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1.
Arch. argent. pediatr ; 120(1): e39-e42, feb 2022. tab, ilus
Article in Spanish | LILACS, BINACIS | ID: biblio-1353777

ABSTRACT

Las nuevas metodologías de secuenciación masiva han permitido caracterizar e identificar variantes genéticas asociadas a diferentes patologías. En este trabajo se presenta el caso de una paciente con una mutación del gen RARS2 que codifica la enzima arginino-ARNt ligasa para la codificación de proteínas. Esta alteración genética se manifiesta en hipoplasia pontocerebelosa tipo 6, con una prevalencia de <1/1 000 0000, caracterizada por un cerebelo y un puente de menor tamaño asociados a un retraso grave en el neurodesarrollo. El análisis de caso permite un mejor conocimiento de enfermedades de origen genético, específicamente, de aquellas con patrones de herencia autosómicos recesivos de padres no consanguíneos. Su estudio sobre todo en lo relacionado con el ámbito familiar y socioeconómico, y su base genética, ayuda a una mejor calidad de vida de los pacientes y su familia.


The latest method of next-generation sequencing has allowed the characterization and identification of genetic variants associated to diverse pathologies. In this article, we present the case of female patient with a mutation of the RARS2 gene that encodes the enzyme for arginyl tRNA synthetase for coding of proteins. This genetic alteration manifests in pontocerebellar hypoplasia type 6, with a prevalence of <1/1,000,0000, characterized by a cerebellum and pons that are smaller in size and are associated with severe neurodevelopmental delay. The analysis of the case of this patient provides better knowledge of diseases of genetic origin; specifically, regarding genetic diseases of autosomal recessive patterns of inheritance from non-consanguineous parents. The impact of these studies; specially within the family, social, economic and genetic aspects helps provide a better quality of life for these patients and their family.


Subject(s)
Humans , Female , Child, Preschool , Arginine-tRNA Ligase/genetics , Quality of Life , Magnetic Resonance Imaging , Sequence Analysis , Colombia , Mutation
2.
Biomédica (Bogotá) ; 41(4): 773-786, oct.-dic. 2021. tab, graf
Article in English | LILACS | ID: biblio-1355749

ABSTRACT

Abstract | Introduction: Next Generation Sequencing (NGS) is cost-effective and a faster method to study genes, but its protocol is challenging. Objective: To analyze different adjustments to the protocol for screening the BRCA genes using Ion Torrent PGM sequencing and correlate the results with the number of false positive (FP) variants. Materials and methods: We conducted a library preparation process and analyzed the number of FP InDels, the library concentration, the number of cycles in the target amplification step, the purity of the nucleic acid, the input, and the number of samples/Ion 314 chips in association with the results obtained by NGS. Results: We carried out 51 reactions and nine adjustments of protocols and observed eight FP InDels in homopolymer regions. No FP Single-Nucleotide Polymorphism variant was observed; 67.5% of protocol variables were jointly associated with the quality of the results obtained (p<0.05). The number of FP InDels decreased when the quality of results increased. Conclusion: The Ion AmpliSeq BRCA1/BRCA2 Community Panel had a better performance using four samples per Ion-314 chip instead of eight and the optimum number of cycles in the amplification step, even when using high-quality DNA, was 23. We observed better results with the manual equalization process and not using the Ion Library Equalizer kit. These adjustments provided a higher coverage of the variants and fewer artifacts (6.7-fold). Laboratories must perform internal validation because FP InDel variants can vary according to the quality of results while the NGS assay should be validated with Sanger.


Resumen | Introducción. La secuenciación de nueva generación es un método rentable y rápido para el estudio de los genes, pero su protocolo entraña desafíos. Objetivo. Investigar diferentes ajustes del protocolo de selección de los genes BRCAmediante secuenciación de Ion Torrent PGM™ y correlacionar los resultados con el número de variantes de falso positivo. Materiales y métodos. El proceso de preparación de la biblioteca, el número de falsos positivos InDels, la concentración de la biblioteca, el número de ciclos en el paso de amplificación de objetivos, la pureza del ácido nucleico, la entrada y el número de muestras por chip del Ion-314 se analizaron en asociación con los resultados obtenidos por secuenciación de nueva generación secuenciación de nueva generación. Resultados. Se hicieron 51 reacciones y nueve ajustes de los protocolos, y se observaron ocho falsos positivos InDels en las regiones de homopolímeros. No se observó ninguna variante de polimorfismo de nucleótido simple falso positivo. En 67,5 % de los casos, las variables de protocolo en su conjunto se asociaron con la calidad de los resultados obtenidos (p<0,05). El número de falsos positivos InDels disminuyó al aumentar la calidad de los resultados. Conclusiones. El panel comunitario Ion AmpliSeq BRCA1/BRCA2 tuvo un mejor rendimiento, con cuatro muestras por chip Ion-314 en lugar de ocho, y el número de ciclos en el paso de amplificación, incluso con ADN de alta calidad, fue mejor con 23. Se observaron mejores resultados con el proceso de ecualización manual y sin el uso del kit Ion Library Equalizer. Estos ajustes proporcionaron una mayor cobertura de las variantes y menos artefactos. Los laboratorios deben realizar la validación interna porque las variantes de falsos positivos InDel pueden variar según la calidad de los resultados. La secuenciación de próxima generación debe validarse con Sanger.


