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1.
Arq. bras. med. vet. zootec. (Online) ; 73(4): 781-790, Jul.-Aug. 2021. tab, ilus
Article in English | ID: biblio-1285278

ABSTRACT

The objective of the present study was to Standardize a Polymerase Chain Reaction (PCR) protocol for the authentication of bovine and buffalo milk, and to detect the presence of Salmonella spp. and Listeria monocytogenes. For this, the target DNA was extracted, mixed, and subjected to a PCR assay. Milk samples were defrauded and experimentally contaminated with microorganisms to assess the detection of target DNA at different times of cultivation, bacterial titers, and concentration of genetic material. In addition, the protocol was tested with DNA extracted directly from food, without a pre-enrichment step. The proposed quadruplex PCR showed good accuracy in identifying target DNA sequences. It was possible to simultaneously identify all DNA sequences at the time of inoculation (0h), when the samples were contaminated with 2 CFU/250mL and with 6h of culture when the initial inoculum was 1 CFU/250mL. It was also possible to directly detect DNA sequences from the food when it was inoculated with 3 CFU/mL bacteria. Thus, the proposed methodology showed satisfactory performance, optimization of the analysis time, and a potential for the detection of microorganisms at low titers, which can be used for the detection of fraud and contamination.


O objetivo do presente estudo foi padronizar um protocolo de reação em cadeia da polimerase (PCR) para a autenticação de leite bovino e bubalino e a detecção da presença de Salmonella spp. e Listeria monocytogenes. Para isso, o DNA-alvo foi extraído, misturado e submetido ao ensaio de PCR. Amostras de leite foram fraudadas e contaminadas experimentalmente com os micro-organismos, para se avaliar a detecção do DNA-alvo em diferentes tempos de cultivo, os títulos bacterianos e a concentração de material genético. Além disso, o protocolo foi testado com DNA extraído diretamente do alimento, sem a etapa de pré-enriquecimento. A PCR quadriplex proposta mostrou boa precisão na identificação de sequências de DNA-alvo. Foi possível identificar simultaneamente todas as sequências de DNA no momento da inoculação (0h), quando as amostras estavam contaminadas com 2 UFC/250mL, e com seis horas de cultura, quando o inóculo inicial foi de 1 UFC/250mL. Também foi possível detectar diretamente as sequências de DNA do alimento quando este foi inoculado com 3 UFC/mL de bactérias. Dessa forma, a metodologia proposta apresentou desempenho satisfatório, otimização do tempo de análise e potencial para detecção de micro-organismos em baixos títulos, podendo ser utilizada para detecção de fraude e contaminação.


Subject(s)
Animals , Cattle , Salmonella/isolation & purification , Buffaloes , Milk/microbiology , Fraud/prevention & control , Listeria monocytogenes/isolation & purification , Food Safety/methods , Multiplex Polymerase Chain Reaction/veterinary
2.
Arq. bras. med. vet. zootec. (Online) ; 73(2): 534-538, Mar.-Apr. 2021. tab, ilus
Article in English | ID: biblio-1248928

ABSTRACT

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)


Subject(s)
Animals , Cattle , Receptors, Prolactin/genetics , DNA Primers/analysis , Polymorphism, Single Nucleotide/genetics , Genotyping Techniques/methods , Multiplex Polymerase Chain Reaction/veterinary
3.
Article in Chinese | WPRIM | ID: wpr-879596

ABSTRACT

OBJECTIVE@#To establish a newborn screening system for Duchenne muscular dystrophy (DMD) through assessment of MM isoenzyme of creatine kinase (CK-MM) activity.@*METHODS@#The CK-MM level was detected using dry blood spot filter paper from 10 252 male newborns. The results were grouped based on their gestational age, sampling time and intervals between the experiments. The threshold value for CK-MM necessitating genetic testing was determined. Next-generation sequencing (NGS) was carried out for those with a CK-MM value over the threshold, and the result was verified by multiplex ligation-dependent probe amplification (MLPA).@*RESULTS@#Based on the result of non-parametric rank sum test, the median CK-MM concentration has increased with the gestational age, and was inversely correlated with the age of the newborns among unaffected specimens. CK-MM on dry blood spot filter paper can be stable for 14 days at 2-8℃. Statistical analysis of CK-MM value of the 10 252 neonates suggested that the threshold may be set as 700 ng/mL. Exonic deletions were found in 2 confirmed cases, whose CK-MM level was greater than 2000 ng/mL.@*CONCLUSION@#Detection of CK-MM in dry blood spot filter paper has provided an effective method for newborn screening of DMD. This simple and inexpensive method can be used for large-scale screening, which is of great value to the early intervention and treatment of the disease.


