Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
권호 표지
DOI QR코드

DOI QR Code

A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR)

  • Bintvihok, Anong (Department of Veterinary Pharmacology, Faculty of Veterinary Science, Chulalongkorn University) ;
  • Treebonmuang, Supitchaya (Research and Development Center for Livestock Production Technology, Faculty of Veterinary Science, Chulalongkorn University) ;
  • Srisakwattana, Kitiya (Research and Development Center for Livestock Production Technology, Faculty of Veterinary Science, Chulalongkorn University) ;
  • Nuanchun, Wisut (Research and Development Center for Livestock Production Technology, Faculty of Veterinary Science, Chulalongkorn University) ;
  • Patthanachai, Koranis (Department of Veterinary Pharmacology, Faculty of Veterinary Science, Chulalongkorn University) ;
  • Usawang, Sungworn (Research and Development Center for Livestock Production Technology, Faculty of Veterinary Science, Chulalongkorn University)
  • Received : 2015.11.13
  • Accepted : 2016.01.07
  • Published : 2016.01.15
Download PDF
⟨ Previous Next ⟩

Abstract

Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.

Keywords

References

  1. Yoshizawa, T., Yamashita, A. and Chokethaworn, N. (1996) Occurrence of fumonisins and aflatoxins in corn from Thailand. Food Addit. Contam., 13, 163-168. https://doi.org/10.1080/02652039609374394
  2. Bhat, R.V. (1991) Aflatoxin: successes and failures of three decades of research in Fungi and Mycotoxins in Stored Products (Champ, B.R., Highley, E., Hocking, A.D. and Pitt, J.I. edition). ACIAR Proc., 36, 80-85.
  3. Norred, W.P. (1986) Occurrence and clinical manifestations of aflatoxicosis in Diagnosis of Mycotoxicosis (Richard, J.L. and Thurston, J.R. edition). Martinus Nijhoff, Dordrecht, pp. 11-29.
  4. Pitt, J.L. and Hocking, A.D. (1985) Fungi and food spoilage, Academic Press, Australia, pp. 259-311.
  5. Bintvihok, A., Kiatipattanasakul, W. and Doi, K. (1997) Acute toxicity of aflatoxin B1 in three species of domestic fowls. J. Toxicol. Pathol., 10, 149-152. https://doi.org/10.1293/tox.10.149
  6. Bintvihok, A., Shoya, S., Nagasawa, S., Sato, M. and Sutherat, S. (1991) Effects of aflatoxin B1 in ducklings: effect on liver lesions in Fungi and Mycotoxins in Stored Products (Champ, B.R., Highley, E., Hocking, A.D. and Pitt, J.I. edition). ACIAR Proc., 36, pp. 233-235.
  7. Bintvihok, A., Nagasawa, S. and Hayashi, M. (1991) Effects of aflatoxin B1 in ducklings: effect on hepatic microsomal drug metabolizing enzyme in Fungi and Mycotoxins in Stored Products (Champ, B.R., Highley, E., Hocking, A.D. and Pitt, J.I. edition). ACIAR Proc., 36, pp. 230-232.
  8. Pitt, J.L., Hocking, A.D. and Glenn, D.R. (1983) An improved medium for the detection of Aspergillus flavus and A. parasiticus. J. Appl. Bacteriol., 54, 109-114. https://doi.org/10.1111/j.1365-2672.1983.tb01307.x
  9. Sugano, K., Bintvihok, A., Thanacharoenwatch, P., Sookthinthai, L. and Intraraksa, R. (1993) Simple and rapid cleanup method for determination of aflatoxin B1 in mixed feed by sep-pak florisil cartridge. Proc. Jpn. Assoc. Mycotoxicol., 37, 43-45.
  10. Chu, F.S., Fan, T.S., Zhang, G.S., Xu, Y.C., Faust, S. and McMahon, P.L. (1987) Improved enzyme-linked immunosorbent assay for aflatoxin B1 in agricultural commodities. J. Assoc. Off. Anal. Chem., 70, 854-857.