Subject(s)
DNA , High-Throughput Nucleotide Sequencing , Sequence Analysis , Genes, BRCA1 , Genes, BRCA2
3.
Electron. j. biotechnol ; 50: 37-44, Mar. 2021. graf, tab
Article in English | LILACS | ID: biblio-1292321

ABSTRACT

BACKGROUND: Short Tandem repeats (STRs) existed as popular elements in both eukaryotic and prokaryotic genomes. RESULTS: In this study, we analyzed the characteristics, distributions, and motif features of STRs within whole-genomes of 140 plant species. The results showed that STR density was negatively correlated with the genome size. Hexanucleotide repeat was the most abundant type of STRs. The distribution of algae shows a preference different from that of other plants. By analyzing GC contents of STRs and genome, it was concluded that STR motif was influenced by GC contents. Analysis of the long STRs in genome (length 1000 bp) found that dicots have the more long STRs. For STR types, di- and tri-nucleotide accounted for the highest proportion. Analyzing and designing long STRs in CDS (length 500 bp) was to verify the role of long STRs in Gossypium hirsutum TM-1 and Solanum tuberosum. By comparing the long STRs found in Fragaria x ananassa with other species, some evolutionary characteristics of the long STRs were obtained. CONCLUSIONS: We got the characteristics, distribution, and motif features of STRs in the whole genome of 140 plants and obtained some evolutionary characteristics of long STRs. The study provides useful insights into STR preference, characteristics, and distribution in plants.


Subject(s)
Plants/genetics , Genetic Variation , Microsatellite Repeats , Base Sequence , Sequence Analysis
4.
Article in Chinese | WPRIM | ID: wpr-921729

ABSTRACT

Multiple methods should be incorporated into the research on pharmacovigilance of traditional Chinese medicine(TCM for a comprehensive and objective evaluation. The arrival of the era of medical big data allows it to be deeply integrated into medical research. The real world study(RWS) represented by hospital information system(HIS) provides a data basis for exploring the pharmacovigilance of TCM. Prescription sequence analysis(PSA) and prescription sequence symmetry analysis(PSSA) developed based on the former serve as a methodological basis for clinical safety evaluation of Chinese patent medicines after marketing. By collating the related studies of HIS, PSA and PSSA and employing the propensity score matching( PSM) method and nested case-control study(NCCS), this paper formed a HIS-, PSA-and PSSA-based technical system for clinical safety evaluation of Chinese patent medicines in the real world, in order to provide a methodological demonstration for the future research on the pharmacovigilance of TCM.


Subject(s)
Case-Control Studies , Drugs, Chinese Herbal , Medicine, Chinese Traditional , Pharmacovigilance , Prescriptions , Sequence Analysis
5.
Article in Chinese | WPRIM | ID: wpr-888402

ABSTRACT

OBJECTIVE@#To explore the molecular basis for a rare case with Para-Bombay AB blood type.@*METHODS@#Serological method was used to determine the blood type of the proband. Exons 6 and 7 of the ABO gene and the coding regions of FUT1 and FUT2 genes were analyzed by direct sequencing.@*RESULTS@#Serological results showed that the proband was a Para-Bombay AB subtype. His genotype was determined as ABO*A1.02/B.01. The proband was also found to harbor c.551-552delAG and c.881-882delTT of the FUT1 gene. For his four children, there were three type B and one type A, though the expression of the H type was normal.@*CONCLUSION@#The double deletions in the coding region of the FUT1 gene probably underlay the Para-Bombay blood type in the proband. Carrier of single-strand deletions may have a normal ABO phenotype.


