Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 20 de 214
Hematol., Transfus. Cell Ther. (Impr.) ; 44(3): 386-391, July-Sept. 2022. tab, ilus
Article in English | LILACS | ID: biblio-1404989


ABSTRACT Introduction: Prevalence of RhD negative phenotype in Nigeria is low; this leads to scarcity of RhD negative red cells for transfusion. Serological and molecular genotyping of RhD negative individuals for weak D types could reduce this scarcity. The aim of this study was to determine the serological prevalence and molecular types of weak D phenotypes among blood donors and pregnant women in Kano, Nigeria. Methods: A total of 4482 blood donors and pregnant women from three hospitals in Kano were recruited. An indirect antiglobulin test was used to determine weak D phenotypes. Molecular genotyping was performed on genomic DNA from whole blood amplified by polymerase chain reaction sequence-specific primers (PCR-SSP) with agarose gel electrophoresis. Results: The mean age of the participants was 26.50 ±5.79 years. The prevalence of the RhD negative phenotype was 4.2% (189/4482). Of the 189 RhD negative phenotypes, 20 (10.6%) were weak D positive. Molecular genotyping of the 20 Weak D positive phenotypes revealed 15 (75%) weak D type 4, of which 11 were due to the RHD*09.03 and RHD*DAR3 (T201R, F223V) polymorphisms and 4, due to RHD* 08.01 and RHD* DFV polymorphisms; 2 (10%) were due to the 602 C>G polymorphism, while the remaining 3 (15%) constituted partial D or other rare weak D types. Conclusion: The prevalence of weak D positive phenotypes is high in this study; weak D type 4 is the most common RhD genetic variant. Routine serologic weak D testing of RhD negative blood and molecular genotyping should be encouraged in resource-limited settings.

Humans , Male , Female , Blood Transfusion , Genotyping Techniques , Phenotype , Serology , Nigeria
Biomédica (Bogotá) ; 42(1): 18-30, ene.-mar. 2022. graf
Article in English | LILACS | ID: biblio-1374504


Introduction: Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations. Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium. Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Hunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods. Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S. Conclusion: The strong correlation between the M13 classification and the sequencing-based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Introducción. Fusarium es un grupo heterogéneo de hongos, difícil de clasificar y con una amplia gama de estilos de vida, que actúa como saprófito, parásito de plantas o patógeno de humanos y animales. La prevalencia de la fusariosis clínica y la falta de tratamientos han incrementado el interés en su diagnóstico preciso, lo que conlleva la caracterización molecular de las poblaciones. Objetivo. Comparar marcadores de genotipificación en la evaluación de la variabilidad genética e identificación de aislamientos clínicos de Fusarium. Materiales y métodos. Se evaluó la huella genética producida por dos cebadores aleatorios: M13, que amplifica una secuencia minisatélite, y (GACA)4, que corresponde a una secuencia repetitiva de ADN. Utilizando el índice discriminatorio de Hunter Gaston (HGDI), el análisis de varianza molecular (AMOVA) y una prueba de Mantel, se comparó la resolución de estos marcadores con métodos de genotipificación basados en secuenciación y PCR. Resultados. El mayor HGDI se asoció con el marcador M13, seguido de (GACA)4. Las pruebas AMOVA y Mantel mostraron correlación entre las clasificaciones obtenidas con M13 y la referencia basada en la secuenciación parcial del factor de elongación de transcripción 1-alfa (TEF1-α) y el ADNr 28S. Conclusión. La fuerte correlación entre la clasificación obtenida con M13 y el método de referencia, así como su alta resolución, demuestran su idoneidad para la caracterización de poblaciones de Fusarium.

Fusarium , DNA Fingerprinting , Bacteriophage M13 , Fusariosis , Genotyping Techniques , Elongin , Genetics, Population
Hematol., Transfus. Cell Ther. (Impr.) ; 43(4): 489-493, Oct.-Dec. 2021. tab
Article in English | LILACS | ID: biblio-1350813


ABSTRACT Objective: Low levels of neutrophils can be an intrinsic condition, with no clinical consequences or immunity impairment. This condition is the benign constitutional neutropenia (BCN), defined as an absolute neutrophils count (ANC) ≤2000 cells/mm. Diagnosis of BCN is of exclusion where patients are submitted to blood tests and possibly to invasive diagnostic search until secondary causes of neutropenia are ruled out. The natural history of the disease suggests benign evolution and Brazilian study showed an overall frequency of 2.59%. The main mechanisms include reduced neutrophil production, increased marginalization, extravasation to the tissues and immune destruction. Genetic studies showed strong association between the single nucleotide variant rs2814778 located on chromosome 1q23.2 in the promoter region of the atypical chemokine receptor 1 (Duffy blood group system) gene (ACKR1, also termed DARC) and BCN. The aim of this study is to evaluate FY phenotypes and genotypes including the analysis of the rs2814778 SNP in Brazilian patients with BCN in order to determine an effective diagnostic tool, allowing reassurance of the patient and cost reduction in their care. Methods: Case control study, with 94 individuals (18 patients and 76 controls). Phenotyping was performed by gel test and genotyping was performed by PCR-RFLP. Results: White blood cell (WBC) and absolute neutrophils (AN) counts showed lower levels in patients compared to controls. In the patient group 83.3% were genotyped as FY*B/FY*B. The SNP rs2814778 (-67T > C) was identified in 77.8% of the patients genotyped as FY*B-67C/FY*B-67C. In the control group, 72.7% were homozygous for the wild type and 23.3% were heterozygous. Conclusion: This study reinforces that FY phenotyping and genotyping can be used to detect most people with BCN, avoiding excessive diagnostic investigation. Besides, this procedure may reduce health costs and be reproductible in clinical practice.

Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Aged , Duffy Blood-Group System , Genotyping Techniques , Neutropenia , Immunophenotyping , Diagnostic Tests, Routine , Neutrophils
Electron. j. biotechnol ; 51: 1-7, May. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1343303


BACKGROUND: This study aimed to explore genetic polymorphisms of the CCKAR gene and their relationship with the growth and development of Qinchuan cattle which could be used as molecular markers for the improvement of the breeding of Qinchuan cattle. RESULTS: Here, we have identified seven single nucleotide polymorphisms (SNPs) at loci g. 1463 C>G; g. 1532 T>A; g. 1570 G>A; g. 1594 C>A; g. 1640 T>C; g. 1677 G>C; and g. 1735 C>T in the coding region of the bovine CCKAR gene. The frequencies identified on allelic and genotypic characteristics have shown that all seven SNPs diverged from the Hardy-Weinberg-Equilibrium. The SNP2, SNP3, SNP6 and SNP7 had the lowest polymorphism information content values, and remaining SNPs were found to be moderate (0.25 < PIC < 0.50). The genotype CG in SNP1 at loci g.1463 C>G had the greatest association with WH, HW, CD and CCF, while the genotype TA at the very same loci was associated with BFT, ULA and IMF content in Qinchuan cattle. The CCKAR gene expression level in adipose tissue, small intestine, liver and skeleton muscle was found to be higher, whereas, the expression level of mRNA in organs of other digestive system including reticulum, abomasum and omasum was moderate. Some expression of CCKAR mRNA was found in the large intestine, kidney and rumen. CONCLUSIONS: In summary, our finding suggested that the CCKAR gene could be used as a potential candidate for the improvement of carcass quality and body measurements of Qinchuan cattle.

Animals , Cattle , Cattle/genetics , Receptor, Cholecystokinin A/genetics , Genetic Variation , Linkage Disequilibrium , Polymerase Chain Reaction/methods , Polymorphism, Single Nucleotide , Digestive System , Livestock , Genotyping Techniques , Gene Frequency , Meat Products
Electron. j. biotechnol ; 51: 58-66, May. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1343388


BACKGROUND: Transmembrane protein 95 (TMEM95) plays a role in male fertility. Previous studies showed that genes with a significant impact on reproductive traits can also affect the growth traits of livestock. Thus, we speculated that the genetic variation of TMEM95 gene may have effects on growth traits of cattle. RESULTS: Two SNPs were genotyped. The rs136174626 and rs41904693 were in the intron 4 and 30 -untranslated region, respectively. The linkage disequilibrium analysis illustrated that these two loci were not linked. The rs136174626 was associated with six growth traits of Nanyang cattle, four traits of Luxi cattle, and three traits of Ji'an cattle. For rs41904693 locus, the GG individuals had greater body height and abdominal girth in Ji' an cattle than TT and TG individuals. In Jinnan cattle, GG and TT individuals had greater body height, height at hip cross, body length, and heart girth than TG individuals. The potential splice site prediction results suggest that the rs136174626 may influence the splicing efficiency of TMEM95, and the miRNA binding site prediction results showed that the rs41904693 may influence the expression of TMEM95 by affecting the binding efficiency of Bta-miR-1584 and TMEM95 30 -UTR. CONCLUSIONS: The findings of the study suggested that the two SNPs in TMEM95 could be a reliable basis for molecular breeding in cattle.

Animals , Cattle , Cattle/genetics , Polymorphism, Single Nucleotide , Membrane Proteins/genetics , Genetic Variation , Cattle/growth & development , DNA Shuffling , Livestock , Genotyping Techniques , Gene Frequency
Rio de Janeiro; SES/RJ; 30/04/2021. 14 p.
Non-conventional in Portuguese | LILACS, SES-RJ | ID: biblio-1392553


O Estado do Rio de Janeiro vem monitorando a evolução das variantes da Covid-19 por meio de três processos de seleção de amostras. O primeiro é o monitoramento realizado pelos municípios que notifica e solicita o sequenciamento, seguindo os critérios e fluxos descritos na Nota técnica da SES-RJ Nº 09/2021. O segundo faz parte da Vigilância Genômica organizada pelo Ministério da Saúde, onde três amostras aleatórias são enviadas pelo Lacen/RJ para FUNED/MG, de acordo com os critérios estabelecidos pela SVS/ FUNED. O terceiro é através de um estudo com financiamento da Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) que iniciou em março de 2021 e irá realizar a genotipagem de um total de 4.800 amostras nos próximos seis meses, sendo 400 a cada 15 dias. Por fim, a Secretaria de Estado de Saúde tem envidado esforços em ações de redução de risco, como a vacinação, ampliação de testagem, monitoramento genômico e promoção de saúde em todo o estado do Rio de Janeiro. E é recomendado manter as medidas de proteção à vida: como evitar aglomeração, usar de máscara, lavar as mãos e fazer higienização das mãos com álcool 70°.

