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1.
Mol Biol Rep ; 48(4): 3629-3635, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33893925

RESUMO

PCR Single-Strand Conformation Polymorphism is a method used to identify and detect mutations and is now well known for its many applications on living beings. This paper will discuss the experimental details, limitations and sensitivity of the PCR Single-Strand Conformation Polymorphism method in relation to all existing literature available to us until today. Genomic DNA extraction, PCR amplification and Single-Strand Conformation Polymorphism conditions (concentration of polyacrylamide slab gel electrophoresis, dissociation treatment of double- stranded DNA) and comparison with PCR Restriction Fragment Length Polymorphism are presented. Since its discovery in 1989, there have been many variations, innovations, and modifications of the method, which makes it very easy, safe, fast and for this reason widely applied in clinical diagnostic, forensic medicine, biochemical, veterinary, microbiological, food and environmental laboratories. One of the possible applications of the method is the diagnosis and identification of mutations in new strains of coronaviruses, because science needs more tools to tackle the problem of this pandemic. The PCR Single-Strand Conformation Polymorphism method can be applied in many cases provided that control samples are available and the required conditions of the method are achieved.


Assuntos
Reação em Cadeia da Polimerase/métodos , Polimorfismo Conformacional de Fita Simples , Animais , Coronavirus/classificação , Coronavirus/genética , Coronavirus/isolamento & purificação , Humanos , Tipagem Molecular/métodos , Patologia Molecular/métodos , Polimorfismo de Fragmento de Restrição , Análise de Sequência/métodos
2.
Medicine (Baltimore) ; 100(13): e25362, 2021 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-33787640

RESUMO

ABSTRACT: We investigated the vaginal flora diversity of preschool-aged (ie, 4-6-year-old) girls in southwest China.Fourteen preschool-aged girls were enrolled in this study. The statuses and differences in their vaginal flora were evaluated by Gram staining, bacterial culturing, and sequencing analysis.Gram staining and microbial culturing showed that the main vaginal flora of the preschool-aged girls were Gram-negative bacilli, whereas the main vaginal flora of healthy adult controls were large Gram-positive bacilli such as Lactobacillus crispatus. Shannon and Simpson indexes indicated that the bacterial diversity tended to decrease with age. The species abundance heat map showed that the vaginal microecology of the girls differed slightly at different ages but mainly comprised Pseudomonas, Methylobacterium, Sphingomona,s and Escherichia. The functional abundance heat map indicated that the bacterial functions increased with age.The vaginal microecology of preschool-aged girls differs from that of adults. A comprehensive understanding of the vaginal flora diversity of preschool-aged girls will aid in clinically diagnosing vulvovaginitis in preschool-aged girls.


Assuntos
Bactérias/isolamento & purificação , Microbiota/genética , Vagina/microbiologia , Vulvovaginite/diagnóstico , Adulto , Fatores Etários , Bactérias/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , China , DNA Bacteriano/isolamento & purificação , Feminino , Voluntários Saudáveis , Humanos , Tipagem Molecular/métodos , Análise de Sequência de DNA , Esfregaço Vaginal , Vulvovaginite/microbiologia
3.
Indian J Med Microbiol ; 39(1): 73-80, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33460733

RESUMO

BACKGROUND: In the initial few months of the COVID-19 pandemic, two distinct strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were identified (L and S strain) based on a tightly linked SNP between two widely separated nucleotides at location 8782 (ORF1ab T8517C) and position 28,144 (ORF8: C251T, codon S84L). MATERIALS AND METHODS: A Type Specific Primer based one step RT-PCR (TSP-PCR) test to distinguish the L and S type strains of SARS-CoV-2 without the need for viral genome sequencing, was developed. The study also analyzed 18,221 whole genome sequences (WGS) available up to April 2020 to know the prevalence of L and S type of strains. Phylogenetic and recombination analysis of SARS-CoV-2 genome with nearest animal and human coronaviruses were analyzed using MEGA X and SimPlot version 3.5.1 software respectively. RESULTS: The rapid TSP-PCR distinguished the L and S type strains of SARS-CoV-2 by amplifying a specific 326 bp and 256 bp fragment of the L and S type strain respectively. The test was used to analyzed 120 random SARS-CoV-2 positive samples from Assam, India among which 118 were found to be of L-type strains only. On analysis of 18,221 WGS, it was found that L type was the predominant strain with an overall prevalence ∼90%. However, pockets of high prevalence of S-type strains (>35%) were still in circulation in Washington region in April 2020. The study did not detect any significant recombination events between closely related coronavirus and SARS-CoV-2. CONCLUSION: TSP-based PCR for identification of circulating strains of SARS-CoV-2, will add in rapid identification of strains of COVID-19 pandemic to understand the spread of the virus, its transmissibility and adaptation into human population. Though, the S-type strains have decreased drastically across the globe since April 2020, the role of TSP-PCR in geographical niches where such strains are still prevalent may help in rapidly distinguishing the strains and study its evolution.


