Development and application of a real-time quantitative PCR for prenatal detection of fetal alpha(0)-thalassemia from maternal plasma.
In order to provide a noninvasive prenatal diagnosis of alpha(0)-Thalassemia (Southeast Asian [SEA] deletion), we have developed a real-time quantitative semi-nested polymerase chain reaction (PCR) method for identifying the fetal alpha(0)-Thalassemia in maternal plasma. Analysis was performed using DNA extracted from 200 muL plasma from 13 pregnant women during 8-20 weeks of gestation who carried fetuses with normal (2), alpha(0)-Thalassemia carrier (8), Hb H disease (1), and homozygous alpha(0)-Thalassemia (Hb Bart's hydrops fetalis (2). The alpha(0)-Thalassemia was detected using a two-step PCR. Plasma DNA was amplified conventionally using alpha(0)-Thalassemia-specific primers and a portion of the first PCR product was subjected to a semi-nested real-time q-PCR using the SYBR green I chemistry for fluorescence detection. Calibration curve for alpha(0)-Thalassemia quantification was prepared by assaying serial dilution of genomic DNA of an alpha(0)-Thalassemia carrier. Differences in the C(T) (threshold cycle) values and calculated concentrations of amplified DNA among normal fetus, alpha(0)-Thalassemia carrier, Hb H disease, and homozygous alpha(0)-Thalassemia were clearly observed, which could help in prenatal prediction of the fetal genotype. This noninvasive prenatal detection of alpha(0)-Thalassemia in maternal plasma should enhance prenatal diagnostic options for this common genetic disorder in routine DNA diagnostic setting.
Tungwiwat
Warunee
W
Biomedical Science Program, Graduate School, Khon Kaen University, Khon Kaen, Thailand.
Fucharoen
Supan
S
Fucharoen
Goonnapa
G
Ratanasiri
Thawalwong
T
Sanchaisuriya
Kanokwan
K
D004247:DNA / Q000097:blood*; D005260:Female; D006801:Humans; D008431:Maternal-Fetal Exchange; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011296:Prenatal Diagnosis; D013785:Thailand; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics*
D004247:DNA