USE OF NULLOMERIC DNA SEQUENCE IN DEVELOPMENT OF REAL-TIME PCR TEST SYSTEMS
Main Article Content
Authors
B.K. Tynysbekov
National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan
A.N. Auganov
National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan
A.S. Akhmetollayeva
National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan
I.A. Akhmetollayev
National Center for Biotechnology, 13/5, Korgalzhyn hwy., Astana, 010000, Kazakhstan
Abstract
This study describes the development of a real-time PCR (qPCR) system for the identification of Pasteurella multocida serotypes using nullomeric DNA sequences. P. multocida is a widespread animal pathogen, and its different serogroups (A, B, D, E, and F) are directly linked to disease severity and distribution. Serotype-specific diagnostics are of great importance for disease prevention and control in veterinary practice.
The main novelty of this approach lies in the use of nullomers in primer and probe design, which minimizes the risk of false-positive signals. Synthetic primers and probes were synthesized and their specificity tested against DNA from Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Optimization experiments showed the most effective conditions were: MgCl₂ – 2.5 mM, dNTP – 0.5 mM and primer/probe concentration – 0.5 pM.
The newly developed qPCR system demonstrated high specificity and sensitivity: it reliably detected different concentrations of P. multocida DNA (ranging from 10⁶ copies down to 1 copy/µL), with a detection threshold of approximately 100,000 copies. Serotype-specific primers produced clear amplification curves with no cross-reactivity observed.
These results confirm that the use of nullomer-based primers is an effective strategy for real-time PCR diagnostics. The advantages of this method highlight its potential for application in epidemiological monitoring, veterinary diagnostics, and integration into portable qPCR platforms. The developed system enables rapid and accurate identification of P. multocida serotypes and may serve as a model for detecting other bacterial pathogens.
Keywords
Pasteurella multocida, serogroups A, B, D, E and F, real-time PCR, qPCR, nullomers, primer designs, fluorescent probes, molecular diagnostics
Article Details
References
Peng Z., Wang X., Zhou R., Chen H., Wilson B. A., Wu B. Pasteurella multocida: Genotypes and Genomics // Microbiology and Molecular Biology Reviews. – 2019. – Vol. 83, №4. – Crossref.
Christensen H., Sajid S. M., Bisgaard M., Magistrali C. F., Massacci F. R., Liman M., Menke T., Bischoff H., Olsen J. E. Prediction of Pasteurella multocida serotypes based on whole genomic sequences // Veterinary microbiology. – 2022. – Vol. 271:109492. - Crossref.
Pasomboon P., Chumnanpuen P., E-kobon T. Comparison of Hyaluronic Acid Biosynthetic Genes From Different Strains of Pasteurella multocida // Bioinformatics and biology insights. – 2021. – Vol. 15. – P. 1-14. - Crossref.
Guan L. J., Yang J. Q., Xu Q. Y., Feng Y. F., Zhang X. C., Tang B., Zhao Z. Q.. Immunogenicity and Efficacy of Serogroup A and D Bacterins against Pasteurella Multocida in Mice // Frontiers in veterinary science. – 2023. – Vol. 10.1132536. - Crossref.
Richardson N. I., Ravenscroft N., Kuttel M. M. Conformational comparisons of Pasteurella multocida types B and E and structurally related capsular polysaccharides // Glycobiology. – 2023. – Vol. 33, №9 – P. 745-754. - Crossref.
Wang J., Sun S., Chen Y., Chen D., Sang L., Xie X. Pathogenic and genomic characterisation of a rabbit sourced Pasteurella multocida serogroup F isolate s4 // BMC Vet Res. – 2022. – Vol. 18(1), №288. – P. 1-12. - Crossref.
Harshitha R., Arunraj D. R. Real-time quantitative PCR: A tool for absolute and relative quantification // Biochemistry and Molecular Biology Education. – 2021. – Vol. 49, №5 . – P. 800–812. - Crossref.
Breeding K. M., Ragipani B., Lee K. D., Malik M., Randis T. M., Ratner A. J. Real-time PCR-based serotyping of Streptococcus agalactiae // Scientific Reports. – 2016. – Vol. 6. - Crossref.
Vergni D., Santoni D. Nullomers and high order nullomers in genomic sequences // PLOS ONE. – 2016. – Vol. 11(12). – P. 1-15. - Crossref.
Hampikian G., Andersen T. Absent sequences: Nullomers and primes // Pacific Symposium on Biocomputing. – 2007. – Vol. 12. – P. 355-366. - Crossref.
Vergni D., Santoni D. Nullomers and High Order Nullomers in Genomic Sequences // PLOS One. – 2016. – Vol. 11(12) . – pp. 1-15. - Crossref.
Acquisti C., Poste G., Curtiss, D., Kumar S. Nullomers: Really a matter of natural selection // PLOS One. – 2007. – Vol. 2(10) . – P. 1-3. - Crossref.
Georgakopoulos-Soares I., Yizhar-Barnea O., Mouratidis I., Hemberg M., Ahituv N. Absent from DNA and protein: genomic characterization of nullomers and nullpeptides across functional categories and evolution // Genome Biology – 2021. – Vol. 22, №245. - Crossref .
Furfaro, L. L., Chang, B. J. & Payne, M. S. A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III // Diagnostic Microbiology and Infectious Disease. – 2017. – Vol. 89, №1. – P. 7-12. - Crossref.
Handigund M., Lee J. Performance Evaluation of NovaplexTM Multiplex Real-Time PCR Assay for Detection of Streptococcus agalactiae Serotypes // Microorganisms. – 2024. – Vol. 12, №10: 2043. - Crossref.
Wang H., Xin L., Wu Y., Liu Y., Yao W., Zhang H., Hu Y., Tong R., Zhu L. Construction of a one-step multiplex real-time PCR assay for the detection of serogroups A, B, and E of Pasteurella multocida associated with bovine pasteurellosis // Frontiers in Veterinary Science. – 2023. – Vol. 28, №10: 1193162. – P. 1-10. - Crossref.
Wang J., Sang L., Sun S., Chen Y., Chen D., Xie X. Characterization of Pasteurella multocida isolated from dead rabbits with respiratory disease in Fujian, China // BMC veterinary research. – 2019. – Vol. 15, №438. - Crossref.
Montgomery A., Tsiatsianis G. C., Mouratidis I., Chan, C. S. Y., Athanasiou M., Papanastasiou A. D., Kantere V., Syrigos N., Vathiotis I., Syrigos K., Yee N. S., Georgakopoulos-Soares I. Utilizing nullomers in cell-free RNA for early cancer detection // Cancer gene therapy. – 2024. – Vol. 31. – P. 861–870. - Crossref.
Casalino G., D'Amico F., Bozzo G., Dinardo F. R., Schiavitto M., Galante D., Aceti A., Ceci E., Romito D., D'Onghia F. P., Dimuccio M. M., Camarda A., Circella E. In field evaluation of impact on clinical signs of an inactivated autogenous vaccine against Pasteurella multocida in rabbits // International Journal of Veterinary science and Medicine. – 2024. – Vol. 12(1). – P. 39–47. - Crossref.