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A.O. Amirgazin

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

V.B. Shvedyuk

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

M.A. Kuibagarov

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

T.B. Karibaev

National Reference Center for Veterinary Medicine, 22/3, 150 let Abaya str., Astana, 010000, Kazakhstan

A.B. Shevtsov

National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan


Polymerase chain reaction (PCR) is a multifunctional tool used extensively in molecular biology. PCR is modified in numerous ways for specific purposes, all of which rely on proper efficiency, specificity and sensitivity. Despite the general acceptance of the "classical" principle, there is no unified optimization scheme that takes into account the diversity of PCR applications. Such an optimization scheme would guide researchers toward the best optimization approaches for their specific PCR application. Currently, researchers are guided only by data from previously published authors who submitted their own PCR optimization schemes. Here, we describe a PCR protocol optimization algorithm for the detection of microorganisms using Pasteurella multocida as an example. P. multocida, which we have use dextensively in the past, allows us to achieve the necessary PCR specificity and sensitivity to demonstrate the application of our algorithm. Our scheme differs from others as it uses an inductive method to learn the specificity of the protocol being developed. Our approach uses real-time PCR, with SYBR Green I, to monitor amplification during the optimization process. Furthermore, the simplicity of this approach means that it can be used to develop and optimize diagnostic PCR for a wide range of researchers and applications. As a proof of concept, PCR conditions were optimized for two pairs of primers which were tested on a collection of samples comprising DNA from 92 species of bacteria, three eukaryotic species, and with the sensitivity of at least five P. multocida genomic copy equivalents.


PCR, optimization, Pasteurella multocida

Article Details


Schildkraut C., Lifson S. Dependence of the Melting Temperature of DNA on Salt Concentration. Biopolymers., 1965, vol. 3, рр. 195-208.

Freier S.M., Kierzek R., Jaeger J.A., SugimotoN., Caruthers M.H., Neilson T., TurnerD.H. Improved free-energy parameters for predictions of RNA duplex stability.Biochemistry Proc. Nati. Acad. Sci. USA, 1986,vol. 83, рр. 9373-9377.

Cobb B.D., CIarkson J.M. A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods.Nucleic Acids Research., 1994,vol. 22, no. 18, pp. 3801-3805.

Box G.E.P., W.G. Hunter and J.S. Hunter. Statistics for Experimenters. John Wiley &Sons.,1978.

Prabhakar P., Thangavelu A., Prabhakar T.G., Kirubaharan J.J., Chandran N.D.J. Rapid virulence typing of P. multocida in sheep isolates of Tamil.Nadu. Indian J. Anim. Sci., 2012, vol. 82 (4), pp. 351-354.

Nurushev M.Z., Kerimbay N.N., Baytanayev O.A., Kerimbay B.N. Actual problems of preservation of the saiga (saiga tatarica l.) in Kazakhstan. International journal of pure and applied zoology, 2019,vol. 4.2, pp.246-254.

Rajeev-Gautam, Dutta T.K., Kumar A.A., Shivachandra S.B. Molecular typing of Indian isolates of P. multocida serogroup-A from different animal species. Indian J. Anim. Sci., 2006,vol. 76 (11), pp. 867-872.

Hall T.A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.Nucl.Acids.Symp.Ser., 1999,vol. 41, pp. 95-98.

US National Library of Medicine National Institutes of Health. Available at: URL.

Calculator for determining the number of copies of a template. Available at: URL.

Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.Mol. Biol. Evol., 2011,vol. 28, pp. 2731-2739.

Ye1 J., Coulouris G., Zaretskaya I., Cutcutache I., Rozen S. Madden T.L. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction.BMC Bioinformatics., 2012, vol. 13, pp. 134.

Domingues L. PCR: Methods and Protocols. Methods in Molecular Biology (Springer-Verlag., New York), 2017, 213 p.

Bergeys Manual of Systematic Bacteriology, second edition. New York, Springer, 2005, vol. 2 (The Proteobacteria), part B (The Gammaproteobacteria).

Sachse K., Frey J.PCR Detection of Microbial Pathogens: Methods and Protocols.Methods in Molecular Biology, Totowa, NJ, © Humana Press Inc., vol. 216.

Pomp D., Medrano J.F. Organic solvents as facilitators of polymerase chain reaction.BioTechniques, 1991, vol. 10, pp. 58-59.

Newton C.R., Graham A. PCR.Bios Scientific Publishers, Oxford, England, 1994.

Susan Frackman, Gary Kobs, Dan Simpson, Doug Storts Betaine and DMSO: Enhancing Agents for PCR Promega Notes Number 65, 1998, pp. 27.

Shevtsov A.B., Kairzhanova A.D., AbishevaG.D., Shevtsova E.S., Kamalova D.K., Dzhailbekova A.S., KaribaevT.B., Sytnik I.I., Ahmetova A.E., MukanovK.K. Development PCR test for specific identification C. coli, C. jejuni, C. fetus Eurasian. Journal of Applied Biotechnology, 2014, no. 3, pp. 59-65.doi: 10.11134/btp.2.2014.8.

Kenneth H. Roux Optimization and Troubleshooting in PCR.PCR Methods and Applications., S186, Mannual Supplement.

Breslauer K.J., Franks R., Blockers H., MarkyL.A. Predicting DNA duplex stability from the base sequence.Proc. Natl. Acad. Sci. USA,June 1986,vol. 83, pp. 3746-3750.

Kamp E.M., Gertie C.A., Bokken M., Thea M.M. Vermeulen, Marten F. de Jong, Herma E., Buys C.M., Reek F.H., SmitsM.A. A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs.J Vet Diagn Invest., 1996,vol. 8, pp. 304-309.

Lichtensteiger C.A., Steenbergen S.M., LeeR.M., Polson D.D., VimrE.R. Direct PCR analysis for toxigenic Pasteurella multocida. J Clin Microbiol., Dec. 1996, vol. 34(12), рр. 3035-3039.

Methodical recommendation. Available at: URL.