Evaluation of the Roche Diagnostics LightCycler-Apo B 3500 Mutation Detection Kit.
Familial defective apolipoprotein (apo) B-100 is an autosomal codominant disorder associated with hypercholesterolemia and an increased risk of coronary artery disease. Two independent mutations affecting the codon 3500 (Arg3500-->Gln and Arg3500-->Trp) have been shown to cause ligand-defective apo B-100. Identification of carriers of these mutations is an important step in the risk stratification of individuals and families with hypercholesterolemia. We evaluated a homogeneous assay for detection of mutations at codon 3500 that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. This single-tube analysis is performed on the LightCycler, a microvolume fluorimeter integrated with a thermal cycler. Continuous acquisition of fluorescence data during a melting curve analysis at completion of PCR allows the detection of mutations, as loss of fluorescence occurs in an allele-specific manner. By plotting melting peaks, the three apo B-100 alleles were readily distinguishable. Using this method, genotyping of 32 samples is completed within 40 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The specific detection of mutations at codon 3500 of the apo B gene on the LightCycler is a rapid and reliable method that is ideally suitable for typing both small and large numbers of samples.
Nauck
M
M
Department of Clinical Chemistry, University Hospital, Freiburg, Germany. msnauck@med1.ukl.uni-freiburg.de
Wieland
H
H
März
W
W
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000235:genetics*; D004247:DNA / Q000032:analysis / Q000235:genetics; D005453:Fluorescence; D005838:Genotype; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000235:genetics*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D011933:Reagent Kits, Diagnostic; D004247:DNA