The signals were induced with the help of a special FRET technique. Determination of the bacterial pathogens Four G + and nine G- bacterial subgroups could be A-1210477 research buy distinguished through a joint consideration of the melting points of the probes and the melting point of the overall PCR product (Figure 1). Figure 1 Differentiation of the bacterial pathogens. The group temperatures indicate the entire Tm of the pathogens. The subgroup temperatures are the melting temperatures of the hybridization probes. S. aureus and S. epidermidis have very close-lying melting temperatures and their species-specific differentiation is not possible via this 16S
XAV-939 research buy rRNA sequence (Figure 2). A comparison of the Gene Bank sequences (S.
aureus and S. epidermidis NCBI Taxonomy ID: NC_009782.1 and JF_799903.1) of these species revealed a variance of only three base-pairs, none of them in the region where the probe is associated with the DNA. Thus, determination of the clinically relevant Staphylococcus species requires other gene sequences in which the antibiotic resistance can be detected . The situation is the same for the two Enterococcus species . At the same time, S. pyogenes and L. monocytogenes are clearly differentiable. Figure 2 Melting-peaks of Staphylococcus aureus and Staphylococcus epidermidis. Revealing that it is impossible to differentiate these Staphylococcus species via the Tm data of the amplicons or probes. Protein Tyrosine Kinase inhibitor Among the G- bacteria, E. coli is one of the most common causative agents of bloodstream infections . Unfortunately, it has almost the same Tm as those of E. cloacae and S. marcescens. Other bacterial strains, such as H. influenza, are clearly differentiable through the melting temperature of the probe (Figure 3) or amplicon. The sensitivity of the reaction was five colony-forming units (CFU) per reaction. Figure 3 Differentiation of Escherichia coli from Haemophilus influenzae
. Although these pathogens have a very similar Tm in the 16S rRNA region, the Tm of the probes are clearly different. Determination of fungal pathogens Fourteen tuclazepam frequently-encountered fungal pathogens could be distinguished. The highly variable ITS 2 target sequence allowed correct identification of all of the clinically relevant fungal strains, through the Tm points on the F1 channel [12, 18]. There was no signal on the F2 or F3 channel. The sensitivity of the reaction was 5 CFU per reaction. The correct differentiation between bacteria and fungi was verified by means of gel electrophoresis, with the help of the amplicon length (fungal amplicons 192–494 bp, bacterial 187 bp). Determination of the co-infection model In case of co-infections, there are some limitations in the detection. If the ratios of the different agents are higher than 1:10, the system does not detect the infectious agent which is in lower quantities.