AiM  Vol.2 No.4 , December 2012
A Novel Non Radioactive PCR-DNA Probe for the Detection of Aflatoxin Producing Aspergillus Species from Major Food Crops Grown in India
ABSTRACT

In the present study, a novel non radioactive digoxigenen labelled PCR-DNA probe was developed targeting nor-1 gene to assess the contamination of aflatoxigenic Aspergillus species in food grain samples from Southern parts of India. The sensitivity of developed PCR-DNA probe was determined to be 10 pg of genomic DNA and 1 pg of purified PCR product. The specificity of the DNA probe was validated by testing against an array of Aspergillus, Fusarium and Penicillium spp. A total of 89 Aspergillus isolates were recovered from 152 grain samples of maize, paddy, and groundnut. Among them, maize had the highest (90%) incidence of toxigenic Aspergillus species. When developed PCR-DNA probe was evaluated onto pure cultures of toxigenic and nontoxigenic Aspergillus species, 60 samples were positive for the nor-1 gene probe. DNA probe results unequivocally matched with the HPLC analysis. In conclusion, the novel PCR-DNA probe developed in this study may find its application in rapid detection of aflatoxin-producing Aspergillus isolates from contaminated cereal grains.


Cite this paper
S. R. Priyanka, M. Venkata Ramana, K. Balakrishna, H. S. Murali and H. V. Batra, "A Novel Non Radioactive PCR-DNA Probe for the Detection of Aflatoxin Producing Aspergillus Species from Major Food Crops Grown in India," Advances in Microbiology, Vol. 2 No. 4, 2012, pp. 577-586. doi: 10.4236/aim.2012.24075.
References
[1]   IARC, “Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins,” IARC Monographs on the Evaluation of Carcinogenic Risks to Human, International Agency for Research on Cancer, Lione, Vol. 56, 1993.

[2]   J. W. Cary and K. C. Ehrlich, “Aflatoxigenicity in Aspergillus: Molecular Genetics, Phylogenetic Relationships and Evolutionary Implications,” Mycopathologia, Vol. 162, No. 3, 2006, pp. 167-177.

[3]   M. Peraica and A. M. Domijan, “Mycotoxins in Food and Human Health,” Archives of Industrial Hygiene and Toxicology, Vol. 52, No. 1, 2001, pp. 23-35.

[4]   T. J. Hansen, “Quantitative Testing for Mycotoxins,” American Association Cereal Chemists, Vol. 38, 1993, pp. 346-348.

[5]   R. Geisen, “Multiplex Polymerase Chain Reaction for the Detection of Potential Aflatoxin and Sterigmatocystin Producing Fungi,” Systematic and Applied Microbiology, Vol. 19, 1996, pp. 388-392. doi.10.1016/S0723-2020(96)80067-1

[6]   G. Criseo, A. Bagnara and G. Bisignano, “Differentiation of Aflatoxin Producing and Non-Producing Strains of Aspergillus flavus Group,” Letters in Applied Microbiology, Vol. 33, No. 4, 2001, pp. 291-295. doi.0.1046/j.1472-765X.2001.00998

[7]   P. Rodrigues, A. Venancio, Z. Kozakiewicz and N. Lima, “A Polyphasic Approach to the Identification of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus Section Flavi Isolated from Portuguese Almonds,” International Journal of Food Microbiology, Vol. 129, No. 2, 2009, pp. 187-193.

[8]   P. Rahimi, B. Sharifnabi and M. Bahar, “Detection of Aflatoxin in Aspergillus Species Isolated from Pistachio in Iran,” Journal of Phytopathology, Vol. 156, No. 1, 2008, pp. 15-20.

[9]   B. Scherm, M. Palomba, D. Serra, A. Marcello and Q. Migheli, “Detection of Transcripts of the Aflatoxin Genes Afld, Aflo, and Aflp by Reverse Transcription-Polymerase Chain Reaction Allows Differentiation of Aflatoxin-Producing and Non-Producing Isolates of Aspergillus Flavus and Aspergillus parasiticus,” International Journal of FoodMicrobiology, Vol. 98, No. 2, 2005, pp. 201-210.

[10]   C. A. Townsend, “Progress towards a Biosynthetic Rationale of the Aflatoxin Pathway,” Pure and Applied Chemistry, Vol. 58, No. 2, 1997, pp. 227-238.

[11]   F. Trail, N. Mahanti, M. Rarick, M. Mehigh, S. H. Liang, R. Zhou and J. E. Linz, “Physical and Transcriptional Map of an Aflatoxin Gene Cluster in Aspergillus parasiticus and Functional Disruption of a Gene Involved Early in the Aflatoxin Pathway,” Applied and Environmental Microbiology, Vol. 61, No. 7, 1995, pp. 2665- 2673.

[12]   A. R. Datta, B. A. Wentz, D. Shook and M. W. Trucksess, “Synthetic Oligodeoxyribonucleotide Probes for Detection of Listeria monocytogenes,” Applied and Environmental Microbiology, Vol. 54, 1988, pp. 2933-2937.

[13]   M. V. Ramana, S. Chandrayanaka, K. Balakrishna, H. S. Murali and H. V. Batra, “A Novel PCR-DNA Probe for the Detection of Fumonisin Producing Fusarium Species from Major Food Crops Grown in Ssouthern India” Mycology: An International Journal on Fungal Biology, Vol. 3, 2012, pp. 167-220.

