Evaluation of the Roche Diagnostics LightCycler-Apo B 3500 Mutation Detection Kit
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Abstract
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.
Copyright (c) 2000 by Walter de Gruyter GmbH & Co. KG
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- The Science of Systematic Reviewing Studies of Diagnostic Tests
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