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Subject: Veterinary Public Health × End date: 2009 × Start date: 2009 × Type: Thesis × Thesis Type: M.V.Sc. ×
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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.
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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.
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    ThesisItemOpen Access
    DETECTION OF LISTERIA MONOCYTOGENES IN LIVESTOCK PRODUCTS BY PCR
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2009-09) SIVA SWETHA, CHINTA; MADHAVA RAO, T(MAJOR); KRISHNAIAH, N; NARASIMHA REDDY, Y
    ABSTRACT : The present study was undertaken to standardize PCR assay for detection of Listeria monocytogenes and Listeriolysin O from livestock products and compare its efficacy with conventional cultural methods. Primers derived from iap and hlyA genes were used which gave specific amplification products of 131 and 456 bp for L. monocytogenes and Listeriolysin O respectively. To determine their suitability to PCR, three different template preparation methods viz. genomic DNA extraction, heat lysis, lysis buffer method were compared, of which heat lysis was found to be efficient and convenient. The specificity of L.monocytogenes and Listeriolysin O were tested using primers from iap and hlyA genes with L.monocytogenes and seven other non-Listeria monocytogenes strains, which gave specific products 131 and 456 bp for L.monocytogenes and Listeriolysin O respectively. The minimum detection level with pure L. monocytogenes culture was found 4 cfu. Evaluation of two non-selective broths (Brain Heart Infusion broth and Tryptone Soya Broth) and two selective broths (LEB and PALCAM) for PCR compatibility, it revealed that LEB was superior followed by PALCAM, BHI and TSB. Spiking studies were carried out by inoculating with pure culture of L.monocytogenes in selective enrichment broths (18 h and 24 h), which revealed that the earliest detection time by PCR for L.monocytogenes was 24 hr and LEB was the most suited selective broth for PCR assay. Screening 200 naturally contaminated samples (25 of milk, 15 of ice cream, 10 of dairy farm water, 25 each of pork, pork swabs, chicken, chicken swabs, fish, fish swabs samples) revealed 23 positive results for L.monocytogenes and Listeriolysin O by PCR.