Optimization of a Method for Detecting Single copies of Hepatitis B Virus DNA using CRISPR/Cas systems
https://doi.org/10.31631/2073-3046-2024-23-6-114-128
Abstract
Relevance. Hepatitis B virus (HBV) is the etiologic agent of acute and chronic hepatitis B in humans. WHO recommends the use of sensitive laboratory assays based on nucleic acid amplification methods to detect HBV DNA. A method for detecting single copies of hepatitis B virus DNA using CRISPR/Cas systems was previously developed for ultrasensitive detection of HBV DNA.
Aims. The aim of present study was to optimize the method for detecting single copies of hepatitis B virus DNA using CRISPR/Cas systems.
Materials and methods. To obtain amplified fragments of the hepatitis B virus genome, 22 oligonucleotides were developed. The preliminary amplification stage was performed by the RPA method using the developed oligonucleotides. The assembly of CRISPR/ Cas ribonucleoprotein complexes specific for fragments of the hepatitis B virus genome was carried out using synthetic guide RNA (oligoribonucleotides). The detection stage was performed in HOLMES 1.
Results and discussion. We maintained the sensitivity of the optimized method at the level of the original one (detection of single copies of hepatitis B virus DNA), when optimizing the method for detecting hepatitis B virus DNA. In addition, we reduced the time required for the analysis. Thus, the time required to detect single copies (6 copies per reaction) of hepatitis B virus DNA using the original method is 83 minutes, while for the optimized method it is 32 minutes.
Conclusions. The optimized method for detecting single copies of hepatitis B virus DNA using CRISPR/Cas systems described in the article can be used in the future to develop new diagnostic kits, including point-of-care kits and/or kits to use in the field.
About the Authors
M. A. Tyumentseva
Central Research Institute of Epidemiology
Russian Federation
Russian Federation
Marina A. Tyumentseva – Cand. Sci. (Biol.), Head of the Laboratory of Genome Editing of the OMD&E,
Moscow
+7 (495) 974-96-46 (ext. 2627)
A. I. Tyumentsev
Central Research Institute of Epidemiology
Russian Federation
Russian Federation
Aleksandr I. Tyumentsev – Cand. Sci. (Biol.), Head of the Laboratory of Experimental Pharmacology of the OMD&E
Moscow
+7 (495) 974-96-46 (ext. 2627)
A. N. Prelovskaya
Central Research Institute of Epidemiology
Russian Federation
Russian Federation
Anna N. Prelovskaya – researcher at the Laboratory of Genome Editing of the OMD&E
Moscow
+7 (495) 974-96-46 (ext. 2627)
V. G. Akimkin
Central Research Institute of Epidemiology
Russian Federation
Russian Federation
Vasily G. Akimkin – Academician of the Russian Academy of Sciences, Dr. Sci. (Med.), Professor, Director
Moscow
+7 (495) 974-96-46 (ext. 2627)
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Review
For citations:
Tyumentseva M.A., Tyumentsev A.I., Prelovskaya A.N., Akimkin V.G. Optimization of a Method for Detecting Single copies of Hepatitis B Virus DNA using CRISPR/Cas systems. Epidemiology and Vaccinal Prevention. 2024;23(6):114-128. (In Russ.) https://doi.org/10.31631/2073-3046-2024-23-6-114-128
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