Rapid typing of the codon 129 polymorphism of the human prion protein gene by combined real-time PCR and melting curve analysis.
Homozygosity of methionine (m/m) at amino acid residue 129 (codon 129) of the human prion protein (PrP) has been reported for all so far analyzed cases of the new variant Creutzfeldt-Jakob disease (vCJD). This contrasts with its general distribution in the healthy Caucasian population of only about 43%. For this reason a predisposition for carriers of the corresponding genotype to develop vCJD after infection with the bovine spongiform encephalopathy (BSE) agent is assumed, and PCR based methods such as allele-specific oligonucleotide hybridization or restriction analysis and sequencing have been developed for codon 129 genotyping. These methods are cumbersome and time-consuming and the need for extensive post-amplification manipulations increases the risk of carry-over contamination with amplified products. To overcome these shortcomings, the authors developed a real-time PCR assay on the LightCycler (LC) instrument combining PCR and temperature melting curve analysis (Tm) in a closed vessel format. Forty-six swabs and blood samples from healthy donors were tested. Of these 23 (50%) were heterozygous at codon 129, 4 (8.7%) homozygous for valine and 19 (41.3%) homozygous for methionine. Accuracy of LC-genotyping was confirmed by automated sequencing of the amplified products. Taken together, genotyping of the codon 129 polymorphism by combined LC-PCR and melting-curve analysis with the LC-instrument is a reliable and easy to perform method even in a screening context with numerous samples. Results can be obtained within 2 hours, including sample preparation.
Schalasta
Gunnar
G
Institute for Virology, Infectiology and Epidemiology and Medical Diagnostic Laboratory, Prof. G. Enders, Stuttgart, Germany. schalasta@labor-enders.de
Roth
Bernhard
B
Enders
Gisela
G
D003062:Codon / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D005838:Genotype; D006801:Humans; D009691:Nucleic Acid Denaturation / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011328:Prions / Q000235:genetics*; D015203:Reproducibility of Results; D013696:Temperature; D013997:Time Factors
D003062:Codon; D011328:Prions