Subject(s)
ABO Blood-Group System/genetics , Alleles , Fucosyltransferases/genetics , Genotype , Humans , Male , Phenotype , Sequence Analysis
6.
Article in Chinese | WPRIM | ID: wpr-942617

ABSTRACT

Objective: To explore the diagnostic significance of the combination of clinical and genetic detection of hereditary hemorrhagic telangiectasia (HHT) by analyzing the clinical and genetic diagnosis of a family with HHT. Methods: Medical history data of the probands and their family members were collected, and the sequence analyses of coding regions of ENG, ACVRL1, SMAD4 and GDF2 genes were performed by PCR-sequencing method, and a comprehensive diagnosis was made based on the clinical features and gene detection results. After the pathogenic gene variation was identified, 11 members of 3 generations of the family were tested for pathogenic gene mutation. Results: There was an ACVRL1 c.715_716delAG mutation in the proband and 9 other family members, which caused p.S239C. Based on the clinical and genetic findings, the 7 suspected were diagnosed and 2 asymptomatic patients were found to carry the mutation site. Conclusion: The combination of clinical features and gene detection can determine the etiology and classification of HHT, which is convenient for the early diagnosis and prevention of the disease.


Subject(s)
Activin Receptors, Type II/genetics , Endoglin/genetics , Genetic Testing , Humans , Mutation , Sequence Analysis , Telangiectasia, Hereditary Hemorrhagic/genetics
7.
Braz. j. med. biol. res ; 54(5): e10274, 2021. graf
Article in English | LILACS | ID: biblio-1153553

ABSTRACT

Prolactin (PRL) plays critical roles in regulation of biological functions with the binding of specific prolactin receptor (PRLR). Revealing the expression patterns of PRLR at different developmental stages is beneficial to better understand the role of PRL and its mechanism of action in striped hamsters. In this study, the cDNA sequence of PRLR (2866-base-pairs) was harvested from the pituitary of mature female striped hamsters (Cricetulus barabensis) that contains an 834-base-pair 5′-untranslated region (1-834 bp), a 1848-base-pair open reading frame (835-2682 bp), and a 184-base-pair 3′-untranslated region (2683-2866). The 1848-base-pair open reading frame encodes a mature prolactin-binding protein of 592 amino acids. In the mature PRLR, two prolactin-binding motifs, 12 cysteines, and five potential Asn-linked glycosylation sites were detected. Our results showed that the PRLR mRNA quantity in the hypothalamus, pituitary, ovaries, or testis was developmental-stage-dependent, with the highest level at sub-adult stage and the lowest level at old stage. We also found that PRLR mRNAs were highest in pituitary, medium level in hypothalamus, and lowest in ovaries or testis. PRLR mRNAs were significantly higher in males than in females, except in the hypothalamus and pituitary from 7-week-old striped hamsters. Moreover, the PRLR mRNAs in the hypothalamus, pituitary, and ovaries or testis were positively correlated with the expression levels of GnRH in the hypothalamus. These results indicated that the PRLR has conserved domain in striped hamster, but also possesses specific character. PRLR has multiple biological functions including positively regulating reproduction in the striped hamster.


Subject(s)
Animals , Male , Female , Prolactin/genetics , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , Pituitary Gland/metabolism , Cricetinae , Sequence Analysis , DNA, Complementary/genetics
8.
Infectio ; 24(4): 217-223, oct.-dic. 2020. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1114872

ABSTRACT

Resumen Candida spp. es un agente etiológico importante en infecciones del tracto urinario, principalmente en población con terapia antimicótica de amplio espectro y con catéteres urinarios. Candida albicans es la especie más frecuente, pero otras especies han surgido como patógenos emergentes. En este trabajo se recolectaron aislamientos de Candida spp. de urocultivos de pacientes que consultaron en Dinamica IPS entre enero 2016 y noviembre 2017. Para estimar la frecuencia de las especies y observar los patrones de sensibilidad, se realizó la identificación fenotípica y su perfil de sensibilidad con el sistema comercial Vitek 2® (BioMérieux, Inc.), adicionalmente se evaluaron mediante análisis de las secuencia y filogenética ITS1-5.8S-ITS2. En el estudio se incluyeron 78 aislamientos de Candida spp. Las frecuencias de especies de Candida identificadas empleando las herramientas moleculares fueron: C. albicans (38,5%), C. tropicalis (23,1%), C. glabrata (21,8%), C. parapsilosis (10,3%), C. metapsilosis y C. krusei (2,5%) y C. guillermondi (1,3%). La identificación por métodos moleculares y por el sistema Vitek 2 fue: C. albicans (93,3%), C. glabrata (94,1%), C. tropicalis (83,3%), C. parapsilosis (75%) C. guilliermondii y C. krusei (100%). La sensibilidad de todos los aislamientos al fluconazol fue 93,6%.


