ISOLATION, IDENTIFICATION AND DIFFERENTIATION OF COMMON VIBRIO SPECIES IN SEA FOOD SAMPLES.
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Date
42837
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PVNR TVU AND HYDERABAD
Abstract
The present study was undertaken to standardize Multiplex PCR assay for detection and differentiation of Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus from sea foods and compare with conventional cultural methods.
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 Vibrios was tested using primers from gyrB and pntA genes for differentiation of Vibrio species using 4 vibrio spp. and 6 other organisms which gave a specific 493/338, 493/409 and 493/656 bp products for Vibrio cholera, Vibrio parahaemolyticus
and Vibrio vulnificus respectively. The minimum detection level with all three pure Vibrio spp cultures was found to be 10 CFU/ml.
APW and SPB enrichment broths were evaluated for m-PCR compatibility; it revealed that APW broth was superior for V.cholerae, while SPB was superior for V.parahaemolyticus and V.vulnificus. Spiking studies were carried out by inoculating with pure culture; it revealed that 18h incubation was better than 8h. The sensitivity of Hichrome and TCBS Agar was 100 and 94.26% respectively for Vibrio organisms by m-PCR technique.
Out of 200 naturally contaminated food samples (40 each of fish, crabs, prawns, oysters and snails) 140 were positive for Vibrio spp by m-PCR, out of which 17, 70 and 16 were positive for V.cholerae, V.parahaemolyticus and V.vulnificus respectively, whereas cultural methods gave 132 positive for vibrio species. Out of 17 positives for V.cholerae by m-PCR, 3, 3, 6, 5 and 0 were from fish, crabs, prawns, oysters and snails respectively. Out of 70 positives for V.parahaemolyticus, 19, 14, 12, 12 and 13 and out of 16 m-PCR positives for V.vulnificus, 6, 3, 4, 3 and 0 are from the above samples respectively.
The mean counts of V.cholerae, V.parahaemolyticus and V.vulnificus were 7.1 x105, 1.07x103 and 5.8x102 in fish, 1.3x106, 6.8x104 and 3.9x102 in crabs, 1.14 x105, 2.18x105 and 2.5x102 in prawns and 4.2x103, 5.01x104 and 4.3x104 in oysters, 0, 2.8x103 and 0 in snails respectively.
V.cholera was highly sensitive to Streptomycin and Gentamicin (100%), followed by Amikacin (93%), Ciprofloxacin and Cotrimoxazole (67%) and Ceftriazone (53%) and highly resistant to Ampicillin (82%) followed by Nalidixic acid (66%), Chloramphenicol (38%), Ceftriazone (26%) and Cephalexin (24%). V.parahaemolyticus was highly sensitive to Ciprofloxacin (100%), followed by Amikacin (95%), Ceftriazone (90%), Gentamicin( 89%) , Streptomycin (88%) and Chloramphenicol (40%), whereas highly resistant to Nalidixic acid (65%) followed by Cephalexin(58%) and Ampicillin (53%). V.vulnificus was highly sensitive to Gentamicin (100%), followed by Amikacin and Ciprofloxacin (98%), Ceftriazone and Cephalexin (90%), Ampicillin (80%), Streptomycin (76%), Chloramphenicol (68%), Nalidixic acid (58%) and Cotrimoxazole (40%).
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