Assuntos
/diagnóstico , Primers do DNA , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase , /genética , /epidemiologia , Genoma Viral , Humanos , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase/métodos , Vigilância em Saúde Pública , RNA Viral , Análise de Sequência de DNA
4.
Methods Mol Biol ; 2182: 187-196, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32894497

RESUMO

Salmonella is recognized as a major human foodborne pathogen and threat to public health world widely. It is important to carry out epidemiological investigations to determine the primary sources of bacterial contamination. Pulsed-field gel electrophoresis (PFGE) is an important method of the molecular typing, and play an important role in tracking the sources of infection and epidemic control. The PFGE is currently considered as "gold standard" of molecular typing methods for bacterial foodborne pathogen. Here, we describe the PFGE protocol to type the Salmonella from pork.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Eletroforese em Gel de Campo Pulsado/métodos , Tipagem Molecular/métodos , Salmonella/genética , DNA Bacteriano/genética , Carne de Porco/microbiologia
5.
PLoS One ; 15(9): e0230547, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32986740

RESUMO

Hydra are freshwater polyps widely studied for their amazing regenerative capacity, adult stem cell populations, low senescence and value as ecotoxicological marker. Many wild-type strains of H. vulgaris have been collected worldwide and maintained effectively under laboratory conditions by asexual reproduction, while stable transgenic lines have been continuously produced since 2006. Efforts are now needed to ensure the genetic characterization of all these strains, which despite similar morphologies, show significant variability in their response to gene expression silencing procedures, pharmacological treatments or environmental conditions. Here, we established a rapid and reliable procedure at the single polyp level to produce via PCR amplification of three distinct microsatellite sequences molecular signatures that distinguish between Hydra strains and species. The TG-rich region of an uncharacterized gene (ms-c25145) helps to distinguish between Eurasian H. vulgaris-Pallas strains (Hm-105, Basel1, Basel2 and reg-16), between Eurasian and North American H. vulgaris strains (H. carnea, AEP), and between the H. vulgaris and H. oligactis species. The AT-rich microsatellite sequences located in the AIP gene (Aryl Hydrocarbon Receptor Interaction Protein, ms-AIP) also differ between Eurasian and North American H. vulgaris strains. Finally, the AT-rich microsatellite located in the Myb-Like cyclin D-binding transcription factor1 gene (ms-DMTF1) gene helps to distinguish certain transgenic AEP lines. This study shows that the analysis of microsatellite sequences, which is capable of tracing genomic variations between closely related lineages of Hydra, provides a sensitive and robust tool for characterizing the Hydra strains.


Assuntos
Hydra/genética , Repetições de Microssatélites/genética , Tipagem Molecular/métodos , Animais , Animais Geneticamente Modificados/genética , Modelos Animais , Reação em Cadeia da Polimerase , Polimorfismo Genético , Reprodutibilidade dos Testes
6.
Arch Virol ; 165(11): 2627-2632, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32776175

RESUMO

Due to the risk of poliovirus importation from Ukraine in 2015, a combined surveillance program monitoring the circulation of enteroviruses (EVs) in healthy children from at-risk areas and in the environment was conducted in Romania. Virological testing of stool samples collected from 155 healthy children aged from two months to six years and of 186 sewage water samples collected from different areas was performed. A total of 58 (37.42%) stool samples and 50 (26.88%) sewage water samples were positive for non-polio EVs, but no poliovirus was detected. A high level of circulation of echovirus (E) types 6 and 7 and coxsackievirus (CV) type B5 was observed.


Assuntos
Enterovirus Humano B/isolamento & purificação , Enterovirus/isolamento & purificação , Fezes/virologia , Esgotos/virologia , Criança , Pré-Escolar , Enterovirus/classificação , Enterovirus/genética , Enterovirus Humano B/genética , Infecções por Enterovirus/virologia , Meio Ambiente , Monitoramento Ambiental/métodos , Voluntários Saudáveis , Humanos , Lactente , Limite de Detecção , Modelos Logísticos , Tipagem Molecular/métodos , Filogenia , Poliovirus/genética , Poliovirus/isolamento & purificação , Romênia , Águas Residuárias/virologia
7.
Gene ; 763: 145048, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32805312