Subject(s)
Dystrophin/genetics , Exons , Humans , Infant, Newborn , Male , Multiplex Polymerase Chain Reaction , Muscular Dystrophy, Duchenne/genetics , Neonatal Screening
4.
Article in Chinese | WPRIM | ID: wpr-879556

ABSTRACT

OBJECTIVE@#To carry out prenatal diagnosis for families with high risk for spinal muscular atrophy (SMA) by using multiplex ligation-dependent probe amplification (MLPA).@*METHODS@#Twenty-one families were enrolled. MLPA was used to detect copy numbers of SMN1 and SMN2 genes. Maternal contamination was excluded by using a short tandem repeat method.@*RESULTS@#For 23 fetuses from the 21 families, 14 were identified as carriers, 1 as SMA patient, and 8 as normal. By linkage analysis of parental samples, three individuals were determined as silent (2+0) carriers.@*CONCLUSION@#MLPA can determine the carrier status of SMA. The identification of three silent (2+0) carriers among the 44 parental samples indicated a risk for such families, for which genetic counseling and reproduction guidance should be provided.


Subject(s)
Female , Genetic Counseling , Heterozygote , Humans , Multiplex Polymerase Chain Reaction , Muscular Atrophy, Spinal/genetics , Pregnancy , Prenatal Diagnosis , Survival of Motor Neuron 1 Protein/genetics
5.
Pesqui. vet. bras ; 41: e06706, 2021. tab
Article in English | ID: biblio-1346697

ABSTRACT

Contamination of the veterinary hospital environment with multiresistant pathogens endangers not only hospitalized animals, but also the workplace safety of veterinarians and nurses, animal guardians and, when in case of a teaching hospital, veterinary students. The objective of this study was to map the main points of bacterial contamination of a veterinary teaching hospital in Brazil to identify multiresistant microorganisms and their antimicrobial resistance genes. Samples were collected from 39 different locations of a veterinary school hospital which comprised a pool according to each hospital environment. In certain environments, more than one pool has been collected. All samples were collected in quadruplicates for the selective isolation of the main multiresistant microorganisms: methicillin-resistant Staphylococcus (MRS), vancomycin resistant Enterococcus (VRE), cephalosporinases and/or extended-spectrum beta-lactamase-producing Gram-negative bacteria (ESBL) and Carbapenemase-producing (CP). After isolation and identification of isolates, multiplex-PCR reactions were performed to detect the main genes for each microorganism and antimicrobial susceptibility tests with the main antibiotics used for each bacterial group according to CLSI. Of the 39 veterinary teaching hospital sites collected, all (100%) had at least one of the microorganisms surveyed, and 17.95% (n=7) of the sites were able to isolate the four pathogens. From the 94 pools collected, it was possible to isolate MRS in 81.91% (n=77), VRE in 12.77% (n=12), cephalosporinases and/or ESBL in 62.77% (n=59) and CP in 24.47%. (n=23). Regarding MRS, the mecA gene was detected in all isolates. All isolated VREs were identified as Enterococcus faecalis and presented the vanA gene. Regarding cephalosporinases and/or ESBL, 89.83% (n=53) of the isolates presented the blaTEM gene, 57.63% (n=34) the blaOXA-1 gene, 37.29% (n=22) blaCTX-M gene from some group (1, 2, 9 ou 8/25) and 20.34% (n=12) the blaSHV gene. It was possible to identify the main microorganisms responsible for causing nosocomial infections in humans (VRE, MRS, ESBL and CP) in the veterinary hospital environment, suggesting a source of infection for professionals and students of veterinary medicine, placing a high risk for public health.(AU)


