A Sample Extraction Method for Faster, More Sensitive PCR-Based Detection of Pathogens in Blood Culture

Abstract

Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenolchloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100-to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples. ( J Mol Diagn

Rapid identification of Gram-positive pathogens and their resistance genes from positive blood culture broth using a multiplex tandem RT-PCR assay

“…Both aerobic and anaerobic blood culture broths were affected. PCR inhibition by blood culture bottle contents such as sodium polyanethol sulfonate and whole blood components including haemoglobin, lactoferrin and immunoglobulins is well described (Regan et al, 2012). These inhibited tests add to the overall costs, with each test costing approximately *Pan-Enterococcus was also positive.…”

Direct identification of Gram-positive bacteria and resistance determinants from blood cultures using a microarray-based nucleic acid assay: in-depth analysis of microarray data for undetermined results

“…Many of the samples with weak signals contained fastidious bacterial species (e.g., viridans group streptococci) that are difficult to identify, even by using conventional culture methods. A concern for molecular testing by using direct blood culture bottles is the inhibitory effects of charcoal, resins, or anticoagulants in the bottles [39,40] . When we analyzed the signal intensities of controls, variations were observed between spots within the same test and between specimens.…”

Evaluación de un método de extracción y purificación de DNA a partir de tejido muscular fijado en formaldehido de cadáveres no identificados

“…La eliminación de la cera de parafina que protege al tejido y la extracción de una buena cantidad de DNA no degradado, son los principales obstáculos en el trabajo con esta clase de muestras (13). En los últimos años, los métodos y protocolos para el aislamiento ácidos de nucléicos a partir de tejidos FFPE han mejorado enormemente (3,4,14).…”

“…Nuestros resultados demuestran que al ampliar el tiempo de acción enzimática de la PK de 1 h a 24 h, aumenta hasta en cuatro veces la concentración de DNA que se extrajo con el kit comercial QIAampR DNA FFPE TISSUE ya que muy probablemente libera los complejos proteicos unidos a este ácido nucléico (6,14,15). También se observa una heterogeneidad en la concentración de DNA en los dos protocolos, posiblemente debido a diferencias en la cantidad de células que se tomaron con las alícuotas, luego de ser maceradas las muestras de músculo supinador.…”