Detection of Fusarium verticillioides by PCR-ELISA based on FUM21 gene.

Fusarium verticillioides is a primary corn pathogen and fumonisin producer which is associated with toxic effects in humans and animals. The traditional methods for detection of fungal contamination based on morphological characteristics are time-consuming and show low sensitivity and specificity. Therefore, the objective of this study was to develop a PCR-ELISA based on the FUM21 gene for F. verticillioides detection. The DNA of the F. verticillioides, Fusarium sp., Aspergillus sp. and Penicillium sp. isolates was analyzed by conventional PCR and PCR-ELISA to determine the specificity. The PCR-ELISA was specific to F. verticillioides isolates, showed a 2.5 pg detection limit and was 100-fold more sensitive than conventional PCR. In corn samples inoculated with F. verticillioides conidia, the detection limit of the PCR-ELISA was 1 × 104 conidia/g and was also 100-fold more sensitive than conventional PCR. Naturally contaminated corn samples were analyzed by PCR-ELISA based on the FUM21 gene and PCR-ELISA absorbance values correlated positively (p < 0.05) with Fusarium sp. counts (CFU/g). These results suggest that the PCR-ELISA developed in this study can be useful for F. verticillioides detection in corn samples.

[1]  N. Thiệu,et al.  Aflatoxins, Fumonisins and Zearalenone Contamination of Maize in the Southeastern and Central Highlands Provinces of Vietnam , 2015 .

[2]  E. Y. Hirooka,et al.  Mycotoxicological quality evaluation of corn samples used by processing industries in the Northern region of Paraná State, Brazil , 2008, Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment.

[3]  K. Rothman,et al.  Exposure to Fumonisins and the Occurrence of Neural Tube Defects along the Texas–Mexico Border , 2005, Environmental health perspectives.

[4]  L. Covarelli,et al.  Infection by mycotoxigenic fungal species and mycotoxin contamination of maize grain in Umbria, central Italy. , 2011, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[5]  Chii-Wann Lin,et al.  Advances in rapid detection methods for foodborne pathogens. , 2014, Journal of microbiology and biotechnology.

[6]  D. Moodley,et al.  Fumonisin B1 induces global DNA hypomethylation in HepG2 cells - An alternative mechanism of action. , 2014, Toxicology.

[7]  M. Busman,et al.  The Fusarium verticillioides FUM Gene Cluster Encodes a Zn(II)2Cys6 Protein That Affects FUM Gene Expression and Fumonisin Production , 2007, Eukaryotic Cell.

[8]  I. Repa,et al.  Acute hepatic effects of low-dose fumonisin B(1) in rats. , 2016, Acta veterinaria Hungarica.

[9]  M S Medina-Martínez,et al.  Mold occurrence and aflatoxin B(1) and fumonisin B(1) determination in corn samples in Venezuela. , 2000, Journal of agricultural and food chemistry.

[10]  H. S. Murali,et al.  Multiplex PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India. , 2011, Journal of the science of food and agriculture.

[11]  S. Swanevelder,et al.  Induction of an altered lipid phenotype by two cancer promoting treatments in rat liver. , 2015, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[12]  Jingao Dong,et al.  Natural incidence of Fusarium species and fumonisins B1 and B2 associated with maize kernels from nine provinces in China in 2012 , 2015, Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment.

[13]  P. E. Nelson,et al.  Fusarium species: an illustrated manual for identification. , 1983 .

[14]  Shaokang Wang,et al.  Stimulation of the proliferation of human normal esophageal epithelial cells by fumonisin B1 and its mechanism , 2013, Experimental and therapeutic medicine.

[15]  P. Battilani,et al.  Mycotoxin occurrence in maize produced in Northern Italy over the years 2009–2011: focus on the role of crop related factors , 2015 .

[16]  D. Zurakowski,et al.  A statistically defined endpoint titer determination method for immunoassays. , 1998, Journal of immunological methods.

[17]  A. González-Córdova,et al.  Capillary electrophoresis for the analysis of contaminants in emerging food safety issues and food traceability , 2010, Electrophoresis.

[18]  S. Piletsky,et al.  Analytical methods for determination of mycotoxins: An update (2009-2014). , 2015, Analytica chimica acta.

[19]  Grimm,et al.  A PCR‐ELISA for the detection of potential fumonisin producing Fusarium species , 1998, Letters in applied microbiology.

[20]  M. Charreyre,et al.  Water-soluble dendrigrafts bearing saccharidic moieties: elaboration and application to enzyme linked OligoSorbent Assay (ELOSA) diagnostic tests. , 2006, Bioconjugate chemistry.

