Quality control system for sample preparation and analysis results in the diagnosis of viral hepatitis B and C by real-time PCR

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Abstract

Introduction. When conducting diagnostic tests for virus Infection, it is crucial to prevent the false negative results. For this, it is necessary to control all stages of the sample preparation process and the quality of the diagnostic reaction itself.

The aim of the work is to create an optimal combination of sample preparation exogenous quality control hepatitis B and C viruses detection system using real–time multiplex PCR to improve the accuracy, reproducibility and reliability of the results.

Material and methods. Hepatitis B virus DNA and hepatitis C virus RNA isolated from blood plasma samples from patients with confirmed diagnoses were used as the object of the study. The following resources were used to perform the in silico study: the online resource Primer-BLAST, integrated into the NCBI database; the online service OligoAnalyzer Tool (Integrated DNA Technologies), as well as the programs Oligo Primer Analysis Software, Clustal Omega and UGENE. The CFX96 Touch amplifier (Bio-Rad Laboratories, USA) was used for real-time PCR. PCR was performed using the "HBV Intifica" and "HCV Intifica" kits (Alkor Bio Group of Companies, Russia).

Results. A stable and robust multiplex PCR system has been developed to control the quality of sample preparation and diagnosis of viral hepatitis B and C from human blood plasma. Application of thermodynamic analysis to primers and probes design improves the efficiency of PCR systems, increases sensitivity and eliminates false negative results.

Conclusions. Thorough optimization of viral primers and probes characteristics, together with application of exogenous internal control in multiplex reaction, significantly improves the accuracy, reproducibility and reliability of analysis results.

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About the authors

A. S. Moroz

Peter the Great St. Petersburg Polytechnic University; Alkor Bio Group of Companies

Author for correspondence.
Email: anny-nice@mail.ru
ORCID iD: 0000-0002-4693-623X
SPIN-code: 4721-1020

Post-graduate Student, Higher School of Biotechnology and Food Production; Research Engineer

Russian Federation, Novorossyskaya str. 48, Saint Petersburg, 194021; Zheleznodorozhny ave., 40a, Saint Petersburg, 192148

V. A. Toropov

Alkor Bio Group of Companies

Email: anny-nice@mail.ru
ORCID iD: 0009-0005-1993-5572

Leading Research Engineer

Russian Federation, Zheleznodorozhny ave., 40a, Saint Petersburg, 192148

V. N. Bolshakov

Peter the Great St. Petersburg Polytechnic University; Alkor Bio Group of Companies

Email: anny-nice@mail.ru
ORCID iD: 0009-0007-5126-6035

Ph.D. (Biol.), Associate Professor, Associate Professor of the Higher School of Biotechnology and Food Production;  Head of the Laboratory of Molecular Diagnostics

Russian Federation, Novorossyskaya str. 48, Saint Petersburg, 194021; Zheleznodorozhny ave., 40a, Saint Petersburg, 192148

E. B. Aronova

Peter the Great St. Petersburg Polytechnic University

Email: anny-nice@mail.ru
ORCID iD: 0000-0003-4376-2972
SPIN-code: 7288-6820

Ph.D. (Tech.), Associate Professor, Associate Professor of the Higher School of Biotechnology and Food Production

Russian Federation, Novorossyskaya str. 48, Saint Petersburg, 194021

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Supplementary files

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2. Fig. 1. Thermodynamic stability for primers F_x20 (A) and F_x16 (B). Along the abscissa axis is the length of the oligonucleotide, along the ordinate axis is ΔG

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3. Fig. 2. Influence of thermodynamic characteristics of primers on the sensitivity and specificity of hepatitis C virus detection in samples with different viral loads. (A) – calculation of amplification curves by regression method; (B) is the result of amplification in logarithmic coordinates. The amplification cycles are on the abscissa axis, and the amplification signal intensity is on the ordinate axis (1 – the result of amplification of a sample with a high viral load (2.5·104 IU/µl) for a system with a direct primer F_x20 without thermodynamically stable secondary structures; 2 – the result of amplification of a sample with a low viral load (5·102 IU/µl) for a system with a direct primer F_x20 without thermodynamically stable secondary structures; 3 – the result of amplification of a sample with a high viral load (2.5·104 IU/µl) for a system with a direct primer F_x16 with thermodynamically stable secondary structures annealed onto a site of nucleic acid with a thermodynamically stable secondary structure; 4 are the result of amplification of a sample with a low viral load (5·102 IU/µl) for a system with a direct primer F_x16 with thermodynamically stable secondary structures annealed onto a site of nucleic acid with a thermodynamically stable secondary structure)

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4. Fig. 3. Results of detection of hepatitis C virus by a multiplex thermodynamically balanced PCR system using an internal control sample. Amplification curves: 1 – concentrated hepatitis C virus matrix; 2 – diluted hepatitis C virus matrix; 3 – internal control sample; 4 – negative control sample. The amplification cycles are on the abscissa axis, and the amplification signal intensity is on the ordinate axis

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5. Fig. 4. Results of hepatitis B virus detection by a multiplex thermodynamically balanced PCR system using an internal control sample.  Amplification curves of concentrated and dilute hepatitis B virus DNA matrix for a system of primers and probes developed taking into account thermodynamic calculations: 1 – concentrated hepatitis B virus matrix; 2 – diluted hepatitis B virus matrix; 3 – internal control sample; 4 – negative control sample. The amplification cycles are on the abscissa axis, and the amplification signal intensity is on the ordinate axis

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