detection of the C282Y mutation associated with hereditary haemochromatosis by conventional and Real Time PCR Degree: MMed Haematology

Audio Recording 08 Oct 2022 at 11:54:50. detection of the C282Y mutation associated with hereditary haemochromatosis by conventional and Real Time PCR Degree: MMed Haematology.

Angled shot of pen on a graph. Executive summary:.

Audio Recording 08 Oct 2022 at 11:59:36. Hereditary haemochromatosis (HH).

Audio Recording 08 Oct 2022 at 12:09:38. HH Iron overload clinically.

Audio Recording 08 Oct 2022 at 12:12:19. Screening and diagnosis.

Audio Recording 08 Oct 2022 at 12:33:34. Available methods of detection of mutation C282Y.

Audio Recording 08 Oct 2022 at 12:20:39. Rationale and research aim.

Real time PCR/ quantitative PCR. The NHLS haematology laboratory in Tshwane district at Prinshof currently has conventional PCR and RT-PCR RT-PCR= Amplification and detection/quantitation of amplicons Conventional PCR requires post amplification processing- i.e. measure DNA concentration using a spectrophotometer, detection of mutation by enzyme digestion of pcr product and visualization of digested fragments of interest by gel electrophoresis The aim of the project is to use conventional PCR and above steps to calibrate the RT-PCR machine to detect mutation by melting curve analysis.

Methods. Design: laboratory based quantitative study to demonstrate the sensitivity and specificity of the method of detecting the C282Y mutation in the laboratory using the existing infrastructure and specimen processing protocols. Population: 30 samples= 10 known (EQA) +10 Known Patients + 10 healthy volunteers Laboratory process 1 Conventional PCR -DNA extraction, PCR amplification, restriction enzyme digestion and visualization of products on gel electrophoresis. Laboratory process 2: Real time PCR- DNA extraction, PCR amplification, Melt curve analysis.

Data analysis. This is an exploratory analysis following testing of real time PCR vs conventional PCR assay for C282Y mutation, diagnostic statistics will be determined using a sample of 30 specimens consisting of 20 positives and 10 negatives. The degree of congruence of the two methods will be determined. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and kappa ( κ ) will be calculated. Results will be analysed according to the below table:.