Thumbnail Image

Thesis

Browse

20101
Reset filters
Subject: Veterinary Public Health × Author: MAHESHWARI, MANI × End date: 2010 × Start date: 2000 × Type: Thesis ×
Reset filters

Search Results

Now showing 1 - 1 of 1
  • Thumbnail Image
    ThesisItemOpen Access
    DETECTION OF VIBRIO CHOLERAE IN FOODS BY PCR TECHNIQUE
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2010-06) MAHESHWARI, MANI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; ANJANEYULU, Y
    ABSTRACT: The present study was undertaken to standardize PCR assay for detection of Vibrio cholerae, its serogroups (O1 and O139) and cholera toxin from foods and compare its efficacy with conventional cultural methods. Primers derived from ompW gene, rfbO1 and rfbO139 genes and ctxA gene were used which gave specific amplification products of 304, 192, 449, 302 bp for V.cholerae, its serogroups (O1 and O139) and cholera toxin 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.cholerae, its serogroups (O1 and O139) and cholera toxin was tested using primers from ompW, rfbO1, rfbO139 and ctxA genes with V.cholerae and nine other non-V.cholerae strains, which gave specific products at 304, 192, 449 and 302 bp for V.cholerae, its serogroups and cholera toxin respectively. The minimum detection level was 2.5 cfu for V.cholerae, its serogroups and cholera toxin. Evaluation of two non-selective broths, i.e Luria Broth (LB) and Tryptic Soy Broth (TSB) and two selective broths, i.e Alkaline Peptone Water Broth (APW) and Salt Polymyxin Broth (SPB) for PCR compatibility, it was revealed that APW was superior followed by SPB, LB and TSB. Spiking studies were carried out by inoculating with pure culture of V.cholerae in selective enrichments broths (8h and 18h), which revealed that earliest detection time for V.cholerae by PCR was 18h and APW was the most suited selective broth for PCR assay. Screening of 245 naturally contaminated samples (35 each of water, fish, crab, shrimp, meat, milk and clinical stool samples) revealed 80 positive for V.cholerae (water-19, fish-16, crab-20, shrimp-6, meat-4, milk-3, and clinical stool samplres-12) out of which 45 belonged to O1 serogroup (water 11, fish-9, crab-12, shrimp-3, meat-2, milk-1 and clinical stool samples-7), 25 belonged to O139 (water-6, fish-5, crab-6, shrimp-2, meat-1, milk-1 and clinical stool samples-4). Out of 45 and 25 positive samples for O1 and O139, 35 and 20 were positive for cholera toxin respectively.