Aim: 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). Methods and 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 9 10 3 CFU g À1 for nor1-positive Aspergillus strains, Tri6 and FUM13-positive Fusarium strains and 2 9 10 4 CFU g À1 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.
Conclusion: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. Significance and Impact of the Study: 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.
Staphylococcal enterotoxin B (SEB), toxic shock syndrome, the superantigens are responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. An easy and quick system is desirable to detect toxin‐producing strains. In this report, we described standardization of a novel multiplex‐polymerase chain reaction (PCR) assay for simultaneous detection of four important genes associated with the Staphylococcus aureus viz., SEB, Tsst, genus‐specific nuclease (nuc) and Fem genes along with an internal amplification control (IAC), which has now become mandatory in diagnostic PCRs, particularly when tested on environmental or food samples. This mPCR method is sensitive enough to detect cells as low as 103 cfu/mL or /g of the food samples. When evaluated on 136 food and environmental samples, the system detected 4 SEB‐positive S. aureus strains. The S. aureus strains that have been identified to contain the SEB gene in the mPCR were unequivocally detected for the toxin expression by the TECRA kit. mPCR produced a 100% correlation with conventional identification method. As SEB and Tsst are qualified as biowarfare molecules, this system is of immense help in detecting them during emergencies of biological war and suspected outbreaks of S. aureus food poisoning directly from the food and environmental samples.
PRACTICAL APPLICATIONS
The virulence‐associated genes targeted in this study are super antigenic in nature and are responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome, and these molecules are also qualified as biowarfare agents. The high throughput and cost‐effective multiplex PCR method reported here could identify all these genes successfully from both artificially spiked and natural food samples and may also find its application in detection of these toxin‐producing Staphylococcus aureus from clinical and environmental samples.
Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the conserved regions of seb and the tsst genes was codonoptimized for expression in Escherichia coli and encoded a 26 kDa recombinant multidomain chimeric protein (r-TE). Hyperimmune antiserum raised against r-TE specifically reacted with SEB (*28 kDa) and Tsst-1 (*22 kDa) components during Western blot analysis and by plate ELISA in confirmed toxin producing strains of S. aureus. The antigenicity of the SEB component of the r-TE protein was also confirmed using TECRA kit. The described procedure of creating a single protein molecule carrying components of two different toxins whilst still retaining the original antigenic determinants of individual toxins proved highly advantageous in the development of rapid, reliable and cost effective immunoassays and may also have the potential to serve as candidate molecule for vaccine studies.
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