A Novel Multiplex Real-Time PCR Assay Using TaqMan MGB Probes for Rapid Detection of Trisomy 21

Authors: Mehrdad Hashemi, Mitra Behrooz Aghdam, Reza Mahdian, Ahmad Reza Kamyab

Abstract:

Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value <0.001). These results represent the presence of 3 copies of target gene in DS samples Vs 2 copies in normal controls. The results of quantitative Real-time PCR were in complete agreement with results of cytogenetic analysis. This study confirms previous reports regarding successful implementation of quantitative Real-time PCR for detection of trisomy 21. However, the assay has been improved by using MGB probes and more accurate data analysis. This assay, in particular, when performed in combination with another molecular assay such as QF-PCR or MLPA, can be used as a reliable technique for rapid prenatal diagnosis of trisomy 21.

Keywords: Trisomy 21, Real-time PCR, MGB-TaqMan Probes, Gene Dosage.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1084208

References:

[1] Yang YH, Nam MS, Yang ES. Rapid prenatal diagnosis of trisomy 21 by real-time quantitative polymerase chain reaction with amplification of small tandem repeats and 5100B in chromosome 21. Yonsei Med J 2005;46:193-197.
[2] Zhu YN, Lu SM, You JF, Zhu B, Yu MY. Novel real-time PCR assay for rapid prenatal diagnosis of Down syndrome: a prospective study of 563 amniocytes. Clin Biochem 2009;42:672-675.
[3] Pertl B, Weitgasser U, Kopp S, Kroisel PM, Sherlock J, Adinolfi M. Rapid detection of trisomies 21 and 18 and sexing by quantitative fluorescent multiplex PCR. Hum Genet 1996;98:55-59.
[4] Cirigliano V, Ejarque M, Canadas MP, Lloveras E, Plaja A, Perez MM, Fuster C, Egozcue J. Clinical application of multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies. Mol Hum Reprod 2001;7:1001-1006..
[5] Nicolini U, Lalatta F, Natacci F, Curcio C, Bui TH. The introduction of QF-PCR in prenatal diagnosis of fetal aneuploidies: time for reconsideration. Hum Reprod Update 2004;10:541-548..
[6] Sun X, Yan M, Zhang Y, Zhou X, Wang C, Zheng F, Xiong C. Practical application of fluorescent quantitative PCR on Trisomy 21 in Chinese Han population. Mol Biol Rep 2006;33:167-173..
[7] Schouten J, Galjaard RJ. MLPA for prenatal diagnosis of commonly occurring aneuploidies. Methods Mol Biol 2008;444:111-122.. Zimmermann B, Levett L, Holzgreve
[8] W, Hahn S. Use of real-time polymerase chain reaction for the detection of fetal aneuploidies. Methods Mol Biol 2006;336:83-100..
[9] Hayat Nosaeid M, Mandian R, Jamali S, Maryami F, Babashah S, Karimipoor M, Zeinali S. Validation and comparison of two quantitative real-time PCR assays for direct detection of DMD/BMD carriers. Clin Biochem 2009;42:1291-1299..
[10] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408..
[11] Hu Y, Zheng M, Xu Z, Wang X, Cui H. Quantitative real-time PCR technique for rapid prenatal diagnosis of Down syndrome. Prenat Diagn 2004;24:704-707..
[12] Dequeker E, Ramsden S, Grody WW, Stenzel TT, Barton DE. Quality control in molecular genetic testing. Nat Rev Genet 2001;2:717-723..