MOLECULAR TECHNIQUES FOR DETECTION OF AFLATOXIGENIC ASPERGILLUS FLAVUS AND DETERMINE THEIR AFLATOXIN IN PISTACHIOS.

Authors

DOI:

https://doi.org/10.36103/ijas.v54i6.1876

Keywords:

Pistachios, Aflatoxin, PCR, ElISA, A. flavus.

Abstract

The better approach to control the contamination of food by aflatoxin (AF), is identifying the fungal strains responsible for producing that toxin. To estimate the presence of aflatoxigenic fungi growth and AFs in pistachios, thirty two pistachios samples (8 fresh and 24 salt roasted) were collected from Sulaimani markets (Iraq).Rapid screening method for AFs production were utilized initially based on culture methods, the positive samples confirmed by polymerase chain reaction (PCR) that were applied using DNA extracted from the isolates of A. flavus targeting the genes (afR, aflP, aflD, and aflQ). For determination of AF production of those isolates, ELISA technique was applied. The results show, contamination of 20 (62.5%) samples of pistachios with fungi, among them A. flavus was the predominant. Ten (71.4% (of the A. flavus isolates were amplified the target genes by applying PCR, indicating their ability to produce AFs. Application of ELISA technique revealed AFs detection in 21(65.6%) samples of pistachios. The mean levels of AFs determined   were 6.06 and 12.08 μg/kg in fresh and salt roasted samples respectively. 19 (59.4%) of pistachio samples were exceed the maximum allowable limit set by the European union of total AFs concentration .Strong correlation obtained between AFs gene expression with AFs production. However, to avoid false negative and false positive results, applying more than one diagnostic molecular technique were suggested.

References

Abd El-Aziz, A. R.M., S.M. Shehata, S.M Hisham and A.A Alobathani.2021. Molecular profile of aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus isolated from stored maize .Saudi Journal of Biological Sciences. 28(1)1383–1391. DOI: 10.1016/j. sjbs.2020.11.073

Agriopoulou ,S., E. Stamatelopoulou and T. Varzakas . 2020.Advances in occurrence, importance, and mycotoxin control strategies: Prevention and detoxification in foods. Foods.9 (2) 10-33.

DOI: 10.3390 /foods9020137

Al-Jaal, B., S. Salama, N. Al-Qasmi and M. Jaganjac, 2019.Mycotoxin contamination of food and feed in the Gulf Cooperation Council countries and its detection. Toxicon . 171(21) 43–50. DOI: 10.1016/j.toxicon.2019.10.003

AL-Zubaidy ,A.M.A., K. I. Hassan, and B. S. Jabbari. 2016 Analysis of naturally occurring penolic compounds of the genera Clinopodium L.,Hymenocrater fish & c. A. Mey. and Melissa L. Iraqi Journal of Agricultural Sciences 47(1) 343-384. https://doi .org/10. 36103/ijas.v47i1.641

Anfossi, L., C. Giovannoli and C. Baggiani.2016. Mycotoxin detection. Curr. Opin. Biotechnol. 37(1) 120–126. DOI: 10.1016/j.copbio.2015.11.005

Al-Shuhaib,M.B., A.H. Albakri, S.H. Alwan, N.B. Almandil, S.AbdulAzeez and J. Borgio, 2018. Optimal PCR primers for rapid and accurate detection of Aspergillus flavus isolates. Microbial Pathogenesis. 116(6) 351-355. https://doi.org/10.1016/j.micpath.2018. 01.049

Ayala, A., M.F. Munoz and S. Arguelles. 2014. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondi aldehyde and 4-hydroxy-2-nonenal. Oxid. Med. Cell. Longev, 38(3) 36-43. Doi: 10.1155/2014/360438

Baazeem, A., E .Garcia-Cela , A. Medina and N. Magan .2021. Interacting abiotic factors affect growth and aflatoxin b1 production profiles of Aspergillus flavus strains on pistachio-based matrices and pistachio nuts. Front. Microbiol. 11(6) 24-37. https://doi.org/10.3389/fmicb.2020.624007

Benkerroum, N. 2019. Retrospective and prospective look at aflatoxin research and development from a practical standpoint. Int. J. Environ. Res. Public Health.16 (19):3633. doi: 10.3390/ijerph16193633

Burns, M., Wiseman, G., Knight,A., Bramley, P., Foster, L. Rollinson, S. Damant, A. and S.Primrose.2016.Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud. Analyst.141(`1) 45–61. https://doi.org/10.1039/C5AN01392E