Abstract Candida spp is an important etiologic agent in urinary tract infections, mainly in patients in broad-spectrum antifungal therapy, with urinary catheters. Candida albicans is the most frequent specie; but other species have arised as emerging pathogens. In this study, isolates of Candida spp. of urine cultures from patients who consulted in Dinamica IPS between January 2016 and November 2017 were evaluated. To estimate the frequency of the species and to observe the sensitivity patterns, the phenotypic identification and its sensitivity profile was performed employed the Vitek 2® commercial system. (BioMérieux, Inc) In addition the isolates were evaluated by sequence analysis and phylogenetics ITS1-5.8S-ITS2. This study included 78 isolates of Candida spp. The frequencies of Candida species identified using the molecular tools were: C. albicans (38.5%), C. tropicalis (23.1%), C. glabrata (21.8%), C. parapsilosis (10.3%), C. guillermondi (1.3%) and C. metapsilosis and C. krusei (2.5%). The identification by molecular methods and by Vitek 2 system were: C. albicans (93.3%), for C. glabrata (94.1%), C. tropicalis (83.3%), C. parapsilosis (75%) and 100% for C. guilliermondii and C. krusei.. fluconazole sensitivity of all isolates was 93.6%


Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Aged , Candida , Diagnostic Techniques, Urological , Candida parapsilosis , Laboratories , Urinary Tract , Urinary Tract Infections , Candida albicans , Fluconazole , Sequence Analysis , Urinary Catheters , Infections
9.
Electron. j. biotechnol ; 43: 23-31, Jan. 2020. ilus, graf
Article in English | LILACS | ID: biblio-1087514

ABSTRACT

Background: Hong Qu glutinous rice wine (HQGRW) is brewed under non-aseptic fermentation conditions, so it usually has a relatively high total acid content. The aim of this study was to investigate the dynamics of the bacterial communities and total acid during the fermentation of HQGRW and elucidate the correlation between total acid and bacterial communities. Results: The results showed that the period of rapid acid increase during fermentation occurred at the early stage of fermentation. There was a negative response between total acid increase and the rate of increase in alcohol during the early fermentation stage. Bacterial community analysis using high-throughput sequencing technology was found that the dominant bacterial communities changed during the traditional fermentation of HQGRW. Both principal component analysis (PCA) and hierarchical clustering analysis revealed that there was a great difference between the bacterial communities of Hong Qu starter and those identified during the fermentation process. Furthermore, the key bacteria likely to be associated with total acid were identified by Spearman's correlation analysis. Lactobacillus, unclassified Lactobacillaceae, and Pediococcus were found, which can make significant contributions to the total acid development (| r| N 0.6 with FDR adjusted P b 0.05), establishing that these bacteria can associate closely with the total acid of rice wine. Conclusions: This was the first study to investigate the correlation between bacterial communities and total acid during the fermentation of HQGRW. These findings may be helpful in the development of a set of fermentation techniques for controlling total acid.


Subject(s)
Bacteria/isolation & purification , Wine/microbiology , Pediococcus/isolation & purification , Pediococcus/genetics , Pediococcus/metabolism , Time Factors , Acetobacter/isolation & purification , Acetobacter/genetics , Acetobacter/metabolism , Cluster Analysis , Sequence Analysis , Computational Biology , Principal Component Analysis , Fermentation , Microbiota , Hydrogen-Ion Concentration , Lactobacillus/isolation & purification , Lactobacillus/genetics , Lactobacillus/metabolism
10.
Journal of Experimental Hematology ; (6): 1059-1063, 2020.
Article in Chinese | WPRIM | ID: wpr-827161

ABSTRACT

Abstract  Single cell sequencing technology is different from traditional sequencing method, which is based on population cell average level. It has been widely used in many fields and made great progress in the application of malignant hematological diseases. In this review, the principle, methodology and application of single cell sequencing technology in malignant hematological diseases are summarized briefly, including the study of the pathogenesis in myelodysplastic syndrome, the mechanism of transformation into leukemia, accurate diagnosis and classification, differential diagnosis, evaluation of targeted drug therapeutic efficacy and exploration of biomarkers; specific diagnostic indicators for myeloproliferative diseases, progression of disease monitoring and epidemiological studies; moreover, the pathogenesis and drug resistance of acute myeloid leukemia (AML), which can provide reference for the diagnosis and research of malignant hematological diseases.


