Prenatal detection of thalassemia by cell-free fetal DNA (cffDNA) in maternal plasma using surface enhanced Raman spectroscopy combined with PCR.

Xiaozhou Li,Hong Lou,Lili Jin,Caesar Siqi Li,Youtao Song,Tianyue Yang

doi:10.1364/boe.9.003167

Abstract

Thalassemias are widely occurring genetic hemoglobin disorders; patients with severe thalassemia often require regular blood transfusions for survival. Prenatal detection of thalassemia is currently invasive and carries the risk of miscarriage and infection. A polymerase chain reaction (PCR)-based surface enhanced Raman spectroscopy (SERS) technique was investigated in this paper for the purpose of detecting prenatal α-thalassemia Southeast Asian (SEA) type deletion using maternal plasma. Couples with the same SEA thalassemia (-SEA/αα) were selected, and the quantification of SEA and wild type (WT) alleles in the maternal plasma sample predicted the fetal genotype. PCR was performed using two pairs of fluorescence tag-labeled primers to produce tag-labeled PCR products for both the SEA (labeled with R6G) and WT (labeled with Cy3) alleles. Then, the labeled PCR products containing the two fluorescence tags were measured by SERS. The ratios between the R6G and Cy3 tags were obtained using multiple linear regressions (MLR), and these ratios corresponded with the physical ratio of WT and SEA concentrations in maternal plasma. After verifying this technique on DNA mixtures with known SEA and WT ratios, the plasma from 24 pregnant women was screened. An accuracy of 91.7% was achieved for detecting the fetal genotypes of Hb Bart's, alpha-trait, and normal trait. The results indicated that the simple PCR-SERS method may be sensitive enough for use on cell free fetal DNA (cffDNA) in maternal plasma for non-invasive prenatal detection (NIPD).

Highlights

Thalassemia is a class of severe inherited single gene blood disorders that occur worldwide
3.1 Verification of polymerase chain reaction (PCR)-SERS First, the SERS spectra of the tags R6G and Cy3, which were labeled to the Southeast Asian deletion (SEA) and wild type (WT) alleles respectively, were measured
Those differences enable the deconvolution of PCR product SERS spectra by multiple linear regressions (MLR) into the spectra of different tags feasible

Summary

Introduction

Thalassemia is a class of severe inherited single gene blood disorders that occur worldwide. It results in poor production of normal hemoglobin which can lead to anemia and less oxygen delivery through blood [1]. The high-incidence areas of thalassemias include tropical and subtropical regions such as the Mediterranean, the Middle East, and Central and Southeast Asia [2]. 56,000 conceptions per year result in a major thalassemia disorder [4]. 9 million pregnant women are carriers annually and 1.33 million pregnancies each year are at risk for a thalassemia major [2]. In China, α and β are the two most prevalent categories of thalassemia, with especially high prevalence in the Southern provinces of Guandong, Guangxi, and Hainan. For example in Guangdong province, the prevalence of SEA thalassemia is 4.14% (compared with thalassemia β of 2.54%, and single globin gene thalassemia of 4.05%) [6]

Methods

Results

Discussion

Conclusion