RESUMO

Cross-contamination of cell lines is a highly relevant and pervasive problem. The analysis of short tandem repeats (STR) is a simple and commercially available technique to authenticate cell lines for more than two decades. At present, STR multiple amplification kits have been developed up to 21 loci while the current STR databases only provide 9-loci STR profiles. Here, we compared the advantages of 21-loci STR methodology using the same algorithm as 9-loci method. The 21-loci method reduced the uncertainty ratio for authentications by 97.5% relative to the 9-loci method and exclude effectively false positive. We show that the additional 12 loci helped to greatly reduce sample-site marker specificity arising from genetic isolation and the occurrence of null alleles, suggesting that inclusion of additional loci in these databases will ultimately improve the efficiency and accuracy of authentication of cell lines. Taken together, we demonstrate the utility of a 21-loci method in human cells, providing a novel marker panel for use as a valuable alternative to 9-loci analyses to minimize cell line authentication errors and reduce costs due to erroneous experiments.


Assuntos
Autenticação de Linhagem Celular/métodos , Repetições de Microssatélites , Linhagem Celular , Autenticação de Linhagem Celular/normas , Linhagem Celular Tumoral , Loci Gênicos , Marcadores Genéticos , Humanos , Tipagem Molecular/métodos , Tipagem Molecular/normas
8.
PLoS One ; 15(8): e0237180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750094

RESUMO

BACKGROUND: Chagas disease, caused by the intracellular parasite Trypanosoma cruzi, is one of the most important parasitological infections in the Americas. It is estimated to infect approximately 6 million people from mostly low income countries in Latin America, although recent infections have been reported in southern US states. Several studies have described an extensive genetic diversity among T. cruzi isolates throughout its geographic distribution in the American continent. This diversity has been correlated with the pathology developed during an infection. However, due to a lack of a single reliable test, current diagnosis practices of the disease are not straightforward since several different tests are applied. The use of current genomic sequence data allows for the selection of molecular markers (MM) that have the ability to identify the Discrete Typing Unit (DTU) of T. cruzi in a given infection, without the need of any sequencing reaction. METHODOLOGY/PRINCIPAL FINDINGS: Applying three criteria on the genomic sequencing data of four different phylogenetic lineages of T. cruzi, we designed several molecular tests that can be used for the molecular typing of the parasite. The criteria used were: (1) single-copy orthologs of T. cruzi, (2) T. cruzi unique loci, and (3) T. cruzi polymorphic loci. All criteria combined allowed for the selection of 15 MM, 12 of which were confirmed to be functional and replicable in the laboratory with sylvatic samples. Furthermore, one MM produced distinct polymerase chain reaction (PCR) amplicon sizes among distinct T. cruzi DTUs, allowing the use of a AFLP-PCR test to distinguish DTUs I, II/IV, V and VI. Whereas two MM can differentiate DTUs I, II, IV and V/VI out of the six current DTUs with a PCR-RFLP test. CONCLUSIONS/SIGNIFICANCE: The designed molecular tests provide a practical and inexpensive molecular typing test for the majority of DTUs of T. cruzi, excluding the need to perform any sequencing reaction. This provides the scientific community with an additional specific, quick and inexpensive test that can enhance the understanding of the correlation between the DTU of T. cruzi and the pathology developed during the infection.


Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Doença de Chagas/diagnóstico , Polimorfismo de Fragmento de Restrição/genética , Trypanosoma cruzi/genética , Doença de Chagas/parasitologia , DNA de Protozoário/genética , Loci Gênicos , Variação Genética , Genoma de Protozoário/genética , Humanos , Tipagem Molecular/métodos , Filogenia , Polimorfismo de Nucleotídeo Único
9.
Cochrane Database Syst Rev ; 8: CD013359, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32853411

