Genetic Polymorphisms of Chicken Antiviral Mx1 and TVB Genes in Indigenous and Giriraja Chicken

2021 ◽  
Author(s):  
S. Nihar ◽  
S. Naveen Kumar ◽  
Wilfred Ruban ◽  
H.M. Yathish ◽  
R. Nagaraja ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Dna Isolation ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Isoamyl Alcohol ◽  
Gene Frequencies ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Recent Developments ◽  
Mx1 Gene

Background: Recent developments in molecular genetics lead to addressing certain poultry diseases via breeding for disease resistance. The present study was carried to identify and compare the genetic polymorphism in Chicken Mx1 and TVB genes among the indigenous and Giriraja chicken using PCR-RFLP technique. Methods: Blood samples were collected from 50 indigenous and 50 Giriraja birds and DNA isolation was done by Phenol: Chloroform: Isoamyl alcohol method. PCR amplification of Chicken Mx1 (exon 14) and Chicken TVB (exon 3) genes was carried out followed by RFLP analysis. Result: PCR product sizes of 301 bp and 303 bp of Mx1 and TVB genes, respectively were successfully amplified. RFLP analysis of Mx1 gene with Hyp8I restriction enzyme revealed three genotypes AA, AB and BB. In indigenous birds genotypic frequencies of AA, AB and BB were 0.314, 0.493 and 0.194, respectively and gene frequencies were 0.56 and 0.44 for alleles A and B, respectively. In Giriraja birds, genotypic frequencies for AA, AB and BB were 0.27, 0.499 and 0.23, respectively and gene frequencies were 0.52 and 0.48 for alleles A and B, respectively. RFLP analysis of TVB gene with NlaIII restriction enzyme revealed two genotypes viz., AA and AB. In indigenous birds genotypic frequencies of AA and AB were 0.81 and 0.18, respectively and gene frequencies were 0.9 and 0.1 for alleles A and B, respectively. In Giriraja birds genotypic frequencies for AA and AB were 0.774 and 0.211, respectively and gene frequencies were 0.88 and 0.12 for alleles A and B, respectively.

scholarly journals Evaluation of the LiPA MYCOBACTERIA Assay for Identification of Mycobacterial Species from BACTEC 12B Bottles

2000 ◽  
Vol 38 (5) ◽  
pp. 1915-1919 ◽  
Author(s):  
Nancimae Miller ◽  
Susanna Infante ◽  
Tim Cleary
Keyword(s):  
Pcr Amplification ◽  
Rflp Analysis ◽  
Culture Fluid ◽  
23S Rrna ◽  
Complete Agreement ◽  
Mycobacterial Species ◽  
Clear Cut ◽  
Colorimetric System ◽  
Pcr Rflp ◽  
Membrane Strip

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacteriumspecies and differentiates M. tuberculosis complex,M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonaegroup, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive forM. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive forMycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

scholarly journals Rapid differentiation of Staphylococcus aureus isolates harbouring egc loci with pseudogenes ψent1 and ψent2 and the selu or selu v gene using PCR-RFLP

2007 ◽  
Vol 56 (2) ◽  
pp. 208-216 ◽  
Author(s):  
Mark M. Collery ◽  
Cyril J. Smyth
Keyword(s):  
Dna Sequencing ◽  
Restriction Enzyme ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Restriction Enzyme Digestion ◽  
Pcr Rflp ◽  
Intergenic Regions ◽  
Unique Restriction ◽  
Genomic Regions

The egc locus of Staphylococus aureus harbours two enterotoxin genes (seg and sei) and three enterotoxin-like genes (selm, seln and selo). Between the sei and seln genes are located two pseudogenes, ψent1 and ψent2, or the selu or selu v gene. While these two alternative sei–seln intergenic regions can be distinguished by PCR, to date, DNA sequencing has been the only confirmatory option because of the very high degree of sequence similarity between egc loci bearing the pseudogenes and the selu or selu v gene. In silico restriction enzyme digestion of genomic regions encompassing the egc locus from the 3′ end of the sei gene through the 5′ first quarter of the seln gene allowed pseudogene- and selu- or selu v-bearing egc loci to be distinguished by PCR-RFLP. Experimental application of these findings demonstrated that endonuclease HindIII cleaved PCR amplimers bearing pseudogenes but not those with a selu or selu v gene, while selu- or selu v-bearing amplimers were susceptible to cleavage by endonuclease HphI, but not by endonuclease HindIII. The restriction enzyme BccI cleaved selu- or selu v-harbouring amplimers at a unique restriction site created by their signature 15 bp insertion compared with pseudogene-bearing amplimers, thereby allowing distinction of these egc loci. PCR-RFLP analysis using these restriction enzymes provides a rapid, easy to interpret alternative to DNA sequencing for verification of PCR findings on the nature of an egc locus type, and can also be used for the primary identification of the intergenic sei–seln egc locus type.

