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

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

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.

OriginalsprogEngelsk
TidsskriftScandinavian Journal of Clinical & Laboratory Investigation
Vol/bind77
Udgave nummer1
Sider (fra-til)60-65
ISSN0036-5513
DOI
StatusUdgivet - feb. 2017

Fingeraftryk

Real-Time Polymerase Chain Reaction
Lactose Intolerance
Eastern Africa
Diagnostic Errors
Population

Citer dette

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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.",
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Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene. / Brasen, Claus Lohman; Frischknecht, Lone; Ørnskov, Dorthe ; Andreasen, Lotte; Madsen, Jonna Skov.

I: Scandinavian Journal of Clinical & Laboratory Investigation, Bind 77, Nr. 1, 02.2017, s. 60-65.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene

AU - Brasen, Claus Lohman

AU - Frischknecht, Lone

AU - Ørnskov, Dorthe

AU - Andreasen, Lotte

AU - Madsen, Jonna Skov

PY - 2017/2

Y1 - 2017/2

N2 - 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.

AB - 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.

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