Davari, E., M. Mohsenzadeh, G. Mohammadi, and R. Rezaeian-Doloei, 2015. Characterization of aflatoxigenic Aspergillus flavus and Aspergillus parasiticus strain isolates from animal feedstuffs in Northeastern Iran. Iranian Journal of Veterinary Research. 16 (2) 150–155. DOI.PMCID: PMC4827679

D’Evoli, L., Lucarini, M., Gabrielli, P., Aguzzi, A. and G.L., Boccia .2015. Nutritional value of Italian pistachios from Bronte (Pistacia vera, L.), their nutrients, bioactive compounds and antioxidant activity. Food Nutr Sci. 101(6) 1267-1276. doi: 10.4236/fns.2015.614132

Dini, A., P. Khazaeli, A. Roohbakhsh, A.Madadlou, M. Pourenamdari, L. Setoodeh, A. Askarian, N. Doraki, and H.Moradi. 2013. Aflatoxin contamination level in Iran’s pistachio nut during the years 2009–2011. Food Control, 30(2) 540–544. DOI: 10.1016 /j.foodcont.2012.08.012

European Commission. 2006 ,Commission regulation (EC) No. 401/2006 . Methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Off. J. Eur. Union .6 (70) 12–34

Fakruddin, M., C. Abhijit, N. H. Md, and M. A. Monzur, 2015. Characterization of aflatoxin producing Aspergillus flavus from food and feed samples. Springerplus Journal. 4(1) 159-173

Gurbuz,Y.,A. Hussein,and E. Ozkose. 2019.Determination of AFLD and AFLQ genes responsible for aflatoxin formation in some livestock concentrated feeds. PakVet J.39(2) 163-168. DOI: 10.29261/pakvetj/2019.057

Hassan,K.I., A.M. Al-Zubaidy and B.S. Jabbari .2015. Assessment of taxonomic relationship between some taxa of clinopodium, hymenocrater. and melissa of Lamiaceae in Iraqi Kurdistan Region using RAPD Marker. Diyala journal for pure science.12 (2) 59-71

Hassan, K. I. and, S.K. HamaSaleh. 2016. A rapid methods for PCR based on detection of Salmonela Spp. and Staphylococcus aureus in spiked and naturally contaminated food. Iraqi Journal of Agricultural Sciences. 47 (Special Issue), 66-73

Hepsag, F., O.Golge and B. Kabak .2014 .Quantitation of aflatoxins in pistachios and groundnuts using HPLC-FLD method. Food Control 38 (2) 75-81.https://doi.org /10.1016/j.foodcont.2013.10.005

Hossain, Md.N., A. Talukder, F. Afroze, M. Rahim, S.Begum, Z.Haque, and M.M. Ahmed. 2018. Identification of aflatoxigenic fungi and detection of their aflatoxin in red chilli (Capsicum annuum) samples using direct cultural method and HPLC. Advances in microbiology. 8(1)42-53.

DOI: 10.4236 /aim.2018.81004

Kabak, B. 2021.Aflatoxins in foodstuffs: Occurrence and risk assessment in Turkey. Journal of Food Composition and Analysis . 96 (5) 23-34. https://doi.org/10.1016/j. jfca.2020.103734

Kaminiaris,M.D. S.Mavrikou, M.Georgiadou, G.Paivana, D.I.Tsitsigiannis, and S. Kintzios. 2020. An Impedance based electrochemical immunosensor for aflatoxin B1 monitoring in pistachio matrices. Chemosensors , 8(14) 121-129. DOI: 10.3390 /chemosensors8040121

.Kizis, D., A. E.Vichou and P. I. Natskoulis. 2021. Recent advances in mycotoxin analysis and detection of mycotoxigenic fungi in grapes and derived products. sustainability, 13(5), 25-37. https://doi.org/10.3390/su13052537

Kolawole,O., J. Meneelya , A. Petchkongkaew and C. Elliott. 2021. A review of mycotoxin biosynthetic pathways: associated genes and their expressions under the influence of climatic factors. Fungal Biology Reviews. 37(18) 8-26.https://doi.org/10.1016/j.fbr.2021.04.003

Liao, J., Z. He, Y. Xia, Y. Lei and B. Liao. 2020. A review on biosynthesis and genetic regulation of aflatoxin production by major Aspergillus fungi. Oil Crop Science. 5 (4) 166–173. https://doi.org/10.1016/j. ocsci.2020.11.001

Mahmoud, M.A., 2015. Detection of Aspergillus flavus in stored peanuts using realtime PCR and the expression of aflatoxin genes in toxigenic and atoxigenic A.flavus isolates. Foodborne Pathog Dis. 12(4) 289–296. DOI: 10.1089/fpd.2014.1854

