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Specificities of multi-primer polymerase chain reaction optimization for the detection of infectious pneumonia agents in human

https://doi.org/10.32362/2410-6593-2021-16-3-225-231

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Abstract

Objectives. The objectives of this work are the development of a multi-primer system based on the polymerase chain reaction (PCR) aimed at the simultaneous detection of six bacterial pathogens that cause human pneumonia and the determination of the parameters important for the optimization of this multi-primer system, including solid-phase PCR systems (biological microarrays).
Methods. To determine the optimal parameters of the system, PCR methods were used in monoplex and multiplex formats.
Results. Primers for Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenza, Legionella pneumophila, Klebsiella pneumoniae, and Streptococcus pneumoniae detection were designed, and the PCR cycling conditions were optimized. The patterns of primer design for solidphase PCR were revealed.
Conclusions. The developed prototype of a system specifically identifies six clinically significant bacterial pathogens. It could be expanded for the analysis of viral and fungal pathogens and used in clinical diagnostics. A prototype of a system for pathogenic agent detection in the immobilized phase (biological microarray) was created.

About the Authors

E. S. Klochikhina
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation

Ekaterina S. Klochikhina, Laboratory Assistant

32, Vavilova ul., Moscow, 119991 



V. E. Shershov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation

Valeriy E. Shershov, Researcher

32, Vavilova ul., Moscow, 119991 



V. E. Kuznetsova
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation

Viktoria E. Kuznetsova, Cand. Sci. (Chem.), Researcher

32, Vavilova ul., Moscow, 119991 



S. A. Lapa
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation

Sergey A. Lapa, Cand. Sci. (Biol.), Senior Researcher

32, Vavilova ul., Moscow, 119991

Scopus Author ID 6603461000 



A. V. Chudinov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences
Russian Federation

Alexander V. Chudinov, Cand. Sci. (Chem.), Head of the Laboratory

32, Vavilova ul., Moscow, 119991

Scopus Author ID 7003833018 



References

1. Nair G.B., Niederman M.S. Community-acquired pneumonia: an unfinished battle. Med. Clin. North Am. 2011;95(6):1143–1161. https://doi.org/10.1016/j.mcna.2011.08.007

2. Harris M., Clark J., Coote N., Fletcher P., Harnden A., McKean M., Thomson A. British Thoracic Society Standards of Care Committee. British Thoracic Society guidelines for the management of community acquired pneumonia in children: update 2011. Thorax. 2011;66:ii1–ii23(Suppl. 2). https://doi.org/10.1136/thoraxjnl-2011-200598

3. Rawson T.M., Wilson R.C., Holmes A. Understanding the role of bacterial and fungal infection in COVID-19. Clin. Microbiol. Infect. 2021;27(1):9–11. https://doi.org/10.1016/j.cmi.2020.09.025

4. Чучалин А.Г., Синопальников А.И., Козлов Р.С., Авдеев С.Н., Тюрин И.Е., Руднов В.А., Рачина С.А., Фесенко О.В. Российское респираторное общество (РРО) Межрегиональная ассоциация по клинической микробиологии и антимикробной химиотерапии (МАКМАХ) Клинические рекомендации по диагностике, лечению и профилактике тяжелой внебольничной пневмонии у взрослых. Пульмонология. 2014;(4):13–48. https://doi.org/10.18093/0869-0189-2014-0-4-13-48 [Chuchalin A.G., Sinopal’nikov A.I., Kozlov R.S., Avdeev S.N., Tyurin I.E., Rudnov V.A., Rachina S.A., Fesenko O.V. Russian Respiratory Society Interregional association on clinical microbiology and antimicrobial chemotherapy Clinical guidelines on diagnosis, treatment and prevention of severe community acquired pneumonia in adults. PULMONOLOGIYA. 2014;(4):13–48 (in Russ.). https://doi.org/10.18093/0869-0189-2014-0-4-13-48

5. Тартаковский И.С. Современные подходы к диагностике атипичных пневмоний. Клинич. Микробиол. и Антимикроб. Химиотер. 2000;2(1):60–68. URL: https://cmac-journal.ru/publication/2000/1/cmac-2000-t02-n1-p060/cmac-2000-t02-n1-p060.pdf [Tartakovskii I.S. Modern approaches to the diagnosis of atypical pneumonia. Klin. Mikrobiol. Antimikrob. Khimioterap. = Clin. Microbiol. Antimicrob. Chemotherapy. 2000;2(1):60–68 (in Russ.). Available from URL: https://cmac-journal.ru/en/publication/2000/1/cmac-2000-t02-n1-p060/cmac-2000-t02-n1-p060.pdf]

6. Бруснигина Н.Ф., Мазепа В.Н., Самохина Л.П. и др. Этиологическая структура внебольничной пневмонии. Мед. Альманах. 2009;2(7):118–121. [Brusnigina N.F., Mazepa V.N., Samokhina L.P., et al. Etiological structure of community-acquired pneumonia. Med. Al’manakh = Med. Alm. 2009;2(7):118–121 (in Russ.).]