Characterization of genetic polymorphism in the goat β-lactoglobulin gene

2000 ◽  
Vol 67 (2) ◽  
pp. 217-224 ◽  
Author(s):  
RAMONA N. PENA ◽  
ARMAND SÁNCHEZ ◽  
JOSEP M. FOLCH
Keyword(s):  
Fragment Length Polymorphism ◽  
Gene Frequencies ◽  
Single Nucleotide ◽  
Pcr Product ◽  
Polymorphic Position ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Β Lactoglobulin ◽  
New Alleles

Two new variants have been detected and characterized for the goat β-lactoglobulin gene at the cDNA level and confirmed at the genomic level. The two polymorphisms are located on exon 7 of the gene. One of the polymorphic sites is produced by a single nucleotide substitution in position +4601, allowing a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) genotyping procedure to be developed using SacII restriction enzyme. The other polymorphic position contains a 10 bp long insertion at position +4641 that can be detected by capillary electrophoresis of the PCR product amplified with a fluorescent primer. The association of these two polymorphisms was also investigated, resulting in the description of two new alleles. Both of these contained the point mutation at the SacII site, with or without the 10 bp insertion at position +4641. The distribution of these new polymorphisms was studied in a population of males of four different goat breeds. The gene frequencies for these variants were similar in Spanish and French breeds.

scholarly journals Polymorphism, Allelic and Genotypic Frequencies of κ-Casein and β-LG genes in Egyptian Buffaloes

2020 ◽  
Vol 12 (4) ◽  
pp. 283
Author(s):  
Z. M. Al-Shawa ◽  
M. F. El-Zarei ◽  
A. A. Ghazy ◽  
M. A. Ayoub ◽  
S. M. Merdan ◽  
...  
Keyword(s):  
Genetic Parameters ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Product ◽  
Heterozygosity Excess ◽  
Pcr Rflp ◽  
Native Populations ◽  
Egyptian Buffalo ◽  
Rflp Assay ◽  
Geographical Locations

With a view to detecting the genotypes of both κ-CN and β-LG genes in native populations of Egyptian buffalo using PCR-RFLP technique, 80 randomly, individuals were selected from five geographical locations of some Egyptian provinces. Also, to estimate the population genetic parameters such as, allelic and genotypic frequencies, heterozygosity, and inbreeding coefficient (FIS) of these studied genes. For genotyping, 453 bp PCR product of κ-CN was digested with AcuI and HpyCH4IV (Isoschizomer for MaeII) restriction enzymes while the 247 bp PCR product of β-LG was digested with HaeIII restriction enzyme. PCR-RFLP results discovered polymorphism at the level of κ-CN gene in all studied Egyptian buffaloes with two distinct alleles “A” and “B”. PCR-RFLP analysis for κ-CN gene using both restriction enzymes successfully detected that polymorphic status of the studied populations. We recommended using AcuI enzyme which was more capable for differentiating between homozygous (17%) and heterozygous (83%) individuals than HpyCH4IV enzyme which defined only 4% of homozygous individuals and the remaining was heterozygous (96%) individuals. Existence of heterozygosity excess in all studied populations referred to higher degree of genetic variability between individuals within these populations. On contrary, results of PCR-RFLP at the level of β-LG gene revealed a monomorphic pattern of Egyptian buffaloes and genotyped as “AA” animals which signified that PCR-RFLP assay with HaeIII enzyme for β-LG gene failed to discover any evidence of polymorphism in Egyptian buffalo under the circumstances of this study or all studied animals possess only one allele.

