Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy.

OBJECTIVE The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy. STUDY DESIGN Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes. RESULTS Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested. CONCLUSION Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

[1]  R. Aebersold,et al.  A systematic approach to the analysis of protein phosphorylation , 2001, Nature Biotechnology.

[2]  M. Adinolfi,et al.  Rapid prenatal diagnosis of aneuploidy by quantitative fluorescent PCR on fetal samples from mothers at high risk for chromosome disorders. , 1999, Molecular human reproduction.

[3]  D. Dressman,et al.  Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Invasive Prenatal Testing for Aneuploidy , 2009 .

[5]  D. Haussler,et al.  Ultraconserved Elements in the Human Genome , 2004, Science.

[6]  K. Hecher,et al.  Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF-PCR in 662 selected pregnancies at risk. , 2000, Molecular human reproduction.

[7]  R. Hochstenbach,et al.  Rapid detection of chromosomal aneuploidies in uncultured amniocytes by multiplex ligation‐dependent probe amplification (MLPA) , 2005, Prenatal diagnosis.

[8]  S. Abbs,et al.  Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis , 2001, The Lancet.

[9]  L. Shaffer,et al.  Molecular cytogenetic and rapid aneuploidy detection methods in prenatal diagnosis , 2007, American journal of medical genetics. Part C, Seminars in medical genetics.

[10]  J. Silver,et al.  Nanoliter scale PCR with TaqMan detection. , 1997, Nucleic acids research.

[11]  Frank Diehl,et al.  Detection and quantification of mutations in the plasma of patients with colorectal tumors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[12]  N. Carter,et al.  Prenatal detection of unbalanced chromosomal rearrangements by array CGH , 2005, Journal of Medical Genetics.

[13]  C. Lundsteen,et al.  Computer-assisted prenatal aneuploidy screening for chromosome 13, 18, 21, X and Y based on multiplex ligation-dependent probe amplification (MLPA) , 2005, European Journal of Human Genetics.

[14]  G. Bonsel,et al.  Bmc Pregnancy and Childbirth Multiplex Ligation-dependent Probe Amplification versus Karyotyping in Prenatal Diagnosis: the M.a.k.e. Study , 2022 .

[15]  S. Goodman,et al.  Research Corrigenda , 2001, Nature Biotechnology.

[16]  K. Kinzler,et al.  Digital PCR. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[17]  James R. Knight,et al.  Genome sequencing in microfabricated high-density picolitre reactors , 2005, Nature.

[18]  L. D. White,et al.  Rapid prenatal diagnosis using uncultured amniocytes and oligonucleotide array CGH , 2008, Prenatal diagnosis.

[19]  N. Niikawa,et al.  Microarray comparative genomic hybridization (CGH)-based prenatal diagnosis for chromosome abnormalities using cell-free fetal DNA in amniotic fluid , 2006, Journal of Human Genetics.

[20]  David Bryder,et al.  Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR , 2006, Proceedings of the National Academy of Sciences.

[21]  E. Mansfield,et al.  Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. , 1993, Human molecular genetics.

[22]  L. Levett,et al.  A large‐scale evaluation of amnio‐PCR for the rapid prenatal diagnosis of fetal trisomy , 2001, Ultrasound in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology.

[23]  B. Ward,et al.  Rapid prenatal diagnosis of 14 cases of triploidy using fish with multiple probes , 1995, Prenatal diagnosis.

[24]  W. Holzgreve,et al.  Molecular methods for rapid detection of aneuploidy. , 2005, Journal of applied genetics.

[25]  B. Brambati,et al.  Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples , 1999, Journal of medical genetics.

[26]  ACOG Practice Bulletin No. 88, December 2007. Invasive prenatal testing for aneuploidy. , 2007, Obstetrics and gynecology.

[27]  Stephen R. Quake,et al.  Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria , 2006, Science.

[28]  L. Westphal,et al.  Aneuploidy in the miscarriages of infertile women and the potential benefit of preimplanation genetic diagnosis. , 2008, Fertility and sterility.

[29]  D. Conrad,et al.  Global variation in copy number in the human genome , 2006, Nature.

[30]  J. Schouten,et al.  Rapid, high throughput prenatal detection of aneuploidy using a novel quantitative method (MLPA) , 2003, Journal of medical genetics.

[31]  S. Dhanjal,et al.  Rapid and simple prenatal diagnosis of common chromosome disorders: advantages and disadvantages of the molecular methods FISH and QF-PCR. , 2003, Reproduction.

[32]  W. Holzgreve,et al.  Novel real-time quantitative PCR test for trisomy 21. , 2002, Clinical chemistry.

[33]  M. Adinolfi,et al.  Rapid molecular method for prenatal detection of Down's syndrome , 1994, The Lancet.

[34]  S. Goodman,et al.  Counting alleles to predict recurrence of early-stage colorectal cancers , 2002, The Lancet.

[35]  W. Kuo,et al.  Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes. , 1991, American journal of human genetics.

[36]  B. Ward,et al.  MATERNAL CELL CONTAMINATION IN UNCULTURED AMNIOTIC FLUID , 1996, Prenatal diagnosis.

[37]  D. Bianchi,et al.  Microarray analysis of cell-free fetal DNA in amniotic fluid: a prenatal molecular karyotype. , 2004, American journal of human genetics.

[38]  Lori J Sokoll,et al.  Assessment of plasma DNA levels, allelic imbalance, and CA 125 as diagnostic tests for cancer. , 2003, Journal of the National Cancer Institute.

[39]  M. Adinolfi,et al.  Quantitative fluorescence polymerase chain reaction for the rapid prenatal detection of common aneuploidies and fetal sex. , 1997, American journal of obstetrics and gynecology.

[40]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .

[41]  D. Zwijnenburg,et al.  Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. , 2002, Nucleic acids research.

[42]  Tak Y. Leung,et al.  Digital PCR for the molecular detection of fetal chromosomal aneuploidy , 2007, Proceedings of the National Academy of Sciences.

[43]  Michael A. Choti,et al.  Counting alleles reveals a connection between chromosome 18q loss and vascular invasion , 2001, Nature Biotechnology.

[44]  D. Bianchi,et al.  Array‐CGH analysis of cell‐free fetal DNA in 10 mL of amniotic fluid supernatant , 2007, Prenatal diagnosis.

[45]  P. Stankiewicz,et al.  Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization , 2006, Genetics in Medicine.

[46]  K. Klinger,et al.  Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: clinical experience with 4,500 specimens. , 1993, American journal of human genetics.

[47]  A. Chao,et al.  Prenatally diagnosed balanced chromosome rearrangements: eight years' experience. , 2006, The Journal of reproductive medicine.

[48]  C. Ogilvie,et al.  Strategies for the rapid prenatal diagnosis of chromosome aneuploidy , 2004, European Journal of Human Genetics.

[49]  Stephen R Quake,et al.  Detection of aneuploidy with digital polymerase chain reaction. , 2007, Analytical chemistry.

[50]  K. Klinger,et al.  Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH). , 1992, American journal of human genetics.

[51]  Ramesh Ramakrishnan,et al.  Studying copy number variations using a nanofluidic platform , 2008, Nucleic acids research.