7. Doyle J.J., Doyle J.L. A Rapid DNA Isolation Procedure for Small Quantities of Fresh Leaf Tissue. Phytochemical Bullettin. 1987;19(1):11–15.

8. Morona J.K., Morona R., Miller D.C., Paton J.C. Streptococcus pneumoniae capsule biosynthesis protein CpsB is a novel manganese-dependent phosphotyrosine-protein phosphatase. J. Bacteriol. 2002;184(2):577–583. https://doi.org/10.1128/JB.184.2.577-583.2002

9. Liu Y., Cao Y., Wang T., Dong Q., Li J., Niu C. Detection of 12 Common Food-Borne Bacterial Pathogens by TaqMan Real-Time PCR Using a Single Set of Reaction Conditions. Front Microbiol. 2019;10:222. https://doi.org/10.3389/fmicb.2019.00222

10. Xirogianni A., Tzanakaki G., Karagianni E., Markoulatos P., Kourea-Kremastinou J. Development of a single-tube polymerase chain reaction assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus spp. directly in clinical samples. Diagn. Microbiol. Infect. Dis. 2009;63(2):121–126. https://doi.org/10.1016/j.diagmicrobio.2008.09.017

11. Huletsky A., Giroux R., Rossbach V., Gagnon M., Vaillancourt M., Bernier M., Gagnon F., Truchon K., Bastien M., Picard F.J, van Belkum A., Ouellette M., Roy P.H., Bergeron M.G. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J. Clin. Microbiol. 2004;42(5):1875–1884. https://doi.org/10.1128/JCM.42.5.1875-1884.2004

12. Morrison K.E., Lake D., Crook J., Carlone G.M., Ades E., Facklam R., Sampson J.S. Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis. J. Clin. Microbiol. 2000;38(1):434–437. https://doi.org/10.1128/jcm.38.1.434-437.2000

13. Binks M.J., Temple B., Kirkham L.-A., Wiertsema S.P., Dunne E.M., et al. Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays. PLoS ONE. 2012;7(3):e34083. https://doi.org/10.1371/journal.pone.0034083

14. De Vos D., Lim A.Jr., Pirnay J.P., et al. Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL. J. Clin. Microbiol. 1997;35(6):1295–1299. https://doi.org/10.1128/jcm.35.6.1295-1299.1997

15. Tabatabaei M., Hemati Z., Moezzi M.O., Azimzadeh N. Isolation and identification of Legionella spp. from different aquatic sources in south-west of Iran by molecular &culture methods. Mol. Biol. Res. Commun. 2016;5(4):215–223. https://dx.doi.org/10.22099/mbrc.2016.3858

16. Turton J.F., Perry C., Elgohari S., Hampton C.V. PCR characterization and typing of Klebsiella pneumoniae using capsular type-specific, variable number tandem repeat and virulence gene targets. J. Med. Microbiol. 2010;59(5):541–547. https://doi.org/10.1099/jmm.0.015198-0


Supplementary files

1. Determination of the pathogen DNA by electrophoretic separation of PCR products. L – GeneRuler 50bp length marker, (1) S. pneumoniae, (2) S. aureus, (3) L. pneumophila, (4) S. aureus + L. pneumophila, (5) H. influenzae, (6) P. aeruginosa, (7) H. influenzae + P. aeruginosa, (8) K. pneumoniae.
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Type Исследовательские инструменты
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2. This is to certify that the paper titled Specificities of multi-primer polymerase chain reaction optimization for the detection of infectious pneumonia agents in human commissioned to us by Ekaterina S. Klochikhina, Valeriy E. Shershov, Viktoria E. Kuznetsova, Sergey A. Lapa, Alexander V. Chudinov has been edited for English language and spelling by Enago, an editing brand of Crimson Interactive Inc.
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  • The article describes the development and optimization of a multi-primer system prototype that can specifically detect six bacterial pathogens that cause human pneumonia (Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenza, Legionella pneumophila, Klebsiella pneumoniae, Streptococcus pneumoniae).
  • The PCR method, in various formats, was used to identify the optimal parameters and patterns of the system.
  • The prototype could be extended for the analysis of viral and fungal pathogens and used in clinical diagnostics.

For citation:


Klochikhina E.S., Shershov V.E., Kuznetsova V.E., Lapa S.A., Chudinov A.V. Specificities of multi-primer polymerase chain reaction optimization for the detection of infectious pneumonia agents in human. Fine Chemical Technologies. 2021;16(3):225-231. https://doi.org/10.32362/2410-6593-2021-16-3-225-231

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