**3.3 Design of oligonucleotides**

In addition to specific oligonucleotide seeds, oligonucleotide seeds were designed to control the amplification reaction; in this case, cattle genes served as DNA markers, namely, the gene encoding the milk protein and the gene encoding the fat milk of cattle.

Within the above loci to designate the genome of the foot-and-mouth disease virus and DNA markers of cattle, primers and probes for PCR were designed (**Table 2**) with the following requirements: the same melting temperature of the primers (±0.5°C); minimum dimers and secondary structures; minimum GC at 3′ end; for the probe, the absence of G at the 5′ end (first nucleotide) and, most importantly, the minimum variability of the nucleotide sequence (maximum two) in the sequence of each oligonucleotide seed.

Result of the design of oligonucleotide seeds, both specific and for amplification control, is presented in **Table 2**.

Thus, the indication of all strains/isolates of all serotypes of FMDV is achieved using three modifications of the oligonucleotide probe; the first probe (P FMDV) is used to identify the main number of virus strains/isolates; the second modification of the probe (Pas FMDV) allows the detection of two isolates not detected by the previous probe viruses; serotype Asia-1, polymorphism features of nine isolates of


#### **Table 2.**

*The nucleotide sequence of primers and probes for PCR.*


**73**

**Figure 1.**

*Genetic Polymorphisms of Foot-and-Mouth Disease Virus*

the third probe is taken into account (Psat FMDV).

serotypes SAT1 and SAT2 and one isolate of serotype A, when they are indicated,

An analysis of the compatibility of specific and controlling PCR oligonucleotide

*Amplification of marker locus for indication of foot-and-mouth disease virus: amplification of plasmid control* 

*(A), amplification of plasmid control and cattle DNA (B), and cattle DNA amplification (C).*

*DOI: http://dx.doi.org/10.5772/intechopen.93351*

seeds is presented in **Table 3**.

#### **Table 3.**

*Homology (%) of target oligonucleotides with test oligonucleotides to control amplification.*

*Some RNA Viruses*

the fat milk of cattle.

**3.3 Design of oligonucleotides**

in the sequence of each oligonucleotide seed.

control, is presented in **Table 2**.

**Analyzed oligonucleotides for internal control of amplification**

*The nucleotide sequence of primers and probes for PCR.*

The gene encoding the milk production of cattle

The gene encoding the fat milk content of cattle DGAT

In addition to specific oligonucleotide seeds, oligonucleotide seeds were designed to control the amplification reaction; in this case, cattle genes served as DNA markers, namely, the gene encoding the milk protein and the gene encoding

Within the above loci to designate the genome of the foot-and-mouth disease virus and DNA markers of cattle, primers and probes for PCR were designed (**Table 2**) with the following requirements: the same melting temperature of the primers (±0.5°C); minimum dimers and secondary structures; minimum GC at 3′ end; for the probe, the absence of G at the 5′ end (first nucleotide) and, most importantly, the minimum variability of the nucleotide sequence (maximum two)

Result of the design of oligonucleotide seeds, both specific and for amplification

Thus, the indication of all strains/isolates of all serotypes of FMDV is achieved using three modifications of the oligonucleotide probe; the first probe (P FMDV) is used to identify the main number of virus strains/isolates; the second modification of the probe (Pas FMDV) allows the detection of two isolates not detected by the previous probe viruses; serotype Asia-1, polymorphism features of nine isolates of

Rp FMDV tgggtgaacgccgtgtgc

Fp kappa ttggcaggcacagtatttgaca Rp kappa attactaccaacagaaaccagttgca

Fp DGAT cctcttcctcaagctgttctcctac Rp DGAT cctcaccagccttggcctt

P FMDV Rox-tttgagattccaagctacagatcactttacctgc-BHQ2 Pas FMDV Rox-ttcgagataccaagctacagatcgctctacctgc-BHQ2 Psat FMDV Rox-tttgagatccctagctacagatcactttacctgc-BHQ2

P kappa Cy5-ttgaagaatttgggcaggtgacctaactg-RTQ3

P DGAT Cy5-acgtcaacctctggtgccgagagc-RTQ3

**Detectable pathogen/marker Primer name Nucleotide sequence 5**′⟶**3**′ Foot-and-mouth disease virus Fp FMDV atctccgtggcaggactcgc

> **Fp FMDF**

*Homology (%) of target oligonucleotides with test oligonucleotides to control amplification.*

Fp kappa — — — — — Rp kappa — — 51.9% — 51.9% P kappa — — — — — Fp DGAT — — — — — Rp DGAT — — 23.2% — — P DGAT — 31.3% — — —

**Rp FMDV** **P FMDV Pas** 

**FMDV**

**Psat FMDV**

**72**

**Table 3.**

**Table 2.**

serotypes SAT1 and SAT2 and one isolate of serotype A, when they are indicated, the third probe is taken into account (Psat FMDV).

An analysis of the compatibility of specific and controlling PCR oligonucleotide seeds is presented in **Table 3**.

#### **Figure 1.**

*Amplification of marker locus for indication of foot-and-mouth disease virus: amplification of plasmid control (A), amplification of plasmid control and cattle DNA (B), and cattle DNA amplification (C).*

To control amplification, a locus was chosen within the framework of the gene encoding the milk production of cattle, since its oligonucleotide seeds are characterized by the absence of homologies with target primers to indicate FMDV, and as a result, will have minimal effect on amplification with specific primers.