Humans , Brazilian Health Surveillance Agency , Epidemiological Monitoring , SARS-CoV-2/pathogenicity , COVID-19/mortality , Respiratory Tract Diseases/prevention & control , Respiratory Tract Infections/diagnostic imaging , Admitting Department, Hospital/standards , Genotyping Techniques/statistics & numerical data , Health Services Research/standards
Arq. bras. med. vet. zootec. (Online) ; 73(2): 534-538, Mar.-Apr. 2021. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1248928


As raças taurinas de origem ibérica Limonero e Carora (Bos primigenius taurus) possuem o fenótipo de pelo curto, liso e com baixa densidade folicular, o que confere a esses animais maior tolerância térmica e melhor produtividade em regiões quentes. Diferentes mutações associadas a esse fenótipo foram descritas no gene do receptor de prolactina PRLR, localizado no cromossomo bovino BTA20. Uma mutação recentemente encontrada é a substituição do nucleotídeo C por T, SNP 39136666 (p. R497*), no exon 11, que gera um códon de parada e, consequentemente, uma menor isoforma desse receptor. Neste trabalho, desenvolveu-se um protocolo rápido e de baixo custo para detecção desse SNP, utilizando-se a técnica de tetra-primer ARMS-PCR. Assim, foi possível detectar essa mutação nas raças brasileiras de origem ibérica localmente adaptadas: Caracu, Crioulo Lageano, Mocho Nacional e Pantaneiro. O alelo T foi mais frequente na raça Caracu (80%), enquanto o alelo C foi mais frequente na raça Crioulo Lageano (84%). Essa simples metodologia pode ser usada para genotipar esse SNP e ajudar na aplicação dessas informações moleculares em programas de melhoramento focados na tolerância térmica em bovinos taurinos e seus mestiços.(AU)

Animals , Cattle , Receptors, Prolactin/genetics , DNA Primers/analysis , Polymorphism, Single Nucleotide/genetics , Genotyping Techniques/methods , Multiplex Polymerase Chain Reaction/veterinary
Electron J Biotechnol ; 49: 72-81, Jan. 2021. tab, graf
Article in English | LILACS | ID: biblio-1291929


BACKGROUND: Persimmon (Diospyros kaki Thunb.) is the most widely cultivated species of the genus Diospyros. In this study, genetic diversity and variations in persimmon genotypes were investigated using single nucleotide polymorphism (SNP) markers identified by genotyping-by-sequencing (GBS) analysis. RESULTS: Ninety-five persimmon accessions grown in the Pear Research Institute, National Institute Horticultural and Herbal Science, were sequenced using the Illumina Hiseq2500 platform and polymorphic SNPs were detected to develop molecular markers. These reliable SNPs were analyzed using the Kompetitive Allele Specific PCR (KASP) assay to discriminate among persimmon genotypes. GBS generated a total of 447,495,724 trimmed reads, of which 89.7% were raw reads. After demultiplexing and sequence quality trimming, 108,876,644 clean reads were mapped to the reference transcriptome. An average of 1,146,070 genotype reads were mapped. Filtering of raw SNPs in each sample led to selection of a total of 1,725,401 high-quality SNPs. The number of homozygous and heterozygous SNPs ranged from 1,933 to 6,834 and from 846 to 5,927, respectively. CONCLUSIONS: Of the 49 SNPs selected for development of an identification system for persimmons, 15 SNPs were used in the KASP assay to analyze 32 persimmon accessions. These KASP markers discriminated among all accessions.

Polymerase Chain Reaction/methods , Diospyros/genetics , Genetic Variation , Genetic Markers , Chromosome Mapping , Polymorphism, Single Nucleotide/genetics , Alleles , Genotyping Techniques , Homozygote
Protein & Cell ; (12): 39-56, 2021.
Article in English | WPRIM | ID: wpr-880896


Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Animals , Alleles , CRISPR-Cas Systems , DNA End-Joining Repair , DNA, Circular/metabolism , Embryo, Nonmammalian , Gene Editing/methods , Gene Knock-In Techniques , Gene Knockout Techniques , Genes, Reporter , Genetic Loci , Genotyping Techniques , Green Fluorescent Proteins/metabolism , Integrases/metabolism , Luminescent Proteins/metabolism , Mutagenesis, Insertional , Single-Cell Analysis , Zebrafish/metabolism
Pesqui. vet. bras ; 41: e06717, 2021. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1250488


The brown howler monkey (Alouatta guariba clamitans) is a primate species widely distributed in South America. Infections by protozoa are common in primates. However, studies on protozoa in primates in Brazil are scarce, so the goal of this study was to investigate DNA from the apicomplexan protozoa Neospora caninum, Sarcocystis spp. and Toxoplasma gondii in tissues of A. guariba clamitans. DNA extraction was performed on tissue samples from the heart, brain, liver, spleen, lung and intestine of six A. guariba clamitans from Santa Maria, Central Region of Rio Grande do Sul, Brazil. Conventional PCR was performed using 18S rRNA gene general primers for Apicomplexa and also specific primers to amplify Neosporaspp. and Toxoplasma gondii DNA. All animals were positive in the 18S PCR and the genetic sequencing confirmed the presence of Sarcocystis spp. DNA in the tissues of four animals belonging to at least two species (S. neurona and S. gigantea) and T. gondii DNA in the other two animals. One positive sample for T. gondii was genotypically characterized as atypical by the restriction fragment length polymorphism technique. N. caninum DNA was not detected in the tested samples. The presence of Apicomplexa protozoan DNA in the tissues of the six animals tested in this study highlights the importance of howler monkeys as maintainers of these pathogens in nature.(AU)