A contaminação do ambiente hospitalar veterinário com patógenos multirresistentes coloca em perigo não apenas os animais hospitalizados, mas também a segurança no local de trabalho de veterinários e enfermeiros, responsáveis por animais e, quando se tratar de um hospital de ensino, estudantes de veterinária. O objetivo deste estudo foi mapear os principais pontos de contaminação bacteriana de um hospital veterinário de ensino no Brasil, identificando microorganismos multirresistentes e seus genes de resistência antimicrobiana. As amostras foram coletadas em 39 locais diferentes de um hospital de escola veterinária, que compreendia um pool de acordo com o ambiente de cada hospital. Em certos ambientes, mais de um pool foi coletado. Todas as amostras foram coletadas em quadruplicados para o isolamento seletivo dos principais microorganismos multirresistentes: Staphylococcus resistente à meticilina (MRS), Enterococcus resistente à vancomicina (VRE), bactérias Gram-negativas produtoras de cefalosporinases e/ou beta-lactamase de espectro estendido (ESBL) e produtoras de carbapenemase (PC). Após o isolamento e identificação dos isolados, foram realizadas reações de PCR multiplex para detectar os principais genes de cada microorganismo e testes de susceptibilidade a antimicrobianos com os principais antibióticos utilizados para cada grupo bacteriano de acordo com o CLSI. Dos 39 locais do VCH coletados, todos (100%) possuíam pelo menos um dos microrganismos pesquisados e 17,95% (n=7) dos locais foram capazes de isolar os quatro patógenos. Dos 94 pools coletados, foi possível isolar MRS em 81,91% (n=77), VRE em 12,77% (n=12), ESBL em 62,77% (n=59) e carbapenemases em 24,47% (n=23). Em relação ao MRS, o gene mecA foi detectado em todos os isolados. Todos os VREs isolados foram identificados como Enterococcus faecalis e apresentaram o gene vanA. Em relação às cefalosporinases e/ou ESBL, 89,83% (n=53) dos isolados apresentaram o gene blaTEM, 57,63% (n=34) o gene blaOXA-1, 37,29% (n=22) o gene blaCTX-M de algum grupo e 20,34% (n=12) o gene blaSHV. Foi possível identificar os principais microrganismos responsáveis por causar infecções nosocomiais em humanos (VRE, MRS, ESBL e CP) no ambiente hospitalar veterinário, sugerindo uma fonte de infecção para profissionais e estudantes de medicina veterinária, colocando alto risco para a saúde pública.(AU)


Subject(s)
Staphylococcus , Cross Infection , Methicillin Resistance , Enterococcus faecalis , Multiplex Polymerase Chain Reaction , Anti-Infective Agents , Anti-Bacterial Agents , beta-Lactamases , Hospitals, Animal
6.
Med. lab ; 25(2): 547-550, 2021. tabs
Article in Spanish | LILACS | ID: biblio-1342899

ABSTRACT

Utilidad clínica de la prueba La relación causal entre el desarrollo de cáncer de cérvix y la infección con genotipos de alto riesgo (AR) del virus del papiloma humano (VPH), ha llevado al desarrollo de estrategias para su detección y caracterización genotípica, como una medida de prevención de este tipo de cáncer. Dado que la presencia del VPH no puede ser determinada mediante los hallazgos clínicos de la paciente, como tampoco en los hallazgos morfológicos en la citología ni en la detección de anticuerpos específicos contra el VPH (pruebas serológicas), su detección y genotipificación recaen en el uso de pruebas moleculares, las cuales en su mayoría están dirigidas a la detección del ADN de los genotipos de alto riesgo, usando la técnica de reacción en cadena de la polimerasa (PCR) convencional y en tiempo real (RT-PCR) [1]. La técnica de PCR permite la amplificación de regiones específicas del ADN del VPH en los genes L1, E6 y E7, los cuales, por sus variaciones en la secuencia, permiten la genotipificación del virus [2,3]. Las pruebas de detección de ADN y/o genotipificación del VPH son consideradas herramientas de tamización en cáncer de cérvix, que detectan la infección causada por VPH. Su aplicación está enfocada en la clasificación de anormalidades citológicas, monitoreo de infecciones persistentes, seguimiento postratamiento de lesiones intraepiteliales de alto grado y vigilancia epidemiológica en salud pública [4-6]. La utilización de la citología y las pruebas de detección de ADN del VPH, aumenta la sensibilidad de la tamización para la detección de cáncer de cérvix y reduce de manera significativa el riesgo de sufrir lesiones cervicales premalignas por un periodo de 5 años [2,7]


Subject(s)
Humans , Alphapapillomavirus , Uterine Cervical Neoplasms , Polymerase Chain Reaction , Multiplex Polymerase Chain Reaction
7.
Rev. med. Risaralda ; 26(1): 28-37, ene.-jun. 2020. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1127001

