RESUMEN

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.

Asunto(s)

Genes Bacterianos , Klebsiella pneumoniae/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Cartilla de ADN/genética , Cartilla de ADN/aislamiento & purificación , Klebsiella pneumoniae/aislamiento & purificación , Límite de Detección , Plásmidos/genética , Plásmidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Temperatura , Factores de Tiempo

RESUMEN

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

Asunto(s)

Cianobacterias/clasificación , Cianobacterias/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Microbiota , Filogenia , Argentina , Simulación por Computador , Cianobacterias/aislamiento & purificación , Cartilla de ADN/genética , Cartilla de ADN/aislamiento & purificación , Ecología , Agua Dulce/microbiología , Variación Genética , Prochlorococcus/clasificación , Prochlorococcus/genética , Prochlorococcus/aislamiento & purificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificación , Agua de Mar/microbiología , Análisis de Secuencia de ADN , Synechococcus/clasificación , Synechococcus/genética , Synechococcus/aislamiento & purificación

RESUMEN

Klebsiella pneumoniae is one of the main pathogenic bacteria that causes nosocomial infections, such as pneumonia, urinary tract infection, and sepsis. Therefore, the rapid and accurate detection of K. pneumoniae is important for the timely treatment of infectious patients. This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of K. pneumoniae-specific gene ureR_1 (Gene ID: 11847803). The ureR_1 gene was obtained through local and online BLAST, and the specific primers were designed for its detection. Positive reactions were observed on all 140 K. pneumoniae clinical isolates while all the 82 non-K. pneumoniae clinical isolates were negative. Plasmids with the specific gene and the mouse blood with K. pneumoniae were used for sensitivity analysis. The detection limit of the LAMP was 1 bacterium/reaction. The results showed that the LAMP targeted to ureR_1 is a fast, specific, sensitive, inexpensive, and suitable method for the detection of K. pneumoniae.

Asunto(s)

Técnicas de Amplificación de Ácido Nucleico/métodos , Genes Bacterianos , Klebsiella pneumoniae/genética , Plásmidos/aislamiento & purificación , Plásmidos/genética , Temperatura , Factores de Tiempo , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , Cartilla de ADN/aislamiento & purificación , Cartilla de ADN/genética , Límite de Detección , Klebsiella pneumoniae/aislamiento & purificación

RESUMEN

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Asunto(s)

Humanos , Piel/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Lepra/microbiología , Mycobacterium leprae/aislamiento & purificación , Valores de Referencia , Piel/patología , Biopsia , ADN Bacteriano/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Cartilla de ADN/aislamiento & purificación , Lepra/patología , Mycobacterium leprae/genética

RESUMEN

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Asunto(s)

Lepra/microbiología , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Piel/microbiología , Biopsia , Cartilla de ADN/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Humanos , Lepra/patología , Mycobacterium leprae/genética , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Piel/patología

RESUMEN

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Asunto(s)

Humanos , Valores de Referencia , Piel/microbiología , Piel/patología , Biopsia , ADN Bacteriano/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Cartilla de ADN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Lepra/microbiología , Lepra/patología , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/genética

RESUMEN

A study was performed to investigate the genomic variations in the shrimp farm isolates of Vibrio alginolyticus and V. harveyi when the isolates were subjected to environmental stress. Samples of shrimps, water and sediment were collected from Southern Indian coastal shrimp farms. Vibrio isolates were biochemically identified and confirmed using 16S rDNA and gyrB gene specific PCR. The bacterial strains were genotyped by PCR fingerprinting using GTG(5) and IS (Insertion Sequence) primers. Seven strains each of V. alginolyticus and V. harveyi were subjected to 10 passages through trypticase soya broth (TSB), which contained different NaCl concentrations (3, 6 and 8%) and trypticase soya agar (TSA). V. alginolyticus was also passaged through TSB with a 12% NaCl concentration. PCR fingerprinting, which was performed on the strains that were passaged through different salt concentrations, confirmed that V. alginolyticus and V. harveyi could affect the genomic variations, depending on the environmental conditions of the culture. The study highlights the complex genotypic variations that occur in Vibrio strains of tropical aquatic environment because of varied environmental conditions, which result in genetic divergence and/or probable convergence. Such genetic divergence and/or convergence can lead to the organismal adaptive variation, which results in their ability to cause a productive infection in aquatic organisms or generation of new strains.

Asunto(s)

Animales/genética , Animales/crecimiento & desarrollo , Animales/aislamiento & purificación , Animales/microbiología , Acuicultura/genética , Acuicultura/crecimiento & desarrollo , Acuicultura/aislamiento & purificación , Acuicultura/microbiología , Cartilla de ADN/genética , Cartilla de ADN/crecimiento & desarrollo , Cartilla de ADN/aislamiento & purificación , Cartilla de ADN/microbiología , ADN Bacteriano/genética , ADN Bacteriano/crecimiento & desarrollo , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/microbiología , Ecosistema/genética , Ecosistema/crecimiento & desarrollo , Ecosistema/aislamiento & purificación , Ecosistema/microbiología , Penaeidae/genética , Penaeidae/crecimiento & desarrollo , Penaeidae/aislamiento & purificación , Penaeidae/microbiología , Reacción en Cadena de la Polimerasa/genética , Reacción en Cadena de la Polimerasa/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa/aislamiento & purificación , Reacción en Cadena de la Polimerasa/microbiología , Vibrio alginolyticus/genética , Vibrio alginolyticus/crecimiento & desarrollo , Vibrio alginolyticus/aislamiento & purificación , Vibrio alginolyticus/microbiología , Vibrio/genética , Vibrio/crecimiento & desarrollo , Vibrio/aislamiento & purificación , Vibrio/microbiología

