• Ешқандай Нәтиже Табылған Жоқ

Selection of an effective DNA extraction method for the detection of Salmonella by the polymerase chain reaction

UDC 579.577.637.1

S.M. Barmak1, 2, Yu.A. Sinyavskiy2, A.B. Berdygaliev2, I.S. Savitskaya1, T.Sh. Sharmanov1, I.Н. Mendenhall3, E.V. Zholdybaeva4

1Al-Farabi Kazakh National University, Almaty, Kazakhstan;

2Kazakh Academy of Nutrition, Almaty, Kazakhstan;

3Duke-NUS Medical School, Singapore;

4NationalCenter for Biotechnology, Nur-Sultan, Kazakhstan (E-mail: sabyr95@mail.ru)

Selection of an effective DNA extraction method for the detection

Selection of an effective DNA extraction method …

The objective of our studies was to select an effective method of DNA extraction for Salmonella detection by the method of polymerase chain reaction.

Materials and Methods

The objects of the study were eight Salmonella bacteria isolated from the foodstuff at the Microbiology Chair of Al-Farabi Kazakh National University.

DNA Extraction. DNA was extracted from Salmonella bacteria by the following methods:

1. Trizol method. The commercial reagent TRizol («Invitrogen», USA) was used in accordance with the manufacturer’s manual.

2. CTAB method. CTAB (cetyl trimethyl ammonium bromide) is a classical cation detergent that is used in DNA extraction. СТАВ lyses the cellular membrane, effectively destroys DNA-protein [7].

3. Sorbent method using silica particles. DNA binds to silica particles under the effect of high concen- trated salt [8].

The quality and quantity of the extracted DNA were controlled with the use of NanoDrop 2000.

For setting PCR used the same amount of DNA (~ 30 ng/μl) obtained by different methods.

PCR-amplification. PCR-fragments of Inv gene from Salmonella bacteria were produced with the use of the following primers: S Inv-1F (direct) and S Inv-1R (inverse). Amplification was carried out in 25 µL of reaction mixture of the following composition: 10 xbuffer of DNA polymerase — 2.5 µL, 10 mM dNTP — 1 µL, MgCl2 — 1 µL, 20–50 ng of DNA-matrix, 20 pM of direct and 20 pM of inverse primers, and 0.5 U of DNA polymerase (Invitrogen), DNA — 1 µL. PCR products were assayed by electrophoresis in 1.5 % agarose gel containing ethidium bromide (1 µg/µL) at the field strength 6 V/cm2. The PCR product sized 500 bp.

Results and Discussion

Comparative study of three different versions of the DNA extraction from Salmonella bacteria was carried out: the trizol method using TRizol commercial reagent, the CTAB method and the sorbent method using silica particles.

The parameters such as quantity and quality of the obtained DNA were taken into consideration to select the DNA extraction method. The results of extracting genome DNA by different methods from 8 Salmonella isolates are shown in Figure 1.

Figure 1. The amount of DNAs extracted from eight Salmonella isolates (ng/µL) by different methods

The implemented studies showed the CTAB method to demonstrate the highest DNA yield. The results of the trizol method are slightly lower. And the lowest DNA yield was demonstrated by the sorbent method.

PCR-assay depends significantly on the quality of the extracted DNA. PCR amplification was conducted with the DNA specimens of equal volume (1 µL) obtained by different extraction methods from samples of the materials under study: the trizol method using TRizol commercial reagent, the CTAB method and the sorbent method using silica particles. The results are shown in Figure 2.

S.M. Barmak, Yu.A. Sinyavskiy et al.

А

В

C

A — TRizol method; B — CTAB method; C — sorbent method;

1–8 — DNAs of Salmonella bacteria; M — 1 kb DNA marker (Invitrogen) Figure 2. Electrophoregram of the DNAs obtained from Salmonella bacteria

Figure 2 shows that the genetic material of high quality and of concentration sufficient for PCR-assay has been obtained by the sorbent method with the use of silica particles (С). At the same time, it should be noted that the DNA concentration in these preparations is 1.61 times less than in preparations obtained by using TRizol (A) and 1.75 times lower versus the preparations obtained by the CTAB method.

Insignificant PCR inhibition in the DNA specimens obtained by the trizol method using TRizol commer- cial reagent and the CTAB method may be caused by the DNA excess, which is a PCR inhibitor.

The same amount of DNA (50 ng/μl) was used to select an effective DNA extraction method for detecting Salmonella in the PCR reaction. The results are presented in Figure 3.

А

В

С

А — 50 ng/μl of DNA isolated with TRizol reagent was used in the PCR reaction; В — 50 ng/μl of DNA isolated with CTAB method was used in the PCR reaction; С — 50 ng/μl of DNA isolated with sorbent method was used in the PCR

reaction; 1–8 — DNAs of Salmonella bacteria; M — 1 kb DNA marker (Invitrogen) Figure 3. PCR products of Salmonella bacteria on an agarose gel using 50 ng/μl of DNA

Selection of an effective DNA extraction method …

As can be seen in Figure 3, optimal results were obtained when PCR was performed using DNA isolated by the sorbent method.

The major criterion in methods of the DNA extraction is that nucleic acid should be the utmostly purified from cellular DNA and protein impurities. The extracted genome DNA should be unfragmented since it is a matrix for the synthesis of the specific product [9]. Such a result is ensured by the sorbent method using silica particles. When this method has used the proteins and cellular components are removed and the utmost purified DNA is left. The advantages of the DNA extraction method with the help of silica particles are as follows: loss minimization in the course of the DNA extraction; lower risk of cross-contamination owing to the linkage of all nucleic acid to the sorbent; high purity of the final product [10]. In contrast to other methods, this one does not require the usage of toxic reagents.

Effectiveness of the process of the DNA extraction is a determining factor in the course of work with a small amount of the material or with the specimens containing a considerable quantity of inhibitors that may result in lower diagnostic sensitivity of the test and inhibition of the amplification process [11]. The properly selected method of the DNA extraction allows achieving the most accurate result of the polymerase chain reaction.

Conclusion

On the basis of the implemented study, one can conclude that the sorbent method using silica particles is the most suitable method for DNA extraction from Salmonella. In the future, this method may be used to kit up the test system for Salmonella identification and genotyping.

The work was carried out within the project of grant financing «Genotyping pathogenic microorganisms in the food raw material and foodstuff realized in the markets and supermarkets of the Republic of Kazakhstan, development of recommendations aimed at reducing the risk of morbidity among the children of preschool and school age», years 2018–2020, No. AP05131147.

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S.M. Barmak, Yu.A. Sinyavskiy et al.

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