dPCR
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The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.
- Klíčová slova
- AAV, RT-qPCR, biodistribution, cell therapy, cellular kinetics, dPCR, gene therapy, qPCR, shedding, transgene expression,
- MeSH
- genetická terapie * MeSH
- polymerázová řetězová reakce MeSH
- tkáňová distribuce MeSH
- vyvíjení léků * MeSH
- Publikační typ
- časopisecké články MeSH
A microfluidic-based digital polymerase chain reaction (dPCR) chip requires precise temperature control as well as uniform temperature distribution to ensure PCR efficiency. However, measuring local temperature and its distribution over thousands of μL/nL-volume samples with minimum disturbance is challenging. Here, we present a method of non-contact localized temperature measurement for determination of the non-uniformity of temperature distribution over a dPCR chip. We filled the dPCR chip with a PCR solution containing amplified DNA fragments with a known melting temperature (T M). We then captured fluorescent images of the chip when it was heated from 70 to 99 °C, plotted the fluorescence intensity of each partition as a function of temperature, and calculated measured T M values from each partition. Finally, we created a 3-D map of the dPCR chip with the measured T M as the parameter. Even when the actual T M of the PCR solution was constant, the measured T M value varied between locations due to temperature non-uniformity in the dPCR chip. The method described here thereby characterized the distribution of temperature non-uniformity using a PCR solution with known T M as a temperature sensor. Among the non-contact temperature measurement methods, the proposed T M-based method can determine the temperature distribution within the chip, instead of only at the chip surface. The method also does not suffer from the undesirable photobleaching effect of fluorescein-based temperature measurement method. Temperature determination over the dPCR chip based on T M allowed us to calibrate the temperature sensor and improve the dPCR configuration and precision. This method is also suitable for determining the temperature uniformity of other microarray systems where there is no physical access to the system and thus direct temperature measurement is not possible.
- Publikační typ
- časopisecké články MeSH
BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection.
- Klíčová slova
- BCR-ABL1 monitoring, CML, chronic myeloid leukemia, Chronic myeloid leukemia, Digital PCR, EAC, Europe Against Cancer, FPR, false positivity rate, GeneXpert BCR-ABL Monitor assay, IS, international scale, LOB, limit of blank, LOD, limit of detection, MR, molecular response, NTC, no template control, RT-qPCR, RT-qPCR, reverse-transcription quantitative PCR, TKI, tyrosine kinase inhibitors, cDNA, complementary DNA, dPCR, digital PCR, pDNA, plasmid DNA, qPCR, quantitative PCR,
- Publikační typ
- časopisecké články MeSH
Lipid nanoparticle-messenger RNA (LNP-mRNA) drug products are a growing class of drug modalities. The unique composition of these drug products requires multiple measurements to account for the different components of these drug modalities. Pharmacokinetic (PK) measurements include measurement of the encapsulated mRNA and components of the LNP in circulation to understand the effectiveness of the therapeutic mRNA. The PK measurements can utilize many different platforms including PCR. Current regulatory guidance documents for bioanalytical method validation are specific to ligand binding and chromatographic assay methods and difficult to interpret for use with molecular workflows. The purpose of this paper is to provide information on considerations for validation of regulated reverse transcription quantitative PCR (RT-qPCR) assays that are used to support the pharmacokinetic analysis of LNP-mRNA drug products.
- Klíčová slova
- LNP, MRNA, PK, RT-dPCR, RT-qPCR,
- MeSH
- kvantitativní polymerázová řetězová reakce * metody MeSH
- lidé MeSH
- lipidy * chemie farmakokinetika MeSH
- liposomy MeSH
- messenger RNA * farmakokinetika aplikace a dávkování MeSH
- nanočástice * chemie MeSH
- polymerázová řetězová reakce s reverzní transkripcí * metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Lipid Nanoparticles MeSH Prohlížeč
- lipidy * MeSH
- liposomy MeSH
- messenger RNA * MeSH
The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from ≈5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent channel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.
- Klíčová slova
- Absolute quantification, Digital polymerase chain reaction, Duplexing methods, Single fluorescence channel duplexing,
- MeSH
- barvicí látky * MeSH
- polymerázová řetězová reakce MeSH
- vyšetření u lůžka * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- barvicí látky * MeSH
Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.
- Klíčová slova
- ACMG/AMP classification, BRCA2, dPCR, functional analysis, quantitation, splicing,
- MeSH
- alternativní sestřih MeSH
- geny BRCA2 * MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- místa sestřihu RNA * MeSH
- myši MeSH
- protein BRCA2 genetika metabolismus MeSH
- sestřih RNA MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- BRCA2 protein, human MeSH Prohlížeč
- messenger RNA MeSH
- místa sestřihu RNA * MeSH
- protein BRCA2 MeSH
The digital polymerase chain reaction (dPCR) multiplexing method can simultaneously detect and quantify closely related deoxyribonucleic acid sequences in complex mixtures. The dPCR concept is continuously improved by the development of microfluidics and micro- and nanofabrication, and different complex techniques are introduced. In this review, we introduce dPCR techniques based on sample compartmentalization, droplet- and chip-based systems, and their combinations. We then discuss dPCR multiplexing methods in both laboratory research settings and advanced or routine clinical applications. We focus on their strengths and weaknesses with regard to the character of biological samples and to the required precision of such analysis, as well as showing recently published work based on those methods. Finally, we envisage possible future achievements in this field.
We demonstrate a smartphone integrated handheld (SPEED) digital polymerase chain reaction (dPCR) device for point-of-care application. The device has dimensions of ≈100 × 200 × 35 mm3 and a weight of ≈400 g. It can perform 45 PCR cycles in ≈49 min. The device also features integrated, miniaturized modules for thermal cycling, image taking, and wireless data communication. These functions are controlled by self-developed Android-based applications. The only consumable is the developed silicon-based dPCR chip, which has the potential to be recycled. The device's precision and accuracy are comparable with commercial dPCR machines. We have verified the SPEED dPCR prototype's utility in the testing of severe acute respiratory syndrome coronavirus 2, the detection of cancer-associated gene sequences, and the confirmations of Down syndrome diagnoses. Due to its low upfront capital investment, as well as its nominal running cost, we envision that the SPEED dPCR device will help to perform cancer screenings and non-invasive prenatal tests for the general population. It will also aid in the timely identification and monitoring of infectious disease testing, thereby expediting alerts with respect to potential emerging pandemics.
- MeSH
- biosenzitivní techniky * MeSH
- chytrý telefon MeSH
- COVID-19 * diagnóza MeSH
- lidé MeSH
- nádory * MeSH
- polymerázová řetězová reakce MeSH
- testování na COVID-19 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The digital polymerase chain reaction (dPCR) is an irreplaceable variant of PCR techniques due to its capacity for absolute quantification and detection of rare deoxyribonucleic acid (DNA) sequences in clinical samples. Image processing methods, including micro-chamber positioning and fluorescence analysis, determine the reliability of the dPCR results. However, typical methods demand high requirements for the chip structure, chip filling, and light intensity uniformity. This research developed an image-to-answer algorithm with single fluorescence image capture and known image-related error removal. We applied the Hough transform to identify partitions in the images of dPCR chips, the 2D Fourier transform to rotate the image, and the 3D projection transformation to locate and correct the positions of all partitions. We then calculated each partition's average fluorescence amplitudes and generated a 3D fluorescence intensity distribution map of the image. We subsequently corrected the fluorescence non-uniformity between partitions based on the map and achieved statistical results of partition fluorescence intensities. We validated the proposed algorithms using different contents of the target DNA. The proposed algorithm is independent of the dPCR chip structure damage and light intensity non-uniformity. It also provides a reliable alternative to analyze the results of chip-based dPCR systems.
- MeSH
- algoritmy MeSH
- DNA * genetika MeSH
- počítačové zpracování obrazu * MeSH
- polymerázová řetězová reakce MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA * MeSH
Quantitative PCR (qPCR) is a widely used method for nucleic acid quantification of various pathogenic microorganisms. For absolute quantification of microbial load by qPCR, it is essential to create a calibration curve from accurately quantified quantification standards, from which the number of pathogens in a sample is derived. Spectrophotometric measurement of absorbance is a routine method for estimating nucleic acid concentration, however, it may be affected by presence of other potentially contaminating nucleic acids or proteins and salts. Therefore, absorbance measurement is not reliable for estimating the concentration of stock solutions of quantification standards, based on which they are subsequently diluted. In this study, we utilized digital PCR (dPCR) for absolute quantification of qPCR plasmid standards and thus detecting possible discrepancies in the determination of the plasmid DNA number of standards derived from UV spectrophotometry. The concept of dPCR utilization for quantification of standards was applied on 45 qPCR assays using droplet-based and chip-based dPCR platforms. Using dPCR, we found that spectrophotometry overestimated the concentrations of standard stock solutions in the majority of cases. Furthermore, batch-to-batch variation in standard quantity was revealed, as well as quantitative changes in standards over time. Finally, it was demonstrated that droplet-based dPCR is a suitable tool for achieving defined quantity of quantification plasmid standards and ensuring the quantity over time, which is crucial for acquiring homogenous, reproducible and comparable quantitative data by qPCR.
- Klíčová slova
- absolute quantification, digital PCR, qPCR, quantification plasmid standard, quantity verification, real time PCR,
- Publikační typ
- časopisecké články MeSH
