bitter is de pil Linos Vandekerckhove, MD, PhD

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1 4//24 Current HIV care HIV copies/ ml plasma Viral load Welcome to the Digital droplet PCR age! bitter is de pil Linos Vandekerckhove, MD, PhD Latent HIV reservoir Time at Ghent University Hospital 2 HIV Life Cycle Content: Principles of digital droplet PCR Plasma RNA Applicability in clinical biology HIV mrna Unspliced RNA Limitations of ddpcr Future directions Spliced RNA Conclusions Entry mrna reverse transcription cdna Integration: proviral DNA Episomal DNA LTR 2LTR plasma cytoplasm nucleus 3 4 PCR, or how to count rabbits! Principle of ddpcr ) Real time PCR 2) Digital PCR 5 6

2 4//24 PCR, or how to count rabbits! 7 8 Real time PCR Real time PCR, major Drawbacks Fluorescence PCR Cycles Indirect quantification: Need for standard curve Need for validated reference material Influence of efficiency: Variation when reactions are inhibited 9 Reaction efficiency qpcr Standard input Fluorescence 8 6 8% % Cycles Plasmid in water Spiked plasmid in gdna. ng/µl ng/µl ng/µl ng/µl ng DNA = equivalent of 5 cells 2 2

3 4//24 Conclusion Real time PCR Digital PCR Great quantification tool However, hampered by high variability in the low ranges More accuracy and sensitivity requiered for the next generation of HIV diagnostics 3 4 Digital PCR Digital PCR 5 Percent positives 8/3=.267 Average amount of rabbits per island (ʎ) ʎ=-ln(-P),2,4,6,8 Percent positive islands (P) 5 6 Microfluidics Digital PCR 7 8 3

4 4//24 Advantages Direct quantification: - No standard curve - LOD = LOQ End point PCR: - Less susceptible to inhibition - Higher flexibility in assay design Sinlge molecule PCR: - Colocalisation of mutations/genes on one DNA strand 9 2 Dilution series This is more representative of a -fold dilution series in ddpcr Quantification of 2LTR across dilution series n= Applicability in clinical biology Examples Digital PCR applications - Copy number variation (CNV) - Rare sequence detection - Mutation detection - Gene expression analysis of rare transcripts - mirna analysis - Next-generation sequencing sample quantification - Diagnosis of ocular Chlamydia trachomatis infections - Quantification of RNA viruses in surface water samples 2 - HER2 expression in breast cancer samples 3 - CMV template quantitation in PCR inhibition-prone stool samples 4 - Detection of KRAS mutations in plasma of colorectal cancer patients associated with poor prognosis 5 - Quantitation of donor DNA in transplant recipients as a surrogate marker of graft injury and rejection 6 Bio-Rad () Roberts et al. 23, (2) Racki et al. 24, (3) Heredia et al. 23, (4) Sedlak et al. 24, (5) Taly et al. 23, (6) Beck et al. 23 4

5 4//24 Comparison of ddpcr and qpcr for enumeration of Cryptosporidium oocysts in faecal samples - Quantitation of DNA without the need for calibration curves - Higher reproducibility - ddpcr is less sensitive to inhibitors than qpcr - The overall cost of ddpcr was two times higher than qpcr. : Yang et al : Yang et al Limitations of ddpcr ) DdPCR inhibition due to: ) DdPCR inhibition 2) False positives 3) Sample preparation 4) Differentiation of positive population droplets - Genomic DNA concentration - Inhibition observed when more than 63 ng of gdna loaded per ddpcr well - Lower droplet formation observed above 3 µg gdna input (Strain et al., 23) - Restriction buffer - The proposed improvement of sensitivity and reproducibility is not yet fully proven: need of thorough evaluation and optimization - Needed for gdna sample preparation prior ddpcr - Inhibition observed above 2 µl restriction buffer added in ddpcr well ) False positives - Independent groups reported positive signals in NTCs - Detected randomly, not assay-dependent and with different fluorescence height - An average of between. and.4 false positive events per well were estimated, analyzing over 5 NTC wells (Strain et al., 23) - Several NTCs with 3 positive events were reported (Kiselinova et al., 24)

6 4//24 Reference: Kiselinova et al Reference: Kiselinova et al ) False positives 3) Sample preparation - Current ddpcr technique does not allow sequencing of the samples to establish whether the false positives represent artefacts - The issue of false positive events in ddpcr and consequent limitation of ddpcr quantification needs to be further investigated, especially for quantification of low abundant templates - has not yet been fully evaluated, especially in patient derived samples - Example: 2-LTR circles quantification: - a modified plasmid DNA isolation has resulted in higher 2-LTR recovery - it enables more cell equivalents to be loaded per ddpcr well - more accurate and sensitive compared to conventional genomic DNA isolation

7 4//24 4) Differentiation of positive population droplets - Not trivial to differentiate the events as positive or negative based on measured fluorescence values - Especially important for low-template quantification - The need of further optimization for setting up the thresholds 37 Reference: Trypsteen et al Future directions Future directions Next generation ddpcr: QX2 Droplet Digital PCR System Benefits: - Optimized for EvaGreen or TaqMan Hydrolysis Probe-based quantification of target DNA or RNA molecules - Flexible ddpcr chemistry and assay setup - Droplet partitioning reduces bias from amplification efficiency and PCR inhibitors Reference: Bio-Rad 39 4 Conclusion DdPCR is a new promising tool in molecular diagnostics Major advantage is the quantification in the low range The technology is hampered by false positive droplets Currently, the technology is in being validated in research settings

8 4//24 HTRU 43 8

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