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A qPCR aptasensor for sensitive detection of aflatoxin M1

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Abstract

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, imposes serious health hazards. AFM1 had previously been classified as a group 2B carcinogen [1] and has been classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) [2]. Determination of AFM1 thus plays an important role for quality control of food safety. In this work, a sensitive and reliable aptasensor was developed for the detection of AFM1. The immobilization of aptamer through a strong interaction with biotin–streptavidin was used as a molecular recognition element, and its complementary ssDNA was employed as the template for a real-time quantitative polymerase chain reaction (RT-qPCR) amplification. Under optimized assay conditions, a linear relationship (ranging from 1.0 × 10−4 to 1.0 μg L−1) was achieved with a limit of detection (LOD) down to 0.03 ng L−1. In addition, the aptasensor developed here exhibits high selectivity for AFM1 over other mycotoxins and small effects from cross-reaction with structural analogs. The method proposed here has been successfully applied to quantitative determination of AFM1 in infant rice cereal and infant milk powder samples. Results demonstrated that the current approach is potentially useful for food safety analysis, and it could be extended to a large number of targets.

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References

  1. International Agency for Research on Cancer (IARC). Some naturally occurring substances: Food items and constituents heterocyclic aromatic amines and mycotoxins. IARC monographs on the evaluation of carcinogenic risk to humans. 1993;56:489–51.

  2. International Agency for Research on Cancer (IARC). Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 2002;82:171–6.

  3. Pei SC, Zhang YY, Eremin SA, Lee WJ. Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies. Food Control. 2009;20(12):1080–5. doi:10.1016/j.foodcont.2009.02.004.

    Article  CAS  Google Scholar 

  4. Anfossi L, Baggiani C, Giovannoli C, Biagioli F, D’Arco G, Giraudi G. Optimization of a lateral flow immunoassay for the ultrasensitive detection of aflatoxin M1 in milk. Anal Chim Acta. 2013;772:75–80. doi:10.1016/j.aca.2013.02.020.

    Article  CAS  Google Scholar 

  5. Liu D, Huang Y, Wang S, Liu K, Chen M, Xiong Y, et al. A modified lateral flow immunoassay for the detection of trace aflatoxin M1 based on immunomagnetic nanobeads with different antibody concentrations. Food Control. 2015;51:218–24. doi:10.1016/j.foodcont.2014.11.036.

    Article  Google Scholar 

  6. Mao J, Lei S, Liu Y, Xiao D, Fu C, Zhong L, et al. Quantification of aflatoxin M1 in raw milk by a core-shell column on a conventional HPLC with large volume injection and step gradient elution. Food Control. 2015;51:156–62. doi:10.1016/j.foodcont.2014.11.022.

    Article  CAS  Google Scholar 

  7. Food and Agriculture Organization of the United Nations. Worldwide regulations for mycotoxins in food and feed in 2003. Food and Nutrition Paper. 2004;81: Rome: FAO.

  8. Hoyos Ossa DE, Hincapié DA, Peñuela GA. Determination of aflatoxin M1 in ice cream samples using immunoaffinity columns and ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Food Control. 2015;56:34–40. doi:10.1016/j.foodcont.2015.03.011.

    Article  CAS  Google Scholar 

  9. Commission Recommendation (EC). Setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union. 2006;364:5–24.

    Google Scholar 

  10. Busman M, Bobell JR, Maragos CM. Determination of the aflatoxin M1 (AFM1) from milk by direct analysis in real time–mass spectrometry (DART-MS). Food Control. 2015;47:592–8. doi:10.1016/j.foodcont.2014.08.003.

    Article  CAS  Google Scholar 

  11. Wang Y, Liu X, Xiao C, Wang Z, Wang J, Xiao H, et al. HPLC determination of aflatoxin M1 in liquid milk and milk powder using solid phase extraction on OASIS HLB. Food Control. 2012;28(1):131–4. doi:10.1016/j.foodcont.2012.04.037.

    Article  Google Scholar 

  12. Lee D, Lee K-G. Analysis of aflatoxin M1 and M2 in commercial dairy products using high-performance liquid chromatography with a fluorescence detector. Food Control. 2015;50:467–71. doi:10.1016/j.foodcont.2014.09.020.

    Article  CAS  Google Scholar 

  13. Pietri A, Fortunati P, Mulazzi A, Bertuzzi T. Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese. Food Chem. 2016;192:235–41. doi:10.1016/j.foodchem.2015.07.006.

