multiplex real-time fluorescence quantitative PCR detection method. This study obtained specific gene sequences of Salmonella, Staphylococcus aureus, and Flavobacterium from a database. This study inoculated the target bacteria into a culture medium for cultivation. DNA extraction was compared between the traditional hot boiling method and commercial kits, and the latter was selected to extract the template. Based on single PCR screening without cross-primers and probes, different fluorescence channels were assigned, and the proportion of key components was adjusted to establish a multiplex PCR method. After enriching the food samples in culture, DNA was extracted for detection. The results show that under different dilution degrees, the intra-batch and inter-batch coefficient of variation of the three target species were all less than 2 %. Compared with the traditional GB4789 national standard method, the multiple real-time fluorescence polymerase chain reaction technology reduced the detection time of three target species in ready-to-eat food samples. For example, by lowering the detection time for Salmonella in braised pork meals from 20.5 h using the traditional method to 6.6 h utilizing the PCR approach, 13.9 h were saved. Similarly, the PCR method allowed for faster identification of Bacillus cereus in rice products and S. aureus in soy products. These results demonstrate that the proposed approach significantly enhances the detection speed of harmful bacteria and improves food safety.
Funding source: Wuhan Municipal Health Commission Research Fund
Award Identifier / Grant number: WG20D11
Acknowledgement
This work was made possible by the generous support from Center for Disease Control and Prevention of Changjiang River Administration and Navigational Affairs, Ministry of Transport, General Hospital of the Yangtze River Shipping. I am particularly thankful to Junfang Wan for her invaluable guidance on my research methodology and her meticulous review of my thesis drafts, which significantly improved the quality of my work.
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Research ethics: Not applicable.
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Informed consent: Informed consent was obtained from all individuals included in this study, or their legal guardians or wards.
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Author contributions: Zhichun Cao, Junfang Wan, is responsible for designing the framework, analyzing the performance, validating the results, and writing the article. Zhonghou Cao, is responsible for collecting the information required for the framework, provision of software, critical review, and administering the process.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors declared no potential conflicts of interest with respect to the research, author-ship, and/or publication of this article.
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Research funding: The project was supported by Wuhan Municipal Health Commission Research Fund (WG20D11).
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Data availability: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
1. Abass, T, Eruaga, MA, Itua, EO, Bature, JT. Advancing food safety through iot: real-time monitoring and control systems. Int Med Sci Res J 2024;4:276–83. https://doi.org/10.51594/imsrj.v4i3.919.Suche in Google Scholar
2. Wu, S, Xia, J, Li, R, Cao, H, Ye, D. Perspectives for the role of single-atom nanozymes in assisting food safety inspection and food nutrition evaluation. Anal Chem 2024;96:1813–24. https://doi.org/10.1021/acs.analchem.3c04339.Suche in Google Scholar PubMed
3. Shymanovich, T, Saville, AC, Mohammad, N, Wei, Q, Rasmussen, D, Lahre, KA, et al.. Disease progress and detection of a California resistance-breaking strain of tomato spotted wilt virus in tomato with LAMP and CRISPR-Cas12a assays. PhytoFrontiers™ 2024;4:50–60. https://doi.org/10.1094/phytofr-05-23-0058-fi.Suche in Google Scholar
4. Pagani, A, Enea, M. Displacement and strain data-driven damage detection in multi-component and heterogeneous composite structures. Mech Adv Mater Struct 2024;31:2053–68. https://doi.org/10.1080/15376494.2022.2149907.Suche in Google Scholar
5. Wang, Y, Liu, X, Chen, C, Chen, Y, Li, Y, Ye, H, et al.. Magnetic nanorobots as maneuverable immunoassay probes for automated and efficient enzyme linked immunosorbent assay. ACS Nano 2022;16:180–91. https://doi.org/10.1021/acsnano.1c05267.Suche in Google Scholar PubMed
6. Yue, Y, Chen, J, Zhang, M, Yin, Y, Dong, Y. Determination of organophosphorus pesticides in vegetables and fruit by an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a lateral-flow immunochromatographic (LFIC) strip assay. Anal Lett 2022;55:1701–18. https://doi.org/10.1080/00032719.2021.2023170.Suche in Google Scholar
7. Wang, Y, Bu, T, Cao, Y, Wu, H, Xi, J, Feng, Q, et al.. A versatile drug bimetallic nano enzyme-integrated enzyme-linked immunosorbent assay for highly sensitive Escherichia coli O157: H7 detection. Anal Chem 2023;95:9237–43. https://doi.org/10.1021/acs.analchem.3c00743.Suche in Google Scholar PubMed
