RESUMEN
OBJECTIVE: This study aimed to establish a highly sensitive and rapid single-tube, two-stage, multiplex recombinase-aided qPCR (mRAP) assay to specifically detect the khe, blaKPC-2, and blaNDM-1 genes in Klebsiella pneumoniae. METHODS: mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, blaKPC-2, and blaNDM-1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. RESULTS: mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, blaKPC-2, and blaNDM-1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, blaKPC-2, and blaNDM-1 genes was 1, 0.855, and 1, respectively (p < 0.05). CONCLUSION: mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, blaKPC-2, and blaNDM-1 genes in K. pneumoniae.
Asunto(s)
Klebsiella pneumoniae , Reacción en Cadena de la Polimerasa Multiplex , beta-Lactamasas , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , beta-Lactamasas/genética , Humanos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/diagnóstico , Sensibilidad y Especificidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Proteínas Bacterianas/genética , Recombinasas/genética , Recombinasas/metabolismoRESUMEN
This study reported a novel highly active alkaline urate oxidase (UOX) and demonstrated its application in reducing uric acid content of food under alkaline conditions. The UOX gene was cloned from Arxula adeninivorans NBRC 10858, and its N-terminally his6-tagged form (rUOX) was overexpressed in Escherichia coli. The rUOX displayed maximal activity at 40⯰C and pH 10, kept more than 90% initial activity under alkaline conditions (pH 9-11) and more than 80% at temperatures below 55⯰C. The apparent Km, turnover number (kcat) and catalytic efficiency (kcat/Km) values for the substrate uric acid were respective 29.15⯵M, 151.16â¯s-1 and 5.19â¯s-1. µM-1, which are improvements over previously reported UOXs. The rUOX efficiently reduced uric acid and purine contents in beer, beef and yeast extract at pH 10, indicating a promising application in food with low purine and uric acid contents to prevent hyperuricemia and gout.