Once all samples are processed, the sample set is analyzed through the qPCR readout portion of the assay. These samples are also analyzed using the appropriate gene-specific qPCR assay as a comparison. The MIC as determined by the molecular AST analyses were compared to the MIC as determined from the predicate macrobroth analysis to determine the agreement between these methods. Rabusertib cell line A brief description of the mechanism
of the ETGA assay is as follows; the ETGA reaction solution bead mill tube is formulated to facilitate microbe-derived DNA polymerase-mediated extension of a primer-template oligonucleotide substrate. Upon bead milling, microbe cell wall lysis allows contact between active microbe derived DNA polymerases and the primer-template substrate. A successful DNA polymerase primer-template extension event of the substrate’s primer oligonucleotide provides a new primer binding site for a subsequent qPCR detection reaction. Thus, DNA polymerase extension activity enables and triggers a downstream qPCR
detection reaction. The subsequent qPCR detection signal is directly proportional to the amount of substrate extended, which is proportional of the amount of microbial DNA polymerase extension activity present, and this is proportional to the amount of viable BAY 11-7082 datasheet proliferating bacteria selleck products present from culture. Complete details regarding the ETGA
assay have been previously described [21] a hyperlink is provided [http://nar.oxfordjournals.org/content/40/14/e109.full.pdf+html?sid=ea56a354-4e91-4515-aec8-ccdc5acfb438]. ETGA and gene-specific qPCR analysis of the time course samples Stored samples were allowed to thaw at room temperature, briefly vortexed, and spun down at 12,000×g for one minute. ETGA readout by qPCR was performed by adding 4 μL of each sample into a reaction well containing 27.2 μL of qPCR reaction mix which has been previously described [21]. For the parallel-run of corresponding gsPCR for either S. aureus or E. coli samples, single reactions were run composed of 3 μL bead mill lysate added to 28 μL of the appropriate qPCR reaction mix into a reaction well. The N-acetylglucosamine-1-phosphate transferase gene targets for the S. aureus and E. coli-specific qPCR assays are nuc and uidA respectively. The primer and probe sequences for these assays have been previously reported [21]. All qPCR analysis was performed on a Roche LightCycler 480 II system (Roche Applied Science, Indianapolis, IN). Cycle values were plotted against time of incubation. The values produced by the overnight samples were plotted as the measured Ct minus 10 to account for the 1000-fold dilution compared to the earlier samples. This assumes that each 10-fold dilution equates to a 3.33 cycle decrease in signal based on an efficient qPCR reaction.