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https://doi.org/10.29013/ESR-19-9.10-11-15
Ikramov Saidazim Adylovich, Junior Researcher, Institute of Chemistry of Plant substances, Academy of Sciences of the Republic of Uzbekistan E-mail: [email protected] Makhnev Artyom Aleksandrovich, Junior Researcher at the Institute of Chemistry of Plant substances, Academy of Sciences of the Republic of Uzbekistan Nasriddinov Khusan Ziyoyiddin ugli, trainee researcher at the Institute of Chemistry of Plant substances, Academy of Sciences of the Republic of Uzbekistan Baimirzaev Asadali Bahrom ugli, laboratory assistant at the Institute of Chemistry of Plant substances, Academy of Sciences of the Republic of Uzbekistan Azimova Shakhnoz Sadykovna, Professor, Institute of Chemistry of Plant substances, Academy of Sciences of the Republic of Uzbekistan
IDENTIFICATION OF HEPATITIS C VIRUS WITH PCR REAL TIME METHOD
Abstract. A set of reagents was developed to detect Hepatitis C virus (HCV) in human plasma by using real-time PCR. For this, the genomes of the 6 main HCV genotypes were analyzed; based on the analysis, primers and a fluorescent probe were designed and synthesized for PCR analysis. The sensitivity and specificity of a set of reagents were investigated on clinical blood plasma samples.
Keywords: Viral Hepatitis C (HCV), PCR, primer, fluorescent probe.
Introduction. Viral hepatitis C is one of the HGV/GBV-A, B and HGV/GBV-C are the only most common viruses, causing serious diseases, be- representatives of the genus Hepacivirus. The HCV coming chronic and leading to cirrhosis, liver cancer chain exhibits a high degree of genetic heterogene-or hepatocellular carcinoma, from which more than ity. HCV is divided into 6 main genotypes, which 100 thousand people die every year [1; 2; 3; 4; 5]. are further subdivided into subtypes [6; 7; 8].In this
Hepatitis C virus consists of single-stranded regard, the task of the study is to develop a test kit for RNA with a positive chain. It belongs to the family the detection of Hepatitis C virus of all genotypes in Flaviviridae. Flaviviridae includes 4 genera: Flavivi- blood plasma using PCR analysis. rus, Pestivirus, Hepacivirus and unclassified viruses. Materials and methods. Design of oligonucle-In addition to HCV, the Flaviviridae family includes otide primers. Primers were designed based on con-yellow fever virus, tick-borne encephalitis virus, served regions of the genome for each genotype of Japanese encephalitis virus, and chronic fever virus. the virus and common to all genotypes. For this, we HCV together with the recently discovered viruses used the sequence of 6 main HCV virus genotypes
from NCBI (http://www.ncbi.nlm.nih.gov). These sequences were then analyzed using UGENE software and conserved regions were identified for all six genotypes. The 5'NCR region of the HCV genome was selected (Table 1). The length of the PCR product was ~ 200 bp To analyze the thermodynamic properties of the primers, the Oligo Analyze program was used.
Synthesis of primers and fluorescent probe. The primers were synthesized by the ASM-2000 DNA synthesizer. In total, 3 pairs of primers were synthesized: 1. ACF92-ACR94; 2. HCV-F1 - HCV-R3; 3. HC1315 - HC1316 and fluorescent probe HCPO2 FAM5I - 3I BHQ1.
Extraction of viral RNA. Sixty different plasma samples were taken in this study. They included 50 samples positive for HCV and 10 negative for HCV from healthy people. Plasma was stored at -60 °C until use. To isolate viral RNA from plasma samples, a set of reagents for extraction of nucleic acids was used
(DNA - Technology, Russia). HCV-positive samples were obtained from the Institute of Virology of the Ministry of Health of the Republic of Uzbekistan. All blood samples are transferred to our institute by qualified personnel in appropriate conditions.
HCV detection using RT-PCR. To evaluate the analysis, an RT-PCR reaction composition was shown in (Table 1).
PCR was performed in accordance with the following temperature program:
37 °C - 30 min 1 cycle;
95 °C - 10 min 1 cycle;
95 °C - 10 sec;
59 °C - 50 sec 40 cycles;
72 °C - 20 sec.
PCR reactions were performed using STRATA-GENE RoboCycler Gradient 96. PCR products were treated on a 2% agarose gel. RT-PCR reaction was performed using a real-time Step ONE PCR system, Applied Biosystems.