O bugio ruivo (Alouatta guariba clamitans) é uma espécie de primata amplamente distribuída na América do Sul. As infecções por protozoários são comuns em primatas. Entretanto, estudos sobre protozoários em primatas no Brasil são escassos, portanto o objetivo deste estudo foi pesquisar DNA dos protozoários Apicomplexa Neospora caninum, Sarcocystisspp. e Toxoplasma gondii em tecidos de A. guariba clamitans. A extração de DNA foi realizada em amostras de tecido do coração, cérebro, fígado, baço, pulmão e intestino de seis A. guariba clamitans oriundos de Santa Maria, Região Central do Rio Grande do Sul, Brasil. Foi realizada PCR convencional utilizando primers geral do gene 18S rRNA para Apicomplexa e também primers específicos para amplificação de DNA de Neospora spp.e Toxoplasma gondii. Todos os animais foram positivos no PCR geral para Apicomplexa e no sequenciamento genético confirmou-se a presença de DNA de Sarcocystis nos tecidos de quatro animais pertencentes a pelo menos duas espécies (S. neurona e S. gigantea), e DNA de T. gondii foi detectado nos outros dois animais. Uma amostra positiva para T. gondii foi caracterizada genotipicamente como atípico pela técnica de polimorfismo do comprimento do fragmento de restrição. Não foi detectado DNA de N. caninum nas amostras testadas. A presença de DNA de protozoários apicomplexa nos tecidos dos seis animais testados neste estudo destaca a importância dos bugios ruivos como mantenedores desses patógenos na natureza.(AU)

Animals , Toxoplasma/pathogenicity , Polymerase Chain Reaction , Apicomplexa/pathogenicity , Alouatta/microbiology , Genotyping Techniques/veterinary , Animals, Wild/microbiology , Protozoan Infections/diagnosis , DNA, Protozoan , Molecular Diagnostic Techniques , Infections
Pesqui. vet. bras ; 41: e06729, 2021. tab, mapas
Article in English | LILACS, VETINDEX | ID: biblio-1250493


Mycobacterium bovis is responsible for bovine and buffalo tuberculosis, an important zoonotic disease with global distribution. The knowledge of the distribution and the precise identification of this disease, including advanced diagnoses such as spoligotyping, allows choosing the best strategies to fight the disease's progress. The present work aimed to investigate mycobacteria's presence, genotype their strains, and evaluate tuberculosis cases' spatial distribution from suggestive lesions in carcasses of bovine and buffalo inspected in slaughterhouses under an official inspection regime in the state of Bahia, Brazil. The study investigated 453,417 animals. Among these, 31 (0.007%) from 17 municipalities were suspected of tuberculosis. Among the culture medium growth, 95% of these were categorized as alcohol-acid resistant bacilli (BAAR). All isolates were subjected to spoligotyping and 95% were confirmed as M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). The strain SB0120 was the most prevalent, and this profile has been described in cases of human tuberculosis by M. bovis, highlighting the zoonotic potential of this profile. This study also identified strains never reported in Bahia, highlighting a distinctive pattern from other parts of Brazil, besides mixed infections. Besides, to identify strains never before described in the state, highlighting a distinctive pattern in Brazil (SB6119 and SB0852, respectively). An unpublished profile was identified and inserted in the international database (, named SB2715.(AU)

O Mycobacterium bovis é o responsável pela tuberculose bovina e bubalina, doença zoonótica importante e com distribuição global. O conhecimento da distribuição e a identificação precisa dessa enfermidade, incluindo diagnósticos mais avançados como o spoligotyping, permite escolher as melhores estratégias de combate ao avanço da doença. O presente trabalho objetivou investigar a presença de micobactérias, genotipar suas estirpes e avaliar a distribuição espacial dos casos de tuberculose a partir de lesões sugestivas nas carcaças de bovinos e bubalinos inspecionadas em frigoríficos sob regime de inspeção oficial no estado da Bahia. Foram investigados 453.417 animais dentre os quais 31 (0,007%) foram suspeitos de doença e provenientes de 17 municípios. Após o crescimento em meio de cultura, 95% foram categorizados como bacilos álcool-ácido resistentes (BAAR). Todos os isolados foram submetidos à spoligotyping e 95% foram confirmados M. bovis (SB0120, SB0121, SB0852, SB0828, SB0295, SB0881, SB1648, SB6119, SB0140, SB1055). A cepa SB0120 foi a mais prevalente e este perfil vem sendo descrito na literatura com casos de tuberculose humana por M. bovis ressaltando o potencial zoonótico deste perfil. Este estudo também identificou cepas nunca relatadas no estado da Bahia, destacando um padrão distinto de outras partes do Brasil, além da existência de infecções mistas. Permitiu ainda relatar linhagens nunca antes descritas no estado com destaque para um padrão novo no Brasil (SB6119 e SB0852 respectivamente). Um perfil inédito identificado foi identificado e inserido no banco de dados internacional (, nomeado SB2715.(AU)

