Loop-mediated isothermal amplification (LAMP) colorimetric phenol red assay for rapid identification of α0-thalassemia: Application to population screening and prenatal diagnosis

Abstract

Background Identification of α0-thalassemia (SEA and THAI deletions) is essential in preventing and controlling of severe thalassemia diseases. We have developed the LAMP colorimetric assays for the detection of these two thalassemia defects and validated them in population screening and prenatal diagnosis. Methods Three LAMP colorimetric assays specific for α0-thalassemia (SEA deletion), α0-thalassemia (THAI deletion) and normal DNA sequence were developed. These assays were validated on 341 subjects who had initial thalassemia screening positive and various thalassemia genotypes. Prenatal diagnosis of α0-thalassemia (SEA deletion) was done on 33 fetuses at risk of having Hb Bart’s hydrops fetalis syndrome. Results The LAMP colorimetric assays for α0-thalassemia (SEA and THAI deletions) could be clearly interpreted by naked eyes. The assay for α0-thalassemia (SEA deletion) showed a 100% (62/62 x 100) sensitivity and 98.2% (274/279 x 100) specificity whereas, that of the α0-thalassemia (THAI deletion) showed 100% (1/1 x 100) sensitivity and 99.7% (339/340 x 100) specificity. We obtained a 100% concordant prenatal diagnosis results using LAMP assays of α0-thalassemia (SEA deletion) in 33 fetuses as compared to the conventional PCR analysis. Conclusions The LAMP colorimetric assays developed are simple, rapid, and do not require sophisticated equipment. Inclusion of the LAMP tests in the existing screening protocol significantly reduce the screening cost and the molecular analysis workload, which should prove useful in the prevention and control program of hemoglobinopathies in the region.

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

“…Diagnosis of the SEA deletion allele by LAMP has been reported using pH-sensitive dyes or fluorescence dyes 18 , 20 , 25 . Recently, methods for detecting the THAI deletion by LAMP using pH-sensitive dyes was reported 20 .…”

“…Diagnosis of the SEA deletion allele by LAMP has been reported using pH-sensitive dyes or fluorescence dyes 18 , 20 , 25 . Recently, methods for detecting the THAI deletion by LAMP using pH-sensitive dyes was reported 20 . The drawback of the pH-sensitive dyes is the requirement of optimal pH for the best results and the difficulty of distinguishing positive readout, changing the color of phenol red from pink to orange.…”

“…The amplification reaction occurs at 60-65°C, allowing the use of a simple water bath or heat block. The LAMP technique has been widely used to diagnose a wide range of infectious diseases 14 , 15 , pharmacogenetic and disease risk alleles 16 , 17 and genetic diseases 18 – 20 . In addition, the amplification products can be visualization using pH-sensitive dyes 21 , a fluorescent metal indicator 22 , fluorescent and non-fluorescent DNA intercalating dye 23 , 24 .…”