Table 1.- The composition of the reaction mixture
Reagent Description Quantity Notes
Virus RNA isolated from plasma Solution containing virus RNA 10 pl
MMLV OT Reverse transcription enzyme 1 pl
RNase inhibitor Enzyme RNazine inhibiting RNase 1 pl
Buffer Enzyme work buffer 4 pl
DNTPs A mixture of 4 nucleotides (A, T, G, C) 0.2 pl
H^O Bidistilled and deionized water 5.6 pl
MgCl2 Solution of MgCl2 1 pl
Primer forward A solution containing a reverse primer 1 pl
Primer reverse Solution containing forward primer 1 pl
Probe A solution containing a primer probe with a fluorescent label 0.1 pl
Taq polymerase Taq polymerase 0.2 pl
Results and discussion. Design and synthesis of primers. The genomes of the 6 main genotypes of Viral Hepatitis C were analyzed. Based on the data obtained, a conserved region was identified for all 6 genotypes of the virus. 3 pairs of primers were designed for this site. After they were synthesized on an oligonucleotide synthesizer ASM-2000. The se-
quence of the selected primers and fluorescent probe are shown in (table 2):
Arrangement of PCR analysis and electrophoresis in 2% agarose gel. Figure 1 shows gel electrophoresis, PCR products in which a particular target was amplified and separated in size.
Table 2.- The nucleotide sequence of the selected primers and probe
№ Identificational the code Sequence
1. HCV-F1 TCACGCAGAAAGCGTCTA
2. HCV-R3 G CAGACCACTATG G CTC
3. HSSHZF FAM-TACAGCCTCCAGGACCCC-BHQ1
With the selected primers, a number of PCR and HCV-R3 give a clear positive result at an anneal-
formulations were performed to select the optimal ing temperature of 59 °C (Figure 1). reaction conditions. PCR results were analyzed by As a result ofstandard PCR analysis, it was found
gel electrophoresis. As a result of the analysis, it was that for viral hepatitis C pairs of primers with num-
found that primers with the numbers: 2.HCV-F1 bers HCV-F1 and HCV-R3 were more effective.
Figure 1. 1 - Primers -ACF92/ACR94; 2 - HCV-F1/HCV-R3; 3 - HC1315/ HC1316; 4 - ACF92/ACR94; 5 - HCV-F1/HCV-R3; 6 - HC1315/HC1316
Amplification Plot
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Figure 2. Determination of the sensitivity of PCR analysis at different concentrations of samples
Specificity and Sensitivity To determine the sensitivity of the method, the lowest cDNA concentration detected by PCR was determined. For this, blood samples were taken from the already known viral load from the Institute of Virology. The samples contained the following amounts of viral particles: blue-10 6, beige-10 5, blue-10 4, yellow-10 3, green-10 2 s a result of the analysis, the smallest amount of RNA that can be detected in the PCR reaction is 100 copies (Fig. 2).
This shows that the sensitivity of the PCR analysis is comparable to its analogues. Using the developed diagnosticum, one can detect cDNA infections in the amount of 100 copies in 100 ^l of a clinical sample.
To determine the specificity of the test kit, the following experiment was conducted. 8 positive samples with hepatitis C virus, 4 samples with negative control, 2 positive samples ofviral hepatitis B, 2 positive samples of HIV were taken.
Amplification Plot
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Figure 3. Test for the specificity of the diagnosticum. Green color - positive samples for hepatitis C, blue color - positive samples for hepatitis B and HIV, red color - samples of negative control
As can be seen from the test results, positive samples with viral hepatitis C (green on the graph) showed a significant increase in the fluorescence signal during PCR cycles, while at the same time when testing positive images for viral hepatitis B and HIV (blue on the graph) and negative control (on the graph is red) received a signal at the level of negative control, therefore, we can conclude that the de-
veloped diagnosticum has the necessary specificity, comparable with analogues.
Thus, we analyzed a database of nucleotide sequences of six viral hepatitis C genotypes in the NCBI. Based on the analysis, a genetic site suitable for the synthesis of primers was identified: 51 - NC -non-coding region. Design and synthesized primers for amplification of DNA fragments by PCR were
developed. To develop real-time PCR, a fluorescent probe was developed and synthesized. The composition of the reaction mixture and the conditions for the PCR reaction were also selected. The tests for
sensitivity and specificity for this set of reagents. This analysis is quick and practical for determining HCV in the patient's plasma and can be used to screen, identify and control the treatment of this disease.
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