The studies was devoted to the construction and testing of a plasmid positive control for real-time polymerase chain reaction to detect the chlamydia genome. A 142-bp region of 16s–23s rRNA operon of the chlamydia was cloned and ligated to the open plasmid vector pTZ19R, and competent E. coli cells were transformed with the resulting construct. The presence of the insert was monitored by ampicillin selection of clones and by PCR. The effectiveness of the application of the obtained structure, the copy number of which was equal to 7.65×1010 DNA molecules per μl, was proven by establishing the correlation of the values of the Ct to the amount of DNA in the sample. Testing a series of multiple dilutions of a positive plasmid control with concentrations from 101 to 107 DNA copies per μl in real-time PCR, a regression line was constructed (R2 = 0.993)
Keywords: DNA, Chlamydia, pTZ19R
Elwell C., Mirrashidi K., Engel J. Chlamydia cell biology and pathogenesis. Nature Reviews. Microbiology. 2016. Vol. 14, iss. 6. P. 385–400. DOI: https://doi.org/10.1038/nrmicro.2016.30.
Borel N., Polkinghorne A., Pospischil A. A review on chlamydial diseases in animals: still a challenge for pathologists? Veterinary Pathology. 2018. Vol. 55, iss. 3. P. 374–390. DOI: https://doi.org/10.1177/0300985817751218.
Cheong H. C., Lee C. Y. Q., Cheok Y. Y., Tan G. M. Y., Looi C. Y., Wong W. F. Chlamydiaceae: diseases in primary hosts and zoonosis. Microorganisms. 2019. Vol. 7, iss. 5. P. 146. DOI: https://doi.org/10.3390/microorganisms7050146.
Vidal S., Kegler K., Greub G., Aeby S., Borel N., Dagleish M. P., Posthaus H., Perreten V., Rodriguez-Campos S. Neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria. BMC Veterinary Research. 2017. Vol. 13, iss. 1. P. 373. DOI: https://doi.org/10.1186/s12917-017-1294-y.
Lienard J., Croxatto A., Aeby S., Jaton K., Posfay-Barbe K., Gervaix A., Greub G. Development of a new chlamydiales-specific real-time PCR and its application to respiratory clinical samples. Journal of Clinical Microbiology. 2011. Vol. 49, iss. 7. P. 2637–2642. DOI: https://doi.org/10.1128/JCM.00114-11.
Rohde G., Straube E., Essig A., Reinhold P., Sachse, K. Chlamydial zoonoses. Deutsches Arzteblatt International. 2010. Vol. 107, iss. 10. P. 174–180. DOI: https://doi.org/10.3238/arztebl.2010.0174.