Mangal M.,F. Khan, S. Bansal and H. Oberoi. 2016.Validation of PCR based detection system for aflatoxin producing molds. Indian J Exp Biol. 54(4) 472-476.PMID: 29466626

Mannaa, M., and K.D.Kim. 2017. Influence of temperature and water activity on deleterious fungi and mycotoxin production during grain storage. Mycobiology. 45(4) 240–254. DOI: 10.5941/MYCO. 2017.45.4.240

Mikaeel, F. B. and A. T. M. Al-Saeed 2020. Serological and molecular diagnosis of toxoplasma gondii among ewes and horses in duhok province-iraq. Iraqi journal of agricultural sciences, 51(4), 1212–1219. https://doi.org/10.36103/ijas.v51i4.1100

Mitchell, N.J., E. Bowers, C. Hurburgh and F.Wu, 2016. Potential economic losses to the USA corn industry from aflatoxin contamination. Food Addit. Contam. Part A Chem Anal Control Expo Risk Assess .33(2) 540–550.

DOI: 10.1080/19440049.2016. 1138545

Montibus, M., L. Pinson-Gadais, F. Richard-Forget, C. Barreau and N. Ponts. 2013. Coupling of transcriptional response to oxidative stress and secondary metabolism regulation in filamentous fungi. Crit. Rev. Microbiol. 41(3) 295-308. DOI: 10.3109/ 1040841X.2013.829416

Mohamed, M. A. and E. I. Al – Shamary , 2022 .Isolation and identification of aflatoxin B1 producing fungi from stored wheat in some silos of Baghdad. Iraqi Journal of Agricultural Sciences, 53(6) 1427–1436 https://doi.org/10.36103/ijas.v53i6.1659

Nadhom, B. N. 2018. Study of molecular composition of virulance bacteria isolated from bovine mastitis with biofilm production, Iraqi Journal of Agricultural Sciences, 49(5):840-846. https://doi.org/10.36103/ijas.v49i5.44

Mohammed , M. F., D. S. Hassawi and N. K. Ibraheem .2023. Extraction, fractionation, and seperatione of cynaroside and scolymoside from Cynara scolymus L. grown in Iraq. Iraqi Journal of Agricultural Sciences, 54(2) 421–430. https://doi.org/10.36103/ijas.v54i2.1716

Niessen, L., J. Bechtner, S. Fodil, , M.H. Taniwaki and R.F. Vogel, 2018. LAMP-based group specific detection of aflatoxin producers within Aspergillus section Flavi in food raw materials, spices, and dried fruit using neutral red for visible-light signal detection. Int. J. Food Microbiol. 266(12) 241–250. DOI: 10.1016/j.ijfoodmicro.2017.12.013

Oliveira, I.S., A.G.D.S.Junior, C.A.S. De Andrade and M.D.L Oliveira. 2019, Biosensors for early detection of fungi spoilage and toxigenic and mycotoxins in food .Current Opinion in Food Science. 29(15) 64-79. https://doi.org/10.1016/j.cofs.2019.08. 004

Ostadrahimi, A., Ashrafnejad,F., Kazemi,A., Sargheini,N., Mahdavi,R., Farshchian, M..and S. Mahluji.2014. Aflatoxin in raw and salt-roasted nuts (pistachios, peanuts and walnuts) sold in markets of Tabriz, Iran Jundishapur J Microbiol.7(1)74-86. DOI: 10.5812/jjm.8674

Ostry, V., F. Malir, J. Toman, and Y. Gross. 2017. Mycotoxins as human carcinogens the IARC Monographs classification. Mycotoxin Research, 33(1), 65-73. DOI: 10.1016/j.ijfoodmicro.2017.12.013

Pakshir,K., Z. Mirshekari, H. Nouraei, Z. Zareshahrabadi, K. Zomorodian, H. Khodadadi, and A. Hadaegh. 2020. Mycotoxins detection and fungal contamination in black and green tea by HPLC-based method. Journal of Toxicology.202(7) 24-31. https://doi.org/10.1155/2020/2456210

Rao, K.R., A.V. Vipin and G. Venkateswaran. 2020. Molecular profile of nonaflatoxigenic phenotype in native strains of Aspergillus flavus. Arch. Microbiol. 202(5) 1143-1155. DOI: 10.1007/s00203-020-01822-1

Riba,A., A.Matmoura1, I.S. Mokrane , F.Mathieu, and A.Sabaou . 2013. Investigations on aflatoxigenic fungi and aflatoxins contamination in some nuts sampled in Algeria. Afr. J. Microbiol. Res.7(42) 4974-4980. DOI: 10.5897/AJMR2013.5867

Rezaei,M.,F.Karimi,M. Parviz, A.Behzadi,M. Javadzadeh and P.Mohammad. 2014. An empirical study on aflatoxin occurrence in nuts consumed in Tehran, Iran 2013. Health. 14(6) 649–653. DOI: 10.4236/health.2014.68084

Schrenk,D.,M. Bignami,L., Bodin,J.K. Chipman, J. del Mazo and B. Grasl-Kraupp. 2020. Risk assessment of aflatoxins in food. EFSA Journal. 18(3):6040.