ABSTRACT

Resumen Introducción: La infección genital por el Virus de Papiloma Humano (VPH) se ha asociado con el cáncer cérvicouterino (CCE) al provocar la aparición de lesiones precursoras de cáncer en la zona de transformación de la unión escamo-columnar del cuello uterino. Existen más de 100 tipos de VPH, clasificados en bajo riesgo oncogénico (VPH-BR) y alto riesgo oncogénico (VPH-AR). Estudios reportan la infección por genotipos de alto riesgo en el 100% de los CCE. En Venezuela, el 67,7% de los CCE, se relacionan con el genotipo de VPH-AR 16. Objetivo: Detectar la presencia de VPH en pacientes con cambios citológicos cervicouterino. Metodología: Se incluyeron 49 pacientes que presentaban cambios citológicos, se tomaron las muestras de la región endocervical y exocervical para la detección y genotipificación del virus mediante la técnica de Multiple PCR. Resultados: Las alteraciones citológicas presentes fueron Células Escamosas Atípicas (69,4%), Células Glandulares Atípicas (4,1%), Lesión Escamosa Intraepitelial de Bajo Grado (16,3%), y Lesión Escamosa Intraepitelial de Alto Grado (10,2%). La detección molecular demostró que 16,3% presentaba VPH, 62,5% correspondían a VPH-AR, 25% a VPH-BR, 12,5% al genotipo 16 y no se detectó el genotipo 18. Se reportó un solo caso de coinfección. Conclusiones: A diferencia de otros estudios, no se encontró una relación estadísticamente significativa entre la presencia del virus y la aparición de cambios citológicos cervicouterino en esta población. No obstante, se detectaron genotipos de alto riesgo oncogénico, lo que puede traducirse en una mayor incidencia de cáncer cervicouterino a futuro.


Abstract Introduction: Genital infection by the Human Papilloma Virus (HPV) has been associated with cervical cancer (CC) since it causes the appearance of precursor cancer lesions in the transformation area of ​​the squamous-columnar junction of the cervix. There are more than 100 types of HPV that are classified as low oncogenic risk (LR-HPV) and high oncogenic risk (HR-HPV). Studies report that the infection by high-risk genotypes is present in 100% of CC. In Venezuela, 67.7% of CC is related to the HPV-16 genotype. Objective: This study seeks to detect the presence of HPV in patients with cervical cytological cell changes. Methodology : Forty-nine patients with cytological changes were studied. The endocervical and ectocervical areas were sampled to detect and genotype the virus by using the Multiplex PCR technique. Results: The cytological alterations presented were: Atypical Squamous Cells (69.4%), Atypical Glandular Cells (4.1%), Low-grade Squamous Intraepithelial Lesion (16.3%) and High-grade Squamous Intraepithelial Lesion (10.2%). Besides, the general molecular detection showed that 16.3% had HPV, 62.5% of it corresponded to HR-HPV, 25% to LR-HPV, and 12.5% ​​to genotype 16. The genotype 18 was not detected, and only one co-infection case was reported. Conclusions: Unlike other studies, a statistically significant relationship was not found between the virus presence and the appearance of cervical cytological cell changes in this population. However, genotypes with high oncogenic risk were detected, which may lead to a higher incidence of cervical cancer in the future.


Subject(s)
Humans , Female , Papillomaviridae , Uterine Cervical Neoplasms , Cervix Uteri , Polymerase Chain Reaction , Cell Biology , Reproductive Tract Infections , Atypical Squamous Cells of the Cervix , Gynecology , Venezuela , Human papillomavirus 16 , Coinfection , Multiplex Polymerase Chain Reaction , Squamous Intraepithelial Lesions , Genitalia , Herpes Zoster
8.
Article in Chinese | WPRIM | ID: wpr-826550

ABSTRACT

OBJECTIVE@#To carry out genetic testing for a male infant suspected for Menkes disease.@*METHODS@#Genomic DNA of the proband and his parents were extracted and subjected to family trio whole exome sequencing (WES). Microduplication and microdeletion of the ATP7A gene were detected by multiplex ligation-dependent probe amplification (MLPA). Suspected variants were subjected to bioinformatic analysis and verified by Sanger sequencing.@*RESULTS@#The proband was found to harbor a de novo c.1870 -13T>G variation of the ATP7A gene, which may alter a splice site and affect its protein product.@*CONCLUSION@#The patient was diagnosed with Menkes disease due to the c.1870 -13T>G variant of the ATP7A gene. Whole exome sequencing of family trios is a powerful tool for the diagnosis of diseases with strong phenotypic heterogeneity.