Animals , Cattle , Buffaloes/genetics , Mycobacterium bovis , Cattle/genetics , Zoonoses , Genotyping Techniques
Braz. j. biol ; 81(2): 392-397, 2021. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1153365


Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disease in cats. However, scarce data on its prevalence are available in Brazil. Persian cats and Persian-related breeds were assessed by molecular genotyping for a C to A transversion in exon 29 of PKD1 gene to determine ADPKD prevalence in a Brazilian population. Genomic DNA extracted from peripheral whole blood or oral swabs samples was used to amplify exon 29 of PKD1 gene employing a PCR-RFLP methodology. From a total of 616 animals, 27/537 Persian and 1/17 Himalayan cats showed the single-nucleotide variant (C to A) at position 3284 in exon 29 of feline PKD1. This pathogenic variation has been identified only in heterozygous state. The prevalence of ADPKD in Persian cats and Persian-related breeds was 5.03% and 1.6%, respectively. There was no significant association between feline breed, gender or age with ADPKD prevalence. Of note, the observed ADPKD prevalence in Persian cats and Persian-related breeds in Brazil was lower than the ones reported in other parts of the world. This finding may be related to genetic counseling and consequent selection of ADPKD-free cats for reproduction.

A doença renal policística autossômica dominante (DRPAD) é a doença genética mais comum em gatos. No entanto, poucos dados sobre sua prevalência estão disponíveis no Brasil. Gatos Persas e de raças relacionadas foram avaliados por genotipagem molecular para a transversão C→A no exon 29 do gene PKD1 felino para determinar a prevalência de DRPAD. DNA genômico extraído de sangue total periférico ou amostras de swabs orais foram utilizados para amplificar o exon 29 do gene PKD1 pela técnica de PCR-RFLP. De um total de 616 gatos, 27/537 Persas e 1/17 Himalaia mostraram a variante de nucleotídeo único (C→A) na posição 3284 no exon 29 do gene PKD1. Esta variante patogênica foi identificada apenas em heterozigose. A prevalência de DRPAD em gatos Persas e raças relacionadas foram de 5,03% e 1,6%, respectivamente. Não houve associações significativas entre raça, gênero ou idade dos felinos e incidência de DRPAD. A prevalência de DRPAD em gatos Persas e raças relacionadas no Brasil foi menor do que em outras partes do mundo, o que pode estar relacionado ao aconselhamento genético e consequente seleção de gatos sem ADPKD para reprodução.

Animals , Cats , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/veterinary , Polycystic Kidney, Autosomal Dominant/epidemiology , Polymorphism, Restriction Fragment Length , Brazil/epidemiology , Polymerase Chain Reaction/veterinary , Prevalence , Genotyping Techniques/veterinary , Mutation
Bol. méd. Hosp. Infant. Méx ; 77(6): 303-311, Nov.-Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1142480


Abstract Background: Astrocytomas are cancer tumors of the central nervous system and represent the most common type of solid tumors during human childhood. In 2016, the World Health Organization established a molecular classification system to regroup tumor entities to achieve a more accurate diagnosis and a better clinical decision-making and selection of treatment in patients with these types of tumors. Methods: We evaluated a genotyping assay for rapid and cost-effective mutation detection in astrocytomas using TaqMan probes in an asymmetric polymerase chain reaction (PCR) assay. Results: Four diffuse astrocytomas (Grade II), three anaplastic astrocytomas (Grade III), and four glioblastomas (Grade IV) were sequenced, and all of them displayed the wild-type (WT) sequence. We tried to set up this melting analysis for the genotyping of pediatric astrocytomas by identifying the specific melting temperatures of the TaqMan probes due to the presence of the WT sequences in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and H3.3 histone A genes (H3F3A). We used an IDH1-TaqMan probe to identify the WT status of IDH1 in two different WT deoxyribonucleic acid (DNA) templates (pilocytic and diffuse astrocytoma) and obtained four melting temperature values ranged from 65.6 to 92.2°C. Furthermore, only four out of 29 reactions displayed amplification of the DNA template. Sanger sequencing was faster and more reliable to detect the gene status in all the sequenced samples. Conclusions: We conclude that conventional Sanger sequencing remains the gold standard for the genotyping of pediatric astrocytomas.

Resumen Introducción: Los astrocitomas son un tipo de cáncer que afecta al sistema nervioso central y representan el tumor sólido más común durante la infancia. En el año 2016, la Organización Mundial de la Salud estableció un sistema de clasificación molecular para reagrupar tumores con identidades genéticas similares y lograr un diagnóstico más preciso, lo que lleva a tomar las decisiones clínicas idóneas al elegir el tratamiento de pacientes con este tipo de tumores. Métodos: Se evaluó un protocolo que involucra el uso de sondas TaqMan en un ensayo de reacción en cadena de la polimerasa asimétrica para la detección de mutaciones en astrocitomas. Se secuenciaron cuatro astrocitomas difusos (Grado II), tres astrocitomas anaplásicos (Grado III) y cuatro glioblastomas (Grado IV). Se intentó establecer las condiciones del análisis para la genotipificación de los astrocitomas pediátricos mediante la identificación de las temperaturas de disociación específicas de las sondas TaqMan producidas por la prescencia de las secuancias WT en los genes isocitrato deshidrogenasa 1 y 2 (IDH1, IDH2) y H3.3 histona A (H3F3A). Resultados: Los astrocitomas mostraron la secuencia wild type (WT) (silvestre) de los genes. Se utilizó una sonda TaqMan IDH1 para identificar el estado de este gen en dos templados WT de DNA (astrocitoma pilocítico y difuso) y se obtuvieron cuatro valores de temperatura de disociación (65.6-92.2 °C). Solo cuatro de las 29 reacciones mostraron amplificación de DNA. La secuenciación de Sanger fue más rápida y confiable para detectar el estado de los genes en todas las muestras. Conclusiones: La secuenciación de Sanger sigue siendo la técnica más práctica para la genotipificación de astrocitomas pediátricos.