Subject(s)
Hematologic Diseases , Humans , Leukemia, Myeloid, Acute , Myelodysplastic Syndromes , Myeloproliferative Disorders , Sequence Analysis
11.
Rev. Investig. Salud. Univ. Boyacá (En línea) ; 7(2): 138-172, 2020. tab, ilust
Article in Spanish | LILACS, COLNAL | ID: biblio-1292512

ABSTRACT

Introducción: el objetivo de la secuenciación es determinar la composición de los nucleótidos presentes en el ADN o el ARN. Desde la finalización del proyecto genoma humano, surgieron diversas tecnologías de secuenciación rápida como Roche 454, SOLiD, Illumina, Ion Torrent, PacBio y Oxford Nanopore, más precisas y costoeficientes, que permiten desarrollar proyectos a gran escala y estudiar genes y genomas, la composición de microbiomas, enfermedades metabólicas y enfermedades genéticas que afectan a la población. Objetivo: describir los fundamentos de los métodos de secuenciación de ADN y sus aplicaciones en las ciencias biomédicas. Métodos: revisión descriptiva de las principales estrategias de secuenciación de ADN de primera, segunda y tercera generación y su aplicación en el entorno biomédico, a partir de la búsqueda de artículos en bases de datos electró-nicas especializadas en investigación científica. Se encontraron 118 documentos, de los cuales se excluyeron 6, por no cumplir con los criterios de inclusión, y se seleccionaron 112, por cumplir con todos los requisitos. Conclusiones:el surgimiento de los métodos de secuenciación de siguiente generación arroja una gran canti-dad de datos, incluidos genomas secuenciados completamente de varias especies, con un rendimiento extenso, tiempos reducidos y costoeficiencia, que lleva a la completa transformación de las ciencias de la vida y logra un progreso sin precedentes en el análisis de genomas, la evaluación de la ecología microbiana y el diagnóstico de enfermedades.


Introduction: The purpose of sequencing is to determine the composition of the nucleotides present in DNA or RNA. Since the completion of the human genome project, several sequencing technologies such as Roche 454, SOLiD, Illumina, Ion Torrent, PacBio and Oxford Nanopore have emerged as tools for rapid sequencing, with greater precision and cost-efficiency, allowing the development of lar-ge-scale projects and the study of genes and genomes, along with the composition of microbiomes and the study of metabolic and genetic diseases that affect the population. Objective: To describe the foundations of the methods of DNA sequencing and their applications in the biomedical sciences. Methods: Descriptive review of the main strategies of first, second and third generation DNA sequencing and their application in the biomedical environment. This review was carried out by searching articles in electronic databases specialized in scientific research. A total of 118 papers were found, of which 6 were excluded as they did not meet the inclusion criteria and 112 were selected as meeting all the requirements. Conclusions: The emergence of next-generation sequencing methods yielding a wealth of data, including fully sequenced genomes of various species, with extensive throughput, reduced time and cost-effec-tiveness that has led to the complete transformation of the life sciences, achieving unprecedented progress in genome analysis, assessment of microbial ecology and disease diagnosis


Introdução: o objetivo do sequenciamento é determinar a composição dos nucleotídeos presentes no DNA ou RNA. Desde a conclusão do projeto do genoma humano, surgiram diversas tecnologias de sequenciamento rápida como Roche 454, SOLiD, Illumina, Ion Torrent, PacBio e Oxford Nanopore, mais precisas e econômicas, que permitem o desenvolvimento de projetos de grande escala e estudo de genes e genomas, composição de microbiomas, doenças metabólicas e genéticas que afetam a popula-ção. Objetivo: descrever os fundamentos dos métodos de sequenciamento de DNA e suas aplicações nas ciências biomédicas. Métodos: revisão descritiva das principais estratégias de sequenciamento de DNA de primeira, segunda e terceira geração e sua aplicação no ambiente biomédico, a partir da busca de artigos em bases de dados eletrônicas especializadas em pesquisa científica. Foram encontrados 118 documen-tos, dos quais 6 foram excluídos por não atenderem aos critérios de inclusão e 112 fo-ram seleciona-dos por atenderem a todos os requerimentos. Conclusões: o surgimento de métodos de sequenciamento de próxima geração rende uma riqueza de dados, incluindo genomas totalmente se-quenciados de várias espécies, com produção extensa, tempos reduzidos e eficiência de custo, levando à transformação completa das ciências da vida e alcançando um progresso sem precedentes no genoma análise, avaliação de ecologia microbiana e diagnóstico de doenças.