RESUMEN

O diagnóstico de certeza da peste baseia-se na identificação e isolamento da Y. pestis pelo cultivo de material de pacientes humanos, de roedores e suas pulgas. No Brasil, os casos humanos e as epizootias de peste ocorrem em locais distantes dos centros de diagnóstico. A competição com a flora cadavérica das carcaças de roedores e procedimentos inadequados de conservação e transporte das amostras aos laboratórios pode resultar na morte do bacilo e resultados falso-negativos. As técnicas moleculares apresentam-se como alternativas para estas situações, particularmente técnicas baseadas em PCR. No presente trabalho foi otimizada para o diagnóstico da peste a técnica N-PCRTbU que se baseia na separação física dos primers por imobilização dos primers internos na tampa do microtubo, dispensando a abertura do mesmo entre as duas etapas de amplificação, o que reduz a possibilidade de falsopositivos pela contaminação cruzada durante a manipulação dos amplicons no Nested-PCR convencional. Dois pares de primers, dirigidos ao gene estrutural (caf1) do antígeno F1 de Y. pestis (um par com homologia a uma região mais externa ao alvo e outro com homologia a uma região mais interna), foram inicialmente testados individualmente por PCR e depois por N-PCR tendo sido obtidos segmentos de tamanho esperado. Foram estabelecidas as concentrações de Taq DNA polimerase, dNTPs e MgCl2, bem como a proporção entre os primers externos e internos para o N-PCRTbU. Nos ensaios para estabelecimento do limiar de detecção a partir de DNA total e da cultura da cepa Y. pestis P. PB 881 a N-PCRTbU foi capaz de detectar respectivamente 10 pg de DNA e 20 bactérias viáveis. Baços de camundongos "Swiss Webster" infectados experimentalmente com a cepa Y. pestis P. PB 881 e amostras de baço, fígado, pulmão e coração conservadas no meio de Cary-Blair por 11 meses foram analisadas em paralelo por cultivo em gelose peptonada e pela técnica NPCRTbU. O fragmento de tamanho esperado (460pb) foi amplificado em todas as amostras mesmo nas que se revelaram multicontaminadas ou negativas pela cultura. Nos ensaios com DNA de outras espécies do gênero Yersinia: Y. pseudotuberculosis e Y. enterocolitica e com baços de camundongos não infectados com a Y. pestis não houve amplificação. Além da especificidade e sensibilidade, como os resultados podem ser obtidos rapidamente, em menos de 24 horas, a N-PCRTbU poderá ser mais uma opção em situações emergenciais [...]

Asunto(s)

Ratones , Peste/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Yersinia pestis/aislamiento & purificación , Estudio de Evaluación , Técnicas de Diagnóstico Molecular , Cartilla de ADN/aislamiento & purificación , Técnicas y Procedimientos Diagnósticos/instrumentación

RESUMEN

We used arbitrarily-primed polymerase chain reaction (AP-PCR) to design and construct a specific primer pair for the identification of Actinobacillus actinomycetemcomitans. We analyzed 25 DNA samples of A. actinomycetemcomitans isolated from patients with localized-juvenile periodontitis. From 90 AP-PCR primers screened, one amplification product was selected, cloned in pCR II vector, and sequenced. The sequence was used to design a single pair of specific primers. The sequence was compared with GenBank entries using BLAST and showed no significant matches. PCR amplification using the new primer pair AA1416 produced a characteristic 3.5-Kb band in all A. actinomycetemcomitans DNAs tested. Primer pair AA16S produced no or different amplicon profiles using DNA samples from bacterial species other than A. actinomycetemcomitans. Our results show that this single primer pair AA1416 can be used in PCR to identify A. actinomycetemcomitans isolates and differentiate them from other periodontal bacteria. These approaches appear promising in facilitating laboratory identification and taxonomy of putative periodontopathogens.

Asunto(s)

Aggregatibacter actinomycetemcomitans/clasificación , Cartilla de ADN , Reacción en Cadena de la Polimerasa/métodos , Aggregatibacter actinomycetemcomitans/genética , Periodontitis Agresiva/microbiología , Clonación Molecular , Cartilla de ADN/aislamiento & purificación , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Escherichia coli/genética , Amplificación de Genes , Vectores Genéticos , Humanos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN

RESUMEN

A autora descreve a metodologia baseada na reaçäo em cadeia da polimerase (PCR) para identificaçäo específica do DNA de Mycobacterium leprae e apresenta uma revisäo das estratégias relacionadas à escolha dos oligonucleotídios iniciadores de síntese do DNA ("primers") e dos critérios de especificidade e sensibilidade necessários à utilizaçäo desse método em estudos clínicos e experimentais

Asunto(s)

Humanos , Cartilla de ADN/aislamiento & purificación , ADN Bacteriano/aislamiento & purificación , Lepra/diagnóstico , Mycobacterium leprae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , ADN Bacteriano/genética , Lepra/microbiología , Mycobacterium leprae/genética , Sensibilidad y Especificidad