REAL-TIME PCR TEST SYSTEM FOR TTV DNA DETECTION IN BIOLOGICAL MATERIAL
Abstract
Background. The study of biological material for the presence of TTV DNA using the PCR method allows for a timely assessment of the functional state of the human liver and immune system. Objective. To develop components for real-time PCR for TTV DNA detection in biological material. Material and methods. The design and selection of optimal primers and probes (taking into account the size (length) of the amplicon, annealing temperature, nucleotide composition, distribution of nucleotides along the length of the primer, length of primers, the possibility of formation of hairpins and dimers by primers) were performed using the Primer-BLAST/Primer3, FastPCR programs. Since primers, even absolutely unique for certain DNA sequences, could anneal at nonspecific sites, not related to the gene analyzed, we checked the correspondence of the primers to the sequences of the target gene. For this purpose, we used the NCBI Primer BLAST online service and assessed the local pairwise alignment of each primer with all nucleotide sequences of the Refseq databases. Results. As the result of studies carried out on the selection of the optimal primer annealing temperature, primer concentrations, as well as the selection of the optimal nucleotide pair, the main parameters of the designed primers were determined. Conclusions. A kit for the detection and quantification of TTV DNA using the polymerase chain reaction method with hybridization-fluorescent detection in real time was created and became the basis for the development of a commercial test system.
References
Bisceglie AMD. TT virus and other anelloviruses [Internet]. UpToDate. 2023. Available from: https://www.uptodate.com/contents/tt-virus-and-other-anelloviruses/print.
Family – Anelloviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ editors. Virus Taxonomy. Ninth Report of the International Committee on Taxonomy of Viruses. Amsterdam: Elsevier; 2012. p. 331-341. doi: https://doi.org/10.1016/B978-0-12-384684-6.00033-1.
Gore EJ, Gard L, Niesters HGM, Van Leer Buter CC. Understanding torquetenovirus (TTV) as an immune marker. Front Med (Lausanne). 2023;10:1168400. https://doi.org/10.3389/fmed.2023.1168400.
Sarairah H, Bdour S, Gharaibeh W. The Molecular Epidemiology and Phylogeny of Torque Teno Virus (TTV) in Jordan. Viruses. 2020;12(2):165. https://doi.org/10.3390/v12020165.
Pachot A, Venet F, Monneret G, Lepape A, Rimmelé T, Tan LK, Brengel-Pesce K, Textoris J. Herpes DNAemia and TTV Viraemia in Intensive Care Unit Critically Ill Patients: A Single-Centre Prospective Longitudinal Study. Front Immunol. 2021;12:698808. https://doi.org/10.3389/fimmu.2021.698808.
Maev IV, Karlovich TI, Burmistrov AI, Chekmazov IA, Andreev DN, Reshetnyak VI. Sovremennye predstavlenija o roli Torque Teno Virus (TTV) pri zabolevanijah pecheni [Current Views of Torque Teno Virus (TTV) in Liver Diseases]. Rossijskij zhurnal gastrojenterologii, gepatologii, koloproktologii [Russian Journal of Gastroenterology, Hepatology, Coloproctology]. 2020;30(4):7-22. https://doi.org/10.22416/1382-4376-2020-30-4-7-22. https://elibrary.ru/ovawzn. (Russian).
Focosi D, Antonelli G, Pistello M, Maggi F. Torquetenovirus: the human virome from bench to bedside. Clin Microbiol Infect. 2016;22(7):589-93. https://doi.org/10.1016/j.cmi.2016.04.007.
Reshetnyak VI, Maev IV, Burmistrov AI, Chekmazov IA, Karlovich TI. Torque teno virus in liver diseases: On the way towards unity of view. World J Gastroenterol. 2020;26(15):1691-1707. https://doi.org/10.3748/wjg.v26.i15.1691.
Kulifaj D, Durgueil-Lariviere B, Meynier F, Munteanu E, Pichon N, Dubé M, Joannes M, Essig M, Hantz S, Barranger C, Alain S. Development of a standardized real time PCR for Torque teno viruses (TTV) viral load detection and quantification: A new tool for immune monitoring. J Clin Virol. 2018;105:118-127. https://doi.org/10.1016/j.jcv.2018.06.010.
Maggi F, Focosi D, Statzu M, Bianco G, Costa C, Macera L, Spezia PG, Medici C, Albert E, Navarro D, Scagnolari C, Pistello M, Cavallo R, Antonelli G. Early Post-Transplant Torquetenovirus Viremia Predicts Cytomegalovirus Reactivations In Solid Organ Transplant Recipients. Sci Rep. 2018;8(1):15490. https://doi.org/10.1038/s41598-018-33909-7.
Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539. https://doi.org/10.1038/msb.2011.75.
Focosi D, Macera L, Pistello M, Maggi F. Torque Teno virus viremia correlates with intensity of maintenance immunosuppression in adult orthotopic liver transplant. J Infect Dis. 2014;210(4):667-8. https://doi.org/10.1093/infdis/jiu209.
1.png)
