Rapid and simultaneous detection of common aneuploidies by quadruplex real-time polymerase chain reaction combined with melting curve analysis.

Jiwu Lou,Yanhui Liu,Dongzhi Li,Zhisong Ji,Yangyang Lin,Ying Zhao,Wanfang Xu,Fenghua Liu,Manna Sun

doi:10.1371/journal.pone.0171886

Abstract

BackgroundDuring the prenatal period, the number variation of chromosomes 13, 18, 21, X and Y accounts for more than 80% of the clinically significant chromosomal abnormalities diagnosed. Rapid tests for prenatal diagnosis of these abnormalities can improve pregnancy management and alleviate parental anxiety. Here, we present a molecular alternative method for detecting common aneuploidies.MethodsThis method is based on co-amplification of segmental duplications located on two different chromosomes using a single pair of primers. Segmental duplications have a high degree of sequence identity, but have single-nucleotide differences in some regions. These sequence differences can be quantified using melting curve analysis of dual-labeled probes to estimate the relative dosages of different chromosomes. We designed two quadruplex real-time PCR assays to detect aneuploidies of chromosomes 13, 18, 21, X and Y.ResultsWe examined 75 aneuploid DNA samples and 56 unaffected DNA control samples using these two assays and correctly identified all samples. Four cases of unbalanced translocation were also accurately detected. The observed averaged ratio for each chromosomal disorder was similar to the theoretically expected value.ConclusionsOur real-time assay is a robust, rapid, and easy to conduct technique for prenatal diagnosis of common aneuploidies, representing a competitive alternative for use in diagnostic laboratories.

Highlights

The number variation of chromosomes 13, 18, 21, X and Y account for more than 80% of the clinically significant chromosomal abnormalities diagnosed [1]
With the widespread use of antenatal screening methods based on maternal serum analysis, fetal ultrasonography, and non-invasive prenatal testing (NIPT) for the more common aneuploidies of chromosomes 13, 18, 21, X and Y, a method for rapid, targeted detection of these aneuploidies may provide more benefit to pregnant women than conventional karyotype analysis [3–5]
Compared to fluorescent in situ hybridization (FISH), quantitative fluorescence PCR (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA) are more suited to a high throughput diagnostic service; they are PCR-based methods that involve the amplification of polymorphic microsatellite markers and ligated probes, respectively

Summary

Introduction

The number variation of chromosomes 13, 18, 21, X and Y account for more than 80% of the clinically significant chromosomal abnormalities diagnosed [1]. A common diagnostic test for chromosomal abnormalities, has been regarded as the standard method for prenatal cytogenetic diagnosis since the early 1970s [2]. Fluorescent in situ hybridization (FISH), quantitative fluorescence PCR (QF-PCR), and multiplex ligation-dependent probe amplification (MLPA) are the three most commonly used rapid, targeted aneuploidy detection methods [6]. All three methods avoid the generation of cultured cells and can rapidly detect (within 1 or 2 days) the most common aneuploidies (trisomy 13, 18, and 21) and aneuploidies of the sex chromosomes. With MLPA, the potential problem of non-informativeness is eliminated, but an overnight step for hybridization lasting approximately 16 hours is required Both QF-PCR and MLPA require a postPCR step for quantitative analysis of the products using an expensive genetic analyzer. We present a molecular alternative method for detecting common aneuploidies

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Conclusion