Subject(s)
Copper-Transporting ATPases , Genetics , Genetic Testing , Humans , Infant , Male , Menkes Kinky Hair Syndrome , Genetics , Multiplex Polymerase Chain Reaction , Mutation , Whole Exome Sequencing
9.
Article in English | WPRIM | ID: wpr-816637

ABSTRACT

Sexually transmitted infections (STIs) are caused by the spread of pathogens via sexual activity and can cause serious complications if left untreated, regardless of their symptoms. Therefore, early diagnosis of STI is important, and molecular diagnostic methods for rapid detection and monitoring are needed. In this study, we evaluated a multiplex polymerase chain reaction (PCR) kit for simultaneously detecting 13 different bacterial, fungal, and viral microorganisms that cause STIs. The kit performance was evaluated for its sensitivity, lot-to-lot variation, and interference in detecting different pathogens. Additionally, its clinical usefulness was evaluated by estimating its sensitivity and specificity for clinical samples. The limit of detection (LOD) was 0.021–50.104 copies for each pathogen. In the tests of lot-to-lot, 100% of positive samples were detected at low concentrations and negative samples all showed negative results. This result confirms that there is no the variation of lot-to-lot. In the test for interference between pathogens, the efficiency of amplification for each pathogen was not significantly reduced and no nonspecific amplification product was formed. We tested 322 vaginal swab samples using the multiplex PCR kit and confirmed that its clinical sensitivity and specificity were 100% for all pathogens. This multiplex PCR kit can be used widely for rapid diagnosis and monitoring of STIs.


Subject(s)
Diagnosis , Early Diagnosis , Limit of Detection , Multiplex Polymerase Chain Reaction , Pathology, Molecular , Sensitivity and Specificity , Sexual Behavior , Sexually Transmitted Diseases
10.
Article in Chinese | WPRIM | ID: wpr-828699

ABSTRACT

OBJECTIVE@#To study the association between clinical phenotypes and genotypes in children with Becker muscular dystrophy (BMD)/Duchenne muscular dystrophy (DMD) so as to provide a theoretical basis for disease management, gene therapy, and prenatal diagnosis.@*METHODS@#A retrospective analysis was performed for the clinical data and gene detection results of 52 children with BMD/DMD. Multiplex ligation-dependent probe amplification (MLPA) was used to detect the DMD gene. The children with negative results of MLPA were further screened by exon chip capture combined with next-generation sequencing (NGS). The mothers of 20 probands were validated by sequencing.@*RESULTS@#The pathogenic genes for BMD/DMD were detected in 50 children by MLPA and NGS, with a detection rate of 96%. Among the 52 children, 36 (69%) had gene deletion, 7 (13%) had duplication, and 7 (13%) had micromutation. Among the 43 children with deletion/duplication, 32 had DMD and 11 had BMD; 37 children (86%) met the reading frame rule, among whom 27 (96%) had DMD and 10 (67%) had BMD. All 7 children with micromutation had DMD.@*CONCLUSIONS@#The reading frame rule has an extremely high predictive value for DMD but a limited predictive value for BMD.


Subject(s)
Child , Dystrophin , Female , Gene Deletion , Genotype , Humans , Multiplex Polymerase Chain Reaction , Muscular Dystrophy, Duchenne , Mutation , Phenotype , Pregnancy , Retrospective Studies
11.
Chinese Journal of Biotechnology ; (12): 908-919, 2020.
Article in Chinese | WPRIM | ID: wpr-826885

ABSTRACT

A multiplex PCR method was developed to detect the main pathogens of Qinghai Tibetan sheep endometritis. First, the genomes of five standard bacterial strains were extracted and specific primers were selected; the multiplex PCR method was established by using the genome of the standard strain as a template. The samples were collected by sterile cotton swab from Tibetan sheep uterus, and then placed in LB medium and numbered. After 48 h, the genomes of cultured bacteria were extracted and detected by single PCR method, then the positive samples were recorded. The positive samples detected by single PCR were selected for multiplex PCR detection and recorded again. The coincidence rate between these two methods was calculated to measure the accuracy of multiplex PCR. In order to identify the species of the pathogen, 30 positive samples verified by single and multiplex PCR were randomly selected for bacterial isolation and identification. In the 600 samples, the infected ratio of Streptococcus agalactiae (GBS) was 47.33%, Escherichia coli 34.83%, Staphylococcus aureus 6.5%, Salmonella and Trueperella pyogenes were negatively detected. Among the positive samples detected by multiplex PCR, the positive ratio of GBS was 45.50%, E. coli 33.50%, S. aureus 6.5%. Comparison of two detection results, Multiplex PCR detection coincidence rate is more than 95%. The isolated pathogens were identified as E. coli, GBS and S. aureus, which was consistent with the results of two methods. The multiplex PCR method was successfully established and the main pathogens of endometritis in Qinghai Tibetan sheep were GBS, E. coli and S. aureus.


