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Digital PCR Market Overview

DIGITAL POLYMERASE CHAIN REACTION

The global real-time PCR and digital PCR (dPCR) collective market size were valued at $2.25 billion as of 2018. The market will expand at a CAGR of 8.5% for the 2019 to 2026 forecast period. The high rate of occurrence of target diseases and rapid technological advancements like high performance as well as superior qPCR and dPCR systems will be among the key factors propelling the growth. According to Viresh Patel, the global marketing director of the Bio-Rad’s Digital Biology Group, digital PCR (dPCR) may finally solve the challenge concerning the accuracy in PCR diagnostics for the monitoring of chronic myeloid leukemia.

1. 0 OVERVIEW OF THE DIGITAL PCR MARKET

  • Countries in the Asia Pacific region, including China, are expected to contribute the highest demand/quota to the global growth of the dPCR (digital PCR) and qPCR (quantitative PCR) markets for the period between 2019 and 2024.
  • The dPCR and qPCR global market will reach $6.3 billion by 2024 from the value of $4.1 billion as of 2019. The growth rate will occur with a CAGR of 8.8% for the forecast time frame (2019 to 2024).

1.1 DESCRIPTION OF DIGITAL POLYMERASE CHAIN REACTION (dPCR)

  • Single-cell genetic approaches like digital polymerase chain reaction (dPCR) enable a detailed investigation of DNA that exists in individual microbial cells. Digital PCR can be used to probe specific/isolated bacteria obtained from the human body for the various “viruses they hosted.”
  • Every cellular life (all living things) and some viruses have specific DNA. Digital PCR leverages on the individual DNA of viruses, bacteria, pathogens, food, etc., to identify the cause of cancer even after the affected area or genes have mutated.
  • Digital PCR is used widely for DNA detection across several fields, such as in cancer biomarker screening, pathogen detection, gene expression analysis, and environmental as well as food presence monitoring.
  • According to the U.S. National Cancer Institute of the United States Department of Health and Human Services, (screen capture available here), some form of cancer gets triggered by excessive secretion of a protein called HER2. When secreted in reasonable amounts, HER2 controls/regulates cell growth; however, when secreted in quantity larger than average values, HER2 leads to cancerous cell growths. The use of dPCR can accurately estimate the quantity of HER2 in cancer tumors.
  • Leukemia is said to affect patients when cancerous tissues grow in their blood, bone marrow, and or lymphatic system. Experts believe that dPCR (described above) holds prospects for the monitoring and treatment of chronic myeloid leukemia and other forms of cancer. Among all types of blood cancers, i.e., lymphoma, leukemia, and lymphoma, leukemia alone accounts for about 36 % of all cases, according to the American Cancer Society.
  • In summary, Digital PCR is a process used to detect the presence of insignificant quantities of nucleic acids that are circulating or may have circulated in the blood of a host. Digital PCR also detects the presence of “DNA of infectious agents.”

1.2 WHO IS USING DIGITAL PCR/WHY IT IS USED

  • Digital PCR is used in clinical microbiology laboratories to absolutely quantify the presence of nucleic acids with improved precision and accuracy.
  • The use of dPCR assay helps in quantifying both the presence of human and HHV-6 DNA in samples. Once a causative virus DNA is not found in cancerous cells, physicians are appropriately guided to “avoid unnecessary treatment with antivirals.
  • Bio-Rad has digital PCR products, systems, and consumables for sensitive “DNA target detection.”
  • Bio-Rad has digital PCR products, information, and services offered in the United States of America, Europe, and Hong Kong (China).
  • Some users of digital PCR equipment include Alonso Puga of the Temple University School of Medicine (USA), Takayo Sasaki of the Florida State University, and David Dobnik of the National Institute of Biology (Slovenia), among other users/laboratory test service providers.
  • According to Select Science web, some users are utilizing dPCR products for cfDNA analysis, Clinical cancer diagnostics, analysis of gene and miRNA copy number, and for absolute “quantification of DNA copy number, CNV, quantitative PCR” to observe small changes.

2. 0 WHAT USERS SAY ABOUT DIGITAL PCR TECHNOLOGY

  • Bio-Rad’s QX200™ Droplet Digital PCR System which includes a droplet generator, laptop computer, droplet reader, software, and associated component consumables, for “EvaGreen or probe-based digital PCR applications” is rated 5-star on a scale of 5 by 18 users. Bio-Rad does not disclose the cost of the entire kit, and interested users need to contact Bio-Rad for the price.
  • Bio-Rad’s AutoDG Droplet Digital PCR System which is a probe-based digital PCR solution, includes an automated droplet generator, a QX200 droplet reader, a laptop computer, and QuantaSoft™ software. It is rated 5-star on a scale of 5 by one user.
  • Digital PCR laboratory equipment reviews made by users from the Temple University School of Medicine (USA), Florida State University, and the National Institute of Biology (Slovenia), reveal that Bio-Rads digital PCR system is easy to use, has sufficient after-sales service, and gives adequate value for money by rating the aspects with 4.7 (approximately 5-stars) on a scale of 5.
  • Overall, Bio-Rads dPCR products are rated 4.7 on a scale of 5.0 by 18 users/reviewers.
  • The Health Europa website reveals that dPCR offers higher sensitivity as well as accuracy in test results and “holds great promise” to advance human healthcare. Health Europa has been collecting the views of diagnostic experts across the globe through technology summits hosted in Europe and America.

