USE OF NULLOMERIC DNA SEQUENCE IN DEVELOPMENT OF REAL-TIME PCR TEST SYSTEMS

2025-10-03T11:42:06+05:00

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.

VIABILITY OF CANINE’S OVARIAN TISSUE AFTER NONEQUILIBRIUM CRYOCONSERVATION

2025-10-02T18:39:01+05:00

Modern approaches to reproductive biotechnology include the development of effective methods for long-term storage of gametes and reproductive tissues of animals. One of the promising areas is cryopreservation of ovarian tissue, which allows preserving fertility, especially in rare and valuable species. However, the effectiveness of this technology directly depends on the type of cryoprotectant, freezing mode, cooling rate and storage method. Nonequilibrium cryopreservation is a method in which biological samples are exposed to liquid nitrogen vapor without strict software control of cooling. This approach allows for a simpler procedure but requires careful optimization of conditions to preserve tissue viability. The aim of this work is to evaluate the effect of various cryoprotectants and the altitude of the samples (4, 5 and 6 cm above the surface of liquid nitrogen) on the morphological preservation of follicles in the ovarian tissue of dogs after nonequilibrium cryopreservation. A study was conducted to investigate the effect of nonequilibrium cryopreservation in liquid nitrogen vapor on the morphofunctional state of ovarian tissue in dogs. Four cryoprotectors (dimethyl sulfoxide, ethylene glycol, propylene glycol, glycerol) and three temperature regimes (4 cm, 5 cm and 6 cm from the liquid nitrogen level) were used. After thawing, a histological analysis was performed to assess the degree of preservation of different types of follicles. It was found that the best viability indicators were observed when using 1.5 M dimethyl sulfoxide (4 and 6 cm), glycerol (4 cm) and propylene glycol (5 cm). The data obtained are important for the development of effective protocols for cryopreservation of ovarian tissue in dogs in order to preserve the gene pool and reproductive potential.

Eurasian Journal of Applied Biotechnology

USE OF NULLOMERIC DNA SEQUENCE IN DEVELOPMENT OF REAL-TIME PCR TEST SYSTEMS

VIABILITY OF CANINE’S OVARIAN TISSUE AFTER NONEQUILIBRIUM CRYOCONSERVATION