  11. Hirano, K., Adachi, Y., Bintvihok, A., Ishibashi, S. and Kumazawa, N.H. (1992) An improved method for extraction and cleanup of aflatoxin B1 from liver. J. Vet. Med. Sci., 54, 567-569. https://doi.org/10.1292/jvms.54.567
  12. Hirano, K., Adachi, Y., Ishibashi, S., Sueyoshi, M., Bintvihok, A. and Kumazawa, N.H. (1991) Detection of aflatoxin B1 in plasma of fowl receiving feed naturally contaminated with aflatoxin B1. J. Vet. Med. Sci. 53, 1083-1085. https://doi.org/10.1292/jvms.53.1083
  13. Shapira, R., Paster, N., Eyal, O., Menasherov, M., Matt, A. and Salomon, R. (1996) Determination of aflatoxigenic molds in grains by PCR. Appl. Environ. Microbiol., 62, 3270-3273.
  14. Levin, R.E. (2012) PCR detection of aflatoxin producing fungi and its limitations. Int. J. Food Microbiol., 156, 1-6. https://doi.org/10.1016/j.ijfoodmicro.2012.03.001
  15. Rodriguez, A., Rodriguez, M., Luque, M.I., Martin, A. and Cordiba, J.J. (2012) Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods. Food Microbiol., 31, 89-99. https://doi.org/10.1016/j.fm.2012.02.009
  16. Bintvihok, A., Thiengnin, S., Patchimasiri, T., Thummabood, S., Shoya, S., Ogura, Y., Kumagai, S. and Doi, K. (1993) Toxic effects of dietary aflatoxin and its residue in tissues and eggs in laying quails. Proc. the 11th Int. Symposium of the World Association of Veterinary Food Hygienists (WAVFH), pp. 299-307.
  17. Chinaphuti, A., Trikarunasawat, C., Wongurai, A. and Kositcharoenkul, S. (2002) Production of in-house ELISA test kit for detection of aflatoxin in agricultural commodities and their validations. Kasetsart J. Nat. Sci., 36, 179-186.
  18. Bintvihok, A., Treebonmuang, S., Patthanachai, K., Srisakwattana, K., Nuanchuen, W. and Usawang, S. (2013) Evaluation of aflatoxin in maize samples feedstuffs by real-time PCR using SYBR Green. Buffalo J., 29, 45-49.
  19. Somashekar, D., Rati, E.R., Chandrashekar, A. (2004) PCRrestriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize. Int. J. Food Microbiol., 93, 101-107. https://doi.org/10.1016/j.ijfoodmicro.2003.10.011
  20. Konietzny. U. and Greiner, R. (2003) The application of PCR in the detection of mycotoxigenic fungi in foods. Braz. J. Microbiol., 34, 283-300. https://doi.org/10.1590/S1517-83822003000400001
  21. Anbazhagan, D., Mui, W.S., Mansor, M., Yan, G.O., Yusof, M.Y. and Sekaran, S.D. (2011) Development of conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens. Braz. J. Microbiol., 42, 448-458. https://doi.org/10.1590/S1517-83822011000200006
  22. Heid, C.A., Stevens, J., Livak, K.J. and Williams, P.M. (1996) Real time quantitative PCR. Genome Res., 6, 986-994. https://doi.org/10.1101/gr.6.10.986
  23. Fittipaldi, M., Codony, F. and Morato, J. (2010) Comparison of conventional culture and real-time quantitative PCR using SYBR Green for detection of Legionella pneumophila in water samples. Available From: http://www.wrc.org.za.
  24. Morrison, T.B., Weis, J.J. and Wittwer, C.T. (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 24, 954-958.
  25. Ririe, K.M., Rasmusses, R.P. and Wittwer, C.T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem., 245, 154-160. https://doi.org/10.1006/abio.1996.9916

Cited by

  1. Shedding New Lights with the Breakthrough Ideas to Understand Current Trends in Modern Toxicology vol.32, pp.1, 2016, https://doi.org/10.5487/TR.2016.32.1.001
  2. Development of Hybrid IgG-Aptamer Sandwich Immunoassay Platform for Aflatoxin B1 Detection and Its Evaluation Onto Various Field Samples vol.9, pp.1663-9812, 2018, https://doi.org/10.3389/fphar.2018.00271