RESUMO

BACKGROUND: Every year, at least one million children become ill with tuberculosis and around 200,000 children die. Xpert MTB/RIF and Xpert Ultra are World Health Organization (WHO)-recommended rapid molecular tests that simultaneously detect tuberculosis and rifampicin resistance in adults and children with signs and symptoms of tuberculosis, at lower health system levels. To inform updated WHO guidelines on molecular assays, we performed a systematic review on the diagnostic accuracy of these tests in children presumed to have active tuberculosis. OBJECTIVES: Primary objectives • To determine the diagnostic accuracy of Xpert MTB/RIF and Xpert Ultra for (a) pulmonary tuberculosis in children presumed to have tuberculosis; (b) tuberculous meningitis in children presumed to have tuberculosis; (c) lymph node tuberculosis in children presumed to have tuberculosis; and (d) rifampicin resistance in children presumed to have tuberculosis - For tuberculosis detection, index tests were used as the initial test, replacing standard practice (i.e. smear microscopy or culture) - For detection of rifampicin resistance, index tests replaced culture-based drug susceptibility testing as the initial test Secondary objectives • To compare the accuracy of Xpert MTB/RIF and Xpert Ultra for each of the four target conditions • To investigate potential sources of heterogeneity in accuracy estimates - For tuberculosis detection, we considered age, disease severity, smear-test status, HIV status, clinical setting, specimen type, high tuberculosis burden, and high tuberculosis/HIV burden - For detection of rifampicin resistance, we considered multi-drug-resistant tuberculosis burden • To compare multiple Xpert MTB/RIF or Xpert Ultra results (repeated testing) with the initial Xpert MTB/RIF or Xpert Ultra result SEARCH METHODS: We searched the Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, the Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, the WHO International Clinical Trials Registry Platform, ClinicalTrials.gov, and the International Standard Randomized Controlled Trials Number (ISRCTN) Registry up to 29 April 2019, without language restrictions. SELECTION CRITERIA: Randomized trials, cross-sectional trials, and cohort studies evaluating Xpert MTB/RIF or Xpert Ultra in HIV-positive and HIV-negative children younger than 15 years. Reference standards comprised culture or a composite reference standard for tuberculosis and drug susceptibility testing or MTBDRplus (molecular assay for detection of Mycobacterium tuberculosis and drug resistance) for rifampicin resistance. We included studies evaluating sputum, gastric aspirate, stool, nasopharyngeal or bronchial lavage specimens (pulmonary tuberculosis), cerebrospinal fluid (tuberculous meningitis), fine needle aspirates, or surgical biopsy tissue (lymph node tuberculosis). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed study quality using the Quality Assessment of Studies of Diagnostic Accuracy - Revised (QUADAS-2). For each target condition, we used the bivariate model to estimate pooled sensitivity and specificity with 95% confidence intervals (CIs). We stratified all analyses by type of reference standard. We assessed certainty of evidence using the GRADE approach. MAIN RESULTS: For pulmonary tuberculosis, 299 data sets (68,544 participants) were available for analysis; for tuberculous meningitis, 10 data sets (423 participants) were available; for lymph node tuberculosis, 10 data sets (318 participants) were available; and for rifampicin resistance, 14 data sets (326 participants) were available. Thirty-nine studies (80%) took place in countries with high tuberculosis burden. Risk of bias was low except for the reference standard domain, for which risk of bias was unclear because many studies collected only one specimen for culture. Detection of pulmonary tuberculosis For sputum specimens, Xpert MTB/RIF pooled sensitivity (95% CI) and specificity (95% CI) verified by culture were 64.6% (55.3% to 72.9%) (23 studies, 493 participants; moderate-certainty evidence) and 99.0% (98.1% to 99.5%) (23 studies, 6119 participants; moderate-certainty evidence). For other specimen types (nasopharyngeal aspirate, 4 studies; gastric aspirate, 14 studies; stool, 11 studies), Xpert MTB/RIF pooled sensitivity ranged between 45.7% and 73.0%, and pooled specificity ranged between 98.1% and 99.6%. For sputum specimens, Xpert Ultra pooled sensitivity (95% CI) and specificity (95% CI) verified by culture were 72.8% (64.7% to 79.6%) (3 studies, 136 participants; low-certainty evidence) and 97.5% (95.8% to 98.5%) (3 studies, 551 participants; high-certainty evidence). For nasopharyngeal specimens, Xpert Ultra sensitivity (95% CI) and specificity (95% CI) were 45.7% (28.9% to 63.3%) and 97.5% (93.7% to 99.3%) (1 study, 195 participants). For all specimen types, Xpert MTB/RIF and Xpert Ultra sensitivity were lower against a composite reference standard than against culture. Detection of tuberculous meningitis For cerebrospinal fluid, Xpert MTB/RIF pooled sensitivity and specificity, verified by culture, were 54.0% (95% CI 27.8% to 78.2%) (6 studies, 28 participants; very low-certainty evidence) and 93.8% (95% CI 84.5% to 97.6%) (6 studies, 213 participants; low-certainty evidence). Detection of lymph node tuberculosis For lymph node aspirates or biopsies, Xpert MTB/RIF pooled sensitivity and specificity, verified by culture, were 90.4% (95% CI 55.7% to 98.6%) (6 studies, 68 participants; very low-certainty evidence) and 89.8% (95% CI 71.5% to 96.8%) (6 studies, 142 participants; low-certainty evidence). Detection of rifampicin resistance Xpert MTB/RIF pooled sensitivity and specificity were 90.0% (67.6% to 97.5%) (6 studies, 20 participants; low-certainty evidence) and 98.3% (87.7% to 99.8%) (6 studies, 203 participants; moderate-certainty evidence). AUTHORS' CONCLUSIONS: We found Xpert MTB/RIF sensitivity to vary by specimen type, with gastric aspirate specimens having the highest sensitivity followed by sputum and stool, and nasopharyngeal specimens the lowest; specificity in all specimens was > 98%. Compared with Xpert MTB/RIF, Xpert Ultra sensitivity in sputum was higher and specificity slightly lower. Xpert MTB/RIF was accurate for detection of rifampicin resistance. Xpert MTB/RIF was sensitive for diagnosing lymph node tuberculosis. For children with presumed tuberculous meningitis, treatment decisions should be based on the entirety of clinical information and treatment should not be withheld based solely on an Xpert MTB/RIF result. The small numbers of studies and participants, particularly for Xpert Ultra, limits our confidence in the precision of these estimates.


