title: Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene.
abstract: BACKGROUND
  Lactase persistence is an autosomal dominant trait commonly distributed in Europe as well as some parts of east Africa and the Arabian Peninsula. Using real-time PCR to detect the -13910C > T variant common in the European population is a reliable analysis although other variants in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants.
  
  
  METHODS
  We genotyped 3395 routine samples using real-time PCR for the -13910C > T-variant. All consecutive samples identified as -13910CC were sequenced using Sanger Sequencing. Using the SDS software we examined various quality value settings to improve on the genetic analysis.
  
  
  RESULTS
  Using real-time PCR resulted in 100% successful genotyping of the -13910C > T variant. By using a quality value of 99% and sequencing the undetermined samples we improved the ability of the assay to identify variants other than -13910C > T. This resulted in a reduction of the diagnostic error rate by a factor of 2.4 while increasing the expenses only 3%.
  
  
  CONCLUSIONS
  We conclude that using a quality value of 99% in the SDS software significantly improves the diagnostic efficiency of the real-time PCR assay for detecting variants associated to lactase persistence.
authors.affiliation: a Department of Clinical Immunology and Biochemistry , Lillebaelt Hospital , Vejle , Denmark.
authors.forename: Claus Lohman
authors.initials: CL
authors.lastname: Brasen
authors.affiliation: a Department of Clinical Immunology and Biochemistry , Lillebaelt Hospital , Vejle , Denmark.
authors.forename: Lone
authors.initials: L
authors.lastname: Frischknecht
authors.affiliation: b Department of Clinical Pathology , Lillebaelt Hospital , Vejle , Denmark.
authors.forename: Dorthe
authors.initials: D
authors.lastname: Ørnskov
authors.affiliation: a Department of Clinical Immunology and Biochemistry , Lillebaelt Hospital , Vejle , Denmark.
authors.forename: Lotte
authors.initials: L
authors.lastname: Andreasen
authors.affiliation: a Department of Clinical Immunology and Biochemistry , Lillebaelt Hospital , Vejle , Denmark.|c Institute of Regional Health Services Research, Faculty of Health Sciences , University of Southern Denmark.
authors.forename: Jonna Skov
authors.initials: JS
authors.lastname: Madsen
mesh_terms: D000483:Alleles; D003718:Denmark / Q000453:epidemiology; D015870:Gene Expression; D005787:Gene Frequency; D005820:Genetic Testing; D005838:Genotype; D006801:Humans; D043322:Lactase / Q000172:deficiency / Q000235:genetics*; D007787:Lactose Intolerance / Q000175:diagnosis* / Q000453:epidemiology / Q000235:genetics* / Q000503:physiopathology; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D015995:Prevalence; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012984:Software
chemical_list: D043322:Lactase
keywords: DNA sequencing; lactose intolerance; molecular diagnostics; real-time PCR; single nucleotide polymorphism