DEVELOPMENT OF HIGH THROUGHPUT REAL-TIME PCR ARRAY TECHNIQUE FOR SCREENING OF FOOD SAMPLES FOR MYCOTOXIN PRODUCING FUNGI CONTAMINATIONK.K. USSUF V. PAREKH AND A.K. SHARMA
A high throughput PCR array technique has been developed for the simultaneous detection of several mycotoxin-producing fungi by using Real - Time PCR. The primers were designed based on the key genes involved in the different mycotoxin producing biosynthetic pathway of aflatoxin, Patulin, Trichothecene, Fumonisins and Ochratoxin A with a uniform Tm (Melting Temperature) to use in a single array simultaneously. These primers were arranged in an array form for the simultaneous detection of 14 mycotoxin-producing fungi in a single reaction set up. The method was validated by using the standard authentic DNA isolated from different mycotoxin producing fungi and real - Time PCR was performed by using SYBR Green 1 fluorescent dye method. The validated protocol was employed for screening the commercially available food samples by using the DNA isolated from the collected food samples. The results from these studies have shown that several commercially available food samples are contaminated with mycotoxin producing fungi. Thus this high throughput Real - Time PCR technology can be employed for the screening of food samples for detecting the contaminating fungi.
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