[14]   M. V. Ramana, K. Balakrishna, H. S. Murali and H. V. Batra, “Multiplex PCR-Based Strategy to Detect Con- tamination with Mycotoxigenic Fusarium Species in Rice and Fingermillet Collected from Southern INDIA,” Jour- nal of the Science of Food and Agriculture, Vol. 91, No. 9, 2011, pp. 1666-73.

[15]   J. Stroka, E. Anklam, U. Jorissen and J. Gilbert, “Immuno-Affinity Column Clean-Up with Liquid Chromatography Post Column Bromination for Determination of Aflatoxins in Peanut Butter, Pistachio Paste, Fig Paste and Paprika Powder: Collaborative Study,” Journal of Association of Analytical Communication and Interaction, Vol. 83, No. 2, 2000, pp. 320-340.

[16]   I. Ferreira, E. Mendes and M. Oliveira, “Quantification of Aflatoxins B1, B2, G1, and G2 in Pepper by HPLC/Fluorescence,” Journal of Liquid of Chromatography, Vol. 27, No. 2, 2004, pp. 325-334.

[17]   J. I. Pitt, R. A. Samson and J. C. Frisvad “List of Accepted Species and Their Teleomorphs in the Family Trichocomaceae,” In: R. A. Samson and J. I. Pitt, Eds., Integration of Modern Taxonomic Methods for Penicillium and Aspergillus, Harwood Academic Publishers, Amsterdam, 2000, pp. 9-47.

[18]   G. A. Payne and M. P. Brown, “Genetics and Physiology of Aflatoxin Biosynthesis,” Annual Review of Phytopathology, Vol. 36, 1998, pp. 329-362.

[19]   J. Yu, J. W. Cary, D. Bhatnagar, T. E. Cleveland, N. P. Keller and F. S. Chu, “Cloning and Characterization of a cDNA from Aspergillus parasiticus Encoding an O-Methyl-Transferase Involved in Aflatoxin Biosynthesis,” Applied and Environmental Microbiology, Vol. 59, No. 11, 1993, pp. 564-3571.

[20]   F. Trail, N. Mahanti, M. Rarick, M. Mehigh, S. H. Liang, R. Zhou and J. E. Linz, “Physical and Transcriptional Map of an Aflatoxin Gene Cluster in Aspergillus parasiticus and Functional Disruption of a Gene Involved Early in the Aflatoxin Pathway,” Applied and Environmental Microbiology, Vol. 61, No. 7, 1995, pp. 2665-2673.

[21]   G. S. Sandhu, B. Kline, L. Stockman and G. D. Roberts, “Molecular Probes for Diagnosis of Fungal Infections,” Journal of Clinical Microbiology, Vol. 33, No. 11, 1995, pp. 2913-2919.

[22]   J. L. Jones, “DNA Probes: Application in the Food Industry,” Trends in Food Science and Technology, Vol. 2, 1991, pp. 28-32.

[23]   C. E. McAlpin and B. Mannarelli, “Construction and Characterization of a DNA Probe for Distinguishing Strains of Aspergillus ?avus,” Applied and Environmental Microbiology, Vol. 61, No. 3, 1995, pp. 1068-1072.

[24]   M. Z. Abdin, M. Malik and A. J. Saleem, “Advances in Molecular Detection of Aspergillus: An Update,” Archives of Microbiology, Vol. 192, No. 6, 2010, pp. 409- 425.

[25]   C. A. Levesque, C. E. Harlton and A. W. A. M. De-Cock, “Identification of Some Oomycetes by Reverse Dotblot Hybridization,” Phytopathology, Vol. 88, No. 9, 1998, pp. 213-222.

[26]   G. R. Janardhana, K. A. Raveesha and H. S. Shetty, “Mycotoxin Contamination of Maize Grains Grown in Karnataka (India),” Food and Chemical Toxicology, Vol. 37, No. 8, 1999, pp. 863-868. doi.:10.1016/S0278-6915(99)00067-8

[27]   S. V. Reddy, D. K. Mayi, M. U. Reddy, K. Thirumala-Devi and D. V. R. Reddy, “Aflatoxins B1 in Different Grades of Chilies (Capsicum annum L.) in India as Determined by Indirect Competitive ELISA,” Food Additives and Contaminants, Vol. 18, No. 6, 2001, pp. 553-558.

[28]   M. Logotheti, A. Kotsovili-Tseleni, G. Arsenis and N. I. Legakis, “Multiplex PCR for the Discrimination of A. fumigatus, A. flavus, A. niger and A. terreus,” Journal of Microbiological Methods, Vol. 76, 2009, pp. 209-211.

[29]   D. Sartori, M. C. Furlaneto, M. K. Martins, M. R. F. Paula, A. Pizzirani-Kleiner, M. H. Taniwaki and M. H. P. Fungaro, “PCR Method for the Detection of Potential Ochratoxin-Producing Aspergillus Species in Coffee Beans,” Research in Microbiology, Vol.157, No. 4, 2006, pp. 350-354. doi.org/10.1016/j.resmic.2005.09.008

 
 
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