DOI 10.2903/j.efsa.2020.6040

Shier,W.T., Y. Lao, T. Steele and H.K Abbas.2005.Yellow pigments used in rapid identification of aflatoxin-producing Aspergillus strains are anthraquinones associated with the aflatoxin biosynthetic pathway.Bioorganic Chemistry. 33(6) 426-438. doi: 10.1016/j.bioorg.2005.09.002

Sohrabi N.and M. Taghizadeh, 2018. Molecular identification of aflatoxigenic Aspergillus species in feedstuff samples. Curr Med Mycol. 4(2) 1-6. DOI: 10.18502 /cmm.4.2.66

Silva, A.S., C. Brites , A.V. Pouca , J. Barbosa and A. Freitas.2019.UHPLC-ToF-MS method for determination of multi-mycotoxins in maize: Development and validation. Current Research in Food Science. 1(5) 1-7. DOI: 10.1016/j.crfs.2019.07.001

Tao, F., Yao, H., Zhu, F., Hruska, Z., Liu, Y., Rajasekaran, K. and D.Bhatnagar.2019. A rapid and nondestructive method for simultaneous determination of aflatoxigenic fungus and aflatoxin contamination on corn kernels. Journal of Agricultural and Food Chemistry. 967 (18) 5230-5239. https://doi.org/10.1021/acs.jafc.9b01044

Wu,Q., L. Xie, , and H.Xu, 2018.Determination of toxigenic fungi and aflatoxins in nuts and dried fruits using imaging and spectroscopic techniques, Food Chemistry. 252(12) 228-242. DOI: 10.1016 /j.foodchem.2018.01.076

Xue, Z., L. Long, Y. Chen, M. Chen and Y. Cheng, 2021.A nanozyme-linked immunosorbent assay based on metal–organic frameworks (MOFs) for sensitive detection of aflatoxin B1,Food Chemistry, 13(12) 338-341.DOI: 10.1016/j.foodchem.2020.128039

Xue, Z., Y.Zhang, W. Yu, J. Zhang, J.Wang, F.Wan, Y. Kim,Y.Liu and X. Kou.2019. Recent vances in aflatoxin B1 detection based on nanotechnology and nanomaterials-A review,Analytica Chimica Acta.106 (9) 1-27. DOI: 10.1016/j.aca. 2019.04.032

Yabe, K., M.Yushita, Y. Yamamoto and M. Kushiro, 2020. Detection of aflatoxigenic fungi in imported raw nuts using the dichlorvos-ammonia (DV-AM) method . JSM Mycotoxins.70(1)7- 13

Yassin, H. Y., A. K. Melconian, and S. S. Mahmood. 2022. Prevalence of exfoliative toxin genes among clinical isolates of Staphylococcus aureus in Iraq, Iraqi Journal of Agricultural Sciences, 53(2) 465–470 https://doi.org/10.36103/ijas.v53i2.1554

Yu J. 2012.Current understanding on aflatoxin biosynthesis and future perspective in reducing aflatoxin contamination. Toxins (Basel). 4(11) 1024-1057. DOI: 10.3390 /toxins4111024

Zhang ,L., Xiao-Wen Dou, ,Cheng Zhang, Antonio F. Logrieco and Mei-Hua Yang 1, 2018 .A Review of Current Methods for Analysis of Mycotoxins in Herbal Medicines .Toxins .10(2) 65-76. DOI: 10.3390/toxins 10020065

Zhan,S., J.Hu, Y. Li, X. Huang and Y. Xiong. 2021. Direct competitive ELISA enhanced by dynamic light scattering for the ultrasensitive detection of aflatoxin B1 in corn samples, Food Chemistry, 16(12)342-351. DOI: 10.1016/j.foodchem.2020.128327

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2023-12-29

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K.I.Hassan. (2023). MOLECULAR TECHNIQUES FOR DETECTION OF AFLATOXIGENIC ASPERGILLUS FLAVUS AND DETERMINE THEIR AFLATOXIN IN PISTACHIOS. IRAQI JOURNAL OF AGRICULTURAL SCIENCES, 54(6), 1773-1783. https://doi.org/10.36103/ijas.v54i6.1876

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