Fast Temperature-Gradient COLD PCR for the enrichment of the paternally inherited SNPs in cell free fetal DNA; an application to non-invasive prenatal diagnosis of β-thalassaemia

被引：9

作者：

Byrou, Stefania ^{[1
,2
]}

Makrigiorgos, G. Mike ^{[3
]}

Christofides, Agathoklis ^{[4
]}

Kallikas, Ioannis ^{[5
]}

Papasavva, Thessalia ^{[1
,2
]}

Kleanthous, Marina ^{[1
,2
]}

机构：

[1] Cyprus Inst Neurol & Genet, Mol Genet Thalassaemia Dept, Nicosia, Cyprus

[2] Cyprus Sch Mol Med, Nicosia, Cyprus

[3] Harvard Med Sch, Dana Farber Canc Inst, Dept Radiat Oncol, Div Med Phys & Biophys, Boston, MA USA

[4] Archbishop Makarios III Hosp, Med Ctr, Fetal Med Dept, Nicosia, Cyprus

[5] Ultrasound & Fetal Med Ctr, Nicosia, Cyprus

来源：

PLOS ONE | 2018年 / 13卷 / 07期

关键词：

MATERNAL PLASMA; CLINICAL-APPLICATION; MONOGENIC DISEASES; RHD STATUS; MUTATIONS; AMNIOCENTESIS; SENSITIVITY; CANCER;

D O I：

10.1371/journal.pone.0200348

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Objective To develop a sensitive, specific, simple, cost-effective and reproducible platform for the non-invasive prenatal detection of paternally inherited alleles for beta-thalassaemia. The development of such an assay is of major significance in order to replace currently-applied invasive methods containing inherent fetal loss risks. Methods We present a fast Temperature-Gradient Co-amplification at Lower Denaturation Temperature Polymerase Chain Reaction (fast TG COLD PCR) methodology for the detection of the paternally-inherited fetal alleles in maternal plasma. Two single-nucleotide polymorphisms (SNPs), rs7480526 (G/T) and rs968857 (G/A) that are located on the beta-globin gene cluster and exhibit a high degree of heterozygosity in the Cypriot population were selected for evaluation. Seventeen maternal plasma samples from pregnancies at risk for beta-thalassemia were analysed for the selected SNPs using the novel fast TG COLD PCR assay. Results Using fast TG COLD PCR, the paternally inherited allele in cell free fetal DNA was correctly determined for all the 17 maternal plasma samples tested, showing full agreement with the Chorionic Villus Sampling (CVS) analysis. Conclusions Our findings are encouraging and demonstrate the efficiency and sensitivity of fast TG COLD PCR in detecting the minor paternally-inherited fetal alleles in maternal plasma for the development of a NIPD assay for beta-thalassaemia.

引用

页数：15

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