Restriction fragment length polymorphism (RFLP) in Exon 2 of the BoLA-DRB3 gene in South American cattle

The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY, and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds.

Taenia saginata: Differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different Dral-RFLP pattern: a two-band pattern (421 and 100 bp) for T saginata and a three-band pattern (234, 188, and 99 bp) for T solium was observed allowing the two species to be separated.. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). of these, three showed a T solium pattern and five a T saginata pattern. (c) 2005 Elsevier B.V. All rights reserved.

Detection and differentiation of Leptospira spp. serovars in bovine semen by polymerase chain reaction and restriction fragment length polymorphism

In view of the importance of venereal transmission of bovine leptospirosis, the objective of the present study was to apply the polymerase chain reaction (PCR) to 26 serovars of Leptospira interrogans, L. borgpetersenii, L. santarosai, L. noguchii and L. biflexa, to determine the detection threshold in semen samples and to evaluate the possibility of differentiation among serovars using 19 restriction endonucleases. The results showed that all serovars were amplified and the detection threshold in semen samples of a bull was 100 bacteria/ml. Using endonucleases we could classify the 26 serovars into eight groups. The present results show that PCR is a method of great potential for the detection of Leptospira spp, at bovine artificial insemination centers. (C) 2000 Elsevier B.V. B.V. All rights reserved.

A new polymerase chain reaction/restriction fragment length polymorphism protocol for Plasmodium vivax circumsporozoite protein genotype (VK210, VK247, and P-vivax-like) determination

For the molecular diagnosis of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) using DNA amplification procedures in the laboratory, the choice of rapid and inexpensive identification products of the 3 different genotypes is an important prerequisite. We report here the standardization of a new polymerase chain reaction/restriction fragment length polymorphism technique to identify the 3 described P. vivax circumsporozoite protein (CSP) variants using amplification of the central immunodominant region of the CSP gene of this protozoan. The simplicity, specificity, and sensitivity of the system described here is important to determine the prevalence and the distribution of infection with these P. vivax genotypes in endemic and nonendemic malaria areas, enabling a better understanding of their phylogeny. (c) 2007 Published by Elsevier B.V.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Use of PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism) in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

Molecular characterization of Giardia duodenalis in dogs from Brazil

The intestinal protozoan parasite Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is a widespread enteric pathogen in human and domestic animals. This organism is one of the most common parasites in domestic dogs in Brazil. In this study, we determined the occurrence and genetic characterization of G. duodenalis isolated from dogs from south-central São Paulo state, Brazil. A total of 300 fecal samples were collected. Fecal specimens were screened for the presence of G. duodenalis using microscopy (zinc sulfate solution flotation technique) and polymerase chain reaction (PCR) targeting the small subunit ribosomal (SSU-rDNA) and glutamate dehydrogenase (GDH) genes. Genetic characterization was performed using restriction fragment length polymorphisms (RFLP) and sequencing analysis of the GDH gene. In addition, selected samples were further characterized by RFLP and sequencing of the beta-giardin gene. The overall occurrence of G. duodenalis was 17.3% (52/300). The occurrence was higher in stray dogs (28%) than in household dogs (6.25%). of the 36 PCR-positive samples that were selected for genotyping, only dog-specific genotype C (20 isolates), D (11 isolates) and mixed C+D (five isolates) isolates were detected in the study. This study provides current information on the infection rates of G. duodenalis genotypes in canine populations and describes for the first time the presence of mixed infections within host-specific C and D genotypes in dogs in Brazil. These genotypes were widespread and commonly found in domestic dogs living in urban and suburban environments of the studied area and confirmed the endemic status of Giardia in this region.

Detecting migrants in populations of Rhizoctonia solani anastomosis group 3 from potato in North Carolina using multilocus genotype probabilities

The relative contribution of migration of Rhizoctonia solani anastomosis group 3 (AG-3) on infested potato seed tubers originating from production areas in Canada, Maine, and Wisconsin (source population) to the genetic diversity and structure of populations of R. solani AG-3 in North Carolina (NC) soil (recipient population) was examined. The frequency of alleles detected by multilocus polymerase chain reaction-restriction fragment length polymorphisms, heterozygosity at individual loci, and gametic phase disequilibrium between all pairs of loci were determined for subpopulations of R. solani AG-3 from eight sources of potato seed tubers and from five soils in NC. Analysis of molecular variation revealed little variation between seed source and NC recipient soil populations or between subpopulations within each region. Analysis of population data with a Bayesian-based statistical method previously developed for detecting migration in human populations suggested that six multilocus genotypes from the NC soil population had a statistically significant probability of being migrants from the northern source population. The one-way (unidirectional) migration of genotypes of R. solani AG-3 into NC on infested potato seed tubers from Canada, Maine, and Wisconsin provides a plausible explanation for the lack of genetic subdivision (differentiation) between populations of the pathogen in NC soils or between the northern source and the NC recipient soil populations.

