Early non-invasive fetal RHD genotyping and sex determination by conventional and multiplex PCR performing a rapid and low-cost cell-free fetal DNA extraction method

摘要

The aims of this investigation were (1) to establish an early non-invasive prenatal genetic diagnosis of fetal sex by analyzing the DYS14 and SRY genes and (2) to determine the fetal RhD factor by examining the RHD gene, performing conventional and multiplex PCR, and a rapid low-cost cell-free DNA isolation method, as effective diagnostic tools. We collected 10 mL of blood from 109 pregnant women at 6-24 weeks of gestation in glass blood tubes containing anticoagulant EDTA. The samples were subjected to a rapid and low-cost cell-free DNA extraction method, from the maternal plasmas obtained by centrifugation at 3500 rpm for 20 min. The plasmas were heated at 90 °C for 10 min, and centrifuged for 20 min at 14,000 rpm to separate the sera. The resulting supernatants (sera) were collected and stored at -20 °C. Two microliters of serum (DNA) was used as a template for the PCR. Each sample was analyzed in at least three replicate settings. The outcomes were considered positive if two or more individual replicates were positive.