Primer-Introduced Restriction Analysis Polymerase Chain Reaction Method for Non-Invasive Prenatal Testing of β-Thalassemia

Abstract

We have developed a new method for non-invasive prenatal testing (NIPT) of paternally inherited fetal mutants for β-thalassemia (β-thal). Specially designed primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) were used to detect four major mutations [IVS-II-654, HBB: c.316-197C > T; codon 17 (A > T), HBB: c.52A > T; −28 (A > G), HBB: c.-78A > G and codons 41/42 (–TTCT), HBB: c.126_129delCTTT] causing β-thal in China. The PIRA-PCR assay was first tested in a series of mixed DNA with different concentrations and mixed proportions. Subsequently, this assay was further tested in 10 plasma DNA samples collected from pregnant women. In the DNA mixture simulation test, the PIRA-PCR assay was able to detect 3.0% target genomic DNA (gDNA) mixed in 97.0% wild-type gDNA isolated from whole blood. For plasma DNA testing, the results detected by PIRA-PCR assay achieved 100.0% consistency with those obtained from the amniocentesis analysis. This new method could potentially be used for NIPT of paternally inherited fetal mutants for β-thal.

• β-Thalassemia (β-thal)
• non-invasive prenatal testing (NIPT)
• primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR)

Declaration of interest

This study was funded by Shenzhen Birth Defect Screening Project Laboratory (to JZF No. [2011] 861) and Shenzhen Engineering Laboratory for Clinical Molecular Diagnostic (to JZF No. [2011] 862). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.