Real-time PCR validation to estimate the number of rickettsias in the biological material under study

Abstract

Using the example of the clinical strain of R. sibirica «Bayevo 105/87», the possibility of quantitative determination of rickettsias in clinical samples from patients with Siberian tick-borne typhus by real-time polymerase chain reaction (PCR-RT) was evaluated. Cultivation was carried out in the yolk sacs of developing chicken embryos, from which a piece of the yolk sac or chorion was taken. A total of 125 samples were examined. A set of reagents “RealBest DNA Rickettsia species (kit1)” was used for PCR-RT. The obtained values of the threshold amplification cycle (Ct) were compared with the results of microscopy of smear preparations stained by the Zdrodovsky method, the values of which were divided into ranks: the I rank - single rickettsias in individual fields of vision, the II rank - single rickettsias in each field of vision, the III rank - from 10 to 25 rickettsias in each field of vision, the IV rank - from 25 to 50 rickettsias in each field of view. The median Ct value for rank I was 17.6 (16.37; 18.58), for the II - 16.0 (15.0; 16.41), for the III - 15.0 (14.0; 15.1) and for the IV - 15.0 (13.7; 14.64). A significant average correlation was established between the number of rickettsias in the preparation under microscopy and the value of the threshold cycle in PCR RT (r=-0, 4849542; p=9.968e-09). When determining the correlation between the pathomorphological characteristic and the value of the threshold cycle, its absence was established. The detection of rickettsias in the blood vessels of the chorion of developing chicken embryos was of interest. In 10 samples, the yolk sac and chorion were taken for the study, and in parallel they were examined by PCR-RT. The use of modern, more sensitive molecular biological methods allows for quantitative analysis of DNA in the chorion, while preserving the volumes of the most valuable material - the yolk sac.