RESUMEN
Klebsiella pneumoniae belongs to Enterobacteriaceae, which is the commonest bacterium causing nosocomial respiratory tract infection. It ranks second in bacteremia and urinary tract infection in gram-negative bacteria. Therefore, the rapid and accurate identification of K. pneumoniae was of great significance for the guide of clinical medication, and timely treatment of patients. The purpose of this study was to establish a rapid and sensitive molecular detection method for K. pneumoniae based on loop-mediated isothermal amplification (LAMP) technology. Firstly, local BLAST and NCBI BLAST were used to analyze the genome of K. pneumoniae. According to the principle of interspecific and intraspecific specificity, CelB (GenBank ID 11847805) was selected as the specific gene. Then, the LAMP and PCR identification systems were established with this target gene. Thirty-six clinical isolates of K. pneumoniae and 50 non-K. pneumoniae were used for the specific evaluation, and both LAMP and PCR could specifically distinguish K. pneumoniae from non-K. pneumoniae. A 10-fold series diluted positive plasmids and simulated infected blood samples were used as the templates in the sensitivity assay, and the results showed that the sensitivity could reach 1 copy/reaction. In summary, a rapid, specific, and sensitive LAMP method was established to detect K. pneumoniae in clinics.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas de Tipificación Bacteriana/métodos , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Bacteriemia/diagnóstico , Bacteriemia/microbiología , Cartilla de ADN/genética , Humanos , Infecciones por Klebsiella/diagnóstico , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Plásmidos/genética , Sensibilidad y EspecificidadRESUMEN
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 TiempoRESUMEN
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.