Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification.

α-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart's hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (--SEA) and the Thai type (--THAI) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia.

Development of DNA controls for detection of ß-thalassemia mutations commonly found in Asian.

INTRODUCTION: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of ß-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe ß-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for ß-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. METHODS: Four recombinant ß-globin plasmids, including a normal sequence and three different mutant panels covering 10 common ß-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis. RESULTS: We demonstrated the applicability of established positive DNA controls for ß-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant ß-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant ß-globin plasmids, are required for detecting 10 common ß-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. CONCLUSION: The ß-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of ß-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.