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Detection Of CCR5 Delta-32 Mutation Using High-Resolution Melting Curve Analysis: Challenges and Facts.
Kiani, Seyed Jalal; Donyavi, Tahereh; Bokharaei-Salim, Farah.
Afiliación
  • Kiani SJ; Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
  • Donyavi T; Department of Medical Biotechnology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran.
  • Bokharaei-Salim F; Department of Virology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Curr HIV Res ; 2024 Sep 13.
Article en En | MEDLINE | ID: mdl-39279712
ABSTRACT

INTRODUCTION:

The C-C chemokine receptor type 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV). Some individuals carry the CCR5 delta-32 genetic polymorphism. People with homozygous CCR5 delta-32 gene are nearly completely resistant to HIV-1 infection. High-resolution melting curve (HRM) analysis is a post-PCR technique utilized for identifying genetic variations in a quick, affordable, and closed-tube assay. The objective of this study was to develop an HRM assay for easy detection of delta-32 mutations. MATERIALS AND

METHODS:

DNA was extracted from peripheral blood mononuclear cells. HRM was performed to detect delta-32 mutation. The study investigated the impact of various factors, including annealing temperature, template concentration, touchdown PCR, additives, amplicon size, and program settings, on HRM Tm differentiation.

RESULTS:

It was expected that there would be a 4°C Tm difference between amplicons with and without delta-32 mutation, but the test showed a difference of only 2.3°C. In attempts to identify heterozygote delta-32 variants, a Tm difference of only 0.4°C could be achieved. Various modifications were applied, such as adjusting the template concentration, using touchdown PCR, and adding DMSO and glycerol. However, none of these changes helped to differentiate the Tm effectively, especially in delta-32 heterozygote samples.

CONCLUSION:

The HRM test identified four samples with heterozygote mutations in each HIV-infected (8.89%) and control (5.72%) groups. More importantly, this study showed that identifying the delta-32 mutation of the CCR5 gene using HRM assay is not as straightforward as previously suggested in some literature, and it requires special setup conditions.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Curr HIV Res Asunto de la revista: SINDROME DA IMUNODEFICIENCIA ADQUIRIDA (AIDS) Año: 2024 Tipo del documento: Article País de afiliación: Irán Pais de publicación: Países Bajos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Curr HIV Res Asunto de la revista: SINDROME DA IMUNODEFICIENCIA ADQUIRIDA (AIDS) Año: 2024 Tipo del documento: Article País de afiliación: Irán Pais de publicación: Países Bajos