scholarly journals IDENTIFIKASI POLIMORFISME GEN GH (GROWTH HORMONE) SAPI BALI DENGAN METODE PCR-RFLP

2006 ◽  
Vol 12 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Sri Rahayu ◽  
Sutiman Bambang Sumitro ◽  
T Susilawati ◽  
Soemarno Soemarno
Keyword(s):  
Growth Hormone ◽  
Restriction Enzyme ◽  
Growth Hormone Gene ◽  
Salting Out ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Reverse Primer ◽  
Total Dna ◽  
Polymerase Chain ◽  
Forward Primer

This study was conducted to identify polymorphism of growth hormone gene of Bali cattle. A PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) procedure was developed for determining polymorphism of growth hormone gene. The DNA was isolated from blood samples by salting out method. Total DNA were amplified with forward primer, 5’-TAGGGGAGGGTGGAAAATGGA-3’ and reverse primer, 5’-GACACCTACTCAGACAATGCG-3’. The PCR product was digested by HaeIII restriction enzyme. Result of the amplification was a specific single band with fragment 450 bp. Restriction with HaeIII restriction enzyme resulted four kinds of haplotype. Haplotype I was not cut by HaeIII restriction enzyme. Haplotype II were cut into two, 225 bp and 150 bp,. Haplotype III were cut into three size, 400 bp, 225 bp and 150 bp. Haplotype IV were cut into five fragments 450 bp, 400 bp, 275 bp, 225 bp and 150 bp.

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia

Parasitology ◽  
1998 ◽  
Vol 117 (1) ◽  
pp. 1-13 ◽  
Author(s):  
K. VICTOIR ◽  
A. L. BAÑULS ◽  
J. AREVALO ◽  
A. LLANOS-CUENTAS ◽  
R. HAMERS ◽  
...  
Keyword(s):  
Gene Locus ◽  
Genetic Characterization ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Natural Isolates ◽  
Phenotype Diversity

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63–RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR–RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni: reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63–RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63–RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63–RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR–RFLP). PCR–RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63–RFLP and PCR–RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

scholarly journals VALIDATION OF PCR-RFLP TESTING METHOD TO DETECT PORCINE CONTAMINATION IN CHICKEN NUGGET

2012 ◽  
Vol 12 (3) ◽  
pp. 302-307 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Winda Cahyaningtyas ◽  
Surajiman Surajiman ◽  
Istini Istini ◽  
Deni Pranowo
Keyword(s):  
Mitochondrial Dna ◽  
Rflp Analysis ◽  
Cytb Gene ◽  
Dna Fragments ◽  
Gene Fragment ◽  
Chicken Nugget ◽  
Testing Method ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Dna Fragment

PCR-RFLP technique to detect porcine contamination in chicken nugget has been developed and validated in this research. Various concentrations of pork were fortified during preparation of the nugget. DNA was then isolated from the nugget followed by PCR employed primers which targeted a 359 bp cytB gene fragment of mitochondrial DNA. For RFLP, the PCR product was digested by means of BamHI and BseDI enzymes. Cutting DNA fragments from nugget containing pork using BseDI enzyme produced DNA fragment with size 228 and 131 bp, while cutting with BamHI enzyme produce DNA fragments with sizes 244 and 115 bp. All of these fragments were not present in RFLP analysis of pork-free nugget. The method shows good specificity and precision and could detect porcine contamination in the nugget up to 5%. The method has been applied to test commercial nugget. Four brand of Halal-labeled commercial nugget as well as four brand of non labeled one gave negative porcine contamination.

scholarly journals Comparative Analysis of the Genomic DNA Isolation Methods on Inula sp. (Asteraceae)

2016 ◽  
Vol 8 (4) ◽  
pp. 451-455
Author(s):  
Emre SEVİNDİK ◽  
Fatih COŞKUN ◽  
Zehra Tuğba MURATHAN ◽  
Mehmet Yavuz PAKSOY ◽  
Veysel UZUN
Keyword(s):  
Genomic Dna ◽  
Dna Isolation ◽  
Low Cost ◽  
Pcr Amplification ◽  
Isoamyl Alcohol ◽  
Dna Amount ◽  
Genomic Dna Isolation ◽  
Asteraceae Family ◽  
Positive Results ◽  
Commercial Kit

