Organizing co mmittee

 
37.3±2.7 
34.6 
65.4 
1.0
 
40.2±2.5 
37.3 
62.7 
5.0
 
59.3±3.6 
55.1 
44.9 
4 
C:SA (1:1) 
0.1 
27.4±4.2 
25.4 
74.6 
0.5 
29.6±3.4 
27.5 
72.5 
1.0 
44.8±2.9 
41.6 
58.4 
2.0 
51.0±3.6 
47.3 
52.7 
5.0 
62.5±4.0 
58.0 
42.0 
 

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Poster presentation 
316 
COMPARISON OF THE OLIGONUCLEOTIDE PROBES OF 
DIFFERENT STRUCTURES FOR THE DIAGNOSIS
OF THE Virus hepatitis B BY PCR ANALYSIS 
 
O.B. Alimukhamedova, O.N. Ashirov, A.A. Makhnev, A.B. Baimirzaev, 
Kh.Z. Nasriddinov, Ikramov S.A., G.A. Piakina 
 
S.Yu. Yunusov Institute of the Chemistry of Plant Substances Academy of sciences of the 
Republic of Uzbekistan st. Mirzo-Ulugbek, 77, 100170 Tashkent 
 
For the diagnosis of infectious diseases such as the hepatitis B virus, a highly 
sensitive quantitative PCR method is widely used using fluorescent probes 
complementary to a specific DNA region of the hepatitis B virus. Fluorescent probes 
are short oligonucleotides of 20–25 nucleotides in length with fluorescent molecules 
and “quencher” molecules attached at the ends. When the probes interact with the DNA 
region of the hepatitis B virus during PCR analysis, a fluorescent signal is formed due 
to the destruction of the probe under the influence of the polymerase enzyme and the 
divergence of the fluorophore and “quencher” molecules. An increase in the fluorescent 
signal indicates the presence of an infection. The sensitivity of PCR analysis directly 
depends on how high the fluorescent signal rises. 
To compare the growth of the fluorescent signal, we designed two fluorescent probes 
with different structures specific to the core gene of the hepatitis B virus DNA. The first 
fluorescent probe A is a single-stranded DNA molecule 21 nucleotides long, to the 5' 
end of which the FAM fluorophore is attached and to the 3' end of the quencher 
molecule BHQ-1. Thus, the FAM fluorophore is 21 nucleotides away from the BHQ-1 
"quencher" molecule. The second fluorescent probe, B, has a double-stranded structure 
in which the FAM fluorophore molecule is in close proximity to the BHQ-1 quencher 
molecule. 
The growth of the fluorescent signal was analyzed on a QuantStudio 5 instrument by 
the relative fluorescence units’ value. To analyze the fluorescent signal, probe A was 
treated with DNase for 15 minutes at 37°C, after which the fluorescence was measured. 
Fluorescent probe B was analyzed using the melt curve method: 60°C - 1 
minute/temperature increase from 60°C every second by 0.5°C (signal reading) to 95°C. 
Fluorescent probes were used at a concentration of 100 nM. 
As a result of the analysis, the growth of the fluorescent signal of probe A began at 
75,000 fl and plateaued at 375,000 fl, for fluorescent probe B, the initial fluorescence 
signal was 50,000 fl and the final fluorescence signal output was 1,000,000 fl. 
Thus, it was found that the increase in fluorescence is 4 times higher when using 
probe B with a double-stranded structure. It was found that the growth of the fluorescent 
signal is inversely proportional to the distance between the molecules of the fluorophore 
and the quencher. The use of more efficient methods for designing fluorescent probes 
can increase the sensitivity of molecular diagnostics of hepatitis B virus.