The main difference between the "ROSSAmed COVID-19 ISOTHERM" and other PCR kits is the detection of virus RNA by colorimetric, loop, isothermal amplification. Unlike the PCR method, RNA amplification occurs at a constant temperature of the reaction mixture, as well as using more productive enzymes, which reduces the analysis time by several times, reduces the risk of errors due to the minimum number of operations. All you need to do is warm up the sample at 95℃ -15 minutes (the enzyme is resistant to inhibition, so no RNA isolation and purification step is required), prepare the reaction mixture, add the sample to it, incubate at 65℃ - 45 minutes. In total, express testing takes 1 hour.
"ROSSAmed COVID-19 ISOTHERM" passed clinical and laboratory tests at the Research Institute of Virology, during which a set of reagents was compared with real-time PCR test systems from Russian manufacturers. The tests included positive samples with low and high virus concentrations, as well as negative samples. Matches were found in 96.1% of cases when testing positive samples, including samples with a low concentration of virus, and in 100% of cases when testing negative samples.
PCR has been the main method for nucleic acid amplification for many years, but modern approaches to molecular diagnostics require simpler and faster methods for obtaining PCR results. The PCR kit “ROSSAmed COVID-19 ISOTHERM” designed to detect and identify specific SARS-CoV-2 coronavirus RNA fragments, uses an innovative method of colorimetric, isothermal, loop sample amplification.
The method of isothermal loop amplification is based on the synthesis of nucleic acids with strand negation catalyzed by Bst-DNA polymerase. The amplification process can be divided into three main stages: 1) the formation of the basic dumbbell structure, 2) the stage of cyclic amplification, 3) the stage of elongation and cycles. As a result of this reaction, many concatemers of inverted repeats of the target fragment are formed, which form cauliflower-like structures in space. A dye sensitive to the pH of the medium is added to the mixture, the amplification products shift the pH of the medium to the acid side, which leads to a change in the color of the mixture, which can be detected with the naked eye.
The analysis time is 1 hour. Registration of results is carried out visually.
Among the distinguishing features of the method, one can note the rapid accumulation of reaction products, low sensitivity to impurities, and high specificity.