Child , Humans , Astrocytoma , Brain Neoplasms , Polymerase Chain Reaction , Sequence Analysis, DNA , Genotyping Techniques , Astrocytoma/diagnosis , Astrocytoma/genetics , Brain Neoplasms/diagnosis , Histones , DNA Probes , Sequence Analysis, DNA/methods , Transition Temperature , Glioma , Isocitrate Dehydrogenase , Mutation
Arq. bras. med. vet. zootec. (Online) ; 72(4): 1339-1345, July-Aug. 2020. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1131509


Free-range chickens may ingest oocysts of T. gondii present in the environment and consequently harbor virulent strains of this parasite in different tissues, without any clinical signs. Isolation of T. gondii through bioassays on mice and cats from naturally infected chicken tissues has been described in several countries, demonstrating the importance of free-range chickens in the transmission of this parasite. The aim of this study was the genotypic characterization of T. gondii isolates obtained from naturally infected free-range chickens in a rural area of the state of Rio Grande do Sul, Brazil. Brain and heart tissue from 12 chickens seropositive for T. gondii were processed using peptic digestion technique for parasite isolation. From 12 samples subjected to mouse bioassay, nine isolates were obtained. RFLP-PCR genotypic characterization was performed using 11 genetic markers: SAG1, 5'-3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico. Genetic characterization of the isolates revealed the presence of five atypical genotypes according to ToxoDB (# 11, # 55, # 64, # 140 and # 163). Our results showed a wide genetic diversity of T. gondii in free-range chickens in this region.(AU)

Galinhas criadas ao ar livre podem ingerir oocistos de T. gondii presentes no ambiente e, com isso, albergar cepas virulentas desse parasita em diferentes tecidos, sem sinais clínicos. O isolamento de T. gondii por meio de bioensaios em camundongos e gatos, a partir de tecidos de galinhas naturalmente infectadas, tem sido descrito em vários países. Isso demonstra a importância das galinhas caipiras na epidemiologia desse parasita. O objetivo deste trabalho foi caracterizar genotipicamente isolados de T. gondii obtidos de galinhas caipiras naturalmente infectadas em uma área rural do município de Santa Maria, estado do Rio Grande do Sul, Brasil. Fragmentos de cérebro e de coração, de 12 galinhas soropositivas para T. gondii, foram processados pela técnica de digestão péptica para isolamento do parasita. Das 12 amostras submetidas a bioensaio com camundongos, nove isolados foram obtidos. A caracterização genotípica por RFLP-PCR foi realizada utilizando-se 11 marcadores genéticos: SAG1, 5'-3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 e Apico e revelou a presença de cinco genótipos atípicos de acordo com o ToxoDB (# 11, # 55, # 64, # 140 e # 163). Os resultados mostraram uma ampla diversidade genética de T. gondii em galinhas caipiras nessa região.(AU)

Animals , Mice , Toxoplasma , Biological Assay/veterinary , Chickens/virology , Toxoplasmosis, Animal , Genotyping Techniques/veterinary , Rural Areas , Polymerase Chain Reaction/veterinary
Electron. j. biotechnol ; 46: 1-7, jul. 2020. ilus, graf, tab
Article in English | LILACS | ID: biblio-1223252


BACKGROUND: Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase (BADH2) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. RESULTS: In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 (badh2-E2) or an 8-bp deletion and 3-bp variation in exon 7 (badh2-E7). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2- E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F2 population. CONCLUSIONS: Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future.

Oryza/enzymology , Oryza/genetics , Betaine-Aldehyde Dehydrogenase/metabolism , Genetic Markers , Alleles , Genotyping Techniques/methods , Genotype , Odorants
An. bras. dermatol ; 95(3): 283-288, May-June 2020. tab
Article in English | LILACS, ColecionaSUS | ID: biblio-1130886


Abstract Background: Alopecia areata is an autoimmune disease that produces non-scarring hair loss around the body. Gene variants of the cytotoxic T-lymphocyte antigen 4 (CTLA4) gene, a negative regulator of T-cell response, have been associated with a predisposition to autoimmune diseases in different populations; however, the involvement of these genetic variants in the development of AA is controversial. Objective: The present study evaluated the potential association of two CTLA4 gene variants with alopecia areata in a Mexican population. Methods: We genotyped +49AG (rs231775) and CT60 (rs3087243) variants in 50 AA patients and 100 healthy control participants through PCR-RFLP. Results: No statistical difference was observed for either of the gene variants regarding allele or genotype frequencies between AA patients and the controls when the parameters of family/personal history of autoimmune diseases or gender were considered (p > 0.05). Study limitations: Small sample size of patients and the data were obtained from Northeast Mexico population. Conclusion: The genetic variants rs231775 and rs3087243 of the CTLA4 gene are not a risk factor for the development of alopecia areata in the analyzed Mexican population.