    Article  CAS  Google Scholar 

  14. Beltrán E, Ibáñez M, Sancho JV, Cortés MÁ, Yusà V, Hernández F. UHPLC–MS/MS highly sensitive determination of aflatoxins, the aflatoxin metabolite M1 and ochratoxin a in baby food and milk. Food Chem. 2011;126(2):737–44. doi:10.1016/j.foodchem.2010.11.056.

    Article  Google Scholar 

  15. Wang X, Li P. Rapid screening of mycotoxins in liquid milk and milk powder by automated size-exclusion SPE-UPLC-MS/MS and quantification of matrix effects over the whole chromatographic run. Food Chem. 2015;173:897–904. doi:10.1016/j.foodchem.2014.10.056.

    Article  CAS  Google Scholar 

  16. Li P, Zhang Q, Zhang W, Zhang J, Chen X, Jiang J, et al. Development of a class-specific monoclonal antibody-based ELISA for aflatoxins in peanut. Food Chem. 2009;115(1):313–7. doi:10.1016/j.foodchem.2008.11.052.

    Article  CAS  Google Scholar 

  17. Kav K, Col R, Kaan TK. Detection of aflatoxin M1 levels by ELISA in white-brined Urfa cheese consumed in Turkey. Food Control. 2011;22(12):1883–6. doi:10.1016/j.foodcont.2011.04.030.

    Article  CAS  Google Scholar 

  18. Anfossi L, Di Nardo F, Giovannoli C, Passini C, Baggiani C. Enzyme immunoassay for monitoring aflatoxins in eggs. Food Control. 2015;57:115–21. doi:10.1016/j.foodcont.2015.04.013.

    Article  CAS  Google Scholar 

  19. Parker CO, Tothill IE. Development of an electrochemical immunosensor for aflatoxin M1 in milk with focus on matrix interference. Biosens Bioelectron. 2009;24(8):2452–7. doi:10.1016/j.bios.2008.12.021.

    Article  CAS  Google Scholar 

  20. Bacher G, Pal S, Kanungo L, Bhand S. A label-free silver wire based impedimetric immunosensor for detection of aflatoxin M1 in milk. Sensors Actuators B. 2012;168:223–30. doi:10.1016/j.snb.2012.04.012.

    Article  CAS  Google Scholar 

  21. Vdovenko MM, Lu CC, Yu FY, Sakharov IY. Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin M1 in milk. Food Chem. 2014;158:310–4. doi:10.1016/j.foodchem.2014.02.128.

    Article  CAS  Google Scholar 

  22. Cruz-Aguado JA, Penner G. Determination of ochratoxin a with a DNA aptamer. J Agric Food Chem. 2008;56(22):10456–61. doi:10.1021/jf801957h.

    Article  CAS  Google Scholar 

  23. Barthelmebs L, Hayat A, Limiadi AW, Marty J-L, Noguer T. Electrochemical DNA aptamer-based biosensor for OTA detection, using superparamagnetic nanoparticles. Sensors Actuators B. 2011;156(2):932–7. doi:10.1016/j.snb.2011.03.008.

    Article  CAS  Google Scholar 

  24. Bonel L, Vidal JC, Duato P, Castillo JR. An electrochemical competitive biosensor for ochratoxin a based on a DNA biotinylated aptamer. Biosens Bioelectron. 2011;26(7):3254–9. doi:10.1016/j.bios.2010.12.036.

    Article  CAS  Google Scholar 

  25. Guo X, Wen F, Zheng N, Luo Q, Wang H, Wang H, et al. Development of an ultrasensitive aptasensor for the detection of aflatoxin B1. Biosens Bioelectron. 2014;56:340–4. doi:10.1016/j.bios.2014.01.045.

    Article  CAS  Google Scholar 

  26. Shim WB, Kim MJ, Mun H, Kim MG. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1. Biosens Bioelectron. 2014;62:288–94. doi:10.1016/j.bios.2014.06.059.

    Article  CAS  Google Scholar 

  27. Wang R, Xiang Y, Zhou X, Liu L-H, Shi H. A reusable aptamer-based evanescent wave all-fiber biosensor for highly sensitive detection of ochratoxin A. Biosens Bioelectron. 2015;66:11–8. doi:10.1016/j.bios.2014.10.079.

    Article  CAS  Google Scholar 

  28. Zhao Q, Geng X, Wang H. Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer. Anal Bioanal Chem. 2013;405(19):6281–6. doi:10.1007/s00216-013-7047-2.

    Article  CAS  Google Scholar 

  29. De Girolamo A, Le L, Penner G, Schena R, Visconti A. Analytical performances of a DNA-ligand system using time-resolved fluorescence for the determination of ochratoxin A in wheat. Anal Bioanal Chem. 2012;403(9):2627–34. doi:10.1007/s00216-012-6076-6.

