Low-cost simultaneous detection of CCR5-delta32 and HLA-B*5701 alleles in human immunodeficiency virus type 1 infected patients by selective multiplex endpoint PCR.
Host genetic traits impact susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, disease progression as well as antiretroviral drug pharmacokinetics and toxicity. Remarkable examples include a 32-bp deletion in the CCR5 coreceptor molecule (CCR5-delta32) impairing attachment of monocytotropic HIV-1 to the host cell membrane and the HLA-B*5701 allele, strongly associated with a potentially fatal hypersensitivity reaction triggered by abacavir, a nucleoside inhibitor of HIV reverse transcriptase. We developed a simple selective multiplex endpoint PCR method for simultaneous analysis of both genetic traits. Two primers were designed for amplification of a region surrounding the CCR5 32-bp deletion site. One common forward primer and two reverse primers with different 3' termini targeting the HLA-B*570101 and HLA-B*570102 alleles were designed for HLA-B*5701 analysis. A panel of 110 reference DNA samples typed in the HLA-B locus was used for development and blind validation of the assay. All the 45 HLA-B*5701 positive and the 55 HLA-B*5701 negative samples were correctly identified. The CCR5-delta32 allele was readily detected in 7 samples and did not interfere with detection of HLA-B*5701 while providing an internal amplification control. Multiplex PCR products were easily identified in agarose gels with no background noise. This simple and low-cost end-point selective multiplex PCR can conveniently screen HIV patients for the protective CCR5-delta32 allele and the risk of developing abacavir hypersensitivity reaction.
Department of Medical Biotechnologies, University of Siena, Italy.
Andrea
A
Rosi
Department of Medical Biotechnologies, University of Siena, Italy.
Genny
G
Meini
Department of Medical Biotechnologies, University of Siena, Italy.
Angelo
A
Materazzi
Department of Medical Biotechnologies, University of Siena, Italy.
Ilaria
I
Vicenti
Department of Medical Biotechnologies, University of Siena, Italy.
Francesco
F
Saladini
Department of Medical Biotechnologies, University of Siena, Italy. Electronic address: maurizio.zazzi@unisi.it.
Maurizio
M
Zazzi
D017931:DNA Primers / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D015658:HIV Infections / Q000235:genetics* / Q000821:virology; D015497:HIV-1 / Q000302:isolation & purification; D015235:HLA-B Antigens / Q000235:genetics*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D019713:Receptors, CCR5 / Q000235:genetics*
C584933:CCR5 protein, human; D017931:DNA Primers; D015235:HLA-B Antigens; C484830:HLA-B*57:01 antigen; D019713:Receptors, CCR5
CCR5 delta32; Detection; HIV-1; HLA-B*5701; PCR