Subject(s)
High-Throughput Nucleotide Sequencing , DNA , Genome, Human , Genetic Techniques , Sequence Analysis
12.
Article in English | WPRIM | ID: wpr-811069

ABSTRACT

PURPOSE: Different characteristics of airway microbiome in asthmatics may lead to differential immune responses, which in turn cause eosinophilic or neutrophilic airway inflammation. However, the relationships among these factors have yet to be fully elucidated.METHODS: Microbes in induced sputum samples were subjected to sequence analysis of 16S rRNA. Airway inflammatory phenotypes were defined as neutrophils (>60%) and eosinophils (>3%), and inflammation endotypes were defined by levels of T helper (Th) 1 (interferon-γ), Th2 (interleukin [IL]-5 and IL-13), Th-17 (IL-17), and innate Th2 (IL-25, IL-33, and thymic stromal lymphopoietin) cytokines, inflammasomes (IL-1β), epithelial activation markers (granulocyte-macrophage colony-stimulating factor and IL-8), and Inflammation (IL-6 and tumor necrosis factor-α) cytokines in sputum supernatants was assessed by enzyme-linked immunosorbent assay.RESULTS: The numbers of operational taxonomic units were significantly higher in the mixed (n = 21) and neutrophilic (n = 23) inflammation groups than in the paucigranulocytic inflammation group (n = 19; p < 0.05). At the species level, Granulicatella adiacens, Streptococcus parasanguinis, Streptococcus pneumoniae, Veillonella rogosae, Haemophilus parainfluenzae, and Neisseria perflava levels were significantly higher in the eosinophilic inflammation group (n = 20), whereas JYGU_s levels were significantly higher in the neutrophilic inflammation group compared to the other subtypes (P < 0.05). Additionally, IL-5 and IL-13 concentrations were correlated with the percentage of eosinophils (P < 0.05) and IL-13 levels were positively correlated with the read counts of Porphyromonas pasteri and V. rogosae (P < 0.05). IL-1β concentrations were correlated with the percentage of neutrophils (P < 0.05). had a tendency to be positively correlated with the read count of JYGU_s (P = 0.095), and was negatively correlated with that of S. pneumoniae (P < 0.05).CONCLUSIONS: Difference of microbial patterns in airways may induce distinctive endotypes of asthma, which is responsible for the neutrophilic or eosinophilic inflammation in asthma.


Subject(s)
Asthma , Colony-Stimulating Factors , Cytokines , Enzyme-Linked Immunosorbent Assay , Eosinophils , Haemophilus parainfluenzae , Inflammasomes , Inflammation , Interleukin-13 , Interleukin-33 , Interleukin-5 , Microbiota , Necrosis , Neisseria , Neutrophils , Phenotype , Pneumonia , Porphyromonas , Sequence Analysis , Sputum , Streptococcus , Streptococcus pneumoniae , Veillonella
13.
Article in English | WPRIM | ID: wpr-762473

ABSTRACT

BACKGROUND: Although the incidence of tuberculosis (TB) is decreasing, cases of multidrug-resistant (MDR) TB and extensively drug-resistant (XDR) TB continue to increase. As conventional phenotype drug susceptibility testing (pDST) takes six to eight weeks, molecular assays are widely used to determine drug resistance. we developed QuantaMatrix Multiplexed Assay Platform (QMAP) MDR/XDR assay (QuantaMatrix Inc., Seoul, Korea) that can simultaneously detect mutations related to both first- and second-line drug resistance (rifampin, isoniazid, ethambutol, fluoroquinolones, second-line injectable drugs, and streptomycin). METHODS: We used 190 clinical Mycobacterium tuberculosis (MTB) strains isolated from Myanmar, compared QMAP and pDST results, and determined concordance rates. Additionally, we performed sequence analyses for discordant results. RESULTS: QMAP results were 87.9% (167/190) concordant with pDST results. In the 23 isolates with discordant results, the QMAP and DNA sequencing results completely matched. CONCLUSIONS: The QMAP MDR/XDR assay can detect all known DNA mutations associated with drug resistance for both MDR- and XDR-MTB strains. It can be used for molecular diagnosis of MDR- and XDR-TB to rapidly initiate appropriate anti-TB drug therapy.