    Article  Google Scholar 

  30. Nguyen BH, Tran LD, Do QP, Nguyen HL, Tran NH, Nguyen PX. Label-free detection of aflatoxin M1 with electrochemical Fe3O4/polyaniline-based aptasensor. Mater Sci Eng C Mater Biol Appl. 2013;33(4):2229–34. doi:10.1016/j.msec.2013.01.044.

    Article  CAS  Google Scholar 

  31. Istamboulie G, Paniel N, Zara L, Granados LR, Barthelmebs L, Noguer T. Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk. Talanta. 2016;146:464–9. doi:10.1016/j.talanta.2015.09.012.

    Article  CAS  Google Scholar 

  32. Malhotra S, Pandey AK, Rajput YS, Sharma R. Selection of aptamers for aflatoxin M1 and their characterization. J Mol Recognit. 2014;27(8):493–500. doi:10.1002/jmr.2370.

    Article  CAS  Google Scholar 

  33. Babu D, Muriana PM. Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B(1). J Microbiol Methods. 2011;86(2):188–94. doi:10.1016/j.mimet.2011.05.002.

    Article  CAS  Google Scholar 

  34. Cavaliere C, Foglia P, Pastorini E, Samperi R, Lagana A. Liquid chromatography/tandem mass spectrometric confirmatory method for determining aflatoxin M1 in cow milk: comparison between electrospray and atmospheric pressure photoionization sources. J Chromatogr A. 2006;1101(1–2):69–78. doi:10.1016/j.chroma.2005.09.060.

    Article  CAS  Google Scholar 

  35. Cucci C, Mignani AG, Dall’Asta C, Pela R, Dossena A. A portable fluorometer for the rapid screening of M1 aflatoxin. Sensors Actuators B. 2007;126(2):467–72. doi:10.1016/j.snb.2007.03.036.

    Article  CAS  Google Scholar 

  36. Neagu D, Perrino S, Micheli L, Palleschi G, Moscone D. Aflatoxin M1 determination and stability study in milk samples using a screen-printed 96-well electrochemical microplate. Int Dairy J. 2009;19(12):753–8. doi:10.1016/j.idairyj.2009.06.004.

    Article  CAS  Google Scholar 

  37. Dinckaya E, Kinik O, Sezginturk MK, Altug C, Akkoca A. Development of an impedimetric aflatoxin M1 biosensor based on a DNA probe and gold nanoparticles. Biosens Bioelectron. 2011;26(9):3806–11. doi:10.1016/j.bios.2011.02.038.

    Article  CAS  Google Scholar 

  38. Larou E, Yiakoumettis I, Kaltsas G, Petropoulos A, Skandamis P, Kintzios S. High throughput cellular biosensor for the ultra-sensitive, ultra-rapid detection of aflatoxin M1. Food Control. 2013;29(1):208–12. doi:10.1016/j.foodcont.2012.06.012.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 21305158), the Special Fund for Agro-scientific Research in the Public Interest (201403071), Modern Agro-Industry Technology Research System of the PR China (nycytx-04-01), Project of risk assessment on raw milk (GJFP2016009), and The Agricultural Science and Technology Innovation Program (ASTIP-IAS12). We thank the University of Liège-Gembloux Agro-Bio Tech and more specifically the research platform AgricultureIsLife for the funding of the scientific stay in Belgium that made this paper possible.

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    Authors and Affiliations

    1. Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2, Yuan Ming Yuan West Road, Haidian District, Beijing, 100193, China

      Xiaodong Guo, Fang Wen, Nan Zheng, Songli Li & Jiaqi Wang

    2. Chimie générale et organique, Gembloux Agro-Bio Tech, Université de Liège, Passage des Déportés 2, 5030, Gembloux, Belgium

      Xiaodong Guo & Marie-Laure Fauconnier

    3. Ministry of Agriculture-Milk and Dairy Product Inspection Center (Beijing), Beijing, 100193, China

      Xiaodong Guo, Fang Wen, Nan Zheng, Songli Li & Jiaqi Wang

    4. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China

      Nan Zheng & Jiaqi Wang

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    1. Xiaodong Guo
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    2. Fang Wen
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    Correspondence to Jiaqi Wang.

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    Xiaodong Guo, Fang Wen and Nan Zheng contributed equally to this work.

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    Guo, X., Wen, F., Zheng, N. et al. A qPCR aptasensor for sensitive detection of aflatoxin M1 . Anal Bioanal Chem 408, 5577–5584 (2016). https://doi.org/10.1007/s00216-016-9656-z

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