Subject(s)
Animals , Bacteria , Genetics , Bacteriological Techniques , Methods , Endometritis , Microbiology , Female , Multiplex Polymerase Chain Reaction , Reference Standards , Polymerase Chain Reaction , Sensitivity and Specificity , Sheep , Sheep Diseases , Microbiology , Tibet
12.
Article in English | WPRIM | ID: wpr-762472

ABSTRACT

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is caused by pathogenic variants in BRCA and other cancer-related genes. We analyzed variants in BRCA gene and other cancer-related genes in HBOC patients to evaluate the clinical validity of next-generation sequencing (NGS) multi-gene panel testing. METHODS: The BRCA1/2 NGS testing was conducted for 262 HBOC patients. Multiplex ligation-dependent probe amplification and direct Sanger sequencing were performed for confirmation. Multi-gene panel testing was conducted for 120 patients who did not possess BRCA1/2 pathogenic variants but met the National Comprehensive Cancer Network criteria. RESULTS: Pathogenic variants in BRCA1/2 were detected in 30 HBOC patients (11.5%). Additionally, four out of the 120 patients possessed pathogenic variants by multi-gene panel testing (3.3%): MSH2 (c.256G>T, p.Glu86*), PMS2 (c.1687C>T, p.Arg563*), CHEK2 (c.546C>A, p.Tyr182*), and PALB2 (c.3351-1G>C). All the four patients had a family history of cancer. CONCLUSIONS: Multi-gene panel testing could be a significant screening tool for HBOC patients, especially for those with a family history of cancer.


Subject(s)
Hereditary Breast and Ovarian Cancer Syndrome , Humans , Mass Screening , Multiplex Polymerase Chain Reaction
13.
Article in English | WPRIM | ID: wpr-762178

ABSTRACT

PURPOSE: Immunological mechanisms underlying asthma exacerbation have not been elucidated. The aim of this study was to assess the associations of various asthma exacerbation traits with selected serum microRNA (miRNA) expression and T-cell subpopulations. METHODS: Twenty-one asthmatics were studied during asthma exacerbation (exacerbation visit [EV] and the follow-up visit [FV] at 6 weeks). At both visits, spirometry was performed, fractional exhaled nitric oxide (FeNO) was measured, and nasopharyngeal and blood samples were collected. In nasopharyngeal samples, respiratory viruses were assayed by multiplex polymerase chain reaction (PCR), and bacterial cultures were performed. Serum miRNAs were assayed with real-time PCR. T-cell surface markers, eosinophil progenitors and intracellular cytokines were assessed by flow cytometry. RESULTS: Two-thirds of patients had moderate or severe exacerbation and the FV, overall improvement in asthma control was observed. The mean expression of serum miRNA-126a, miRNA-16 and miRNA-21 was significantly lower at the EV than at the FV. At EV, miRNA-29b correlated with FeNO (r = 0.44, P < 0.05), and 5 of 7 miRNA tested correlated with pulmonary function tests. The number of cluster of differentiation (CD)45+CD4+interleukin (IL)4+ cells was significantly higher at the EV than at the FV, and positive correlations of T-regulatory cells and eosinophil progenitors with asthma control was found. At the EV, serum miRNAs negatively correlated with the number of T cells expressing IL-4, IL-17, IL-22 and interferon gamma, while at the FV both positive and negative correlations with T-cell subsets were observed. No association of detected pathogen (viruses and bacteria) in nasopharyngeal fluid with clinical, functional and immunological parameters was found. CONCLUSIONS: Epigenetic dysregulation during asthma exacerbation could be related to respiratory function, airway inflammation and T-cell cytokine expression.


Subject(s)
Asthma , Cytokines , Disease Progression , Eosinophils , Epigenomics , Flow Cytometry , Follow-Up Studies , Humans , Inflammation , Interferons , Interleukin-17 , Interleukin-4 , MicroRNAs , Multiplex Polymerase Chain Reaction , Nitric Oxide , Real-Time Polymerase Chain Reaction , Respiratory Function Tests , Spirometry , T-Lymphocyte Subsets , T-Lymphocytes
14.
Article in Korean | WPRIM | ID: wpr-811321

ABSTRACT

PURPOSE: To report a case of Epstein-Barr virus-related corneal endotheliitis accompanied by secondary glaucoma.CASE SUMMARY: A 73-year-old male presented with blurred vision in his right eye. In the ophthalmic evaluation, there were dispersed keratic precipitates overlying corneal edema. The anterior chamber showed trace ~1+ graded inflammation and an endothelial density decrease. His best-corrected visual acuity and intraocular pressure in the right eye were 0.2 and 34 mmHg, respectively. Paracentesis was performed on the anterior chamber of the right eye to confirm the diagnosis under the suspicion of corneal endotheliitis with trabeculectomy for the intraocular pressure control. Epstein-Barr virus was confirmed using a multiplex polymerase chain reaction (PCR), and oral and eye drops of Acyclovir were used to treat the patient. There was no evidence of a recurrence over 2 years and his intraocular pressure was 12 mmHg and best-corrected visual acuity was maintained at 0.5.CONCLUSIONS: A case of Epstein-Barr virus-related corneal endotheliitis was diagnosed using PCR of the aqueous humor. The patient was treated with an oral antiviral agent and eyedrops without a recurrence.


