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

Lou, Jiwu; Sun, Manna; Zhao, Ying; Ji, Zhisong; Li, Fenghua; Li, Dong-Zhi; Xu, Wanfang; Lin, Yangyang; Liu, Qing

doi:10.1371/journal.pone.0171886

Cited by 5 publications

(14 citation statements)

References 17 publications

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“…In clinical practice, quantitative fluorescence PCR (QF-PCR), fluorescent in situ hybridization (FISH), and multiplex ligation-dependent probe amplification (MLPA) are the most frequently used rapid, targeted aneuploidy detection techniques. FISH is an expensive, relatively labor-intensive technique and requires an overnight hybridization step, which delays getting the result [ 4 , 6 ]. PCR-based methods that involve the amplification of polymorphic microsatellite markers or ligated probes are more suited to high throughput testing.…”

Section: Discussionmentioning

confidence: 99%

“…NTCs included nuclease-free water used in reaction mixes. (5) and aneuploidy status determination (6). The hexagons represent the thousands of reaction wells on the chip.…”

Section: Workflow Of Chip-based Digital Pcrmentioning

confidence: 99%

“…Thus, a rapid and precise molecular method for aneuploidy detection is desirable. Currently, the most popular rapid diagnostics is based on real-time PCR [ 4 , 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

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Clinical Validation of Novel Chip-Based Digital PCR Platform for Fetal Aneuploidies Screening

2021

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Fetal aneuploidy is routinely diagnosed by karyotyping. The development of techniques for rapid aneuploidy detection based on the amplification reaction allows cheaper and rapid diagnosis. However, the currently available solutions have limitations. We tested a novel approach as a diagnostic tool in clinical practice. The objective of this study was to provide a clinical performance of the sensitivity and specificity of a novel chip-based digital PCR approach for fetal aneuploidy screening. The study was conducted in 505 pregnant women with increased risk for fetal aneuploidy undergoing invasive prenatal diagnostics. DNA extracted from amniotic fluid or CVS was analyzed for the copy number of chromosomes 13, 18, 21, X, and Y using a new chip-based solution. Performance was assessed by comparing results with findings from karyotyping. Aneuploidy was confirmed in 65/505 cases positive for trisomy 21, 30/505 cases positive for trisomy 18, 14/505 cases positive for trisomy 13 and 21/505 with SCAs. Moreover, 2 cases with triploidy and 2 cases with confirmed mosaicisms of 21 and X chromosomes were detected. Clinical sensitivity and specificity within this study was determined at 100% for T21 (95% CI, 99.26–100%), T18 (95% CI, 99.26–100%), and T13 (95% CI, 99.26–100%). Chip-based digital PCR provides equally high sensitivity and specificity in rapid aneuploidy screening and can be implemented into routine prenatal diagnostics.

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Section: Discussionmentioning

confidence: 99%

“…NTCs included nuclease-free water used in reaction mixes. (5) and aneuploidy status determination (6). The hexagons represent the thousands of reaction wells on the chip.…”

Section: Workflow Of Chip-based Digital Pcrmentioning

confidence: 99%

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Clinical Validation of Novel Chip-Based Digital PCR Platform for Fetal Aneuploidies Screening

2021

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“…FISH is a labor-intensive technique, relatively costly. In addition, it requires technical expertise and overnight hybridization step, which extends the procedure [7,8]. Compared to FISH, PCR-based methods are more suited to a high throughput diagnostic.…”

Section: Introductionmentioning

confidence: 99%

“…The MPLA method overcomes the potential problem with amplification of polymorphic microsatellite markers, yet overnight hybridization step precludes a single-day mode. In addition, both QF-PCR and MLPA methods require a post-PCR step for quantitative analysis of the products using an expensive genetic analyzer [8].…”

Section: Introductionmentioning

confidence: 99%

Chip-Based Digital PCR Approach Provides A Sensitive and Cost-Effective Single-Day Screening Tool for Common Fetal Aneuploidies—A Proof of Concept Study

Nykel

Kaszkowiak

Fendler

et al. 2019

IJMS

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In the prenatal period, the copy number aberrations of chromosomes 13, 18, 21, X and Y account for over 80% of the clinically significant chromosome abnormalities. Classical cytogenetic analysis is the gold standard in invasive prenatal diagnostics but the long test waiting time affects its clinical utility. Several molecular rapid tests have been developed and employed in clinical practice, however all have substantial drawbacks. The aim of the study was to design and evaluate an optimized tool for rapid molecular detection of fetal aneuploidies. We established a novel single-day method using a chip-based platform, the QuantStudio 3D Digital PCR system. In order to assess the clinical usefulness of our screening test, we analyzed 133 prenatal samples. The difference in distributions of euploid and aneuploid samples identified the ploidy of each of the target chromosomes with high precision. The distribution of the chromosome ratio for euploid and aneuploid samples showed a statistically significant result (p = 0.003 for trisomy 13, p = 0.001 for trisomies 18 and 21, Mann–Whitney U test). Our results suggest that this novel chip-based approach provides a tool for rapid, technically simple, cost-effective screening for common fetal aneuploidies.

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Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies

Chen

Huang

et al. 2021

EBioMedicine

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Background: Segmental duplication (SD) regions are distinct targets for aneuploidy detection owing to the virtual elimination of amplification bias. The difficulty of searching SD sequences for assay design has hampered their applications. Methods: We developed a computational program, ChAPDes, which integrates SD searching, refinement, and design of specific PCR primer/probe sets in a pipeline to remove most of the manual work. The generated primer/probe sets were first tested in a multiplex multicolour melting curve analysis for the detection of five common aneuploidies. The primer/probe sets were then tested in a digital PCR assay for the detection of trisomy 21. Finally, a digital PCR protocol was established to quantify maternal plasma DNA sequences for the non-invasive prenatal detection of fetal trisomy 21. Findings: ChAPDes could output 21,772 candidate primer/probe sets for trisomy 13, 18, 21 and sex chromosome aneuploidies within 2 working days. Clinical evaluation of the multiplex multicolour melting curve analysis involving 463 fetal genomic DNA samples revealed a sensitivity of 100% and specificity of 99.64% in comparison with the reference methods. Using the established digital PCR protocol, we correctly identified two trisomy 21 fetuses and thirteen euploid foetuses from the maternal plasma samples. Interpretation: The combination of ChAPDes with digital PCR detection could facilitate the use of SD as potential biomarkers for the non-invasive prenatal testing of fetal chromosomal aneuploidies.

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

Cited by 5 publications

References 17 publications

Clinical Validation of Novel Chip-Based Digital PCR Platform for Fetal Aneuploidies Screening

Clinical Validation of Novel Chip-Based Digital PCR Platform for Fetal Aneuploidies Screening

Chip-Based Digital PCR Approach Provides A Sensitive and Cost-Effective Single-Day Screening Tool for Common Fetal Aneuploidies—A Proof of Concept Study

Segmental duplication as potential biomarkers for non-invasive prenatal testing of aneuploidies

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