Rapid Detection of HDC Gene in Enterobacter aerogenes from Fish Products Using In Silico PCR for Food Safety and Allergy Risk Assessment
Deteksi Cepat Gen HDC pada Enterobacter aerogenes dari Produk Perikanan Menggunakan In Silico PCR untuk Penilaian Keamanan Pangan dan Risiko Alergi
Abstract
Histamine poisoning is a significant food safety concern associated with fish and fishery products. Enterobacter aerogenes is one of the histamine-producing bacteria capable of converting L-histidine into histamine through the histidine decarboxylase (HDC) enzyme. The accumulation of histamine in fish and fishery products can lead to allergic reactions and foodborne illnesses, highlighting the need for rapid detection methods. This study aimed to design and optimize primers targeting the HDC gene in E. aerogenes and validate their specificity using in silico PCR. The HDC gene sequence was retrieved from the NCBI GenBank database, and primers were designed using Primer3Plus software. The in silico PCR analysis was performed to assess the specificity of the primers against the genome of E. aerogenes and other histamine-producing bacteria species. The optimized Primer Pair 1 successfully amplified a 230 bp target region, showing high specificity for E. aerogenes in product size and annealing position with no cross-reactivity to other histamine-producing bacteria species. These findings demonstrate the potential of in silico PCR as a rapid and cost-effective screening tool for detecting histamine-producing bacteria in fish and fishery products. However, further in vitro validation is required to confirm the applicability of these primers in real-world food safety and allergy risk assessments.
References
Ayodeji Ahmed, A.Y.E.L.O.J.A., 2020. Glimpse of fish as perishable staple. Al-Qadisiyah Journal For Agriculture Sciences, 10(2), pp.349-375.
Betsy, C.J. and Siva, C., 2023. Sequence Retrieval, Analysis, and Manipulation. In Fisheries Biotechnology and Bioinformatics (pp. 193-203). Singapore: Springer Nature Singapore.
Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T.G., Bertoni, M., Bordoli, L. and Schwede, T., 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic acids research, 42(W1), pp.W252-W258.
Bikandi, J., Millán, R.S., Rementeria, A. and Garaizar, J., 2004. In silico analysis of complete bacterial genomes: PCR, AFLP–PCR and endonuclease restriction. Bioinformatics, 20(5), pp.798-799.
Björnsdóttir-Butler, K., Bolton, G.E., Jaykus, L.A., McClellan-Green, P.D. and Green, D.P., 2010. Development of molecular-based methods for determination of high histamine producing bacteria in fish. International Journal of Food Microbiology, 139(3), pp.161-167.
Bjornsdottir-Butler, K., Jones, J.L., Benner, R. and Burkhardt III, W., 2011. Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish. Food microbiology, 28(3), pp.356-363.
Bjornsdottir?Butler, K., Green, D.P., Bolton, G.E. and McClellan?Green, P.D., 2015. Control of Histamine?Producing Bacteria and Histamine Formation in Fish Muscle by Trisodium Phosphate. Journal of Food Science, 80(6), pp.M1253-M1258.
Comi, G. and Iacumin, L., 2025. Spoilage of meat and fish. In The microbiological quality of food (pp. 221-248). Elsevier Science Ltd.
Delgado, C.L., Wada, N., Rosegrant, M.W., Meijer, S. and Ahmed, M., 2003. The future of fish: issues and trends to 2020.
Economou, V., Gousia, P., Kemenetzi, D., Sakkas, H. and Papadopoulou, C., 2017. Microbial quality and histamine producing microflora analysis of the ice used for fish preservation. Journal of Food Safety, 37(1), p.e12285.
Enache, E., Kataoka, A.I., Black, D.G., Weddig, L., Hayman, M. and Bjornsdottir-Butler, K., 2013. Heat resistance of histamine-producing bacteria in irradiated tuna loins. Journal of food protection, 76(9), pp.1608-1614.
Ethica, S.N., Sri, D., Sri, S.D. and Sulistyaningtyas, A.R., 2020. Streptolysin encoding genes sagC and sagD as biomarkers of fish pathogen Streptococcus iniae: an in silico study.
Ethica, S.N., Hidayati, N., Fuad, H., Arham, C., Ariyadi, R., Purwaningrum, E. and Zillur Rahman, K.M., 2020. Detection of rtxA gene as a biomarker of seafood-borne pathogen Vibrio cholerae using in silico PCR assay. Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology, 15(2), pp.10-15578.
Feng, C., Teuber, S. and Gershwin, M.E., 2016. Histamine (scombroid) fish poisoning: a comprehensive review. Clinical reviews in allergy & immunology, 50, pp.64-69.
García-Tapia, G., Barba-Quintero, G., Gallegos-Infante, J.A., Aguilar, R.P., Ruíz-Cortés, J.A. and Ramírez, J.A., 2013. Influence of physical damage and freezing on histamine concentration and microbiological quality of yellowfin tuna during processing. Food Science and Technology, 33, pp.463-467.
Guo, X., Rao, Y., Guo, L., Xu, H., Lv, T., Yu, X., Chen, Y., Liu, N., Han, H. and Zheng, B., 2019. Detection and genomic characterization of a Morganella morganii isolate from China that produces NDM-5. Frontiers in Microbiology, 10, p.1156.
Gupta, S., Dongre, A., Saxena, J. and Jyoti, A., 2017. Computation and in silico validation of a real-time PCR array for quantitative detection of pathogens isolated from blood sample in sepsis patients.
Hassan, M., Shaltout, F.A. and Saqur, N., 2020. Histamine in some fish products. Archives of Animal Husbandry & Dairy Science, 2(1), pp.1-3.
