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Author: SHASHIKALA, SANKARAMADDI × End date: 2009 × Type: Thesis ×
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    ThesisItemOpen Access
    DETECTION OF YERSINIA ENTEROCOLITICA IN LIVESTOCK PRODUCTS BY PCR TECHNIQUE
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-09) SHASHIKALA, SANKARAMADDI; KRISHNAIAH, N(MAJOR); MADHAVA RAO, T; GOPALA REDDY, A
    ABSTRACT : The present study was undertaken to standardize PCR assay for detection of Yersinia enterocolitica and Yersinia heat stable enterotoxin from livestock products and compare its efficacy with conventional cultural methods. Primers derived from ail gene and yst B genes used which gave specific amplification products of 425 and 146bp for Y. enterocolitica and yersinia heat stable enterotoxin (yst B) respectively. To determine their suitability to PCR, three different template preparation methods viz. heat lysis, lysis buffer-1and 2 were compared, of which heat lysis was found to be efficient and convenient. The specificity for Y. enterocolitica and yersinia heat stable enterotoxin were tested using primers from ail and yst B genes with Y. enterocolitica and seven other non-Y. enterocolitica strains, which gave specific products at 425 and 146bp for Y. enterocolitica and Yersinia heat stable enterotoxin respecitvely. The minimum detection level with pure Y. enterocolitica culture was 2.0 cfu. Evaluation of two non-selective broths (Tryptic Soya broth and Brain heart infusion broth) and two selective broths (Yersinia selective enrichment broth and Irgasan-ticarcillin-potassium chloride broth) for PCR compatibility, it revealed that ITC was superior followed by YSE, BHI and TSB. Spiking studies were carried out by inoculating with pure culture for Y. enterocolitica in selective enrichment broths (24hr), which revealed that earliest detection time by PCR of Y. enterocolitica was 24 hr and ITC was the most suited selective broth for PCR assay. Screening 300 naturally contaminated samples (25 each of pork, pork tonsils, pork tongues, pork surface swabs, swine faeces, swine farm water, milk, beef, cattle faeces, dairy farm water, chicken and poultry farm water samples) revealed 132 (14, 23, 23, 10, 9, 21, 4, 5, 4, 8, 6 and 5 respectively for the above samples) positive for Y. enterocolitica, out of which 56 samples (5,10,9,4,2,9,2,2,1,5,4 and 3 respectively) were positive for yst B gene.