The designed primers for indicating FMDV have a melting point (58.6°C ± 0.1°C), and the primers for amplification control have a melting point (55.45°C ± 0.15°C), and such a temperature difference implies more favorable amplification conditions for specific primers (at an annealing temperature of 58.5°C) and minimizes the effect of oligonucleotides to control amplification on the course of the reaction.

To control the amplification result, a positive control was created. Specific marker of positive control has the following nucleotide sequence (5′-atctccgtggcaggactcgccgtccactctggacctgacgagtaccggcgtctctttgagcccttccagggtctctttgagattccaagctacagatcactttacctgcgttgggtgaacgccgtgtgc-3′) for the indication of FMDV in plasmid DNA. The insertion of the marker sequence into the plasmid "pAL2-T" was ordered at ZAO Evrogen. To test the operability of the developed oligonucleotide seeds and plasmid control, cattle DNA and plasmid control were amplified with the primers and probes, developed above (specific for amplification control). The amplification result is shown in the **Figure 1**.

Amplification with oligonucleotide seeds for the indication of FMDV was effective both in separate PCR with positive plasmid control and in combination with cattle DNA. Amplification of the FMDV genetic markers and the control locus was performed in a single test tube. The plasmid DNA concentration was 1 × 108 DNA copies/μl (designations in **Figure 1A** and **B**). With separate amplification with cattle DNA, cross-reactions with primers/probes for indication and for foot-andmouth disease did not occur. Amplification with oligonucleotide seeds for detecting FMDV was indicated on the Rox channel, and internal amplification was controlled on the Cy5 channel.

#### **4. Conclusion**

An analysis of the variability of the nucleotide sequences of the genomes of the different FMDV strains/isolates within each serotype revealed a locus characterized by maximum conservatism. In the sequence of oligonucleotide seeds, a sufficient level of polymorphism in the genomes of the virus isolates was found only with respect to the PCR probe (in 12 isolates by serotypes A, Asia-1, SAT1, and SAT2), and the effect of such variability on the number of detected virus isolates allows modification of the PCR probe (Pas FMDV and Psat FMDV).

**75**

**Author details**

Russian Federation

Khammadov Nail Ildarovich

Federal Center for Toxicological, Radiation and Biological Safety, Kazan City,

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: nikhammadov@mail.ru

provided the original work is properly cited.

*Genetic Polymorphisms of Foot-and-Mouth Disease Virus*

*DOI: http://dx.doi.org/10.5772/intechopen.93351*

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Bioethics**

This work was carried out without the use of animals.

*Genetic Polymorphisms of Foot-and-Mouth Disease Virus DOI: http://dx.doi.org/10.5772/intechopen.93351*

*Some RNA Viruses*

course of the reaction.

result is shown in the **Figure 1**.

on the Cy5 channel.

**Conflict of interest**

**Bioethics**

**4. Conclusion**

To control amplification, a locus was chosen within the framework of the gene encoding the milk production of cattle, since its oligonucleotide seeds are characterized by the absence of homologies with target primers to indicate FMDV, and as a

(58.6°C ± 0.1°C), and the primers for amplification control have a melting point (55.45°C ± 0.15°C), and such a temperature difference implies more favorable amplification conditions for specific primers (at an annealing temperature of 58.5°C) and minimizes the effect of oligonucleotides to control amplification on the

To control the amplification result, a positive control was created. Specific marker of positive control has the following nucleotide sequence (5′-atctccgtggcaggactcgccgtccactctggacctgacgagtaccggcgtctctttgagcccttccagggtctctttgagattccaagctacagatcactttacctgcgttgggtgaacgccgtgtgc-3′) for the indication of FMDV in plasmid DNA. The insertion of the marker sequence into the plasmid "pAL2-T" was ordered at ZAO Evrogen. To test the operability of the developed oligonucleotide seeds and plasmid control, cattle DNA and plasmid control were amplified with the primers and probes, developed above (specific for amplification control). The amplification

Amplification with oligonucleotide seeds for the indication of FMDV was effective both in separate PCR with positive plasmid control and in combination with cattle DNA. Amplification of the FMDV genetic markers and the control locus was performed in a single test tube. The plasmid DNA concentration was 1 × 108 DNA copies/μl (designations in **Figure 1A** and **B**). With separate amplification with cattle DNA, cross-reactions with primers/probes for indication and for foot-andmouth disease did not occur. Amplification with oligonucleotide seeds for detecting FMDV was indicated on the Rox channel, and internal amplification was controlled

An analysis of the variability of the nucleotide sequences of the genomes of the different FMDV strains/isolates within each serotype revealed a locus characterized by maximum conservatism. In the sequence of oligonucleotide seeds, a sufficient level of polymorphism in the genomes of the virus isolates was found only with respect to the PCR probe (in 12 isolates by serotypes A, Asia-1, SAT1, and SAT2), and the effect of such variability on the number of detected virus isolates allows

modification of the PCR probe (Pas FMDV and Psat FMDV).

This work was carried out without the use of animals.

The author declares no conflict of interest.

result, will have minimal effect on amplification with specific primers. The designed primers for indicating FMDV have a melting point

**74**
