microbeПроблемы особо опасных инфекцийProblems of Particularly Dangerous Infections0370-10692658-719XRussian Research Anti-Plague Institute “Microbe”10.21055/0370-1069-2017-3-53-57microbe-412Research ArticleМИКРОБИОЛОГИЯMICROBIOLOGYРАЗРАБОТКА АЛГОРИТМА ОПРЕДЕЛЕНИЯ УРОВНЯ ЭКСПРЕССИИ ГЕНОВ CTXA И TOXR VIBRIO CHOLERAE МЕТОДОМ ОТ-ПЦР С ГИБРИДИЗАЦИОННО-ФЛУОРЕСЦЕНТНЫМ УЧЕТОМ РЕЗУЛЬТАТОВ В РЕЖИМЕ РЕАЛЬНОГО ВРЕМЕНИDEVELOPMENT OF THE ALGORITHM FOR IDENTIFICATION OF THE LEVEL OF VIBRIO CHOLERAE CTXA AND TOXR GENE EXPRESSION USING RT-PCR WITH REAL-TIME HYBRIDIZATION-FLUORESCENT REGISTRATION OF RESULTSКрицкийА. А.KritskyA. A.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

rusrapi@microbe.ruЧелдышоваН. Б.CheldyshovaN. B.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

ТучковИ. В.TuchkovI. V.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

СмирноваН. И.SmirnovaN. I.

410005, Саратов, ул. Университетская, 46

46, Universitetskaya St., Saratov, 410005

ФКУЗ «Российский научно-исследовательский противочумный институт «Микроб»РоссияRussian Research Anti-Plague Institute “Microbe”Russian Federation201720092017035357Copyright © Крицкий А.А., Челдышова Н.Б., Тучков И.В., Смирнова Н.И., 20172017Крицкий А.А., Челдышова Н.Б., Тучков И.В., Смирнова Н.И.Kritsky A.A., Cheldyshova N.B., Tuchkov I.V., Smirnova N.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.microbe.ru/jour/article/view/412Цель

Цель. Создание алгоритма оценки экспрессии структурных и регуляторных генов вирулентности Vibrio cholerae на модели генов ctxA и toxR, кодирующих и контролирующих биосинтез холерного токсина.

Материалы и методы

Материалы и методы. В работе использовано 10 штаммов Vibrio choleraе классического и Эль Тор биоваров. Клонирование фрагментов генов проводили путем лигирования и трансформации. ОТ-ПЦР проводили на амплификаторах «БИС М112» и «Rotor-GeneQ». Обработку результатов осуществляли с помощью программного обеспечения к прибору RotorGeneQ (Software 1.8.17.5).

Результаты и выводы

Результаты и выводы. Разработан алгоритм оценки уровня экспрессии генов ctxА и toxR V. cholerae методом ОТ-ПЦР с гибридизационно-флуоресцентным учетом результатов в режиме реального времени. Указанный алгоритм позволяет быстро и эффективно проводить статистически значимое определение экспрессии структурных и регуляторных генов вирулентности V. cholerae и может быть использован для оценки вновь выделяемых штаммов холерного вибриона. 

Objective of the study is to design the algorithm for assessment of expression of the structural and regulatory virulence Vibrio cholerae genes by the model of ctxA and toxR genes encoding and controlling biosynthesis of cholera toxin.

Materials and methods

Materials and methods. Utilized were 10 strains of Vibrio cholerae, classical and El Tor biovars. Cloning of gene fragments was carried out through transformation and ligation. RT-PCR was done in “BIS M112” and “Rotor-GeneQ” amplifiers. Processing of the results was performed by means of the software package in set with Rotor-GeneQ (Software 1.8.17.5).

Results and conclusions

Results and conclusions. Developed has been the algorithm for evaluation of the level of expression of V. cholerae ctxA and toxR genes applying RT-PCR with real-time hybridization-fluorescent registration of results. The stated algorithm allows for rapid and effective statistically significant specification of the expression of structural and regulatory virulence V. cholerae genes and can be used for evaluation of newly discovered cholera vibrio strains. 