Assuntos
Tipagem Molecular/métodos , Tuberculose dos Linfonodos/diagnóstico , Tuberculose Meníngea/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Pulmonar/diagnóstico , Adolescente , Antibióticos Antituberculose/uso terapêutico , Viés , Criança , Fezes/microbiologia , Conteúdo Gastrointestinal/microbiologia , Humanos , Tipagem Molecular/normas , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/uso terapêutico , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose dos Linfonodos/tratamento farmacológico , Tuberculose dos Linfonodos/microbiologia , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/tratamento farmacológico , Tuberculose Meníngea/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia
10.
Arch Virol ; 165(10): 2335-2340, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32719956

RESUMO

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.


Assuntos
Infecções por Caliciviridae/virologia , Primers do DNA/química , Gastroenterite/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sapovirus/genética , Proteínas Estruturais Virais/genética , Sequência de Bases , Infecções por Caliciviridae/diagnóstico , Primers do DNA/genética , Fezes/virologia , Gastroenterite/diagnóstico , Expressão Gênica , Genótipo , Humanos , Tipagem Molecular/métodos , Filogenia , Sapovirus/classificação , Sapovirus/isolamento & purificação , Alinhamento de Sequência
11.
Sci Rep ; 10(1): 6737, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317653

RESUMO

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.


Assuntos
Bactérias/virologia , Inoviridae/classificação , Myoviridae/classificação , Podoviridae/classificação , RNA Ribossômico 16S/genética , Siphoviridae/classificação , Líquido Ascítico/microbiologia , Líquido Ascítico/virologia , Bactérias/classificação , Bactérias/genética , Hemocultura/métodos , Capsídeo/química , Líquido Cefalorraquidiano/microbiologia , Líquido Cefalorraquidiano/virologia , Filtração/métodos , Humanos , Inoviridae/genética , Inoviridae/isolamento & purificação , Lisogenia/fisiologia , Tipagem Molecular/métodos , Myoviridae/genética , Myoviridae/isolamento & purificação , Podoviridae/genética , Podoviridae/isolamento & purificação , Soro/microbiologia , Soro/virologia , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Urina/microbiologia , Urina/virologia
12.
Lett Appl Microbiol ; 71(2): 138-145, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32333808

RESUMO

Salmonellosis is a leading bacterial cause of foodborne illness, and numerous Salmonella enterica serovars have been responsible for foodborne outbreaks. In the United States outbreaks are often linked to poultry and poultry-related products. The prevalence of Salmonella serovar Infantis has been increasing in poultry processing facilities over the past few years and in 2018 was identified as the causative agent for a large multistate outbreak linked to raw chicken. CRISPR-typing is a subtyping approach based on PCR and the sequencing of two Salmonella loci, CRISPR1 and CRISPR2. CRISPR-typing was used to interrogate 138 recent (2018-2019) isolates and genomes of ser. Infantis. Results show that the CRISPR elements are remarkably conserved in this serovar. The most conserved spacers, and those also unique to ser. Infantis, were used as targets to develop a ser. Infantis-specific qPCR assay. This assay was able to detect ser. Infantis in mixed serovar cultures of Salmonella, down to 0·1% of the population, highlighting the utility of this molecular approach in improving surveillance sensitivity for this important food safety pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of human salmonellosis cases caused by Salmonella enterica serovar Infantis (ser. Infantis) has been increasing, as has its prevalence in broiler chickens, which are a frequent reservoir of Salmonella. A cluster of ser. Infantis genetically linked to an outbreak strain have been identified in numerous processing facilities. A qPCR assay targeting CRISPR elements that are unique to ser. Infantis has been developed and can detect this serovar directly from mixed cultures. This assay is sensitive enough to reveal ser. Infantis within a mixed Salmonella population where it constitutes only 0·1% of the population. The rapid nature of qPCR lends this assay to high-throughput screening of poultry samples to detect this important pathogen.


