Accurate detection of α-globin gene copy number variants with two reactions using droplet digital PCR.

Background: The α-thalassemia is a highly prevalent disease in tropical and subtropical regions, including southern China, and is mainly caused by deletion in α-globin genes ( and ). The clinical manifestation of α-thalassemia is highly correlated with the copy number of α-globin genes. The decrease in copy number results in α-thalassemia, while duplication or triplication compounded with β-thalassemia may aggravate the clinical manifestation. However, the common methods used to measure the copy number variants can only detect the three common types: -, -α, and -α, and may easily miss the rare deletional type and duplication or triplication cases. Therefore, a new method that allows the detection of different copy number variants in α-globin genes simultaneously and accurately needs to be established.

Methods: A total of 428 peripheral-blood and fetal chorionic villus or amniotic fluid samples were used in this study. We employed a pair of primers and two probes, one for and another for , to perform droplet digital polymerase chain reaction (ddPCR). Each reaction needed the ddPCR of as a reference gene to calculate the copy number.

Results: We accurately detected the copy number variants in α-globin genes, including the common form α-thalassemia, triplications such as ααα, and trisomy 16, by performing only two reactions. The accuracy rate for detecting the copy number of α-globin genes was up to 100%.

Conclusion: In conclusion, ddPCR served as an accurate and rapid method for detecting copy number variations in the clinical screening for α-thalassemia.