ИСТИНА

Post-transplantation infection due to the bacterial contamination of cellular therapy products is a potential risk in regenerative medicine, therefore the sterility tests are crucial within the quality control. These tests are usually performed by automated blood culture systems, which yield accurate results, but take several days, and cannot provide the sterility data before transplantation. It’s possible to detect bacteria in cellular therapy products using polymerase chain reaction (PCR) with universal primers, amplifying the 16S rDNA gene of all eubacteria species. This method is fast and cost-effective, but often gives false-positive results due to high PCR sensitivity and reagents contamination with traces of bacterial DNA. There are various ways to deal with false-positive results, but all of them require additional time and cannot prevent failure due to occasional contamination during PCR setup. We have developed a simple method, which allows to eliminate false-positive results and adjust the sensitivity of PCR-based sterility test. For PCR we used 0,4 μM of each primer for 16S rDNA gene (5ʹ-TCCTACGGGAGGCAGCAGT-3ʹ; 5ʹ-GGACTACCAGGGTATCTAATCCTGTT-3ʹ) and 0,4 μM of each primer for human beta-actin gene (5ʹ-GCGCCGTTCCGAAAGTT-3ʹ; 5ʹ-CGGCGGATCGGCAAA-3ʹ). The reaction conditions were 95°C for 5 min, 50 cycles of 95°C for 10 s and 54°C or 55°C for 40 s. Results were detected by agarose gel electrophoresis. The sensitivity was determined by six 10-fold serial dilutions of the E.coli suspension (105-1 CFU) added to 1ml aliquots of the medium containing 105 of human adipose-derived mesenchymal stromal cells. DNA was isolated from these mixtures and PCR was performed as described. When PCR was performed with an annealing temperature of 54°C sensitivity of the method was 1000 CFU/ml and with an annealing temperature of 55°C sensitivity was 10-100 CFU/ml, which considered to be sufficient to prevent adverse reactions. No positive results were found in negative controls containing human cells only, but false-positive results were detected in no template controls. Thus, our method has the internal control, which allows to avoid false-positive results due to the competition between the reactions. The method is also configurable to adjust the sensitivity, which can be useful for various applications.