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RU-2861442-C1 - METHOD FOR DIFFERENTIATING STRAINS OF CAUSATIVE AGENT OF CANINE CORONAVIRUS INFECTION, CANINE CORONAVIRUS

RU2861442C1RU 2861442 C1RU2861442 C1RU 2861442C1RU-2861442-C1

Abstract

FIELD: molecular biology. SUBSTANCE: method for differentiating strains of the causative agent of canine coronavirus infection, Canine Coronavirus , using designed primers and analysis of PCR product melting curves is described. EFFECT: high sensitivity, specificity, and reliability of the differentiation of Canine Coronavirus strains. 3 cl, 2 dwg, 3 tbl, 3 ex

Inventors

  • Doronin Maksim Igorevich
  • Galkina Tatiana Sergeevna
  • Komarova Anna Aleksandrovna
  • Matveev Ivan Sergeevich
  • Kara Dmitrii Igorevich

Dates

Publication Date
20260505
Application Date
20250915

Claims (6)

  1. 1. A method for differentiating strains of the pathogen causing coronavirus infection in dogs (Canine Coronavirus) , comprising the following steps:
  2. - extraction of RNA of the causative agent of canine coronavirus infection from the sample;
  3. - conducting reverse transcription and real-time PCR of a specific 68 bp fragment of the cDNA of the S gene of the canine coronavirus pathogen using SEQ ID NO: 1 and SEQ ID NO: 2 and SYBR Green;
  4. - differentiation of coronavirus infection strains by analyzing the melting temperature curves of PCR products, which revealed the following melting temperature peaks for the strains: "SWU-SSX4/2021/CCoVIIa" - 70.62±0.01°C, "B906_ZJ_2019" - 74.02±0.01°C, "B617_ZJ_2019" - 69.87±0.01°C, "B001_AH_2018" - 73.27±0.01°C, "B825_ZJ_2019" - 70.31±0.01°C, "CCoV/dog/HCM27/2014" - 70.11±0.01°C, "CCoV/wolf/2016/IT" - 70.99±0.01°С, “Elmo/02” - 70.85±0.01°С, “1-71” - 69.54±0.01°С, “TN449” - 69.21±0.01°С, “NVSL” - 71.09±0.01°С and the differences in these temperatures are used to differentiate the specified strains of coronavirus infection.
  5. 2. The method according to item 1, wherein the analytical specificity is 100%, the diagnostic sensitivity in the 95% confidence interval is 99.12-100.00%, and the diagnostic specificity is 98.26-100.0%.
  6. 3. The method according to paragraph 1, wherein the analysis time is reduced to 2.5 hours.

Description

The invention relates to veterinary virology, to molecular diagnostic tools, namely to the differentiation of strains of the pathogen causing coronavirus infection in dogs Canine Coronavirus . The causative agent of coronavirus infection in dogs belongs to the species Canine Coronavirus (CCoV), genus Alphacoronavirus , subfamily Orthocoronavirinae , family Coronaviridae [1]. The nucleic acid of the infectious agent is represented by a single-stranded RNA molecule of positive polarity and a size of approximately 29,400-29,800 nt. About 65% of the viral ribonucleic acid is occupied by two large, partially overlapping open reading frames (ORFs): ORF1a and ORF1b, which encode two polyproteins. From the 3´-end, 1/3 of the genome (approximately 9,000 nt) consists of other ORFs carrying information on structural and non-structural viral proteins. Structural proteins of alphacoronavirus include S-, E-, M- and N-proteins, encoded by ORF2-, ORF4-, ORF5- and ORF6-genes, respectively [2-6]. The system of measures to combat canine coronavirus enteritis and its prevention includes immunization of animals, as well as monitoring the level of tension of post-vaccination immunity [4-10]. Given that vaccine strains of the canine coronavirus virus are used to produce inactivated culture preparations but differ from each other in immunobiological parameters, it is essential to use methods that can quickly differentiate strains of the Canine Coronavirus pathogen during the virus-containing material control stage. This stage is essential for quality control of raw materials used in large-scale vaccine production. Molecular biological analysis methods, particularly PCR, are widely used in the diagnosis of various infectious diseases, including C-CoV. By analyzing single-nucleotide polymorphisms of the target region of the pathogen's gene in the presence of one or more unique substitutions, it is possible to differentiate genetic forms of viruses from each other, even within the same species [11]. Single nucleotide polymorphism is a difference in DNA sequences in the same regions of the genome by one nucleotide (adenine (A), thymine (T), guanine (G) or cytosine (C)). High-resolution melting (HRM) analysis is a simple, rapid, and inexpensive real-time polymerase chain reaction (RT-PCR)-based method used to identify genetic variations between populations and detect single-nucleotide polymorphisms (SNPs) in nucleic acid sequences [12]. This method is effective and allows for the determination of differences between the melting temperatures of SNP alleles using a fluorescent dye embedded in the double-stranded DNA structure. To obtain accurate and reliable results, it is necessary to optimize the primer design, PCR mixture, and thermal cycling modes before conducting HRM analysis [13, 14]. Analysis of maximum melting temperatures for various strains of viruses and other microorganisms involves studying the melting curve of amplicons containing allele-specific mutations over a wide temperature range with a narrow step size. The combination of improved quantitative amplicon detection tools and the latest generation of fluorescent dyes has enabled the detection of genetic changes in nucleic acid sequences through controlled melting of double-stranded nucleic acid molecules [12], in particular, CCoV cDNA. The essence of the presented method is that after PCR of the target gene region using a fluorescent dye, the mixture is gradually heated. Upon reaching a certain temperature, the reaction products begin to dissociate, the fluorophore is gradually released, and the fluorescence level (Fl), detected by the thermal cycler's optical system, decreases. The analysis sensitivity reaches one nucleotide based on the change in the cDNA melting temperature peak, enabling the developed method to achieve high overall accuracy. Sequencing is used to differentiate strains of the canine coronavirus pathogen [15, 16]. However, this method is expensive, labor-intensive, and time-consuming. The closest prototype is the “Method for differentiating the Rich vaccine strain from other strains and field isolates of the canine coronavirus pathogen using the analysis of the maximum extrema of the amplicon melting temperature graphs using the SuperNova v428 violet laser dye” [17], however, this method allows for the differentiation of only the Rich vaccine strain from other strains and isolates of canine coronavirus and does not provide the ability to differentiate other production strains of the canine coronavirus pathogen, in particular, “SWU-SSX4/2021/CCoVIIa”, “B906_ZJ_2019”, “B617_ZJ_2019”, “B001_AH_2018”, “B825_ZJ_2019”, “CCoV/dog/HCM27/2014”, “CCoV/wolf/2016/IT”, “Elmo/02”, "1-71", "TN449", "NVSL" CCoV. The objective of the present invention is to develop a sensitive and specific, simple and rapid method for differentiating strains of the pathogen causing coronavirus infection in dogs Canine Coronavirus in order to eliminate the above-mentioned disadvantages