8. Charernchai, S, Chikae, M, Phan, TT, Wonsawat, W, Hirose, D, Takamura, Y. Automated paper-based femtogram sensing device for competitive enzyme-linked immunosorbent assay of aflatoxin B1 using sub microliter samples. Anal Chem 2022;94:5099–105. https://doi.org/10.1021/acs.analchem.1c05401.Suche in Google Scholar PubMed PubMed Central
9. Gao, S, Abd El-Aty, AM, Xu, L, Zhao, J, Li, J, Lei, X, et al.. Rapid detection of prometryn residues in agricultural samples: method comparison of the Ic-ELISA and colloidal gold immunochromatographic test strips. ACS Agric Sci Technol 2024;4:345–55. https://doi.org/10.1021/acsagscitech.3c00490.Suche in Google Scholar
10. Zhang, CQ, Wan, Y, Shi, ZW, Luo, JC, Li, HY, Li, SS, et al.. Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection. Appl Microbiol Biotechnol 2023;107:4903–15. https://doi.org/10.1007/s00253-023-12604-2.Suche in Google Scholar PubMed
11. Karimi Kakh, M, Golchin, M, Kazemi Arababadi, M, Daneshvar, H. Application of the Leishmania infantum 21-kDa recombinant protein for the development of an immunochromatographic test. Parasite Immunol 2020;42:6–9. https://doi.org/10.1111/pim.12770.Suche in Google Scholar PubMed
12. Tsugami, Y, Nagasawa, Y, Sugiyama, A, Ando, Y, Oda, K, Maehana, K, et al.. Performance evaluation of a rapid immunochromatographic test kit in detecting bovine mastitis-causing streptococci. J Vet Med Sci 2024;86:474–9. https://doi.org/10.1292/jvms.23-0438.Suche in Google Scholar PubMed PubMed Central
13. James, AE, Gulley, T, Kothari, A, Holder, K, Garner, K, Patil, N. Performance of the BinaxNOW coronavirus disease 2019 (COVID-19) antigen card test relative to the severe acute respiratory coronavirus virus 2 (SARS-CoV-2) real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assay among symptomatic and asymptomatic healthcare employees. Infect Control Hosp Epidemiol 2022;43:99–101. https://doi.org/10.1017/ice.2021.20.Suche in Google Scholar PubMed PubMed Central
14. Blumenfeld, NR, Bolene, MAE, Jaspan, M, Ayers, AG, Zarrandikoetxe, S, Freudman, J, et al.. Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis. Nat Nanotechnol 2022;17:984–92. https://doi.org/10.1038/s41565-022-01175-4.Suche in Google Scholar PubMed
15. Wright, WF, Simner, PJ, Carroll, KC, Auwaerter, PG. Progress report: next-generation sequencing, multiplex polymerase chain reaction, and broad-range molecular assays as diagnostic tools for fever of unknown origin investigations in adults. Clin Infect Dis 2022;74:924–32. https://doi.org/10.1093/cid/ciab155.Suche in Google Scholar PubMed
16. Saad, A, Hussein, AE, Tarabees, R. Real-time PCR versus conventional methods for detection of viable but NonCulturable E. coli O157 isolated from food of animal origin. J Current Vet Res 2023;5:106–20. https://doi.org/10.21608/jcvr.2023.320433.Suche in Google Scholar
17. Britt, NS, Khader, K, He, T, Willson, TM, Effiong, A, Timbrook, TT, et al.. Examining the clinical impact of rapid multiplex polymerase chain reaction-based diagnostic testing for bloodstream infections in a national cohort of the veterans health administration. Pharmacotherapy 2023;43:24–34. https://doi.org/10.1002/phar.2747.Suche in Google Scholar PubMed
18. Sharma, K, Gupta, A, Sharma, M, Singh, S, Sharma, A, Singh, R, et al.. Detection of viable Mycobacterium tuberculosis in ocular fluids using mRNA-based multiplex polymerase chain reaction. Indian J Med Microbiol 2022;40:254–7. https://doi.org/10.1016/j.ijmmb.2021.12.019.Suche in Google Scholar PubMed
19. Vishwakarma, SK, Patil, A, Pandey, A, Arya, M. Biosorption of heavy metal (Mn2+) by thermophilic bacterial strains isolated from Surya Kund hot spring, Yamunotri, Uttarakhand. Appl Biochem Biotechnol 2024;196:2518–33. https://doi.org/10.1007/s12010-023-04682-9.Suche in Google Scholar PubMed
20. Kumbhari, A, Cheng, TNH, Ananthakrishnan, AN, Kochar, B, Burke, KE, Shannon, K, et al.. Discovery of disease-adapted bacterial lineages in inflammatory bowel diseases. Cell Host Microbe 2024;32:1147–62. https://doi.org/10.1016/j.chom.2024.05.022.Suche in Google Scholar PubMed PubMed Central
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Artikel in diesem Heft
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- Advance in brewing process, flavour detection technologies and functional components of fruit wine: a review
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- Detection technology for food pathogenic bacteria based on multiplex real-time fluorescence quantitative PCR
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Artikel in diesem Heft
- Frontmatter
- Critical Review
- GABA as a functional ingredient: current research on biosynthesis, analytical methods, and health benefits
- Review
- Advance in brewing process, flavour detection technologies and functional components of fruit wine: a review
- Articles
- Detection technology for food pathogenic bacteria based on multiplex real-time fluorescence quantitative PCR
- Molecular dynamics simulation of the mass transfer process for the drying of sugary fruits and vegetables
- The characterization of size-controlled nanocrystalline cellulose from soy hulls with ultrasonic assisted extraction
- Comparative study of continuous, intermittent, and interval starting-accessibility drying; case of green onion (Allium cepa L.) leaves
