Abstract
The aim of the present study was to develop a multiplex PCR (mPCR) assay for the simultaneous detection of five major metabolic pathway genes viz. aflr (Aflatoxin), pks (Ochratoxin A), tri5 (Trichothecene), pks13 (Zearalenone) and fum13 (Fumonisin), producing Aspergillus, Penicillium and Fusarium species. The mPCR assay with competitive internal amplification control to eliminate false negative results employing specific primers for each of the above mentioned five genes was optimized and validated using standard strains. The standardized mPCR assay detected all five major mycotoxin metabolic genes along with artificially inoculated maize seeds with mycotoxigenic Fusarium, Penicillium and Aspergillus spores. The detection limit of this mPCR assay was 1 × 103 spores per gram of artificially inoculated samples upon 48 h of incubation at room temperature. When the developed mPCR assay was applied on to 177 contaminated maize, paddy and sorghum, many of the samples (100 out of 177) were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high performance liquid chromatography and in general both the methods provided unequivocal results. The developed mPCR based assay was found to be rapid, cost effective and user friendly and can be used for diagnosis of major mycotoxigenic fungi.
Similar content being viewed by others
References
Abdin MZ, Ahmad MM, Javed S (2010) Advances in molecular detection of Aspergillus: an update. Arch Microbiol 192(6):409–425
Amadi JE, Adeniyi DO (2009) Mycotoxin production by fungi isolated from store grains. Afr J Biotechnol 8:1219–1221
Bhat RV, Vasanthi S (2003) Mycotoxin food safety risks in developing countries . In: Food safety in food security and food trade. Focus 10 Vision 2020. Food, Agriculture and Environment 1–2
Butchko RA, Plattner RD, Proctor RH (2003) FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis. J Agri Food Chem 51:3000–3006
CAC (Codex alimentarius commission) (2001) Comments submitted on the proposed draft maximum level for ochratoxin A in cereal and cereal products.CX/FAC 01/22, Feb. 2001:1–4
Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB (2001) Simultaneous detection of Neisseria meningitidis, Haemophilusinfluenzaeand Streptococcus pneumoniaein suspected cases of meningitis and septicaemia using realtime PCR. J Clin Microbiol 39:1553–1558
Dao HP, Mathieu F, Lebrihi A (2005) Two primer pairs to detect OTA producers by PCR method. Int J Food Microbiol 104:61–67
Dickison JH, Kroll RG, Grant KA (1995) The direct application of the polymerase chain reaction to DNA extracted from foods. Lett Appl Microbiol 20:212–216
Ehrlich KC, Cotty PJ (2004) An isolate of Aspergillus flavus used to reduce aflatoxins contamination in cottonseed has a defective polyketide synthase gene. Appl Microbiol Biotechnol 65:473–478
EU (2006) Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002. In: Official Journal of the European Communities, 1.2.2002. http://www.bfr.bund.de/cm/209/2002_178_en_efsa.pdf. Accessed 29 June 2006
Hayat A, Paniel N, Rouhati A, Marty JL, Barthelmebs L (2012) Recent advances in ochratoxin A-producing fungi detection based on PCR methods and ochratoxin A analysis in food matrices. Food Control 26:401–415
Hoorfar J, Malorny B, Abdulmawjood A, Cook N, Wagner M, Fach P (2004) Practical considerations in design of internal amplification controls for diagnostic PCR assays. J Clin Microbiol 42:1863–1868
ICMSF International Commission on Microbiological Specifications for Foods (1996) Microbiological specifications of food pathogens, London. Blackie Acad Professional, London, pp 347–381
Jarvis B (1971) Factors affecting the production of mycotoxins. J Appl Microbiol 34:199–213
Kampelmacher EH (1973) Since Eve ate apples. Cannad J Pub Health 64:231–237
Kumar S, Balakrishna K, Batra HV (2006) Detection of Salmonella enteric serovarTyphi(S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in multiplex format. Lett Appl Microbiol 42:149–154
Levin RE (2012) PCR detection of aflatoxin producing fungi and its limitations. Int J Food Microbiol 156(1):1–6
Lopez MM, Bertolini E, Olmos A, Caruso P, Gorris MT, Llop P (2003) Innovative tools for detection of plant pathogenic viruses and bacteria. Int J Food Microbiol 6:233–243
Lopez MM, Llop P, Olmos A, Marco-Noales E, Cambra M, Bertolini EV (2009) Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses? Curr Issu in Mol Bio 11:13–46
Niessen L (2007) PCR-based diagnosis and quantification of mycotoxin producing fungi. Int J Food Microbiol 119:38–46
Niessen L (2008) PCR-based diagnosis and quantification of mycotoxin-producing fungi. Adv Food Nutr Res 54:81–138
Pittet A (1998) Natural occurrence of mycotoxins in food and feeds—an update review. Rev Med Vet 149:479–492
Ramana MV, Balakrishna K, Murali HS, Batra HV (2011) Multiplex PCR based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India. J Sci Food Agric 91(9):1666–1673, 106:291–296
Ratti C, Budge G, Ward L, Clover G, Rubies-Autonell C, Henry C (2004) Detection and relative quantitation of soil-borne cereal mosaic virus (SBCMV) and Polymyxagraminisin winter wheat using real-time PCR (TaqMan (R)). J Virol Methods 122:95–103
Richard JL, Bennett GA, Ross PF, Nelson PE (1993) Analysis of naturally occurring mycotoxins in feedstuffs and food. Presented at symposium titled “Residues of natural toxin in feedstuffs and food” at the ASAS 84th Annu. Mtg. Pittsburgh, PA
Rioadan MJ, Wilinson MG (2008) A survey of the incidence and level of Aflatoxin contamination in a range of imported spice preparations on the Irish retail market. Food Chem 107:1429–1435
Russell R, Paterson M (2006) Identification and quantification of mycotoxigenic fungi by PCR. Proc Biochem 41:1467–1474
Shapira R, Paster N, Eyal O, Menasherov M, Mett A, Salomon R (1996) Detection of aflatoxigenicmolds in grains by PCR. Appl Environ Microbiol 62:3270–3273
Suanthiea Y, Cousinb MA, Woloshuk CP (2009) Multiplex real-time PCR for detection and quantification of mycotoxigenic Aspergillus, Penicillium and Fusarium. J Stored Prod Res 45:139–145
Wu CL, Cheng XW, He HF, Lv X, Wang JW, Deng RQ, Long QX, Wang XZ (2008) A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1. J Virol Methods 148:81–88
Acknowledgments
Authors are thankful to the Director DFRL, for providing necessary facilities to carry out the present study.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(DOCX 24 kb)
Rights and permissions
About this article
Cite this article
Priyanka, S.R., Venkataramana, M., Balakrishna, K. et al. Development and evaluation of a multiplex PCR assay for simultaneous detection of major mycotoxigenic fungi from cereals. J Food Sci Technol 52, 486–492 (2015). https://doi.org/10.1007/s13197-013-1001-3
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13197-013-1001-3