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Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry
Published on: June 21, 2018
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Clinically accessible amplitude-based multiplex ddPCR assay for tryptase genotyping.
Manca Svetina1,2, Julij Šelb1,3, Jonathan J Lyons4
1University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia.
Scientific Reports
|January 29, 2024
Summary
Hereditary alpha tryptasemia (HαT) testing is improved with a new multiplex ddPCR assay. This method accurately quantifies tryptase genes, offering significant cost and time savings for clinical use.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Hereditary alpha tryptasemia (HαT) is an autosomal dominant trait linked to TPSAB1 copy number.
- HαT is a potential biomarker for mastocytosis and anaphylaxis risk assessment.
- Current genetic testing for tryptase gene composition is limited due to locus complexity.
Purpose of the Study:
- To develop and validate a multiplex droplet digital PCR (ddPCR) assay.
- To reliably quantify alpha- and beta-tryptase encoding sequences in a single reaction.
- To provide a more efficient method for tryptase genotyping.
Main Methods:
- Optimization of ddPCR conditions including primers, probes, and thermal gradients.
- Testing various DNA quantities and primer/probe concentrations.
- Validation of the multiplex ddPCR assay against original duplex assays.
Main Results:
- A multiplex ddPCR assay was successfully developed and optimized.
- Results from the multiplex ddPCR assay were identical to duplex assays for all 114 samples.
- The assay reliably quantifies alpha- and beta-tryptase encoding sequences simultaneously.
Conclusions:
- The multiplex ddPCR assay offers a reliable and efficient method for tryptase genotyping.
- This assay reduces material costs by threefold and saves considerable time.
- The developed assay is highly suitable for routine clinical implementation in HαT determination.
