Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene

Claus Lohman Brasen, Lone Frischknecht, Dorthe Ørnskov, Lotte Andreasen, Jonna Skov Madsen

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
JournalScandinavian Journal of Clinical & Laboratory Investigation
Volume77
Issue number1
Pages (from-to)60-65
ISSN0036-5513
DOIs
Publication statusPublished - Feb 2017

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