Rapid determination of the Delta32 deletion in the human CC-chemokine receptor 5 (CCR5) gene without DNA extraction by lightcycler real-time polymerase chain reaction.
CCR5 is a major coreceptor for cellular entry of macrophage-tropic isolates of the human immunodeficiency virus (HIV). A 32-base pair deletion of the CCR5 gene (CCR5Delta32) protects against HIV infection because the frame shift leads to a truncated protein not expressed on the cell surface. CCR5Delta32 also delays progression in heterozygous HIV-infected patients and improves responses to antiretroviral therapy. Available methods for CCR5 genotyping, however, are cost expensive and/or time consuming. To improve CCR5 genotyping we studied four primer sets flanking the CCR5Delta32 deletion site using real-time polymerase chain reaction (PCR) on a LightCycler device. Primers amplified fragments of different length depending on the presence or absence of the Delta32 mutation. Next, melting curves of the amplified fragments were analyzed using SYBR green, a conventional double-strand DNA dye. To circumvent initial DNA extraction, we also studied serially diluted "interphase" leukocytes as PCR templates after centrifugation of EDTA blood. Finally, the validity of the new method was checked by analyzing 100 blood samples with known CCR5 genotypes. Amplicons of 82 bp:50 bp and 97 bp:65 bp fragment ratios could easily be discriminated due to the differences in their melting temperatures (3 degrees C and 2 degrees C, respectively). Furthermore, CCR5 genotyping was possible without initial DNA extraction and yielded optimal results at 1:400 to 1:600 dilution of the "interphase" leukocytes. Results of the new LightCycler PCR protocol were identical to conventional CCR5 genotyping but required considerably less time and expenditures. We have established a new real-time PCR protocol, which enables fast, cost-saving, and reliable CCR5 genotyping.
Department of Internal Medicine I, Rheinische Friedrich Wilhelms Universität, D-53105 Bonn, Germany. nischalke@uni-bonn.de
Hans Dieter
HD
Nischalke
Jacob
J
Nattermann
Mathias
M
Lichterfeld
Rainer P
RP
Woitas
Jürgen K
JK
Rockstroh
Tilman
T
Sauerbruch
Ulrich
U
Spengler
D017931:DNA Primers; D015658:HIV Infections / Q000276:immunology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D019713:Receptors, CCR5 / Q000235:genetics* / Q000276:immunology; D012016:Reference Values; D017384:Sequence Deletion; D013816:Thermodynamics
D017931:DNA Primers; D019713:Receptors, CCR5