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Genotypning av laktostolerans (LCT-13910C>T) direkt på blod med realtids-PCR: Utvärdering av Kapa Probe Force
Jönköping University, School of Health and Welfare, HHJ, Dep. of Natural Science and Biomedicine.
Jönköping University, School of Health and Welfare, HHJ, Dep. of Natural Science and Biomedicine.
2016 (Swedish)Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesisAlternative title
Genotyping of lactase persistence (LCT-13910C>T) directly on blood with real time PCR : Evaluation of Kapa Probe Force (English)
Abstract [sv]

Hos vuxna individer förekommer två fenotyper gällande produktionen av laktas, vilka kallas laktostolerans och laktosintolerans. Vid laktosintolerans produceras otillräckliga mängder laktas vilket framkallar symptom som magsmärtor och flatulens vid intagandet av mjölkprodukter. En enbaspolymorfism (LCT-13910C>T) har kopplats till laktostolerans hos nordvästeuropéer och kan genotypas med smältkurveanalys i realtids-PCR. På Laboratoriemedicin vid Länssjukhuset Ryhov används idag en metod vid genotypning av LCT-13910C>T där extraktion av DNA från blod krävs innan analys. Anledningen till detta är att DNA-polymeraset som ingår enzymmixen LightCycler® FastStart DNA Master HybProbe endast fungerar med rent DNA-templat. Med en annan enzymmix, Kapa Probe Force, ska analys kunna göras direkt på blod. För att utvärdera enzymmixen jämfördes resultat från befintlig metod och resultat från metod med Kapa Probe Force, gällande förmågan att identifiera genotyperna LCT-13910C/C, C/T och T/T samt med avseende på imprecision. Vid jämförelse mellan metoderna samstämde resultatet i avseende på genotyp till 100 % utifrån specificerade smälttemperaturer (Tm) för respektive genotyp angivna i kitet för primer/prober. Däremot syntes lägre fluorescensnivå på smältopparna i metod med Kapa Probe Force, men påverkade inte tolkning av smältkurvorna. En lägre prov-till-prov-variation sågs även i resultatet från metod med Kapa Probe Force gentemot befintlig metod.

Abstract [en]

Among adults two phenotypes are found with regards to production of lactase, these are termed lactase persistence and lactose intolerance. Lactose intolerance is characterized by a low production of lactase, which leads to symptoms such as stomach ache and flatulence after the consumption of dairy products. A single nucleotide polymorphism (LCT-13910C>T) has been correlated with the occurrence of lactase persistence in northwestern Europeans. Genotyping of LCT-13910C>T is possible with melting curve analysis in real time PCR. The currently used method for genotyping of LCT-13910C>T at Ryhov County Hospital requires the extraction of DNA template from blood, due to the fact that the DNA-polymerase in the kit LightCycler® FastStart DNA Master HybProbe requires pure DNA template for analysis. With another DNA-polymerase, included in the kit Kapa Probe Force, analysis on crude samples such as pure blood should be possible. Evaluation of Kapa Probe Force included comparison of the results from both methods with regards to identification of genotypes LCT-13910C/C, C/T and T/T and with regard to imprecision. The results from Kapa Probe Force were 100 % consistent with the results from existing method and acquired melting temperatures (Tm) were all within the accepted ranges specified in the kit of primers and probes. The fluorescence of melting curves acquired with Kapa Probe Force was significantly lower, however this had no effect when it came to interpreting the results. A lower variation could also be seen between samples with Kapa Probe Force compared to existing method.

Place, publisher, year, edition, pages
2016. , 39 p.
Keyword [en]
Polymorphism, lactose intolerance, PCR, DNA-polymerase, blood
Keyword [sv]
Polymorfism, laktosintolerans, PCR, DNA-polymeras, blod
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:hj:diva-30807ISRN: JU-HHJ-BLA-1-20160028OAI: oai:DiVA.org:hj-30807DiVA: diva2:941678
Subject / course
HHJ, Biomedical Laboratory Science
Supervisors
Examiners
Available from: 2016-06-29 Created: 2016-06-22 Last updated: 2016-06-29Bibliographically approved

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