Development of a technique for analyzing the lengths of restriction fragments to assess the mefloquine resistance of plasmodium falciparum based on the identification of single-nucleotide polymorphisms (A958146T, A961013G, G961625T) of the PFMDR1 gene
- 作者: Ariukov A.R.1, Solovev A.I.1, Krutikova A.A.2, Kovalenko A.N.1, Kapatsyna V.A.3, Romanenko V.A.1
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隶属关系:
- Kirov Military Medical Academy
- All-Russian Research Institute of Genetics and Animal Breeding
- Clinical Infectious Hospital named after S.P. Botkin
- 期: 卷 25, 编号 2 (2023)
- 页面: 249-260
- 栏目: Research paper
- ##submission.dateSubmitted##: 05.05.2023
- ##submission.dateAccepted##: 27.05.2023
- ##submission.datePublished##: 13.07.2023
- URL: https://journals.eco-vector.com/1682-7392/article/view/387427
- DOI: https://doi.org/10.17816/brmma387427
- ID: 387427
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A method for identifying single nucleotide polymorphisms of the PfMDR1 gene (A958146T, A961013G, G961625T) based on the analysis of the lengths of restriction fragments using polymerase chain reaction technology with specific primers is presented. To identify the polymorphism A958146T (amino acid substitution N86Y), it was proposed to amplify a fragment of parasitic deoxyribonucleic acid with a length of 417 bp, including the 86th codon of the PfMDR1 gene, followed by treatment of the amplicon with ApoI endonuclease, the restriction site of which includes the sequence of the analyzed mutation. With the Plasmodium falciparum genotype unchanged, the isolated deoxyribonucleic acid site is divided into two fragments of size 239 and 179 bp. In the case of an altered genotype containing a single nucleotide missense mutation A > T, the original fragment 417 bp will be preserved. When designing the system to detect the amino acid substitution N1042D caused by the A961013G mutation, a pair of specific primers were selected that limit the 404 bp-long section of parasitic deoxyribonucleic acid. For restriction analysis, the most optimal was the use of AseI endonuclease, which in the case of an unchanged Plasmodium falciparum genotype divides the initial amplicon into 4 fragments (132, 116, 99 and 25 bp), and in the presence of A > G mutation — into 3 (248, 99, 25 bp). It was found that a fragment of the PfMDR1 gene, including the G961625T mutation leading to the amino acid substitution D1246Y, contains 1 site corresponding to the restriction site of endonuclease BglII. Therefore, in the case of the wild genotype of Plasmodium falciparum, the initial fragment of deoxyribonucleic acid is cut into 2 short sections (300 and 269 bp). With the D1246 mutation, the replacement of the nucleotide G > T leads to the disappearance of the restriction site, so only one source fragment (509 bp) will be recorded on the electrophoregram. Based on the analysis of the data obtained, criteria for evaluating the drug resistance of Plasmodium falciparum have been developed. The decrease in sensitivity to mefloquine and its derivatives of pathogens of tropical malaria can be evidenced by positive results obtained using the developed methods for detecting haplotypes A > T (N86Y (band 417 bp)), A > G (N1042D (bands 248, 99 and 25 bp)), G > T (D1246Y (509 bp)). The developed methods can be used to analyze the spread of drug-resistant tropical malaria.
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作者简介
Artem Ariukov
Kirov Military Medical Academy
编辑信件的主要联系方式.
Email: arukov.artem@yandex.ru
ORCID iD: 0000-0001-8774-5467
SPIN 代码: 4073-6487
Researcher ID: IAO-0519-2023
graduate student
俄罗斯联邦, Saint PetersburgAlexey Solovev
Kirov Military Medical Academy
Email: solopiter@gmail.com
ORCID iD: 0000-0002-3731-1756
SPIN 代码: 2502-8831
MD, Dr. Sci. (Med.), professor
俄罗斯联邦, Saint PetersburgAnna Krutikova
All-Russian Research Institute of Genetics and Animal Breeding
Email: anntim2575@mail.ru
ORCID iD: 0000-0003-2561-145X
candidate of biological sciences, senior researcher
俄罗斯联邦, Saint PetersburgAlexander Kovalenko
Kirov Military Medical Academy
Email: ank561@mail.ru
ORCID iD: 0000-0002-7553-0634
SPIN 代码: 9131-6360
MD, Dr. Sci. (Med.), associate professor
俄罗斯联邦, Saint PetersburgVladimir Kapatsyna
Clinical Infectious Hospital named after S.P. Botkin
Email: ingashi@mail.ru
ORCID iD: 0000-0002-8959-0873
SPIN 代码: 6401-4611
infectious disease doctor
俄罗斯联邦, Saint PetersburgVladimir Romanenko
Kirov Military Medical Academy
Email: Arukov.artem@yandex.ru
SPIN 代码: 9855-9483
student
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