PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize.
Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.
Somashekar
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Food Microbiology Department, Central Food Technological Research Institute, Mysore-570 013, Karnataka, India.
Rati
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ER
Chandrashekar
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A
D001230:Aspergillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001231:Aspergillus flavus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000032:analysis; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis; D005656:Fungal Proteins / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013172:Spores, Fungal; D013997:Time Factors; D014157:Transcription Factors / Q000302:isolation & purification; D003313:Zea mays / Q000382:microbiology*
C113837:AFLR protein, Aspergillus; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors