Droplet Digital PCR Rapidly Detects Alpha-Thalassemia Variants

Alpha (α)-thalassemia is a highly prevalent genetic disease worldwide and it is characterized by various degrees of alpha globin chain deficit caused by either deletional or non-deletional mutations although the former are more common.

Recently, there have been more reported cases of rare α-thalassemia mutations due to the advancement of molecular techniques involved in thalassemia detections. Therefore, it is essential to develop a new method, which allows the detection of different α-thalassemia mutations including the rare ones simultaneously and accurately.


Scientists at the University Putra Malaysia (Serdang, Malaysia) designed an assay for the detection of triplications, common and rare deletional α-thalassemia using droplet digital PCR (ddPCR). Eleven archived DNA samples with known genotypes and one DNA sample with heterozygous deletion spanning from HS-40 region to alpha1 globin region were used in the study.

Four copy number variations (CNV) assays were carried out in duplications for each sample, which includes the upstream alpha globin regulatory element HS- 40, Hemoglobin, alpha 2 (HBA2), HBA3.7 and HBA1 genes. A total of 20 µL of each polymerase chain reaction (PCR) mixture was loaded into a sample well of a DG8TM Cartridge for QX100 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, USA), followed by 70 µL of droplet generation oil into the oil wells. After the thermal cycling process, the sealed 96-well plate was placed in the Bio-Rad QX100 Droplet Digital Reader.

The team found that found that the use of ddPCR is convenient as it allows direct quantification without the requirement of a calibration curve unlike quantitative PCR. They showed that showed that ddPCR is accurate and precise in the detection of alpha thalassemia deletions and triplications based on the gene dosages using absolute quantification. Twelve samples with different α-thalassemia deletions at different regions located at HS-40 to HBA1 and triplications of α-globin genes were detected accurately. The investigators also showed that ddPCR saves time with less turnaround time and minimize the labor work required as compared to other techniques. The study was published in the August 2016 issue of the International Journal of Laboratory Hematology.

Related Links:
University Putra Malaysia
Bio-Rad Laboratories