Subject(s)
Acyclovir , Aged , Anterior Chamber , Aqueous Humor , Corneal Edema , Diagnosis , Glaucoma , Herpesvirus 4, Human , Humans , Inflammation , Intraocular Pressure , Male , Multiplex Polymerase Chain Reaction , Ophthalmic Solutions , Paracentesis , Polymerase Chain Reaction , Recurrence , Trabeculectomy , Visual Acuity
15.
Braz. j. infect. dis ; 23(6): 468-470, Nov.-Dec. 2019. tab
Article in English | LILACS | ID: biblio-1089315

ABSTRACT

ABSTRACT The precise diagnosis of bacterial meningitis is essential. Cytological and biochemical examination of cerebrospinal fluid (CSF) are not specific. Conventional methods for bacterial meningitis lack sensitivity or take too long for a final result. Therefore, other methods for rapid and accurate diagnosis of central nervous system infections are required. FilmArray meningitis/encephalitis (ME) panel is a PCR multiplex for simultaneous and rapid identification of 14 pathogens, including 6 bacteria, 7 viruses, and Cryptococcus. We evaluated 436 CSF samples submitted to FilmArray ME Panel. Among them, 25 cases were positive for bacteria, being Streptococcus pneumonia the most frequent (48 %). Among positive cases for bacteria, 60 % were positive only with FilmArray. All the bacterial meningitis cases in which the only positive test was FilmArray had CSF findings suggestive of bacterial meningitis, including neutrophilic pleocytosis, increased CSF protein and lactate, and decreased CSF glucose. These findings suggest that FilmArray may increase the diagnostic sensitivity for bacterial meningitis.


Subject(s)
Humans , Cerebrospinal Fluid/microbiology , Cerebrospinal Fluid/virology , Meningitis, Bacterial/diagnosis , Multiplex Polymerase Chain Reaction/methods , Bacteria/isolation & purification , Viruses/isolation & purification , Microbial Sensitivity Tests/methods , Sensitivity and Specificity , Meningitis, Bacterial/cerebrospinal fluid
16.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 17(2): 71-76, ago. 2019. tab, ilus
Article in Spanish | LILACS, BDNPAR | ID: biblio-1008486

ABSTRACT

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)


STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)


Subject(s)
Shiga-Toxigenic Escherichia coli/isolation & purification , Multiplex Polymerase Chain Reaction , Cross-Sectional Studies , Escherichia coli O157/isolation & purification , Escherichia coli O157/genetics , Shiga-Toxigenic Escherichia coli/genetics , Escherichia coli O104/isolation & purification , Escherichia coli O104/genetics
17.
An. bras. dermatol ; 94(4): 429-433, July-Aug. 2019. tab
Article in English | LILACS | ID: biblio-1038308

ABSTRACT

Abstract: Background: Behçet disease is a prototypical systemic autoimmune disease, caused by a complex interplay between environmental and genetic factors. The transmembrane immunoglobulin and mucin domain-3 (TIM-3) is a distinct member of the TIM family that is preferentially expressed on Th1 cells and plays a role in Th1-mediated autoimmune or inflammatory diseases, such as Behçet disease. Objective: The aim of this study was to test the potential association between TIM-3 gene polymorphisms and Behçet disease. Methods: Two single-nucleotide polymorphisms of TIM-3 (rs9313439 and rs10515746) were genotyped in 212 patients with Behçet disease and 200 healthy controls. Typing of the polymorphisms was performed using multiplex PCR amplification. Results: There were no significant differences in allele and genotype frequencies between the Behçet disease patients and controls who were successfully genotyped. Similar results were also found after stratification by gender, age, or clinical features. Study limitations: Lack of studies on various racial or ethnic groups and small sample size. Conclusion: This study failed to demonstrate any association between the tested TIM-3 polymorphisms and Behçet disease.


