Thumbnail Image

Thesis

Browse

20081
Reset filters
Author: BINDU KIRANMAYI, Ch × Start date: 2003 × Type: Thesis ×
Reset filters

Search Results

Now showing 1 - 1 of 1
  • Thumbnail Image
    ThesisItemOpen Access
    DETECTION OF ESCHERICHIA COLI O157:H7 FROM LIVESTOCK PRODUCTS BY PCR TECHNIQUE
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2008-10) BINDU KIRANMAYI, Ch; KRISHNAIAH, N(MAJOR); VENKATESWARA RAO, L; DHANA LAKSHMI, K
    ABSTRACT: The present study was undertaken to standardize PCR assay for detection of Escherichia coli O157:H7 and Shiga-Toxigenic Escherichia coli (STEC) from livestock products and compare its efficacy with conventional cultural methods. Primers derived from HlyA gene and Stx1 and Stx2 genes were used which gave specific amplification products of 361, 614 and 779 bp for Escherichia coli O157:H7 and STEC (Stx1 and Stx2) 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 Escherichia coli O157:H7 and STEC were tested using primers from HlyA, Stx1 and Stx2 genes with Escherichia coli O157:H7 and nine other non-Escherichia coli O157:H7 strains, which gave specific products at 361, 614 and 779 bp for Escherichia coli O157:H7 and STEC respectively. The minimum detection level with pure Escherichia coli O157:H7 culture was found 1.7 cfu. Evaluation of two non-selective broths (Brain Heart Infusion broth and Buffered Peptone Water) and two selective broths (modified E.coli broth and modified Tryptic Soy Broth) for PCR compatibility, it revealed that mTSB was superior followed by mEC, BHI and BPW. Spiking studies were carried out by inoculating with pure culture of Escherichia coli O157:H7 in selective enrichments broths (10h and 18h), which revealed that earliest detection time by PCR of Escherichia coli O157:H7 was 18h and mTSB was the most suited selective broth for PCR assay. Screening 600 naturally contaminated samples (50 each of mutton, mutton swabs, sheep faeces, sheep farm water, beef, beef swabs, beef and dairy cattle faeces, milk, dairy farm water, chicken and poultry farm water samples) revealed 110 positive for Escherichia coli O157:H7 out of which 40 carried only Stx1, 18 carried only Stx2 and 9 carried both Stx1 and Stx2 simultaneously. Out of 600 samples 248 samples were positive for STEC of which 178 were Stx1, 70 Stx2 and 40 Stx1 and Stx2 simultaneously.