Simple, fast, low-cost and high throughput protocols are required for DNA isolation of plant species. In this study, phenol chloroform isoamyl alcohol and commercial (Sigma) DNA isolation kit methods were applied on some Inula species that belong to Asteraceae family. Genomic DNA amounts, A260, A280, A260/A230 and purity degrees (A260/A280) that were obtained through both methods were measured through electrophoresis and spectrophotometer. Additionally, PCR amplification was realized by primer pairs specific to nrDNA ITS, cpDNA ndhF (972F-1603R) and trnL-F regions. Results showed that maximum genomic DNA in nanograms obtained by phenol chloroform isoamyl alcohol method. The study also revealed that I. macrocephala had the maximum DNA and I. heterolepis had the minimum DNA amount. A260/A280 purity degrees showed that the highest and lowest purity in gDNAs obtained through phenol-choloform isoamyl alcohol method were in I.aucheriana and I. salicina, respectively. The highest and lowest purity degrees of gDNAs obtained through commercial kit was observed in I. fragilis and I. macrocephala samples, respectively. PCR amplification results showed that while band profiles of each three regions (ITS, trnL-F and ndhF) did not yield positive results in PCR amplifications using phenol-choloform isoamyl alcohol method; PCR band profiles obtained through commercial kit yielded positive results. As a result, it is fair to say that the relation of genomic DNA with PCR was found to be more efficient although the maximum amount of genomic DNA was obtained through phenol chloroform isoamyl alcohol method. 

Studies on wild and vaccine strains of poliovirus isolated from water and sewage

1995 ◽  
Vol 31 (5-6) ◽  
pp. 317-321 ◽  
Author(s):  
Jane Sellwood ◽  
Pamela A. Litton ◽  
Jacqueline McDermott ◽  
Jonathon P. Clewley
Keyword(s):  
Cell Line ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
River System ◽  
Eradication Programme ◽  
Typing Method ◽  
Efficient System ◽  
Poliovirus Receptor ◽  
Pcr Rflp

The WHO Poliovirus Eradication Programme has renewed interest in the identification of wild and vaccine strains of poliovirus circulating in the community. One method of monitoring these strains is to study poliovirus isolates detected in sewage. To facilitate the isolation of poliovirus from sewage and eliminate the possibility of detecting the other enteroviruses, sewage was inoculated onto a transfected Mouse L cell line. This cell line contains the gene for the poliovirus receptor which allows poliovirus infection to take place but not that of other enteroviruses. This cell line is, however, too sensitive to the toxic elements of concentrated sewage to be of practical use. Polioviruses have also been isolated from river and seawater as part of three year surveys of sewage discharges into a river system and a coastal harbour. These isolates have been characterised using PCR amplification of a region of the VP1 gene followed by restriction fragment length polymorphism (RFLP) analysis. All isolates were vaccine-like although many poliovirus type 2 isolates had distinct PCR-RFLP profiles. The RFLP typing method is an efficient system for rapidly monitoring poliovirus isolates from the environment.

scholarly journals An RFLP-Based Technique for Identifying Fungi in the Sooty Blotch and Flyspeck Complex on Apple

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 794-799 ◽  
Author(s):  
K. B. Duttweiler ◽  
G. Y. Sun ◽  
J. C. Batzer ◽  
T. C. Harrington ◽  
M. L. Gleason
Keyword(s):  
Agar Plate ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Its Region ◽  
Morphological Description ◽  
Sooty Blotch ◽  
Pcr Rflp ◽  
Sooty Blotch And Flyspeck ◽  
Rflp Assay

A restriction fragment length polymorphism (RFLP)-based technique was developed to identify members of the sooty blotch and flyspeck (SBFS) disease complex on apple because these fungi are difficult to identify using agar-plate isolation and morphological description. The method includes polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) using a fungal-specific forward primer (ITS1-F) and an SBFS-specific reverse primer (Myc1-R), followed by digestion of the PCR product by the HaeIII restriction enzyme. When applied to previously identified isolates of 24 SBFS-causing species in nine genera, the PCR-RFLP assay produced 14 unique banding patterns. Different genera never shared the same RFLP pattern. To evaluate performance in vivo, the technique was applied to DNA extracted directly from SBFS colonies on apple fruit from three Iowa orchards. The primers amplified the rDNA of only SBFS fungi, with the exception of a Cladosporium sp.; however, its RFLP banding pattern was distinct from those of SBFS fungi. The majority (60%) of SBFS colonies in the in vivo trial were identified to genus by RFLP analysis. The PCR-RFLP assay greatly streamlined the identification process by minimizing the need for culturing, indicating its value as a tool for field studies of the SBFS complex.

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