Subject(s)
Humans , Male , Female , Adult , Behcet Syndrome/genetics , Polymorphism, Single Nucleotide , Hepatitis A Virus Cellular Receptor 2/genetics , Case-Control Studies , Logistic Models , Risk Factors , Risk Assessment , Alleles , Genetic Association Studies , Multiplex Polymerase Chain Reaction , Gene Frequency , Iran
19.
Arq. neuropsiquiatr ; 77(4): 224-231, Apr. 2019. tab, graf
Article in English | LILACS | ID: biblio-1001360

ABSTRACT

ABSTRACT This study evaluated the operational characteristics of the multiplex polymerase chain reaction (PCR) for cerebrospinal fluid (CSF) from patients with cellular and biochemical characteristics of acute bacterial meningitis and positive or negative CSF cultures. Methods: Multiplex PCR was performed for 36 CSF samples: culture-proven acute bacterial meningitis (n = 7), culture-negative acute bacterial meningitis (n = 17), lymphocytic meningitis (n = 8), and normal CSF (n = 4). The operational characteristics of multiplex PCR were evaluated with definite and probable bacterial meningitis, using culture positive, cytological and biochemical CSF characteristics as the gold standard. Results: Multiplex PCR for CSF was efficient in the group with CSF cellular and biochemical characteristics of acute bacterial meningitis but with a negative CSF culture. This group demonstrated high specificity, positive predictive value, and efficiency. Conclusions: Multiplex PCR for CSF can improve the speed and accuracy of acute bacterial meningitis diagnosis in a clinical setting as a complement to classical immunological and bacteriological assays in CSF. It is also useful for CSF culture-negative acute bacterial meningitis.


RESUMO Este estudo avaliou as características funcionais da reação em cadeia da polimerase (PCR) multiplex para amostras de líquido cefalorraquidiano (LCR) de pacientes com características celulares e bioquímicas de meningite bacteriana aguda e culturas de LCR positivas ou negativas. Métodos: O PCR multiplex foi realizado em 36 amostras de LCR: meningite bacteriana aguda comprovada por cultura (n = 7), meningite bacteriana aguda com cultura negativa (n = 17), meningite linfocítica (n = 8) e LCR normal (n = 4). As características funcionais do PCR multiplex foram avaliadas para meningite bacteriana definitiva e provável, utilizando cultura positiva, características citológicas e bioquímicas do LCR como padrão-ouro. Resultados: O PCR multiplex do LCR foi eficiente no grupo com características celulares e bioquímicas do LCR de meningite bacteriana, mas com cultura do LCR negativa. Este grupo demonstrou especificidade, valor preditivo positivo e eficiência altos. Conclusões: Os autores concluíram que o PCR multiplex do LCR pode melhorar a velocidade e a precisão do diagnóstico de meningite bacteriana em um ambiente clínico como complemento aos ensaios imunológicos e bacteriológicos clássicos no LCR. Também é útil para meningite bacteriana aguda com cultura de LCR negativa.


Subject(s)
Humans , Male , Female , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Young Adult , Cerebrospinal Fluid/microbiology , Meningitis, Bacterial/diagnosis , Meningitis, Bacterial/cerebrospinal fluid , Multiplex Polymerase Chain Reaction/methods , Reference Standards , Acute Disease , Predictive Value of Tests , Reproducibility of Results , Bacteriological Techniques/methods , Sensitivity and Specificity , Meningitis, Bacterial/microbiology , Statistics, Nonparametric
20.
Article in English | WPRIM | ID: wpr-785637

ABSTRACT

Mucopolysaccharidosis type II (MPS II) is a rare X-linked recessive lysosomal storage disease caused by mutation of the iduronate-2-sulfatase gene. The mutation results in iduronate-2-sulfatase deficiency, which causes the progressive accumulation of heparan sulfate and dermatan sulfate in cellular lysosomes. The phenotype, age of onset, and symptoms of MPS II vary; accordingly, the disease can be classified into either the early-onset type or the late-onset type, depending on the age of onset and the severity of the symptoms. In patients with severe MPS II, symptoms typically first appear between 2 and 5 years of age. Patients with severe MPS II usually die in the second decade of life although some patients with less severe disease have survived into their fifth or sixth decade. Here, we report the establishment of a preimplantation genetic diagnosis (PGD) strategy using multiplex nested polymerase chain reaction, direct sequencing, and linkage analysis. Unaffected embryos were selected via the diagnosis of a single blastomere, and a healthy boy was delivered by a female carrier of MPS II. This is the first successful application of PGD in a patient with MPS II in Korea


Subject(s)
Age of Onset , Blastomeres , Dermatan Sulfate , Diagnosis , Embryonic Structures , Female , Heparitin Sulfate , Humans , Korea , Lysosomal Storage Diseases , Lysosomes , Male , Mucopolysaccharidoses , Mucopolysaccharidosis II , Multiplex Polymerase Chain Reaction , Parturition , Phenotype , Polymerase Chain Reaction , Preimplantation Diagnosis , Prostaglandins D
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