title: Evaluation of a multiplex PCR assay for concurrent detection of four major mycotoxigenic fungi from foods.
abstract: OBJECTIVE
  To develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of four major mycotoxin metabolic pathway genes, viz. nor1 (aflatoxin), Tri6 (trichothecene), FUM13 (fumonisin) and otanps (ochratoxin A).
  
  
  RESULTS
  A mPCR assay with competitive internal amplification control, employing specific primers for each of the aforementioned four genes, was optimized and validated using 10 reference strains and 60 pure culture isolates. The standardized mPCR assay detected all four mycotoxin metabolic genes in artificially contaminated maize samples with a sensitivity of 2 × 10(3) CFU g(-1) for nor1-positive Aspergillus strains, Tri6 and FUM13-positive Fusarium strains and 2 × 10(4) CFU g(-1) for otanps-positive Penicillium strains. When the developed mPCR assay was applied to 40 natural foods, 35% (14 of 40) of the samples were contaminated with either one or more mycotoxins. The mPCR results were further evaluated with high-performance liquid chromatography (HPLC), and in general, both the methods provided unequivocal results.
  
  
  CONCLUSIONS
  The current mPCR assay is a rapid and reliable tool for simultaneous specific and sensitive detection of aflatoxigenic Aspergillus strains, trichothecene- and fumonisin-producing Fusarium strains, and ochratoxigenic Penicillium species from naturally contaminated foods.
  
  
  CONCLUSIONS
  This mPCR assay could be a supplementary strategy to current conventional mycotoxin analytical techniques such as thin-layer chromatography (TLC), high performance thin layer chromatography, HPLC, etc., and a reliable tool for high-throughput monitoring of major mycotoxin-producing fungi during the processing steps of food and feed commodities.
authors.affiliation: Microbiology Division, Defence Food Research Laboratory, Mysore, Karnataka, India.
authors.forename: R
authors.initials: R
authors.lastname: Rashmi
authors.forename: M V
authors.initials: MV
authors.lastname: Ramana
authors.forename: R
authors.initials: R
authors.lastname: Shylaja
authors.forename: S R
authors.initials: SR
authors.lastname: Uppalapati
authors.forename: H S
authors.initials: HS
authors.lastname: Murali
authors.forename: H V
authors.initials: HV
authors.lastname: Batra
mesh_terms: D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology / Q000379:methods*; D037341:Fumonisins / Q000032:analysis; D005670:Fusarium / Q000235:genetics / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009183:Mycotoxins / Q000032:analysis; D009793:Ochratoxins / Q000032:analysis; D010407:Penicillium / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014255:Trichothecenes / Q000032:analysis
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D037341:Fumonisins; D009183:Mycotoxins; D009793:Ochratoxins; D014255:Trichothecenes; C025589:ochratoxin A