Subject(s)
Diagnosis , DNA , Drug Resistance , Drug Therapy , Ethambutol , Extensively Drug-Resistant Tuberculosis , Fluoroquinolones , Incidence , Isoniazid , Myanmar , Mycobacterium tuberculosis , Phenotype , Seoul , Sequence Analysis , Sequence Analysis, DNA , Tuberculosis , Tuberculosis, Multidrug-Resistant
14.
Article in English | WPRIM | ID: wpr-762458

ABSTRACT

BACKGROUND: Mutations in the quinolone resistance-determining regions (QRDRs) of Acinetobacter baumannii DNA gyrase (gyrA) and topoisomerase IV (parC) are linked to fluoroquinolone (FQ) resistance. We developed a mismatched PCR-restriction fragment length polymorphism (RFLP) assay to detect mutations in the gyrA and parC QRDRs associated with FQ resistance in A. baumannii. METHODS: Based on the conserved sequences of A. baumannii gyrA and parC, two primer sets were designed for mismatched PCR-RFLP to detect mutations in gyrA (codons 83 and 87) and parC (codons 80 and 84) by introducing an artificial restriction enzyme cleavage site into the PCR products. This assay was evaluated using 58 A. baumannii strains and 37 other Acinetobacter strains that have been identified by RNA polymerase β-subunit gene sequence analysis.


Subject(s)
Acinetobacter baumannii , Acinetobacter , Conserved Sequence , DNA Gyrase , DNA Topoisomerase IV , DNA-Directed RNA Polymerases , Polymerase Chain Reaction , Sequence Analysis
15.
J. venom. anim. toxins incl. trop. dis ; 26: e20190075, 2020. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1101266

ABSTRACT

Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)


Subject(s)
Animals , Spider Venoms , Introns , Polymerase Chain Reaction , Sequence Analysis , Cysteine , Nucleotides
17.
Article in Korean | WPRIM | ID: wpr-816605

ABSTRACT

BACKGROUND: Rapid and accurate detection of Mycobacterium tuberculosis (MTB) is of primary importance for infection control and selection of anti-tuberculosis drugs. However, most clinical laboratories report MTB complex (MTC) without reporting MTB because MTC comprising MTB, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti, Mycobacterium caprae and Mycobacterium pinnipedii have 99.9% similarity at the nucleotide level and identical 16S rRNA sequences. This study was conducted to analyze the species frequency of MTC isolates obtained from clinical specimen.METHODS: Of 310 MTC isolates obtained from clinical samples in a tertiary care hospital from February 2017 to August 2018, MolecuTech Real TB-Taq (YD Diagnostics, Korea) real-time PCR was performed, specifically to detect MTB. For DNA showing MTB negative results by MTB-specific real-time PCR or pyrazinamide-resistant strains, PCR-based MTC typing, spoligotyping, and exact tandem repeat D gene sequencing were performed.RESULTS: All the 310 MTC isolates were identified to be MTB. Two MTB strains of East-African-Indian 4-Vietnam genotype, which have not been reported in Korea, were also found.CONCLUSION: There was no zoonotic tuberculosis in this study. Since we investigated only 310 MTC isolates detected in only one medical institution, multi-center study is needed to accurately know the prevalence of zoonotic tuberculosis in Korea.


Subject(s)
DNA , Genotype , Goats , Infection Control , Korea , Mycobacterium bovis , Mycobacterium tuberculosis , Mycobacterium , Prevalence , Real-Time Polymerase Chain Reaction , Sequence Analysis , Tandem Repeat Sequences , Tertiary Healthcare , Tuberculosis
18.
São Paulo; SES/SP; 2020. 142 p. tab.(Tese).
Thesis in Portuguese | LILACS, ColecionaSUS, SES-SP, CONASS, SESSP-CTDPROD, SES-SP, SESSP-TESESESSP, SES-SP | ID: biblio-1283114

