RESUMO
Killer cell immunoglobulin-like receptor (KIR) genes encode cell surface molecules that recognize HLA molecules and modulate the activity of natural killer (NK) cells. KIR genes exhibit presence and absence polymorphism, which generates a variety of gene-content haplotypes in worldwide populations. KIR gene-content variation is implicated in many diseases and is also important for placentation and transplantation. Because of the complexity of KIR polymorphism, variation in this family is still mostly studied at the gene-content level, even with the advent of next-generation sequencing (NGS) methods. Gene-content determination is generally expensive and/or time-consuming. To overcome these difficulties, we developed a method based on multiplex polymerase chain reaction with specific sequence primers (PCR-SSP) followed by melting curve analysis that allows cost-effective, precise and fast generation of results. Our method was 100% concordant with a gel-based method and 99.9% concordant with presence and absence determination by NGS. The limit of detection for accurate typing was 30 ng of DNA (0.42 µM) with 260/230 and 260/280 ratios as low as 0.19 and of 0.44. In addition, we developed a user-friendly Java-based computational application called killerPeak that interprets the raw data generated by Viia7 or QuantStudio 7 quantitative PCR machines and reliably exports the final genotyping results in spreadsheet file format. The combination of a reliable method that requires low amount of DNA with an automated interpretation of results allows scaling the KIR genotyping in large cohorts with reduced turnaround time.
Assuntos
Genótipo , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Polimorfismo Genético , Receptores KIR/genética , Primers do DNA/química , Primers do DNA/metabolismo , Expressão Gênica , Técnicas de Genotipagem/economia , Técnicas de Genotipagem/instrumentação , Técnicas de Genotipagem/normas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Limite de Detecção , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/instrumentação , Reação em Cadeia da Polimerase Multiplex/normas , Desnaturação de Ácido Nucleico , Receptores KIR/classificação , Receptores KIR/imunologia , SoftwareRESUMO
Diarrhea is considered the second most common cause of infant mortality worldwide. The disease can be caused by many different pathogens, including diarrheagenic Escherichia coli (DEC), which includes the pathotypes enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC). To develop a multiplex PCR system for the safe and accurate identification of the five main pathotypes of DEC, seven pairs of primers were determined for the following genes: aaiC, escV, bfpA, ipaH, elt, stx1, and stx2. To validate the system, 413 isolates from different sources (water and both animal and human stool) were analyzed that had been characterized previously. The sensitivity data were grouped by pathotype, in which 92.7% of the atypical EPEC were correlated, as were 92.8% of the STEC, 91.35% of the EAEC, and 100% of the typical EPEC, ETEC, and EIEC. These findings indicate that it is possible to detect the major five pathotypes of DEC from different sources, which can aid in determining the epidemiology of diarrhea with a low cost, high sensitivity and specificity, and the easy and safe viewing of the resulting PCR products.
Assuntos
Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Microbiologia da Água , Animais , Escherichia coli Enterotoxigênica/isolamento & purificação , Escherichia coli Enterotoxigênica/patogenicidade , Infecções por Escherichia coli/diagnóstico , Proteínas de Escherichia coli/genética , Humanos , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/normas , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. METHODS: Based on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. RESULTS: DNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. CONCLUSIONS: The results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.
Assuntos
Humanos , Antituberculosos/farmacologia , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , DNA Bacteriano/análise , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase Multiplex/economia , Mycobacterium tuberculosis/efeitos dos fármacos , Mutação Puntual , Sensibilidade e Especificidade , Análise de Sequência de DNA , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
We hereby propose a novel sensitive, specific, and cost-efficient method to detect Ehrlichia canis and Anaplasma platys DNA from canine whole blood samples by multiplex PCR. Blood samples from hemoparasited dogs attending the Veterinary Hospital at the Universidade Federal Rural da Amazônia-UFRA, Belém, Brazil, were collected in tubes containing EDTA. Amplification of E. canis and A. platys 16S rDNA by nested (n) PCR was successfully achieved by using primers specific to the Anaplasmataceae in the first round of PCR, followed by a second round of PCR using E. canis-specific primers in conjunction with A. platys-specific primers. The amplicons obtained were cloned and sequenced, yielding sequences of 478 and 473 bp (including primers) pertaining to regions of the 16S rDNA of E. canis and A. platys, respectively. The protocol we here propose may help to measure the prevalence of canine monocytic ehrlichiosis (CME) and canine cyclic thrompocytopenia, not only in northern Brazil, where there is no data available, but also elsewhere.
Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/diagnóstico , DNA Bacteriano/sangue , Ehrlichia canis/isolamento & purificação , Ehrlichiose/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Anaplasma/genética , Anaplasmose/epidemiologia , Animais , Sequência de Bases , Brasil/epidemiologia , Análise Custo-Benefício , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Cães , Ehrlichia canis/genética , Ehrlichiose/epidemiologia , Feminino , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Multiplex/economia , Reação em Cadeia da Polimerase Multiplex/normas , Prevalência , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/veterinária , Fatores de TempoRESUMO
Approximately 170 million people are chronic carriers of hepatitis C virus (HCV). Patients with chronic hepatitis C are currently treated with pegylated interferon and ribavirin (PEG-IFN/RBV). A genome-wide association with PEG-IFN/RBV treatment response and a single nucleotide polymorphism (rs12979860) has been identified near the interleukin 28B gene that encodes interferon-λ-3. In this paper, we describe an innovative, fast, and low-cost multiplex polymerase chain reaction with confronting two-pair primers that detects the rs12979860 polymorphism. The assay is internally controlled and does not require the use of restriction endonucleases or special equipment. Moreover, the assay decreases costs, being about 40% cheaper than direct sequencing methods.
Assuntos
Hepatite C Crônica/genética , Interleucinas/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Polimorfismo de Nucleotídeo Único , Adulto , Primers do DNA/genética , Feminino , Estudo de Associação Genômica Ampla , Hepatite C Crônica/economia , Humanos , Interferons , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex/economiaRESUMO
OBJECTIVE: To evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. METHODS: Based on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. RESULTS: DNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. CONCLUSIONS: The results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.