Optimization and validation of the method lactose intolerance genotyping with real-time PCR

• Main Author: Stenberg, Jenny
• Format: Others
• Language: English
• Published: Uppsala universitet 2011
• Subjects: lactose intolerance; single nucleotide polymorphism; real-time PCR; genotype; optimization.
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-150810

Abstract Primary lactose intolerance has been associated with a single nucleotide polymorphism located upstream of the lactase gene. The most common diagnostic tests for lactose intolerance are time-consuming and the patient is not allowed to eat and drink for 12 hours before the test is carried out. A method that can establish the genotype would be an easier way of diagnosing lactose intolerance compared to fenotypic lactose intolerance tests. Optimization and validation of a previously published method was performed with real-time polymerase chain reaction. We used whole blood from de-identified blood donors. During the optimization and validation we used a positive control, genotype C/T from Laboratoriemedicin Västernorrland, Sundsvall. The whole-blood was extracted using the MagNa Pure LC instrument. The reagent used was KAPA PROBE FAST qPCR Master Mix. The optimized program for real-time PCR was established to be 95°C 3min [95°C x 3sec, 55°C x 20sec, detection, 72°C x 15sec] x 50 cycles. Optimal probe concentration was found to be 0.2µM and primer concentration will be 0.5µM. This genotyping method is a good first-stage screening test for lactoseintolerance. Before it can be used as a routine method further validation will be necessary in order to ensure that the evaluation of the results can be done in an easy and secure way.