1 50.0 1 0 (15.7) (17.6) (0) (0) 69.6 % 18.6 (17.6) 100.0 “( )” – in the parentheses Multiplex-qPCR results. Discussion Molecular diagnostics of microbial etiological agents of sepsis is currently
at an initial stage and is limited more to scientific research than to diagnostic practice. Only few kits for the detection of microorganisms that cause sepsis are available on the market: SeptiFast (Roche) and SeptiTest (Molzym), but in no way do they satisfy the needs of molecular sepsis diagnostics [8, 9]. The SeptiFast (Roche) AZD1480 system enables the detection of more than a dozen specific microbial species, while SeptiTest (Molzym) theoretically allows to detect every possible microorganism species, but sequencing of the PCR product is required, which Omipalisib Selleck Compound C increases the cost and prolongs the wait for the result. The starting point for the design of
the described nested-multiplex qPCR method was the work describing the application of the qPCR method to detect bacteria and fungi in biological materials separately – Bispo et al. described the PCR methodology in the detection of bacteria with Gram differentiation in the vitreous humor, and Sugita et al. described the PCR method for the detection of yeast and filamentous fungi in the eyeball when it is inflamed [10, 11]. During the work carried out by our team, it was possible to combine the sequences of primers and probes described by the authors into a multiplex reaction for simultaneous detection of bacteria and fungi with their differentiation into Gram-negative bacteria, Gram-positive bacteria, yeast fungi, and filamentous fungi. The results of sensitivity determination of such a method
in the multiplex system has shown that it is possible to achieve the detection threshold of 9.9 × 102 CFU/ml to 5.4 × 103 CFU/ml depending on the group of microorganisms (Table 3). The resulting sensitivity was lower than the one obtained using SeptiFast (Roche) test with which one can detect the presence of individual microorganisms at the level of: 3 × 100 CFU/ml for E. coli, 3 × 101 CFU/ml for S. aureus, 3 × 101 CFU/ml for C. albicans and 3 × 100 CFU/ml for A. fumigatus[12]. In order to increase the sensitivity of the DOK2 detection method in the multiplex qPCR system, a preliminary amplification procedure (I) was designed so as to gain an opportunity to carry out detection of the presence of bacteria and fungi in the nested multiplex qPCR system. The designed primer sequences and amplification procedure related to their use allowed to reduce the detection threshold to approximately 101 CFU/ml for all of the four examined groups of microorganisms (Table 3). The resulting sensitivity is slightly lower than in the case of SeptiFast (Roche) test, but it should be taken into account that the number of cells of bacteria and fungi amplified in the PCR reaction oscillate at a maximum of 7.