Assuntos
Tipagem Molecular/métodos , Doenças das Aves Domésticas/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Intoxicação Alimentar por Salmonella/epidemiologia , Salmonella enterica/classificação , Animais , Galinhas/microbiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Surtos de Doenças , Humanos , Aves Domésticas/microbiologia , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Sorogrupo , Estados Unidos/epidemiologia
13.
Cancer Cytopathol ; 128(7): 482-490, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32129949

RESUMO

BACKGROUND: High-risk human papillomavirus-positive (hrHPV+) oropharyngeal squamous cell carcinomas comprise a subset of head and neck squamous cell carcinomas (HNSCCs) with a distinct biology and prognosis. Commonly, the diagnosis of HNSCC is rendered on fine-needle aspiration (FNA). Because cell blocks may be insufficient for determining HPV status using microscopy-based techniques, the ability of liquid-based assays was examined in the current study. METHODS: The performance of the Roche cobas 4800 platform was evaluated on the FNA material from the cell pellet and corresponding cell-free supernatant fluid specimens of primary and metastatic HNSCCs. These results were compared with the p16 immunostain result from the histologic material obtained from the same patient. Discrepant cases were adjudicated using hrHPV RNA in situ hybridization. RESULTS: A total of 41 samples (23 primary tumors and 18 lymph node metastases) were acquired from 34 patients with HNSCC. Primary tumors included the oropharynx (20 samples), oral cavity (13 samples), larynx (3 samples), and skin (3 samples). In 2 cases, a primary tumor could not be identified. Twenty-three samples (56%) were found to be p16 positive by immunohistochemistry. Twenty-two samples were found to be positive on cobas hrHPV testing from both cell pellet and cell-free supernatant fluid. Two cell-free supernatant fluid specimens yielded indeterminate cobas results. At the time additional hrHPV RNA in situ hybridization analysis was performed, one cobas-positive cell pellet was deemed to be a false-positive result. The sensitivity of the cobas assay was 100% for pellet material and cell-free supernatant fluid, with specificities of 94.7% and 100%, respectively. CONCLUSIONS: cobas hrHPV testing of HNSCC specimens demonstrated high concordance with p16 immunohistochemistry on the corresponding cell block and/or tissue specimen. Using the cell-free supernatant fluid in this platform could provide accurate HPV results while conserving material for cytomorphologic analyses.


Assuntos
Técnicas Citológicas/métodos , DNA Viral/análise , Neoplasias de Cabeça e Pescoço/diagnóstico , Tipagem Molecular/métodos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Feminino , Seguimentos , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Prognóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia
14.
Parasit Vectors ; 13(1): 122, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32143704

RESUMO

BACKGROUND: Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS: We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS: This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS: Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.


Assuntos
Cyclospora/genética , Cyclospora/isolamento & purificação , Ciclosporíase/diagnóstico , Genoma Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Tipagem Molecular/métodos , Sequência de Bases , Análise por Conglomerados , Biologia Computacional , Cyclospora/classificação , Ciclosporíase/parasitologia , DNA de Protozoário/genética , Fezes/parasitologia , Técnicas de Genotipagem/métodos , Humanos , Oocistos/genética , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
15.
Sci Rep ; 10(1): 5125, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198413

RESUMO

Nanopore based DNA-sequencing delivers long reads, thereby simplifying the decipherment of bacterial communities. Since its commercial appearance, this technology has been assigned several attributes, such as its error proneness, comparatively low cost, ease-of-use, and, most notably, aforementioned long reads. The technology as a whole is under continued development. As such, benchmarks are required to conceive, test and improve analysis protocols, including those related to the understanding of the composition of microbial communities. Here we present a dataset composed of twelve different prokaryotic species split into four samples differing by nucleic acid quantification technique to assess the specificity and sensitivity of the MinION nanopore sequencer in a blind study design. Taxonomic classification was performed by standard taxonomic sequence classification tools, namely Kraken, Kraken2 and Centrifuge directly on reads. This allowed taxonomic assignments of up to 99.27% on genus level and 92.78% on species level, enabling true-positive classification of strains down to 25,000 genomes per sample. Full genomic coverage is achieved for strains abundant as low as 250,000 genomes per sample under our experimental settings. In summary, we present an evaluation of nanopore sequence processing analysis with respect to microbial community composition. It provides an open protocol and the data may serve as basis for the development and benchmarking of future data processing pipelines.


