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

• Published in: PloS one 2017-02, Vol.12 (2), p.e0171886-e0171886
• Main Authors: Lou, Jiwu, Sun, Manna, Zhao, Ying, Ji, Zhisong, Liu, Fenghua, Li, Dongzhi, Xu, Wanfang, Lin, Yangyang, Liu, Yanhui
• Format: Article
• Language: English
• Subjects: Abnormalities; Analysis; Aneuploidy; Anxiety; Assaying; Biology and Life Sciences; Case-Control Studies; Children & youth; Childrens health; Chromosome aberrations; Chromosome Disorders - diagnosis; Chromosome Disorders - genetics; Chromosomes; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 18; Chromosomes, Human, Pair 21; Chromosomes, Human, X; Chromosomes, Human, Y; Clinical medicine; Deoxyribonucleic acid; Diagnosis; Diagnostic systems; DNA; DNA - analysis; DNA probes; Female; Fetuses; Fluorescent Dyes - chemistry; Genetic testing; Hospitals; Humans; Karyotyping; Male; Maternal & child health; Medicine and Health Sciences; Melting; Melting curve; Nucleotides; Physical Sciences; Polymerase chain reaction; Pregnancy; Prenatal development; Prenatal diagnosis; Primers; Real time; Real-Time Polymerase Chain Reaction; Reproduction (copying); Research and Analysis Methods; Translocation; Trisomy; Womens health
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0171886

During 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. This 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. We 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. Our 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.