Broad-range real-time PCR and sequencing from the 16S rRNA gene region
May 15, 2017
Broad-range real-time PCR and sequencing from the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. a sensitivity and specificity of 100%. The probe specific for showed eight discrepancies resulting in a sensitivity of 100% and a specificity of SB-408124 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore this assay significantly reduced the time needed for identification. In conclusion using pathogen-specific probes offers a faster substitute for pathogen recognition and could enhance the analysis of blood stream infections. Blood stream attacks certainly are a main reason behind loss of life in the global globe and want an intensive and adequate therapeutic technique. Inadequate antibiotic therapy can be connected with higher mortality prices the looks of antibiotic level of resistance and much longer hospitalization measures (14). Regular susceptibility and identification testing have many limitations such as for example Rabbit polyclonal to PCBP1. insufficient rapidity and sensitivity. The current yellow metal regular i.e. bloodstream culture usually needs 6 to 12 h of incubation before development is recognized and an additional 24 to 48 h for the definitive recognition from the infectious agent and its own susceptibility to antibiotics (1 9 Schedule diagnostics already make use of molecular approaches for the immediate recognition of viral and bacterial pathogens. Nevertheless most in-house assays are targeted against one particular bacterium and/or pathogen and don’t present broad-range pathogen recognition. Recently many PCR assays have already been developed focusing on a panel of the very most relevant bacterial and fungal blood stream pathogens which may be performed straight with blood such as for example SeptiFast (Roche Diagnostics GmbH Mannheim Germany) SepsiTest (Molzym GmbH & Co. KG Bremen Germany) and VYOO (SIRS-Lab GmbH Jena Germany) or using positive bloodstream cultures like the microarray-based program Prove-it Sepsis (Mobidiag Helsinki Finland). As talked about in our earlier work immediate recognition in whole bloodstream can be hampered by many factors like the existence of PCR inhibitors and history DNA low bacterial fill insufficient level of sensitivity and problems of creating an assay with the capacity of detecting an array of pathogens (5). On the other hand molecular tests of growth-positive bloodstream cultures will not need highly delicate assays due to the current presence of a higher bacterial fill. Furthermore as yet the usage of culturing continues to be necessary to determine the microorganism’s antimicrobial profile. Which means role of blood cultures continues to be very important to the identification and detection of causative bacterial agents. Molecular tests of blood ethnicities possibly in conjunction with regular testing could enable more rapid identification and consequently more rapid diagnosis and start of correct therapy. Molecular approaches such as broad-range real-time PCR and sequencing of the 16S rRNA gene region are widely known methods for the detection and identification of bacteria in clinical samples (4 7 10 15 However because of the need for sequencing the identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The assay is particularly intended for the identification of positive blood cultures for which Gram staining results are known. In this proof-of-concept study priority was given to the genera or species most frequently found in blood cultures and/or those that could direct the choice of a suitable antibiotic therapy. Therefore we SB-408124 selected a panel of eight species- or genus-specific probes. The Gram-negative bacteria were divided into spp. spp. spp. spp. SB-408124 and spp. from other GPCs a catalase test was performed by adding 1 colony to a drop of 3% H2O2. For the identification of spp. catalase-positive strains were tested for coagulase and DNase production. If both tests were SB-408124 negative the strain was identified as coagulase-negative (CoNS). To discern spp. from other catalase-negative GPCs bile esculin Tellur diagnostic tablets (Rosco Diagnostica Taastrup Denmark) and an API 20 Strep test.