Showing papers by "Chaitanya G. Joshi published in 2007"

Use of cytochrome b gene variability in detecting meat species by multiplex PCR assay

Abstract: ‘Use of cytochrome b gene variability in detecting meat species by multiplex PCR’ Name of student Major Advisor Shally Jain. Dr.M. N. Brahmbhatt Assistant Professor Department of Veterinary Public Health College of Veterinary Science and Animal Husbandry Anand Agricultural University Anand 388001 Food authenticity is currently an issue of major concern for food authorities, since incorrect labeling of animal foods may have remarkable negative consequences. To circumvate this problem, molecular methods had been developed. The present study was carried out for detection of meat species with the use of cytochrome b gene variability by multiplex PCR. Meat samples from cattle, buffalo, sheep, goat, chicken, pig and horse were utilized for molecular analysis. Genomic DNA was isolated from six samples of each species as per method described by Ausubel et al. (1987) with some modifications. Mitochondrial cyotchrome b gene was amplified by conventional and multiplex PCR using a common forward primer and species-specific reverse primer. Multiplex PCR was carried by mixing of primers in the different ratio viz. 1: 0.2: 3: 0.6: 0.6: 3: 0.6: 2 for forward: Chicken: Goat: Cattle: Cattle: Sheep: Pig: and horse specific reverse primers. PCR cycling protocol included initial denaturation at 94 C for 5 minutes then followed by 34 cycles of 94  C for 30 seconds, 62 C for 30 seconds 72 C for 30 seconds and final extension at 72 for 10 minutes. PCR amplicons were resolved by agarose gel electrophoresis and for each species produce a characteristic band pattern in conventional and multiplex PCR was obtained. The PCR products showed species-specific DNA fragments of 157, 227, 274, 274, 331, 398 and 439 bps from goat, chicken, cattle, buffalo, sheep, pig and horse respectively. Species-specific DNA fragments were amplified from the tissue samples cooked at 100 oC and 120 oC except for horse meat cooked at 120o C. The cattle and buffalo fragment was weakly amplified from DNA extracted from cooked meat at 120 oC sample. Putrefied meat samples showed successful amplification of mitochondrial cytochrome b gene using species specific primers by multiplex PCR. Putrefication did not inhibit detection potential of the test and same results were obtained as in fresh meat samples. Semi – quantification of pork and beef DNA mixed in different ratio could be done by this method. The intensity of amplified product band increased as the concentration of DNA template increased. The PCR efficiencies calculated was 1.00 with correlation 0.82 and 0.99, respectively for cattle and pig. The multiplex PCR could detect upto 0.9 ng of DNA of meat species. Thus, the multiplex PCR was found to be a simple reliable and sensitive and highly specific test for simultaneous detection of meat species. Dr. M. N. Brahmbhatt M.V.Sc; Ph.D. Assistant Professor Department of Veterinary Public Health College of Veterinary Science and Animal Husbandry Anand Agricultural University

Growth hormone gene polymorphism and its association with lactation yield in dairy cattle

Abstract: Kankrej, Gir and Holstein were typed for 3 growth hormone loci to identify the expected RFLP (restriction fragment length polymorphisms) markers in exon fifth (GH1 locus), intron third (GH2 locus) and 3' region (GH3 locus) of the gene with AluI MspI and HaeIII restriction enzymes, respectively. The Kankrej breed was monomorphic at GH1 locus. The alleles A, D and F at 3 GH loci were more frequent in Kankrej and Gir cows, while the frequencies of their alternative alleles were comparatively more in Holstein cows. B and C alleles of GHI and GH2 loci, respectively, appeared to be significantly associated with higher lactation milk yield. The cows with AB and BB genotypes at GHI and CD and DD genotypes at GH2 locus yielded more or less same first lactation milk indicating complete dominance of B and D alleles over A and C alleles, respectively. The genotypes at GH3 locus were independent of lactation yields. The phylogenie tree based on genetic distances indicated that Kankrej and Gir belonged to close cluster while Holstein belonged to different cluster.

Analysis of genetic relationship among three varieties of indigenous Kadaknath breed using 25 chicken microsatellite markers

Abstract: The genetic variability of three varieties of indigenous Kadaknath breed of poultry was evaluated using 25 microsatellite markers. All the 25 microsatellite markers were found to be polymorphic in all the three varieties. Number of alleles observed for all the markers varied from 3 to 10. Allele size of polymorphic loci ranged from 98-356 bp and showed very large variation across loci. Average heterozygosity values were found to be 0.721, 0.694 and 0.689 in Jet black, Golden and Pencilled varieties, respectively. Overall heterozygosity value for all the microsatellite loci among all the three varieties was found to be 0.701. Average polymorphic information content (PIC) values were found to be 0.671, 0.699, and 0.617 in Jet black, Golden and Pencilled varieties, respectively. Overall PIC values for all the microsatellite loci among all the three varieties were found to be 0.662. The Nei’s standard genetic distance (Ds) values between Jet black and Golden, Jet black and Pencilled, and Golden and Pencilled varieties were found to be 0.1678, 0.0951 and 0.1943 respectively. Phylogenetic consensus tree constructed using the boostrapped data and neighbour-joining method grouped all the three varieties into one cluster. Therefore, it is concluded that all the three varieties of Kadaknath breed had negligible genetic distance between each other.

Prolactin genotyping of indian buffalo breeds using pcr-rflp

Abstract: Prolactin plays an important regulatory function in milk production and reproduction. Prolactin gene was explored in three buffalo breeds by PCR-RFLP. Genomic DNA was isolated from blood samples of 21 Jaffarabadi, 44 Mehsani and 29 Surti buffaloes (Bubalus bubalis) and also from semen samples of 2 Jaffarabadi bulls. A 156 bp PRL gene exon III segment was amplified by PCR using bovine specific primers. RFLPs in this segment was studied using Rsa I restriction enzyme. Genotype AA was not detected in any of the breeds studied. The frequencies of AB and BB genotypes in Jaffarabadi, Mehsani and Surti buffaloes were 0.87 and 0.13; 1.0 and 0.0; 0.965 and 0.035, respectively. Frequencies of allele A and B were 0.435 and 0.565 in Jaffarabadi; 0.5 each in Mehsani; and 0.482 and 0.518 in Surti respectively. INTRODUCTION Lactation is under the physiological influence of the endocrine system. The milk protein and hormone genes are excellent candidate genes for linkage analysis with Quantitative Trait Loci (QTL) because of their biological significance on the quantitative traits of interest. Among several hormones that regulate lactation and reproduction in bovines, prolactin is an important anterior pituitary hormone.

Detection of meat species by polymerase chain reaction of actin gene family.

Abstract: The aim of this study was to develop a simple method for simultaneous identification of multiple meat species. During this study, six raw meat samples from each species including cattle, buffalo, sheep, goat, pig and poultry in fresh and cooked state were subjected for DNA extraction. The amplification of extracted DNA was done through conventional polymerase chain reaction with actin gene primers. The results of conventional polymerase chain reaction with actin gene primers revealed a clear cut differentiation of pig and poultry with that of bovine, ovine and caprine meat. However, it could not differentiate the meat of sheep from that of goat and meat of cattle from that of buffalo.