Fluorescent amplified fragment length polymorphism genotyping of human and animal Staphylococcus aureus isolates from dairy farms with manual milking

Fluorescence amplified fragment length polymorphism (fAFLP) was used to assess the genetic relatedness of 40 Staphylococcus aureus strains isolated from human and animal skin samples in seven dairy farms with manual milking. S. aureus was isolated from 11 out of 30 (36%) human skin samples and from 29 out of 100 (29%) teat skin samples from apparently healthy cows. Genomic DNA from each isolate was double-digested with EcoRI and MseI and complementary oligonucleotide adaptors were ligated to the restriction fragments. Pre-selective and selective, amplification reactions were performed, the amplified fragments were separated by electrophoresis in an ABI377 sequencer and analysed using GeneScan 3.1 and Genotyper 2.5. Three single isolates (a-c), a predominant cluster with 35 isolates (d) and another cluster with two isolates (e) were identified. Both clusters d and e included human and animal isolates genetically related, because the profiles had 90-100% homology. Since no cluster was comprised uniquely of human or animal isolates and given the close genetic relatedness among human and animal samples in the farms, the present findings support the. hypothesis that dairy workers can spread S. aureus through manual milking. (C) 2005 Elsevier B.V. All rights reserved.

PSA and androgen-related gene (AR, CYP17, and CYP19) polymorphisms and the risk of adenocarcinoma at prostate biopsy

The aim of the present study was to examine the impact of polymorphisms in prostate-specific antigen (PSA) and androgen-related genes (AR, CYP17, and CYP19) on prostate cancer (PCa) risk in selected high-risk patients who underwent prostate biopsy. Blood samples and prostate tissues were obtained for DNA analysis. Single-nucleotide polymorphisms in the 50-untranslated regions (UTRs) of the PSA (substitution A > G at position -158) and CYP17 (substitution T > C at 50-UTR) genes were detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism assays. The CAG and TTTA repeats in the AR and CYP19 genes, respectively, were genotyped by PCR-based GeneScan analysis. Patients with the GG genotype of the PSA gene had a higher risk of PCa than those with the AG or AA genotype (OR = 3.79, p = 0.00138). The AA genotype was associated with lower PSA levels (6.44 +/- 1.64 ng/mL) compared with genotypes having at least one G allele (10.44 +/- 10.06 ng/mL) (p = 0.0687, 95% CI - 0.3146 to 8.315, unpaired t-test). The multivariate analysis confirmed the association between PSA levels and PSA genotypes (AA vs. AG+GG; chi(2) = 0.0482) and CYP19 (short alleles homozygous vs. at least one long allele; chi(2) = 0.0110) genotypes. Genetic instability at the AR locus leading to somatic mosaicism was detected in one PCa patient by comparing the length of AR CAG repeats in matched peripheral blood and prostate biopsy cores. Taken together, these findings suggest that the PSA genotype should be a clinically relevant biomarker to predict the PCa risk.

Relationship of IL-1 and TNF-alpha polymorphisms with Helicobacter pylori in gastric diseases in a Brazilian population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polymorphisms and Haplotypes in the Interleukin-4 Gene are Associated With Chronic Periodontitis in a Brazilian Population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polymorphisms of the TLR2 and TLR4 genes are associated with risk of gastric cancer in a Brazilian population

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

GSTT1, GSTM1 and CYP2E1 genetic polymorphisms in gastric cancer and chronic gastritis in a Brazilian population

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and grastic cancer

Aim: To evaluate the association between polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk for chronic gastritis and gastric cancer, in a Southeastern Brazilian population. Methods: Genotyping by PCR-RFLP was carried out on 202 patients with chronic gastritis (CG) and 160 patients with gastric cancer (GC), matched to 202 (C1) and 150 (C2) controls, respectively. Results: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codons 194 and 399 and of XRCC3 codon 241. However, the combined analyses of the three variant alleles (194Trp, 399Gln and 241Met) showed an increased risk for chronic gastritis when compared to the GC group. Moreover, an interaction between the polymorphic alleles and demographic and environmental factors was observed in the CG and GC groups. XRCC1 194Trp was associated with smoking in the CG group, while the variant alleles XRCC1 399Gln and XRCC3 241Met were related with gender, smoking, drinking and H pylori infection in the CG and GC groups. Conclusion: Our results showed no evidence of a rela-tionship between the polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk of chronic gastritis and gastric cancer in the Brazilian population, but the combined effect of these variants may interact to increase the risk for chronic gastritis, considered a premalignant lesion. Our data also indicate a gene-environment interaction in the susceptibility to chronic gastritis and gastric cancer. © 2005 The WJG Press and Elsevier Inc. All rights reserved.