RESUMO
Pyrosequencing technology is a sequencing method that screens DNA nucleotide incorporation in real time. A set of coupled enzymatic reactions, together with bioluminescence, detects incorporated nucleotides in the form of light pulses, which produces a profile of characteristic peaks in a pyrogram. We used this technology to identify the warfare agent Bacillus anthracis by sequencing 4 single nucleotide polymorphisms (SNPs) in the rpoB gene as chromosomal markers for B. anthracis. In addition, 1 segment in each of the B. anthracis plasmids pXO1 and pXO2 was analyzed to determine the virulence status of the bacterial strains. Pyrosequencing technology is a powerful method to identify B. anthracis.
Assuntos
Bacillus anthracis/classificação , Bacillus anthracis/genética , Técnicas de Tipagem Bacteriana , RNA Polimerases Dirigidas por DNA/genética , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA/métodos , Animais , Bacillus/classificação , Bacillus/genética , Bacillus anthracis/patogenicidade , Genótipo , Humanos , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Especificidade da Espécie , VirulênciaRESUMO
BACKGROUND: Genetic information is becoming increasingly important in diagnosis and prognosis of infectious diseases. In this study we investigated the possibility of using a single technology, the Pyrosequencing trade mark technology (Biotage AB, Uppsala, Sweden), to gather several kinds of important genetic information from the human pathogen Helicobacter pylori, as well as from the carrier of the H. pylori infection. MATERIALS AND METHODS: DNA from 87 clinical isolates of H. pylori, 50 isolates from H. pylori-infected transgenic mice and nine gastric biopsies from H. pylori-infected patients was analyzed for targets in the 16S rRNA, 23S rRNA and cytotoxin associated gene A (cagA) genes to determine species identity, clarithromycin susceptibility and virulence level, respectively. In addition, three single nucleotide polymorphisms in the human interleukin-1B (IL-1B) gene, reported to affect the risk of developing gastric cancer, were analyzed in the gastric biopsy samples. RESULTS: All DNA targets were processed and analyzed in parallel, enabling convenient genetic characterization of both pathogen and host. All genotypes were easily and accurately assigned. In the 16S rRNA analysis, 99.83% of the bases were correctly called. CONCLUSIONS: We conclude that genetic analysis using Pyrosequencing trade mark technology was nonlaborious, and gave highly accurate data for different kinds of target. We therefore believe that this technology has the potential to complement or in the future substitute the time-consuming traditional microbial identification and typing methods, as well as enabling rapid typing of relevant host genetic markers.
Assuntos
Infecções por Helicobacter/genética , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Interleucina-1/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Animais , Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Biópsia , Claritromicina/farmacologia , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Ribossômico/química , DNA Ribossômico/isolamento & purificação , Farmacorresistência Bacteriana/genética , Mucosa Gástrica/química , Genes Bacterianos , Genes de RNAr , Ilhas Genômicas/genética , Genótipo , Infecções por Helicobacter/microbiologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação Puntual , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Virulência/genéticaRESUMO
Resistance development is a significant clinical problem in Helicobacter pylori and represents the major cause of treatment failure. Today the problem is most focused on the macrolide clarithromycin that is an essential component of the H. pylori treatment. Traditional methods for resistance determination, e.g., disc diffusion tests or E-tests, could in the next 5 years be replaced by DNA-based methods. The most commonly used molecular methods available today are not used in the daily routine work. Rapid and reliable DNA-based methods for prediction of antimicrobial resistance phenotype are currently available within research. As fabrication costs reduce and validated targeted assays are developed with easy hands-on procedures, it is most likely that such assays will become important tools for clinical diagnosis of resistant H. pylori strains.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Helicobacter pylori/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , DNA Bacteriano/genética , Helicobacter pylori/classificação , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Two different enzyme assays, both based on the interaction of native reverse transcriptase (RT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymes from 18 HIV-1 isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC(50)), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC(50) values and the sensitivity of the corresponding virus to AZT in cell culture (r=0.60, P<0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT-TP giving 50% residual primer function, or CT(50)), revealed a more than 600-fold difference between the different isolate RTs. For the majority of enzymes there was a strict correlation between the results from the two assays; however, four isolates exhibited significantly higher CT(50)/IC(50) ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT-specific substitutions, including Thr-215-->Tyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39-->Ala (isolates 80 and 157). The Thr-39-->Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.
