Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Sep;62(9):3270–3273. doi: 10.1128/aem.62.9.3270-3273.1996

Detection of aflatoxigenic molds in grains by PCR.

R Shapira 1, N Paster 1, O Eyal 1, M Menasherov 1, A Mett 1, R Salomon 1
PMCID: PMC168121  PMID: 8795215

Abstract

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillus flavus group in grains and floods. Three genes, ver-1, omt-1, and apa-2, coding for key enzymes and a regulatory factor in aflatoxin biosynthesis, respectively, have been identified, and their DNA sequences have been published. In the present study, three primer pairs, each complementing the coding portion of one of the genes, were generated. DNA extracted from mycelia of five Aspergillus species, four Penicillium species, and two Fusarium species was used as PCR template for each of the primer pairs. DNA extracted from peanut, corn, and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds Aspergillus parasiticus and A. flavus in all three primer pairs. The detection limit of the PCR was determined by using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were obtained only after a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10(2) spores per g). No DNA spores per g). It is concluded that genes involved in the aflatoxin biosynthetic pathway may form the basis for an accurate, sensitive, and specific detection system, using PCR, for aflatoxigenic strains in grains and foods.

Full Text

The Full Text of this article is available as a PDF (679.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnheim N., Erlich H. Polymerase chain reaction strategy. Annu Rev Biochem. 1992;61:131–156. doi: 10.1146/annurev.bi.61.070192.001023. [DOI] [PubMed] [Google Scholar]
  2. Atmar R. L., Metcalf T. G., Neill F. H., Estes M. K. Detection of enteric viruses in oysters by using the polymerase chain reaction. Appl Environ Microbiol. 1993 Feb;59(2):631–635. doi: 10.1128/aem.59.2.631-635.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang P. K., Cary J. W., Bhatnagar D., Cleveland T. E., Bennett J. W., Linz J. E., Woloshuk C. P., Payne G. A. Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis. Appl Environ Microbiol. 1993 Oct;59(10):3273–3279. doi: 10.1128/aem.59.10.3273-3279.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang P. K., Skory C. D., Linz J. E. Cloning of a gene associated with aflatoxin B1 biosynthesis in Aspergillus parasiticus. Curr Genet. 1992 Mar;21(3):231–233. doi: 10.1007/BF00336846. [DOI] [PubMed] [Google Scholar]
  5. Ellis W. O., Smith J. P., Simpson B. K., Oldham J. H. Aflatoxins in food: occurrence, biosynthesis, effects on organisms, detection, and methods of control. Crit Rev Food Sci Nutr. 1991;30(4):403–439. doi: 10.1080/10408399109527551. [DOI] [PubMed] [Google Scholar]
  6. Kaji H., Xu Y., Takahashi Y., Abe H., Tamaki N., Chihara K. Human TRH receptor messenger ribonucleic acid levels in normal and adenomatous pituitary: analysis by the competitive reverse transcription polymerase chain reaction method. Clin Endocrinol (Oxf) 1995 Mar;42(3):243–248. doi: 10.1111/j.1365-2265.1995.tb01871.x. [DOI] [PubMed] [Google Scholar]
  7. Kapperud G., Vardund T., Skjerve E., Hornes E., Michaelsen T. E. Detection of pathogenic Yersinia enterocolitica in foods and water by immunomagnetic separation, nested polymerase chain reactions, and colorimetric detection of amplified DNA. Appl Environ Microbiol. 1993 Sep;59(9):2938–2944. doi: 10.1128/aem.59.9.2938-2944.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. McAlpin C. E., Mannarelli B. Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus. Appl Environ Microbiol. 1995 Mar;61(3):1068–1072. doi: 10.1128/aem.61.3.1068-1072.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Payne G. A., Nystrom G. J., Bhatnagar D., Cleveland T. E., Woloshuk C. P. Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl Environ Microbiol. 1993 Jan;59(1):156–162. doi: 10.1128/aem.59.1.156-162.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Payne G. A., Woloshuk C. P. Transformation of Aspergillus flavus to study aflatoxin biosynthesis. Mycopathologia. 1989 Sep;107(2-3):139–144. doi: 10.1007/BF00707551. [DOI] [PubMed] [Google Scholar]
  11. Skory C. D., Chang P. K., Cary J., Linz J. E. Isolation and characterization of a gene from Aspergillus parasiticus associated with the conversion of versicolorin A to sterigmatocystin in aflatoxin biosynthesis. Appl Environ Microbiol. 1992 Nov;58(11):3527–3537. doi: 10.1128/aem.58.11.3527-3537.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Yu J., Cary J. W., Bhatnagar D., Cleveland T. E., Keller N. P., Chu F. S. Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis. Appl Environ Microbiol. 1993 Nov;59(11):3564–3571. doi: 10.1128/aem.59.11.3564-3571.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Yu J., Chang P. K., Cary J. W., Wright M., Bhatnagar D., Cleveland T. E., Payne G. A., Linz J. E. Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus. Appl Environ Microbiol. 1995 Jun;61(6):2365–2371. doi: 10.1128/aem.61.6.2365-2371.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES