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Subject: Veterinary Public Health × Author: SIVA SWETHA, CHINTA × End date: 2009 × Start date: 2009 × Type: Thesis × Thesis Type: M.V.Sc. ×
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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.