A multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus

Aims: To develop a multiplex reverse transciption-polymerase chain reaction (RT-PCR) protocol to discriminate aflatoxin-producing from aflatoxin-nonpro-ducing strains of Aspergillus flavus . Methods and Results: The protocol was first optimized on a set of strains obtained from laboratory collections and then validated on A. flavus strains isolated from corn grains collected in the fields of the Po Valley (Italy). Five genes of the aflatoxin gene cluster of A. flavus , two regulatory ( aflR and aflS ) and three structural ( aflD , aflO and aflQ ), were targeted with specific primers to highlight their expression in mycelia cultivated under inducing conditions for aflatoxins production. 48-h-old cultures expressed the complete set of the genes analysed here whereas 24-h-old ones did not. Genomic PCR (quadruplex PCR) was also performed in parallel using chromosomal DNA extracted from the same set of strains to correlate the integrity of the genes with their expression. Conclusions: We show that a good correlation exists between gene expression of the aflatoxin genes, here analysed by multipex RT-PCR, and aflatoxin production, except for one strain that apparently transcribed all the relevant genes but did not produce aflatoxin in the medium.

[1]  R. Russell M. Paterson,et al.  Identification and quantification of mycotoxigenic fungi by PCR , 2006 .

[2]  Deepak Bhatnagar,et al.  The aflatoxin pathway regulator AflR induces gene transcription inside and outside of the aflatoxin biosynthetic cluster. , 2006, FEMS microbiology letters.

[3]  A. Chandrashekar,et al.  Detection of aflatoxigenic fungi in selected food commodities by PCR , 2005 .

[4]  Q. Migheli,et al.  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. , 2005, International journal of food microbiology.

[5]  A. Chandrashekar,et al.  Isolation, enumeration and PCR characterization of aflatoxigenic fungi from food and feed samples in India , 2004 .

[6]  John E. Linz,et al.  Clustered Pathway Genes in Aflatoxin Biosynthesis , 2004, Applied and Environmental Microbiology.

[7]  P. Chang.  Lack of interaction between AFLR and AFLJ contributes to nonaflatoxigenicity of Aspergillus sojae. , 2004, Journal of biotechnology.

[8]  Jens Christian Frisvad,et al.  Introduction to food- and airborne fungi. , 2004 .

[9]  R. Geisen,et al.  Quantification of the copy number of nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its correlation to the cfu of Aspergillus flavus in foods. , 2003, International journal of food microbiology.

[10]  P. Chang.  The Aspergillus parasiticus protein AFLJ interacts with the aflatoxin pathway-specific regulator AFLR , 2003, Molecular Genetics and Genomics.

[11]  R. Geisen,et al.  Monitoring the Production of Aflatoxin B1 in Wheat by Measuring the Concentration of nor-1 mRNA , 2003, Applied and Environmental Microbiology.

[12]  K. Ehrlich,et al.  Sequence comparison of aflR from different Aspergillus species provides evidence for variability in regulation of aflatoxin production. , 2003, Fungal genetics and biology : FG & B.

[13]  Richard A. Wilson,et al.  Relationship between Secondary Metabolism and Fungal Development , 2002, Microbiology and Molecular Biology Reviews.

[14]  Jiujiang Yu,et al.  Nonfunctionality of Aspergillus sojae aflR in a Strain of Aspergillus parasiticus with a Disrupted aflR Gene , 2002, Applied and Environmental Microbiology.

[15]  G. Criseo,et al.  Differentiation of aflatoxin‐producing and non‐producing strains of Aspergillus flavus group , 2001, Letters in applied microbiology.

[16]  A. Dobson,et al.  The use of reverse transcription-polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439. , 2000, International journal of food microbiology.

[17]  Biing-Hui Liu,et al.  Regulation of aflR and Its Product, AflR, Associated with Aflatoxin Biosynthesis , 1998, Applied and Environmental Microbiology.

[18]  G. Payne,et al.  Characterization of aflJ, a Gene Required for Conversion of Pathway Intermediates to Aflatoxin , 1998, Applied and Environmental Microbiology.

[19]  G. Payne,et al.  Overexpression of aflR Leads to Upregulation of Pathway Gene Transcription and Increased Aflatoxin Production in Aspergillus flavus , 1997, Applied and environmental microbiology.

[20]  W. Holzapfel,et al.  Detection of aflatoxinogenic fungi in figs by a PCR reaction. , 1997, International journal of food microbiology.

[21]  N. Paster,et al.  Detection of aflatoxigenic molds in grains by PCR , 1996, Applied and environmental microbiology.

[22]  John E. Linz,et al.  Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus , 1995, Applied and environmental microbiology.

[23]  J. Bennett,et al.  Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi , 1995, Applied and environmental microbiology.

[24]  G. Payne,et al.  Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis , 1994, Applied and environmental microbiology.

[25]  G. Payne,et al.  Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus , 1993, Applied and environmental microbiology.

[26]  U. L. Diener,et al.  Improved method of screening for aflatoxin with a coconut agar medium , 1987, Applied and environmental microbiology.

[27]  S. Arseculeratne,et al.  Coconut as a medium for the experimental production of aflatoxin. , 1969, Applied microbiology.