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Development and Application of a Real‐Time Quantitative PCR for Prenatal Detection of Fetal α 0 ‐Thalassemia from Maternal Plasma
Author(s) -
TUNGWIWAT WARUNEE,
FUCHAROEN SUPAN,
FUCHAROEN GOONNAPA,
RATANASIRI THAWALWONG,
SANCHAISURIYA KANOKWAN
Publication year - 2006
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1368.013
Subject(s) - hydrops fetalis , thalassemia , prenatal diagnosis , fetus , real time polymerase chain reaction , genotype , polymerase chain reaction , microbiology and biotechnology , beta thalassemia , medicine , andrology , biology , genetics , pregnancy , gene
In order to provide a noninvasive prenatal diagnosis of α 0 ‐Thalassemia (Southeast Asian [SEA] deletion), we have developed a real‐time quantitative semi‐nested polymerase chain reaction (PCR) method for identifying the fetal α 0 ‐Thalassemia in maternal plasma. Analysis was performed using DNA extracted from 200 μL plasma from 13 pregnant women during 8–20 weeks of gestation who carried fetuses with normal (2), α 0 ‐Thalassemia carrier (8), Hb H disease (1), and homozygous α 0 ‐Thalassemia (Hb Bart's hydrops fetalis (2). The α 0 ‐Thalassemia was detected using a two‐step PCR. Plasma DNA was amplified conventionally using α 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 α 0 ‐Thalassemia quantification was prepared by assaying serial dilution of genomic DNA of an α 0 ‐Thalassemia carrier. Differences in the C T (threshold cycle) values and calculated concentrations of amplified DNA among normal fetus, α 0 ‐Thalassemia carrier, Hb H disease, and homozygous α 0 ‐Thalassemia were clearly observed, which could help in prenatal prediction of the fetal genotype. This noninvasive prenatal detection of α 0 ‐Thalassemia in maternal plasma should enhance prenatal diagnostic options for this common genetic disorder in routine DNA diagnostic setting.