Humans , Male , Female , Adolescent , Adult , Young Adult , Genetic Variation/genetics , Alopecia Areata/genetics , CTLA-4 Antigen/genetics , Case-Control Studies , Risk Factors , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Genetic Association Studies , Genotyping Techniques , Gene Frequency , Mexico , Middle Aged
Arq. bras. med. vet. zootec. (Online) ; 72(1): 33-39, Jan.-Feb. 2020. graf
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1088915


A biópsia embrionária associada à genotipagem permite a obtenção de informações genômicas antes mesmo da transferência dos embriões. Neste estudo, foram avaliadas amostras biopsiadas de blastocistos bovinos transferidos para receptoras (n=47), sob a hipótese de que a raça (Gir ou Girolando), o estádio embrionário (blastocisto ou blastocisto expandido) e a competência para estabelecimento de prenhez (positiva ou negativa) afetariam a quantidade e a qualidade do DNA da amostra obtida. O DNA foi extraído, amplificado, quantificado por eletroferograma e genotipado. O parâmetro call rate (CR) foi adotado para mensurar a qualidade da genotipagem. Obteve-se concentração de DNA de 86,07±171,66ng/µL e CR 0,73±0,17. O CR não variou em função da quantidade de DNA nas amostras. As variáveis raça e estádio embrionário não influenciaram a concentração de DNA, nem o CR. Houve efeito da prenhez sobre o CR (P=0,0187), mas, como houve maior CR nas amostras provenientes do grupo prenhez negativa, não foi possível associar esse parâmetro à qualidade embrionária. Concluiu-se que a raça e a qualidade embrionária não afetam os parâmetros aqui estudados em amostras embrionárias, ou seja, embriões com maiores chances de implantação não refletem alta qualidade nas amostras de biópsia genotipadas.(AU)

Embryo biopsy associated with genotyping allows genomic information before embryo transfer. In this study, blastocyst biopsy samples from embryos transferred to recipients (n= 47) were evaluated, under the hypothesis that breed (Gyr or Girolando), embryonic stage (blastocyst or expanded blastocyst) and competence to establish pregnancy (positive or negative) would affect the quantity and DNA quality of samples. DNA was extracted, amplified, quantified by electropherogram and genotyped. The parameter call rate (CR) was used to measure the quality of genotyping. DNA concentration of 86.07±171.66ng/µl, and CR 0.73±0.17 was obtained. CR did not vary according to the amount of DNA in the samples. The variables breed and embryonic stage had no influence on DNA concentration or CR. There was pregnancy effect on the CR (P= 0.0187), but since there was a higher CR in the samples from the negative pregnancy group, it was not possible to associate this parameter with the embryonic quality. We conclude that the breed and embryo quality do not affect the evaluated parameters in embryonic samples. Embryos with higher chances of implantation do not reflect high quality in embryo biopsy genotyped samples.(AU)

Animals , Cattle , Selection, Genetic , Biopsy/veterinary , Sequence Analysis, DNA/veterinary , Embryo, Mammalian , Genotyping Techniques/veterinary , In Vitro Techniques/veterinary
Rio de Janeiro; s.n; 2020. xv, 78 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1128734


O Pegivirus humano 1 (HPgV-1) é um vírus de RNA de fita simples de polaridade positiva membro da família Flaviviridae, e que possui similaridade genômica com o vírus da hepatite C (HCV). No entanto, diferentemente do HCV, o HPgV-1 é linfotrópico e estabelece uma infecção subclínica. Vários estudos relataram que a infecção pelo HPgV-1 está associada à progressão tardia da doença pelo HIV, com indivíduos infectados demonstrando maiores contagens de células TCD4+, menor carga viral do HIV, uma progressão mais lenta para AIDS e, consequentemente, uma expectativa de vida prolongada. Em pacientes com coinfecção crônica por HCV e HIV, o RNA do HPgV-1 foi associado a níveis significativamente mais baixos de ALT e AST e a uma melhora na sobrevida livre de cirrose, sugerindo um efeito benéfico da infecção pelo HPgV-1 em ambas as infecções. Para a melhor compreensão do impacto do HPgV-1 nas co-infecções, se faz necessário conhecer as características epidemiológicas desse vírus. O objetivo deste estudo foi determinar a prevalência e distribuição genotípica do HPgV-1 em doadores de sangue e pacientes HCV e HIV positivos atendidos em um hospital no Rio de Janeiro entre os anos de 2017 a 2018.

Um ensaio de RT-PCR para amplificação específica da região 5'UTR do genoma viral foi realizado em 236 amostras de soro, sendo 56 amostras de coinfecção HCV/HIV, 60 de HCV mono-infectadas, 60 de HIV mono-infectadas e 60 de doadores de sangue. Todas as amostras positivas foram submetidas ao seqüenciamento direto do genoma viral para genotipagem e caracterização molecular. A prevalência geral de HPgV-1 foi de 15,7% (37/236). Maiores frequências de HPgV-1 foram encontradas no grupo de indivíduos com HIV 28,3% (17/60), seguido pelos grupos de indivíduos co-infectados com HCV/HIV (14,3%), doadores de sangue (11,6%) e em co-infecção com HCV (8,3%). A análise filogenética revelou a presença dos genótipos 2a (22,8%), 2b (57,1%), 3 (8,5%) e 1 (8,5%) de HPgV-1. Este é o primeiro estudo que caracteriza a infecção pelo HPgV-1 em pacientes com HCV e HCV/HIV na cidade do Rio de Janeiro e que visa contribuir com maiores informações sobre características epidemiológicas e clínicas do HPgV-1 no curso natural da infecções pelo HCV e/ou HIV. (AU)