V. choleraeхолерный токсингены ctxА и toxRОТ-ПЦРуровень экспрессииV. choleraecholera toxinctxA and toxR genesRT-PCRlevel of expressionReferencesСмирнова Н.И., Челдышова Н.Б., Заднова С.П., Кутырев В.В. Молекулярно-генетические особенности штаммов Vibrio cholerae classica, вызвавших эпидемию азиатской холеры в России в 1942 г. Мол. генет., микробиол. и вирусол. 2001; 4:12–6.Smirnova N.I., Cheldyshova N.B., Zadnova S.P., Kutyrev V.V. [Molecular-genetic peculiarities of Vibrio cholerae classica strains that caused epidemic of Asian cholera in Russia in 1942]. Mol. Genet. Mikrobiol., Virusol. 2001; 4: 12–6.Creating Standard Curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. Applied Biosystems; 2003.Creating Standard Curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. Applied Biosystems; 2003.Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using Real-Time quantitative PCR and the 2–ΔΔCt Method. Methods. 2001; 25(4):402–8. DOI: 10.1006/meth.2001.1262.Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using Real-Time quantitative PCR and the 2–ΔΔCt Method. Methods. 2001; 25(4):402–8. DOI: 10.1006/meth.2001.1262.Malinen E., Kassinen A., Rinttila Т., Palva A. Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology. 2003; 149(Pt 1):269–77. DOI: 10.1099/mic.0.25975-0.Malinen E., Kassinen A., Rinttila Т., Palva A. Comparison of real-time PCR with SYBR Green I or 5’-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology. 2003; 149(Pt 1):269–77. DOI: 10.1099/mic.0.25975-0.Marashi S.M., Bakhshi B., Fooladi A.A., Tavakoli A., Sharifnia A., Pourshafie M.R. Quantitative expression of cholera toxin mRNA in Vibrio cholerae isolates with different CTX cassette arrangements. J. Med. Microbiol. 2012; 61(Pt 8):1071–3. DOI: 10.1099/jmm.0.038752-0.Marashi S.M., Bakhshi B., Fooladi A.A., Tavakoli A., Sharifnia A., Pourshafie M.R. Quantitative expression of cholera toxin mRNA in Vibrio cholerae isolates with different CTX cassette arrangements. J. Med. Microbiol. 2012; 61(Pt 8):1071–3. DOI: 10.1099/jmm.0.038752-0.Matson J.S., Withey J.H., DiRita V.J. Regulatory networks controlling Vibrio cholerae virulence gene expression. Infect. Immun. 2007; 75(12):554–29. DOI: 10.1128/IAI.01094-07.Matson J.S., Withey J.H., DiRita V.J. Regulatory networks controlling Vibrio cholerae virulence gene expression. Infect. Immun. 2007; 75(12):554–29. DOI: 10.1128/IAI.01094-07.Muller P.Y., Janovjak H., Miserez A.R., Dobbie Z. Processing of gene expression data generated by quantitative Real-Time RTPCR. Biotechniques. 2002; 32(6):1372–9.Muller P.Y., Janovjak H., Miserez A.R., Dobbie Z. Processing of gene expression data generated by quantitative Real-Time RT-PCR. Biotechniques. 2002; 32(6):1372–9.Palmer S., Wiegand A.P., Maldarelli F., Bazmi H., Mican J.M., Polis М., Dewar R.L., Planta A., Liu S., Metcalf J.A., Mellors J.W., Coffin J.M. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 2003; 41(10):4531–6.Palmer S., Wiegand A.P., Maldarelli F., Bazmi H., Mican J.M., Polis М., Dewar R.L., Planta A., Liu S., Metcalf J.A., Mellors J.W., Coffin J.M. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 2003; 41(10):4531–6.Sharkey F.H., Banat I.M., Marchant R. Detection and quantification of gene expression in environmental bacteriology. Appl. Environ. Microbiol. 2004; 70(7):3795–806. DOI: 10.1128/AEM.70.7.3795–3806.2004.Sharkey F.H., Banat I.M., Marchant R. Detection and quantification of gene expression in environmental bacteriology. Appl. Environ. Microbiol. 2004; 70(7):3795–806. DOI: 10.1128/AEM.70.7.3795–3806.2004.Smith C.J., Osborn A.M. Advantages and limitations of quantitative PCR(Q-PCR)-based approaches in microbial ecology. FEMS Microbiol. Ecol. 2009; 67(1):6–20. DOI: 10.1111/j.15746941.2008.00629.x.Smith C.J., Osborn A.M. Advantages and limitations of quantitative PCR(Q-PCR)-based approaches in microbial ecology. FEMS Microbiol. Ecol. 2009; 67(1):6–20. DOI: 10.1111/j.1574-6941.2008.00629.x.Wang Т., Brown M.J. mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal. Biochem. 1999; 269(1):198–201. DOI: 10.1006/abio.1999.4022.Wang Т., Brown M.J. mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal. Biochem. 1999; 269(1):198–201. DOI: 10.1006/abio.1999.4022.Fykse E.M., Skogan G., Davies W., Olsen J.S., Blatny J.M. Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 2007; 73(5):1457–66. DOI: 10.1128/AEM.01635-06.Fykse E.M., Skogan G., Davies W., Olsen J.S., Blatny J.M. Detection of Vibrio cholerae by real-time nucleic acid sequence-based amplification. Appl. Environ. Microbiol. 2007; 73(5):1457–66. DOI: 10.1128/AEM.01635-06.

The authors declare that there are no conflicts of interest present.