Experience in developing reagent kits for identifying pathogens of tick-borne infections using isothermal amplification

The risk of infection from the bite of an infected tick leads to the adoption of emergency preventive measures. The prophylactic use of antibiotics and antiviral immunoglobulins to all those bitten carries an unjustified risk of developing antibiotic resistance and adverse reactions, since the frequency of tick bites in Russia (up to 500 thousand per year), and ticks are carriers of Borrelia (25—50%) and tick-borne encephalitis virus (up to 3%) less than half. In this regard, the development of rapid tests for use directly when removing a tick will be in demand. We previously developed a diagnostic kit for visual detection of Borrelia burgdorferi s.l. DNA in isothermal LAMP method. The purpose of this work was to develop kits for multiplex isothermal detection of nucleic acids from Borreliosis pathogens Borrelia burgdorferi s.l. and Borrelia miyamotoi, as well as a for the detection of tick-borne encephalitis virus. Materials and methods. We used 178 DNA and 325 RNA samples isolated from ticks, as well as cerebrospinal fluid samples from patients. To create LAMP, we chose the primer design and reaction buffer composition. LAMP products are detected using CFX96 Touch and FLUORITE devices in isothermal mode, and visual detection as options are also available. Comparative testing of the RT-PCR method was carried out using commercial kits «Vector-Best» and «Central Research Institute of Epidemiology» (Russia). Results. The selected reagents for LAMP in the «NT MxLAMP BOR+MIY» kit in one testing made it possible to detect Borrelia DNA with a limit of analytical sensitivity 46 copies/μl to B. burgdorferi s.l., and 361 copies/µl to B. miyamotoi. The «NT LAMP TBEV1» tick-borne encephalitis virus detection kit showed a sensitivity limit of 36 copies/μl. In comparative tests, all positive results coincided with the effectiveness of a commercial PCR test, however, the relative sensitivity of the «NT MxLAMP BOR19+MIY1» kit was inferior to the results obtained using the «Vector-Best» kits. An additional option for visual detection of results allows you to determine the DNA of borreliosis pathogens in ticks and cerebrospinal fluid, including without prior isolation of nucleic acids. Conclusions. For the practical use of diagnostic kits, it is necessary to increase their sensitivity, adapt them for use with portable devices with thermostatting and simple visual detection, and also resolve issues regulating the performance of these analyzes at points for emergency prevention of tick-borne infections.