DETECTION OF SALMONELLA TYPHIMURIUM IN LIVESTOCK PRODUCTS BY PCR TECHNIQUE
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Date
2007-09
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT :
The present study was undertaken to standardize PCR assay for detection of Salmonella spp. and Salmonella typhimurium from livestock products and compare its efficacy with conventional cultural methods. Two sets of primers derived from invA and fliC genes were used, which gave specific amplification products of 389bp and 620bp for Samonella spp. and Salmonella typhimurium respectively. To determine their suitability to PCR, four different template preparation methods viz. genomic DNA extraction, heat lysis, and two lysis buffers were compared, of which heat lysis was found to be efficient and convenient. The specificity for Salmonella spp was tested using primers from invA gene with five strains of Salmonella and 6 other non salmonella strains, which gave a specific 389bp product for all Salmonella strains only. Primers from fliC gene gave a specific 620bp product only for Salmonella typhimurium when tested with five strains of Salmonella. The minimum detection level with pure Salmonella typhimurium culture was found to be 3.7cfu/ml. Evaluation of two non selective broths (Buffered Peptone Water and Brain Heart Infusion agar) and four selective enrichment broths (Rappaport vassiliadis, Tetrathionate, Selenite Cystine, Selenite F broths) for PCR compatibility, it revealed that BHI, BPW and TT were superior followed by SF, RV and SC. Spiking studies were carried out by inoculating with pure culture of Salmonella typhimurium in BPW pre enrichment broth (8h and 16h) followed by selective enrichment in four different selective broths(12h and 18h), which revealed that earliest detection time by PCR of Salmonella was 26h(8h pre enrichment+18h selective enrichment). TT was the most suited selective broth for PCR assay. Screening of 262 naturally contaminated samples (milk, meat, poultry feed, cloacal, faecal, eggs and fish) revealed 78 and 97 positive for Salmonella spp. by Cultural and PCR methods respectively. Out of 97 positive for Salmonella spp. by PCR, it has given 15 positive for Salmonella typhimurium.
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