Оценка возможностей метода RAPD-PCR для генетической идентификации штаммов B. subtilis

The natural features of Bacillus subtilis strains (wide biodiversity within the species, the ability for sustainable growth in various media, symbiotic properties, high antagonistic activity, production of a number of hydrolytic enzymes and antibiotics of different chemical classes, resistance to adverse environmental factors and ecological plasticity) determined their prospects to obtain new biological products to protect plants from diseases of fungi and bacterial origin

Revealing genetic variations in bacteria populations can be achieved by application of genotyping techniques. In addition, bacterial genotyping is used to identify strains, identify antibiotic resistance genes and virulence genes. One of the most effective and fast method for genotyping is PCR, which allows the amplification of polymorphic regions in the genomes being studied. This approach makes it possible to quickly identify bacterial strains, that makes it possible to identify the spread of infections, determine the sources of the pathogen in environmental objects, and certify commercially used strains of antagonistic bacteria that are used in the development of environmentally friendly biopreparations. PCR-based genotyping methods have significant advantages over many other methods for identifying genetic profiles of strains. In particular, the pulsed-field gel electrophoresis (PFGE) method requires expensive specialized equipment and analysis takes a long time. Methods for identifying bacterial strains using sequencing of specific DNA regions, such as multilocus sequence typing (MLST), often do not provide a sufficiently high resolution, since they are based on sequencing only several conserved housekeeping genes. Work is constantly being carried out to develop genotyping methods based on the use of polymerase chain reaction. Among the fastest and simplest genotyping methods is RAPD-PCR, which is based on the use of short primers for carrying out a polymerase chain reaction

The research object was 14 B. subtilis isolates isolated from environmental objects and grown in the laboratory of Microbiological Plant Protection of the All-Russian Institute of Plant Protection. A literature search was conducted to get optimal primers for PCR. We analyzed genotyping data, discriminatory ability, quantity and quality of amplified DNA fragments obtained previously on a number of strains of this bacterial species. As a result of the search, three short RAPD-PCR primers were identified (OPA-3, OPL-12 and OPM-15), along with forward and reverse primers ERIC, which are used to amplify a conserved region of the genome of enterobacteria of different species (ERIC1 and ERIC2), as well as primer M13, which detects minisatellite DNA in bacteria and animals (see table 1).

The primers were first checked simultaneously against several publications in the scientific literature to eliminate errors in the nucleotide sequence, and then checked against the NCBI database to confirm their specificity with respect to the strains of bacteria being studied and additional control of the nucleotide sequence.

The mechanism for detecting genomic DNA variability using PCR with ERIC primers is the amplification of a DNA section that has different lengths in individual bacterial strains. RAPD primers detect differences in DNA at the species level and often discriminate even strains at subspecies level in bacteria. The mechanism for detecting differences lies in different primer binding sites in different species and strains, which leads to the amplification of DNA fragments of various lengths.

The reaction mixture for PCR for 10 samples contained the following components:

103 µl of distilled water

14 µl of producer suggested PCR buffer

14 µl MgCL2 (25 mM, 2.5 mM of final concentration)

3 µl dNTP (5 mM, 100 µM of final concentration)

3 µl primer (20 µM, 0.4 µM of final concentration)

3 µl Hot Start Taq polymerase (5u/µl).

After mixing the components, the mixture was added to 10 PCR tubes in a volume of 14 μl in each. Then 1 μl of genomic DNA isolated from bacterial strains was added to these tubes. At the primer annealing temperature recommended in the literature for ERIC primers of 52°C, surpisingly, no amplification occurred, and this circumstance required lowering the temperature to 37°C. Before the first PCR cycle, a long primary denaturation step was carried out (once) at 95°C for 5 minutes, and after amplification, a long elongation step was carried out at 72°C for 3 minutes. The results of genotyping using the RAPD method were carried out twice, and in both cases the results were the same, which indicates the reproducibility of the selected conditions for conducting the DNA fragment amplification reaction. Preliminary experiments have shown that the optimal concentration of magnesium chloride in the mixture is 2.5 mM. At this concentration, active synthesis of PCR products occurred without the appearance of an excessive trail of nonspecific fragments (Fig.1). Optimal annealing temperature for all primers was 37°C.

After completion of PCR, the amplified DNA was transferred into wells for agarose electrophoresis in the presence of ethidium bromide (1.5% agarose, Tris-acetate buffer). Electrophoresis is carried out at 100V for 3 hours in a chamber with a distance between electrodes of 20 cm (voltage gradient 5 V/cm). GeneRuler (ThermoFisher™) was used as a marker to deterrmine the lengths of DNA fragments that were amplified as a result of PCR. Visualization of the results of separation of amplified fragments was carried out in a gel documentation system under ultraviolet light. Analysis of the number and distribution of DNA fragments in the gel was carried out visually in relation to DNA fragments of marker DNA.

The OPM-15 primer did not result in the amplification of discernible DNA fragments in the Bacillus subtilis genome, and the ERIC primer pair produced a monomorphic pattern consisting of two DNA fragments in all isolates. Primer M13 also did not lead to the formation of clearly distinguishable amplifications. The primers OPA-3 and OPL-12 turned out to be the most informative for B.subtilis (fig. 1).

Figure 1 - Genotyping B.subtilis strains using primer OPA-3 (left, A) and primer OPL-12 (right, B)

Note: M - marker of DNA fragment length (GeneRuler 100 bp, Thermo Fisher Scientific)

DOI:10.23649/JAE.2023.39.5.2

No amplification occurred in isolate number 3. The OPA-3 primer revealed 4 different genotypes among all isolates, with the dominant cluster comprising isolates 1, 2, 4, and 8, while isolates 7, 9, and 10 were unique, i.e. they differed from the dominant cluster and from each other. A similar picture was observed when using the OPL-12 primer. The difference was that in addition to isolates 1, 2, 4, and 8, the dominant cluster also included isolate 7. Accordingly, the remaining isolates 9 and 10 were unique. In total, the latter primer revealed three genotypes.

Thus, these two primers can be successfully used for genotyping and identification (certification) of Bacillus subtilis strains, since they are capable to amplify several polymorphic DNA fragments for subsequent visualization.

1. The optimal temperature for genotyping B. subtilis using primers OPA-3 and OPL-12 is 37°C, the concentration of magnesium ions is 2.5 mM;

2. When genotyping B. subtilis strains, the short RAPD primers OPA-3 and OPL-12 were the most informative as compared with three other primers;

3. PCR genotyping of B. subtilis strains used as an active component in biopreparations allows certification of these strains to confirm their purity and genetic uniformity.