[21]  R. Proctor,et al.  FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis. , 2003, Journal of agricultural and food chemistry.

[22]  Y. Ueno,et al.  A limited survey of fumonisins in corn and corn-based products in Asian countries , 1993, Mycotoxin Research.

[23]  J. Frisvad,et al.  Production of Fumonisin B2 and B4 by Aspergillus niger on grapes and raisins. , 2010, Journal of agricultural and food chemistry.

[24]  S. Uppalapati,et al.  Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods , 2013, Journal of applied microbiology.

[25]  C. P. Vendruscolo,et al.  Leukoencephalomalacia Outbreak in Horses due to Consumption of Contaminated Hay , 2016, Journal of veterinary internal medicine.

[26]  V. Scussel,et al.  Fumonisins in corn (Zea mays L.) from Southern Brazil , 2014, Food additives & contaminants. Part B, Surveillance.

[27]  R. A. Butchko,et al.  Deletion analysis of FUM genes involved in tricarballylic ester formation during fumonisin biosynthesis. , 2006, Journal of agricultural and food chemistry.

[28]  Hami Alpas,et al.  Rapid and standardized methods for detection of foodborne pathogens and mycotoxins on fresh produce , 2014 .

[29]  M. Y. Sreenivasa,et al.  Molecular Detection of Fumonisin Producing Fusarium Species of Freshly Harvested Maize Kernels Using Polymerase Chain Reaction (PCR) , 2006 .

[30]  I. Repa,et al.  A comparative pathological finding in pigs exposed to fumonisin B1 and/or Mycoplasma hyopneumoniae , 2016, Toxicology and industrial health.

[31]  Daren W Brown,et al.  Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. , 2003, Fungal genetics and biology : FG & B.

[32]  G. Tantillo,et al.  Detection of Vibrio parahaemolyticus in shellfish using polymerase chain reaction–enzyme‐linked immunosorbent assay , 2012, Letters in applied microbiology.

[33]  J. Frisvad,et al.  Fumonisin B2 production by Aspergillus niger. , 2007, Journal of agricultural and food chemistry.

[34]  J. Kaczmarek,et al.  Molecular diagnostics on the toxigenic potential of Fusarium spp. plant pathogens , 2014, Journal of applied microbiology.

[35]  M. M. Teixeira,et al.  Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil. , 2011, International journal of food microbiology.

[36]  A. Omar,et al.  Application of PCR-ELISA in Molecular Diagnosis , 2014, BioMed research international.

[37]  W. Gelderblom,et al.  Quantitative Determination of Fumonisins B1 and B2 by High-Performance Liquid Chromatography with Fluorescence Detection , 1990 .

[38]  Leslie T. Stayner,et al.  IARC Monographs on the evaluation of carcinogenic risks to humans: Some traditional herbal medicines, some mycotoxins, naphthalene and styrene , 2002 .

[39]  R. Krska,et al.  Natural mycotoxin contamination of maize (Zea mays L.) in the South region of Brazil , 2017 .

[40]  B. Odhav,et al.  The effect of thermal processing on fumonisin B1 (FB1) levels in maize-based foods , 2013 .

[41]  J. Cenis,et al.  Rapid extraction of fungal DNA for PCR amplification. , 1992, Nucleic acids research.

[42]  S. Tanumihardjo,et al.  Maize: A Paramount Staple Crop in the Context of Global Nutrition. , 2010, Comprehensive reviews in food science and food safety.

[43]  L. V. Cota,et al.  Prevalence of fumonisin-producing Fusarium species in Brazilian corn grains , 2014 .

[44]  J. Flaherty,et al.  Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal. , 2002, Journal of food protection.

[45]  P. Garapati,et al.  Mould incidence and mycotoxin contamination in freshly harvested maize kernels originated from India. , 2014, Journal of the science of food and agriculture.

[46]  P. Hariprasad,et al.  Molecular identification and characterization of Fusarium spp. associated with sorghum seeds. , 2014, Journal of the science of food and agriculture.

[47]  B. Turcotte,et al.  A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins , 2006, Microbiology and Molecular Biology Reviews.

[48]  N. Lima,et al.  Mycotoxin contamination of Maize and Guinea corn from markets in Plateau State, Nigeria , 2015 .

[49]  W. Marasas,et al.  Natural occurrence of Fusarium and subsequent fumonisin contamination in preharvest and stored maize in Benin, West Africa. , 2005, International journal of food microbiology.

[50]  T. Utterback,et al.  Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides. , 2005, Fungal genetics and biology : FG & B.