ABSTRACT

No mundo são registrados 1,7 bilhões de casos de doença diarreica aguda (DDA) por ano, sendo considerada a segunda maior causa de morte em crianças menores de 5 anos de idade. Após a introdução da vacina contra rotavírus (RVA) em diversos países do mundo e o aprimoramento das técnicas de diagnóstico molecular, outros vírus emergiram como importantes causadores de DDA, como os sapovírus (SaV), sendo frequentemente associados à surtos e casos esporádicos em adultos e crianças. No Brasil, pouco se sabe sobre a ocorrência do SaV e seu impacto em Saúde Pública. O presente estudo teve por objetivo a padronização, implantação e caracterização molecular de SaV em amostras de fezes de pacientes com gastroenterite. Foram selecionadas amostras negativas para RVA, norovírus e adenovírus entéricos, totalizando 3974 amostras coletadas de pacientes de 0 a 91 anos, provenientes das regiões Sudeste, Sul e Centro-Oeste do Brasil. Os ensaios de PCR convencional e rRT-PCR foram padronizados para implantação no diagnóstico laboratorial dos SaV. Os SaV foram detectados em 149 (3,7%) das amostras analisadas: a faixa etária de 0 a 2 anos foi a mais acometida (5,7%). Este foi o primeiro estudo a detectar SaV no estado de SP, com detecção expressiva no ano de 2016 (8,9%). A variabilidade genotípica dos SaV nas amostras estudadas foi detectada após a análise de sequencia parcial da VP1: genogrupo GI (GI.1, GI.2, GI.3, GI.6), genogrupo GII (GII.1, GII.2, GII.3, GII.4 GII.5) e genogrupo GIV (GIV.1).


Subject(s)
Sequence Analysis , Diagnosis , Sapovirus
19.
Rev. argent. microbiol ; 51(2): 170-178, jun. 2019.
Article in English | LILACS | ID: biblio-1013369

ABSTRACT

Steroids, including testosterone, estrone, 17β-estradiol, estriol and 17β-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17β-estradiol (E2), 17β-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48 h.


Los esferoides-que incluyen la testosterona, la estrona, el 17 β-estradiol, el estriol y el 17 p-etinilestradiol-son nocivos no solo para la población dinámica de las formas de vida acuática, sino también para la salud pública. En este estudio se aisló una bacteria marina degradadora de testosterona de la isla de Nanao, en el Mar del Sur de China, a la que se denominó cepa N3. Se determinó que esta cepa también podría usar 17 β-estradiol (E2), 17 p-etinilestradiol (EE2), estriol (E3) o colesterol como únicas fuentes de carbono. De acuerdo con el análisis de la secuencia del gen 16S rRNA, la cepa N3 se identificó como Vibrio sp. La caracterización adicional mostró que dicha bacteria es un organismo aerobio, gram negativo y móvil, y que presenta resistencia a ampicilina, carbenicilina, penicilina y espectinomicina. Para optimizar la condición de cultivo en relación con su capacidad de degradar la testosterona, se utilizaron el diseño factorial Plackett-Burman y el diseno compuesto central. En condiciones óptimas, el 92% de la testosterona fue degradada por Vibrio sp. N3 en 48 h.


Subject(s)
Testosterone/antagonists & inhibitors , Vibrio/isolation & purification , Vibrio/genetics , Marine Environment/analysis , Sequence Analysis/methods
20.
Article in English | WPRIM | ID: wpr-760648

ABSTRACT

OBJECTIVE: Human papillomaviruses (HPVs) are among the agents responsible for infection and cancer of the skin and mucous membranes in the human body. The aim of this study was to investigate the frequency and type distribution of HPVs in married female patients with gynecological complaints, who had visited the Maternity Hospital in Erzurum, Turkey. METHODS: In this study, 263 cervical swab samples were taken from married women using the Pap smear method and were investigated for positive reactivity against HPV. The L1 gene region of HPV was investigated using molecular methods. For this purpose, polymerase chain reaction (PCR) assays and sequence analysis of positive samples were performed. Phylogenetic analyses were performed using a bioinformatics approach after sequencing. RESULTS: HPV-DNA was detected in 17 (6.5%) samples. Highest positive reactivity to HPV-DNA was found in the 35–44 age group at 9.2%. Fourteen out of seventeen positive samples were included in the phylogenetic analysis. All isolates clustered in the Alphapapillomavirus genus. Six samples were found to be HPV 70 positive, four were HPV 16 positive, and the rest were HPV 54, 72, 81, and 114 positive. When genotyping data were evaluated according to the risk group, we found that 28.6% of the 14 samples were found to be high risk-HPV, and 71.4% were low risk-HPV. CONCLUSIONS: As per our knowledge, this is the first report on the phylogenetic analysis of HPV genotypes isolated from women in Turkey. The prevalence of low- and-high risk HPV was determined in married women in Erzurum, and these results contribute to the epidemiological data on the distribution of HPV types for this region.


Subject(s)
Alphapapillomavirus , Computational Biology , Female , Genotype , Hospitals, Maternity , Human Body , Human papillomavirus 16 , Humans , Methods , Mucous Membrane , Papanicolaou Test , Papillomavirus Infections , Polymerase Chain Reaction , Prevalence , Sequence Analysis , Skin Neoplasms , Turkey , Vaginal Smears
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