RESUMEN
The data presented in this paper supports the research article "A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment" ( Vutukuru et al., 2016) [1]. We compared a list of sepsis causing pathogens to the ApoH binding data given to us by ApoH technologies. The data highlights the binding of ApoH beads to sepsis causing pathogens.
RESUMEN
Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~200µL or less. We have also demonstrated that this method of enrichment allows detection of 1CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30min to complete and can be easily integrated with various downstream molecular and microbiological techniques.
Asunto(s)
Patógenos Transmitidos por la Sangre/aislamiento & purificación , Sangre/microbiología , Técnicas Microbiológicas/métodos , Patología Molecular/métodos , Sepsis/diagnóstico , Sepsis/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/patogenicidad , Candida tropicalis/genética , Candida tropicalis/aislamiento & purificación , Células Cultivadas , Recuento de Colonia Microbiana/métodos , ADN Bacteriano/sangre , ADN de Hongos/sangre , Enterococcus/genética , Enterococcus/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Hongos/clasificación , Hongos/genética , Hongos/aislamiento & purificación , Hongos/patogenicidad , Genoma Humano , Humanos , Límite de Detección , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Sepsis/sangre , Factores de Tiempo , beta 2 Glicoproteína I/administración & dosificaciónRESUMEN
The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt(-)) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS(+) phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt(-) phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6 We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt(-) mutants. We determine that the lys2-128∂ Spt(-) phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt(-) transcription in tested Spt(-) mutants.
Asunto(s)
ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Elongación Transcripcional/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación Fúngica de la Expresión Génica , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Retroelementos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Sitio de Iniciación de la Transcripción , Factores de Elongación Transcripcional/genéticaRESUMEN
Using anti-human CD45 antibody coated beads, we show a 98% reduction of WBCs from spiked blood samples in 1h, thereby enriching it for pathogens. This enrichment allowed the detection of <10CFU of Escherichia coli in 1mL blood using quantitative PCR; something not observed in unenriched samples.