Assuntos
Bactérias/genética , Benchmarking/métodos , Genoma Bacteriano/genética , Tipagem Molecular/métodos , Sequenciamento por Nanoporos/métodos , Análise de Sequência de DNA/métodos , Bactérias/classificação , Sequência de Bases , DNA Bacteriano/genética , Metagenômica/instrumentação , Metagenômica/métodos , Sequenciamento por Nanoporos/instrumentação
16.
BMC Res Notes ; 13(1): 97, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093758

RESUMO

OBJECTIVES: Molecular typing methods are useful for rapid detection and control of a disease. Recently, the use of high-resolution melting (HRM) for spa typing of MRSA isolates were reported. This technique is rapid, inexpensive and simple for genotyping and mutation screening in DNA sequence. The aim of this study was to evaluate the ability of HRM-PCR to analysis spa genes amongst MRSA isolates. RESULTS: A total of 50 MRSA isolates were collected from two teaching hospitals in Shiraz, Iran. The isolates were confirmed as MRSA by susceptibility to cefoxitin and detection of mecA gene using PCR. We used HRM analysis and PCR-sequencing method for spa typing of MRSA isolates. In total, 15 different spa types were discriminate by HRM and sequencing method. The melting temperature of the 15 spa types, using HRM genotyping were between 82.16 and 85.66 °C. The rate of GC % content was 39.4-46.3. According to the results, spa typing of 50 clinical isolates via PCR-sequencing and HRM methods were 100% similar. Consequently, HRM method can easily identify and rapidly differentiate alleles of spa genes. This method is faster, less laborious and more suitable for high sample at lower cost and risk of contamination.


Assuntos
Infecção Hospitalar/tratamento farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Meticilina/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Temperatura de Transição , Antibacterianos/uso terapêutico , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecção Hospitalar/microbiologia , Genótipo , Humanos , Irã (Geográfico) , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana/métodos , Tipagem Molecular/métodos , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Reação em Cadeia da Polimerase , Infecções Estafilocócicas/microbiologia
17.
Breast Cancer Res ; 22(1): 23, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075687

RESUMO

BACKGROUND: Experimental and epidemiological studies demonstrate a role for 27-hydroxycholesterol (27HC) in breast cancer development, though results are conflicting. Cholesterol 27-hydroxylase (CYP27A1) and oxysterol 7-alpha-hydroxylase (CYP7B1) regulate 27HC concentrations, while differential expression of the liver X receptor (LXR) and estrogen receptor beta (ERß) may impact the association between 27HC and breast cancer risk. METHODS: We evaluated correlates of tumor tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß and the association between circulating prediagnostic 27HC concentrations and breast cancer risk by marker expression in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg cohort including 287 breast cancer cases with tumor tissue available. Tumor protein expression was evaluated using immunohistochemistry, and serum 27HC concentrations quantified using liquid chromatography-mass spectrometry. Conditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: A higher proportion of CYP7B1-positive cases were progesterone receptor (PR)-positive, relative to CYP7B1-negative cases, whereas a higher proportion of ERß-positive cases were Bcl-2 low, relative to ERß-negative cases. No differences in tumor tissue marker positivity were observed by reproductive and lifestyle factors. We observed limited evidence of heterogeneity in associations between circulating 27HC and breast cancer risk by tumor tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß, with the exception of statistically significant heterogeneity by LXR-ß status in the subgroup of women perimenopausal at blood collection (p = 0.02). CONCLUSION: This exploratory study suggests limited associations between tumor marker status and epidemiologic or breast cancer characteristics. Furthermore, the association between circulating 27HC and breast cancer risk may not vary by tumor expression of CYP27A1, CYP7B1, LXR-ß, or ERß.


Assuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Hidroxicolesteróis/sangue , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Colestanotriol 26-Mono-Oxigenase/metabolismo , Família 7 do Citocromo P450/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Alemanha/epidemiologia , Humanos , Receptores X do Fígado/metabolismo , Pessoa de Meia-Idade , Tipagem Molecular/métodos , Gradação de Tumores , Avaliação Nutricional , Estudos Prospectivos , Fatores de Risco , Esteroide Hidroxilases/metabolismo
18.
DNA Cell Biol ; 39(4): 579-598, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32069124

RESUMO

The genus Schizothorax is one of the most diverse groups of schizothoracine fish. Many species within this genus possess highly similar morphological characters and are very difficult to be identified accurately only based on morphology. The present study aims to test the effectiveness of mitochondrial cytochrome c oxidase subunit I (COI) gene and cytochrome b (Cytb) gene for discriminating the Schizothorax fish. A total of 185 individuals of 11 species for COI gene and 264 individuals of 23 species for Cytb gene were used for analyzing, respectively. According to the genetic distances, only one species based on COI gene and five species based on Cytb gene had "barcoding gaps," respectively. The tree-based analysis displayed that four species based on COI gene and six species based on Cytb gene clustered monophyletic group with strong support, respectively. The optimal threshold value of Schizothorax is 0.005 based on COI gene and 0.008 based on Cytb gene. The results of genetic similarity tests performed through online BLAST showed that 108 of 185 similarity searches succeeded in identifying conspecific sequences based on COI gene and 199 of 264 succeeded in identifying conspecific sequences based on Cytb gene. Considering greater interspecific genetic distance in Kimura 2-parameter (K2P) analysis and many clades with higher supporting values in tree-based analysis, we suggest that Cytb gene has better resolution in discrimination of Schizothorax species than COI gene. However, there are still many confused clustering relationships based on molecular data currently available. Incomplete lineage sorting, the existence of possible cryptic species and problematic morphological identification, etc. might have greatly weakened the resolution of Cytb gene in discrimination of Schizothorax species.


Assuntos
Cyprinidae/classificação , Citocromos b/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Animais , Sequência de Bases , Cyprinidae/genética , Variação Genética/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Tipagem Molecular/métodos , Análise de Sequência de DNA , Tibet
19.
Epidemiol Infect ; 148: e51, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32052718

RESUMO

In June 2017, an outbreak of Salmonella Kottbus infection was suspected in Germany. We investigated the outbreak with whole-genome sequencing (WGS) and a case-control study. Forty-six isolates from 69 cases were subtyped. Three WGS clusters were identified: cluster 1 (n = 36), cluster 2 (n = 5) and cluster 3 (n = 3). Compared to controls, cluster 1 cases more frequently consumed raw smoked ham (odds ratio (OR) 10, 95% confidence interval (CI) 1.2-88) bought at supermarket chain X (OR 36, 95% CI 4-356; 9/10 consumed ham Y). All four cluster 2 cases interviewed had consumed quail eggs. Timely WGS was invaluable in distinguishing concurrent outbreaks of a rare Salmonella serotype.


Assuntos
Surtos de Doenças , Doenças Transmitidas por Alimentos/epidemiologia , Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Infecções por Salmonella/epidemiologia , Salmonella enterica/classificação , Sequenciamento Completo do Genoma/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Análise por Conglomerados , Comportamento Alimentar , Feminino , Doenças Transmitidas por Alimentos/microbiologia , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Salmonella/microbiologia , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação
20.
Sci Rep ; 10(1): 2205, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32042063

RESUMO

Some pathogens and toxins have the potential to be used as weapons of mass destruction and instigate population-based fear. Efforts to mitigate biothreat require development of efficient countermeasures which in turn relies on fast and accurate methods to detect the biological agents in a range of complex matrices including environmental and clinical samples. We report here an mass spectrometry (MS) based methodology, employing both targeted and shot-gun approaches for the verification of biological agents from the environmental samples. Our shot-gun methodology relied on tandem MS analysis of abundant peptides from the spiked samples, whereas, the targeted method was based on an extensive elucidation of marker proteins and unique peptides resulting in the generation of an inclusion list of masses reflecting relevant peptides for the unambiguous identification of nine bacterial species [listed as priority agents of bioterrorism by Centre for Disease Control and Prevention (CDC)] belonging to phylogenetically diverse genera. The marker peptides were elucidated by extensive literature mining, in silico analysis, and tandem MS (MS/MS) analysis of abundant proteins of the cultivated bacterial species in our laboratory. A combination of shot-gun MS/MS analysis and the targeted search using a panel of unique peptides is likely to provide unambiguous verification of biological agents at sub-species level, even with limited fractionation of crude protein extracts from environmental samples. The comprehensive list of peptides reflected in the inclusion list, makes a valuable resource for the multiplex analysis of select biothreat agents and further development of targeted MS/MS assays.


Assuntos
Proteínas de Bactérias/análise , Armas Biológicas/classificação , Bioterrorismo/prevenção & controle , Tipagem Molecular/métodos , Espectrometria de Massas em Tandem , Biomarcadores/análise , Cromatografia Líquida de Alta Pressão , Simulação por Computador , Mineração de Dados , Peptídeos/análise
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