Detection and quantification of biotargets are important analytical tasks, which are solved using a wide range of various methods. In recent years, methods based on the isothermal amplification of nucleic acids (NAs) have been extensively developed. Among them, a special place is occupied by rolling circle amplification (RCA), which is used not only for the detection of a specific NA but also for the analysis of other biomolecules, and is also a versatile platform for the development of highly sensitive methods and convenient diagnostic devices. The present review reveals a number of methodical aspects of RCA-mediated analysis; in particular, the data on its key molecular participants are presented, the methods for increasing the efficiency and productivity of RCA are described, and different variants of reporter systems are briefly characterized. Differences in the techniques of RCA-mediated analysis of biotargets of various types are shown. Some examples of using different RCA variants for the solution of specific diagnostic problems are given.

Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3′-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3′-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.

The use of biocontrol agents based on endophytic bacteria against phloem-feeding insects is limited by a lack of knowledge and understanding of the mechanism of action of the endophyte community that makes up the plant microbiome. In this work, the mechanisms of the additive action of endophytic strains B. subtilis 26D and B. subtilis 11VM on the resistance of bread spring wheat against greenbug aphid Schizaphis graminum, was studied. It was shown that B. subtilis 26D secreted lipopeptide surfactin and phytohormones cytokinins, and B. subtilis 11VM produced iturin and auxins into the cultivation medium. Both strains and their lipopeptide-rich fractions showed direct aphicidal activity against greenbug aphid. For the first time, it was shown that B. subtilis 26D and B. subtilis 11VM in the same manner, as well as their lipopeptide-rich fractions, activated the expression of salicylate- and ethylene-dependent PR genes, and influenced plant redox metabolism, which led to an increase in plant endurance against aphids. The composition of endophytic strains B. subtilis 26D + B. subtilis 11VM had an additive effect on plant resistance to aphids due to an increase in the number of endophytic bacterial cells, and, as well as due to the synergistic effect of their mixture of lipopeptides − surfactin + iturin, both on the aphid mortality and on the expression of PR1 and PR3 genes. All these factors can be the reason for the observed increase in the growth of plants affected by aphids under the influence of B. subtilis 26D and B. subtilis 11VM, individually and in composition. The study demonstrates the possibility of creating in the future an artificial composition to enhance plant microbiome with endophytic bacteria, which combines growth-promoting and plant immunity stimulating properties against phloem-feeding insects. This direction is one of the most promising approaches to green pesticide discovery in the future.