Humans , Epidemiology , HIV , Hepatitis C , Flaviviridae Infections , Genotyping Techniques
Biol. Res ; 53: 15, 2020. tab, graf
Article in English | LILACS | ID: biblio-1100921


BACKGROUND: Current South American populations trace their origins mainly to three continental ancestries, i.e. European, Amerindian and African. Individual variation in relative proportions of each of these ancestries may be confounded with socio-economic factors due to population stratification. Therefore, ancestry is a potential confounder variable that should be considered in epidemiologic studies and in public health plans. However, there are few studies that have assessed the ancestry of the current admixed Chilean population. This is partly due to the high cost of genome-scale technologies commonly used to estimate ancestry. In this study we have designed a small panel of SNPs to accurately assess ancestry in the largest sampling to date of the Chilean mestizo population (n = 3349) from eight cities. Our panel is also able to distinguish between the two main Amerindian components of Chileans: Aymara from the north and Mapuche from the south. RESULTS: A panel of 150 ancestry-informative markers (AIMs) of SNP type was selected to maximize ancestry informativeness and genome coverage. Of these, 147 were successfully genotyped by KASPar assays in 2843 samples, with an average missing rate of 0.012, and a 0.95 concordance with microarray data. The ancestries estimated with the panel of AIMs had relative high correlations (0.88 for European, 0.91 for Amerindian, 0.70 for Aymara, and 0.68 for Mapuche components) with those obtained with AXIOM LAT1 array. The country's average ancestry was 0.53 ± 0.14 European, 0.04 ± 0.04 African, and 0.42 ± 0.14 Amerindian, disaggregated into 0.18 ± 0.15 Aymara and 0.25 ± 0.13 Mapuche. However, Mapuche ancestry was highest in the south (40.03%) and Aymara in the north (35.61%) as expected from the historical location of these ethnic groups. We make our results available through an online app and demonstrate how it can be used to adjust for ancestry when testing association between incidence of a disease and nongenetic risk factors. CONCLUSIONS: We have conducted the most extensive sampling, across many different cities, of current Chilean population. Ancestry varied significantly by latitude and human development. The panel of AIMs is available to the community for estimating ancestry at low cost in Chileans and other populations with similar ancestry.

Humans , Male , Female , Ethnicity/genetics , Indians, South American/genetics , Polymorphism, Single Nucleotide/genetics , Population Groups/genetics , Genetics, Population/organization & administration , Saliva , Genetic Markers/genetics , Chile , Phylogeography , Genotyping Techniques , Gene Frequency/genetics , Genotype
Rio de Janeiro; s.n; 2020. 159 p. ilus.
Thesis in Portuguese | LILACS | ID: biblio-1145658


A tuberculose (TB), provocada pelo Mycobacterium tuberculosis, é uma doença crônica contagiosa em aglomerados populacionais como as unidades prisionais. Neste trabalho realizou-se a identificação molecular de isolados de M. tuberculosis e investigação de infecção mista utilizando spoligotyping e MIRU-VNTR em 224 amostras de uma coorte do estado de Rondônia, populações geral e carcerária e análises de bioinformática em dois grupos distintos de genomas de micobactérias, sendo 30 genomas de Moçabique e 88 genomas da coleção do nosso laboratório. Amostras de escarro foram submetidas à baciloscopia, cultura para micobactérias, teste de sensibilidade aos antimicrobianos e identificação molecular pela técnica de Spoligotyping e MIRU-VNTR demonstraram a diversidade dos isolados de M. tuberculosis circulantes na região havendo a predominância de 63,8% da família LAM, seguido da família X com 11,6%, Haarlem 6,3% e família T com 4%, além de dois isolados da família Ural e cinco da família EAI.

Ainda, 18 novos perfis foram identificados, incluindo um perfil que há indícios de exclusividade do Brasil. Os resultados indicam que as cadeias de transmissão entre as populações geral e carcerária estão interligadas. As unidades prisionais apresentaram formação de grupos específicos de aglomerações e exclusividade das subfamílias T3 e X3. Entre a coorte de Rondônia, a taxa de resistência foi de 28% (4,8% MDR) e 46,6% entre os genomas analisados. Além disso, sugere-se que os isolados resistentes estão mais associados a população geral desse estudo e que processos evolutivos potencialmente associados ao surgimento de resistência. Foi detectada uma frequência de 19,3% de infecção mista pelo MIRU-VNTR e 80% por WGS; casos suscetíveis foram mais associados a infecção mista, exceto para casos MDR/XDR da coorte Brasil/Moçambique. Portanto, verifica-se a manutenção dos índices de TB em Rondônia, mas com uma diversidade maior do que o esperado, incluindo famílias com notificação em baixa frequência, bem como a indicação de que a presença da infecção mista pode ter importância clínica de acordo com a população. (AU)

Humans , Prisons , Tuberculosis , Coinfection , Genotyping Techniques , Molecular Biology