Hu, J.W., Cao, M.J., Guo, S.C., Zhang, L.J., Su, W.J. and Liu, G.M., 2015. Identification and inhibition of histamine-forming bacteria in blue scad (Decapterus maruadsi) and chub mackerel (Scomber japonicus). Journal of Food Protection, 78(2), pp.383-389.
Hung, J.H. and Weng, Z., 2016. Designing polymerase chain reaction primers using Primer3Plus. Cold Spring Harbor Protocols, 2016(9), pp.pdb-prot093096.
Hungerford, J.M., 2010. Scombroid poisoning: a review. Toxicon, 56(2), pp.231-243.
Hungerford, J.M., 2021. Histamine and scombrotoxins. Toxicon, 201, pp.115-126.
Kalendar, R., Shevtsov, A., Otarbay, Z. and Ismailova, A., 2024. In silico PCR analysis: A comprehensive bioinformatics tool for enhancing nucleic acid amplification assays. Frontiers in bioinformatics, 4, p.1464197.
Kanki, M., Yoda, T., Ishibashi, M. and Tsukamoto, T., 2004. Photobacterium phosphoreum caused a histamine fish poisoning incident. International journal of food microbiology, 92(1), pp.79-87.
Kariyanna, B., Prabhuraj, A., Asokan, R., Babu, P., Jalali, S.K., Venkatesan, T., Gracy, R.G. and Mohan, M., 2020. Identification of suitable reference genes for normalization of RT-qPCR data in eggplant fruit and shoot borer (Leucinodes orbonalis Guenée). Biologia, 75, pp.289-297.
Lehane, L. and Olley, J., 2000. Histamine fish poisoning revisited. International journal of food microbiology, 58(1-2), pp.1-37.
Milicevic, B., Danilovic, B., Zdolec, N., Kozachinski, L., Dobranic, V. and Savic, D., 2014. Microbiota of the fermented sausages: influence to product quality and safety.
Mohd Afendy, A.T., Noor Azlina, M. and Radu, S., 2013. Evaluation of primer set targeting Inva gene of Salmonella sp. by in silico PCR simulation against major foodborne pathogens.
Nevado, D.L., Santos, S.D., Bastian, G., Deyta, J., Managuelod, E.J., Fortaleza, J.A. and De Jesus, R., 2023. Detection, identification, and inactivation of histamine-forming bacteria in seafood: a mini-review. Journal of Food Protection, 86(3), p.100049.
Novoslavskij, A., Terentjeva, M., Eizenberga, I., Valci?a, O., Bartkevi?s, V. and B?rzi?š, A., 2016. Major foodborne pathogens in fish and fish products: a review. Annals of microbiology, 66, pp.1-15.
Oktariani, A.F., Ramona, Y., Sudaryatma, P.E., Dewi, I.A.M.M. and Shetty, K., 2022. Role of marine bacterial contaminants in histamine formation in seafood products: A review. Microorganisms, 10(6), p.1197.
de Oliveira Andrade, L.J., de Oliveira, G.C.M., de Oliveira, L.C.M. and de Oliveira, L.M., 2024. Homologies between Chloroplast, plasma membrane, plastid, chloroplast envelope, putative [Theobroma cacao] and PEG10 protein [Homo sapiens]: a starting point for to deliver drugs to cells?.
Sahu, L., Panda, S.K., Paramithiotis, S., Zdolec, N. and Ray, R.C., 2015. Biogenic amines in fermented foods: overview. Fermented Foods—Part I: Biochemistry and Biotechnology; Montet, D., Ramesh, CR, Eds, pp.318-332.
San Millán, R.M., Martínez-Ballesteros, I., Rementeria, A., Garaizar, J. and Bikandi, J., 2013. Online exercise for the design and simulation of PCR and PCR-RFLP experiments. BMC Research Notes, 6, pp.1-4.
Sheng, L. and Wang, L., 2021. The microbial safety of fish and fish products: Recent advances in understanding its significance, contamination sources, and control strategies. Comprehensive Reviews in Food Science and Food Safety, 20(1), pp.738-786.
Surya, T., Sivaraman, B., Alamelu, V., Priyatharshini, A., Arisekar, U. and Sundhar, S., 2019. Rapid methods for histamine detection in fishery products. Int. J. Curr. Microbiol. Appl. Sci, 8, pp.2035-2046.
Tidwell, J.H. and Allan, G.L., 2001. Fish as food: aquaculture's contribution. EMBO reports.
Visciano, P., Schirone, M., Tofalo, R. and Suzzi, G., 2014. Histamine poisoning and control measures in fish and fishery products. Frontiers in microbiology, 5, p.500.
Watahiki, M., Kawahara, R., Suzuki, M., Aoki, M., Uchida, K., Matsumoto, Y., Kumagai, Y., Noda, M., Masuda, K., Fukuda, C. and Harada, S., 2020. Single-tube multiplex polymerase chain reaction for the detection of genes encoding Enterobacteriaceae carbapenemase. Japanese journal of infectious diseases, 73(2), pp.166-172.
Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L. and Lepore, R., 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic acids research, 46(W1), pp.W296-W303.
Wongsariya, K., Bunyapraphatsara, N., Yasawong, M. and Chomnawang, M.T., 2016. Development of molecular approach based on PCR assay for detection of histamine producing bacteria. Journal of food science and technology, 53, pp.640-648.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S. and Madden, T.L., 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC bioinformatics, 13, pp.1-11.
Yu, B. and Zhang, C., 2011. In silico PCR analysis. In Silico Tools for Gene Discovery, pp.91-107.
