title: Semi-nested Taqman real-time quantitative PCR for noninvasive prenatal diagnosis of Bart's hydrops fetalis.
abstract: BACKGROUND
  Non-invasive prenatal diagnosis based on detection of fetal cell-free DNA is limited when mother and father are both carriers for the same autosomal recessive mutation.
  
  
  OBJECTIVE
  Develop the semi-nested Taqman real-time PCR for quantification of alpha-thalassemia-1 SEA type deletion allele in plasma of alpha-thalassemia-1 SEA carriage pregnancies.
  
  
  METHODS
  Plasma DNA was extracted from six women who carried fetuses with normal, 11 with heterozygote alpha-thalassemia-1 SEA type deletion and seven with Bart's hydrops fetalis. DNA was amplified using conventional PCR with the primary specific primer set for alpha-thalassemia-1 SEA type deletion. PCR product was then subjected to the semi-nested real-time PCR using the secondary specific primer and Taqman probe set for alpha-thalassemia-1 SEA type deletion. The standard curve was constructed using ten-fold serial dilutions of conventional PCR product of the heterozygote alpha-thalassemia-1 SEA type deletion.
  
  
  RESULTS
  Women who carried fetuses with Bart's hydrops fetalis displayed a trend toward higher mean copy number of alpha-thalassemia-1 SEA type deletion allele vs. women who carried fetuses with normal and heterozygote, albeit not reaching statistical significance.
  
  
  CONCLUSIONS
  The maternally inheritedfetal allele present in maternal plasma is difficult to discern the fetal cell-free DNA from a higher background DNA of the mother Thus, further investigation is needed to improve the diagnosis ofBart's hydrops fetalis using this technique.
authors.affiliation: Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand. sakornmi001@yahoo.com
authors.forename: Sakorn
authors.initials: S
authors.lastname: Pornprasert
authors.forename: Kanyakan
authors.initials: K
authors.lastname: Sukunthamala
authors.forename: Naowarat
authors.initials: N
authors.lastname: Kunyanone
authors.forename: Sririchai
authors.initials: S
authors.lastname: Sittiprasert
authors.forename: Khanungnit
authors.initials: K
authors.lastname: Thungkham
authors.forename: Sumeth
authors.initials: S
authors.lastname: Junorse
authors.forename: Khachonsilp
authors.initials: K
authors.lastname: Pongsawatkul
authors.forename: Wisut
authors.initials: W
authors.lastname: Pattanaporn
authors.forename: Chantip
authors.initials: C
authors.lastname: Jitwong
mesh_terms: D000328:Adult; D000483:Alleles; D003937:Diagnosis, Differential; D005260:Female; D006801:Humans; D015160:Hydrops Fetalis / Q000175:diagnosis* / Q000235:genetics*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017085:alpha-Thalassemia / Q000097:blood / Q000235:genetics*