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.

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.

Differentiation of Acidithiobacillus ferrooxidans and A-thiooxidans strains based on 16S-23S rDNA spacer polymorphism analysis

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli. (C) 2004 Elsevier SAS. All rights reserved.

RFLP and cytogenetic evidence on the origin and evolution of allotetraploid domesticated peanut, Arachis hypogaea (Leguminosae)

Nuclear restriction fragment length polymorphism (RFLP) analysis was used to determine the wild diploid Arachis species that hybridized to form tetraploid domesticated peanut. Results using 20 previously mapped cDNA clones strongly indicated A. duranensis as the progenitor of the A genome of domesticated peanut and A. ipaensis as the B genome parent. A large amount of RFLP variability was found among the various accessions of A. duranensis, and accessions most similar to the A genome of cultivated peanut were identified. Chloroplast DNA RFLP analysis determined that A. duranensis was the female parent of the original hybridization event. Domesticated peanut is known to have one genome with a distinctly smaller pair of chromosomes ('A'), and one genome that lacks this pair. Cytogenetic analysis demonstrated that A. duranensis has a pair of 'A' chromosomes, and A. ipaensis does not. The cytogenetic evidence is thus consistent with the RFLP evidence concerning the identify of the progenitors. RFLP and cytogenetic evidence indicate a single origin for domesticated peanut in Northern Argentina or Southern Bolivia, followed by diversification under the influence of cultivation.

Fluorescent amplified fragment length polymorphism (fAFLP) analyses and genetic diversity in Litopenaeus vannamei (Penaeidae)

The Pacific white shrimp, Litopenaeus vannamei (Penaeidae), represents about 95% of all Brazilian shrimp production. The Brazilian L. vannamei foundation broodstock was made up of specimens collected from different American Pacific sites, but little information was collected on the genetic structure of the broodstock. We used the fluorescence amplified fragment length polymorphism (fAFLP) method to study the genetic diversity of L. vannamei broodstock lines 03CMF1 and 03CBF1 originally produced by breeder-shrimps imported mainly from Panama and Ecuador, although wild individuals from other localities may also have been used in producing these two lines. Our results showed a total of 93 polymorphic bands ranging from 50 to 500 bp, the mean Nei's genetic diversity calculated for the total sample was 13.4% and identity and genetic distance analyses indicated high genetic homogeneity within and between both the broodstock lineages studied which suggests that they had similar genetic structure. These results may represent an important tool for the appropriate management of L. vannamei broodstocks. Copyright by the Brazilian Society of Genetics.

Head and neck carcinogenesis: Impact of MTHFD1 G1958A polymorphism

Objective: To investigate the MTHFD1 G1958A polymorphism involved in the folate metabolism as a risk for head and neck cancer, and to find the association of the polymorphism with the risk factors and clinical and histopathological characteristics. Methods: Retrospective study investigating MTHFD1 G1958A polymorphism in 694 subjects (240 patients in the Case Group and 454 in the Control Group) by Restriction Fragment Length Polymorphism (RFLP) Analysis. Multiple logistic regression and chi-square tests were used in the statistical analysis. Results: Multivariable analysis showed that smoking and age over 42 years were disease predictors (p < 0.05). MTHFD1 1958GA or AA genotypes were associated with smoking (p = 0.04) and alcoholism (p = 0.03) and were more ofen found in more advanced stage tumors (p = 0.04) and in patients with a shorter survival (p = 0.03). Conclusion: Te presence of MTHFD1 G1948A polymorphism associated with smoking and alcoholism raises the head and neck cancer risk.

Genotipagem do Mycobacterium tuberculosis utilizando RFLP e Spoligotyping em associação com Miru para avaliar a epidemiologia molecular da tuberculose no município de Araraquara-SP

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

Genotyping, serotyping and determination of mating-type of Cryptococcus neoformans clinical isolates from São Paulo State, Brazil

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

Prevalence and antifungal susceptibility of Candida parapsilosis complex isolates collected from oral cavities of HIV-infected individuals

At present, few data are available on the prevalence and antifungal susceptibility of Candida parapsilosis complex isolates from HIV-infected individuals. The C. parapsilosis complex comprises three species, C. parapsilosis sensu stricto, C. metapsilosis and C. orthopsilosis. Fifteen of 318 Candida isolates were identified as members of the C. parapsilosis complex by PCR and restriction fragment length polymorphism (RFLP). The prevalence of C. parapsilosis complex isolates was 4.7 %, 2.2 % being identified as C. parapsilosis sensu stricto and 2.5% as C. metapsilosis, while no C. orthopsilosis was isolated. This is believed to be the first study that has identified isolates of C. metapsilosis obtained from the oral cavity of HIV-infected individuals. Antifungal susceptibility tests indicated that all the isolates were susceptible to amphotericin B (AMB), fluconazole (FLC), ketoconazole (KTC), itraconazole (ITC), voriconazole (VRC) and caspofungin (CASPO). Although isolates of C. parapsilosis sensu stricto and C. metapsilosis were susceptible to FLC, isolates of C. metapsilosis showed a tendency for higher MICs (>= 1.0 mu g ml(-1)). Based upon the frequency of candidiasis and the fact that certain isolates of the C. parapsilosis complex respond differently to FLC therapy, our data may be of therapeutic relevance with respect to susceptibility and potential resistance to specific antifungal agents. Our data suggest that C. metapsilosis can be a human commensal; its importance as a pathogen has yet to be confirmed.

Genetic characterisation of Giardia duodenalis in dairy cattle in Brazil

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