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Subject: Veterinary Public Health × Author: SUBHASHINI, NELAPATI × End date: 2009 × Start date: 2003 × Type: Thesis ×
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    ThesisItemOpen Access
    DETECTION OF VIBRIO PARAHAEMOLYTICUS IN FOODS BY PCR TECHNIQUE
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-09) SUBHASHINI, NELAPATI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, Y
    ABSTRACT : The present study was undertaken to standardize PCR assay for detection of Vibrio parahaemolyticus and their toxins from foods and compare its efficacy with conventional cultural methods. Primers derived from toxR gene and tdh and trh genes were used which gave specific amplification products of 368, 623 and 460 bp for V. parahaemolyticus and toxins (tdh and trh) respectively. To determine their suitability to PCR, four different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffers-1 and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for V. parahaemolyticus and toxins were tested using primers from toxR, tdh and trh genes with V. parahaemolyticus and nine other non-V. parahaemolyticus strains, which gave specific products at 368, 623 and 460 bp for V. parahaemolyticus and toxins respectively. The minimum detection level was 2.5 cfu for both V. parahaemolyticus and toxins. Evaluation of two non-selective broths, i.e Brain Heart Infusion Broth (BHI) and Tryptic Soy Broth (TSB) and two selective broths, i.e Alkaline Peptone Water Broth (APW) and Salt Polymixin Broth (SPB) for PCR compatibility, it was revealed that SPB was superior followed by APW, BHI and TSB. Spiking studies were carried out by inoculating with pure culture of V. parahaemolyticus in selective enrichments broths (8h and 18h), which revealed that earliest detection time for V. parahaemolyticus by PCR was 18h and SPB was the most suited selective broth for PCR assay. Screening of 280 naturally contaminated samples (35 each of fresh water fish, sea fish, fresh water prawn, marine shrimp, crab, fresh water, marine water and mutton samples) revealed 165 positive for V. parahaemolyticus (fresh water fish-17, sea fish-20, fresh water prawn-24, marine shrimp-33, crab-27, fresh water-14, marine water-28 and mutton-2) out of which 24 carried only tdh (fresh water fish-2, sea fish-4, fresh water prawn-3, marine shrimp-4, crab-4, fresh water-2, marine water-5 and mutton-nil), 95 carried only trh (fresh water fish-12, sea fish-16, fresh water prawn-9, marine shrimp-13, crab-15, fresh water-12, marine water-18 and mutton-nil) and 15 carried both tdh and trh (fresh water fish-1, sea fish-2, fresh water prawn-1, marine shrimp-3, crab-2, fresh water-2, marine water-4 and mutton-nil) simultaneously.