3.0 FACTORS: DETERMINING WHICH PROVIDER’S PRODUCT TO PURCHASE

  • As of 2019, Bio-Rad released the “first FDA-Cleared Digital PCR System” and testing equipment for the monitoring and treatment of chronic myeloid leukemia. Bio-Rad is at the front of innovations in digital PCR testing products and consumables.
  • We have assumed that the status of a dPCR product such as any health agency clearance and certification details should be the first consideration when determining which provider’s dPCR product to purchase.
  • FDA clearance of a consumer medical device reveals that it has been reviewed and is considered safe for sale, purchase, and use in America.
  • Bio-Rad has several patents that relate to the Chinese, European, and American economies.
  • Bio-rad does not provide the price of its digital PCR devices and solutions on its website.

Digital PCR Process and Benefits

Some benefits of using dPCR to detect leukemia include the ability to measure nucleic acid concentrations within 10% and the ability to detect rare nucleic acid variants in the background of complex mixtures of wild-type (normal) molecules.

Helpful findings

dPCR Process

  • The technique of a qPCR process consists of end-point detection, absolute qualification, and quantitative assay.
  • “In qPCR, the amount of amplified DNA is measured at each cycle during the PCR reaction, i.e., in real-time. The ‘absolute’ quantity of target sequence is interpolated using a standard curve generated with a calibrator.”
  • The PCR cycle includes three steps: Denaturation of double-stranded DNA by heat, Annealing of primers to their complementary target DNA sequences and Extension of primers by a thermostable DNA polymerase.

dPCR Benefits

  • According to Virish Patel, the global marketing director for Bio-Rad’s Digital Biology Group, dPCR offers several advantages over real-time PCR for BCR-ABL monitoring, including the ability to measure nucleic acid concentrations within 10% and the ability to detect rare nucleic acid variants in the background of complex mixtures of wild-type (normal) molecules, among others.

The Key properties of digital PCR compared to real-time PCR.

Digital PCR: BioRad

BioRad’s QXDx BCR-ABL %IS Kit uses QXDx Droplet Digital PCR System in quantifying leukemia, specifically Chronic Myeloid Leukemia (CML). It takes 4.5 to 5.5 hours to complete a workflow. Bio-Rad also offers Expert Care plans for all its products which include services such as on-site installation, training, preventative maintenance, validation and calibration to repair services.

QXDx BCR-ABL %IS Kit for the Droplet Digital PCR System.

  • Among the dPCR products of Bio-Rad, the QXDx BCR-ABL %IS Kit is specific for quantifying leukemia.
  • The QXDx BCR-ABL %IS Kit is a digital PCR test that monitors the p210 BCR-ABL major translocation in the peripheral blood of patients with Chronic Myeloid Leukemia (CML)”.
  • It is run on Bio-Rad’s QXDx Droplet Digital PCR System.
  • It elevates chronic myeloid leukemia (CML) monitoring to a new level of sensitivity (0.001%IS, MR 5.0 with four wells), precision and reproducibility.
  • BioRad claims that their Droplet Digital PCR System workflow is simple and easy-to-use.

IN THE US:

  • Bio-Rad released its first FDA-cleared Digital PCR System and Test for monitoring Chronic Myeloid Leukemia on February 14, 2019.

IN EUROPE and CHINA:

  • On December 4, 2017, BioRad announced the launch of the QXDx BCR-ABL %IS Kit, the first CE-IVD digital PCR test that can monitor the molecular response of patients with chronic myeloid leukemia (CML) to therapy.
  • The kit is available for diagnostic use in Europe, Hong Kong, and New Zealand with its CE-IVD mark.
  • Browsing through the Bio-Rad China website, the product QXDx BCR-ABL %IS Kit is not available.

How it works

  • “The QXDx AutoDG ddPCR System and QXDx BCR-ABL %IS Kit represents the first-ever digital PCR solution that can monitor and directly quantitate the molecular response of patients with chronic myeloid leukemia under tyrosine kinase inhibitor therapy”.
  • There are 20,000 partitioned droplets and the droplets containing the target sequence are detected by fluorescence and scored as positive and those without are scored as negative.
  • Poisson statistical analysis of positive and negative droplets yields absolute quantification of sequence unlike the RT-PCR which relies on a standard curve.
  • Results are reported on the International Scale (IS).

WORKFLOW

How long it takes

Bio-Rad dPCR Certified Service Provider in the US

  • In the US, Centrillion Technologies (CA), UF — ICBR Gene Expression & Genotyping (FL), and ATCC (VA) are the three certified service providers of BioRad for its Droplet Digital PCR.

Bio-Rad Support

  • Bio-Rad customer support includes product support document, tutorials, webinars, customer care, and technical support.
  • For their dPCR, Bio-Rad has a Certified Service Provider Program where they offer authorized use of ddPCR for contract research, verification of Bio-Rad’s standard of quality, and co-marketing partnerships and highest quality data for customers.
  • Bio-Rad also have the Expert Care Program where they offer a wide range of solutions from on-site installation, training, preventative maintenance, validation and calibration to repair services.
  • Bio-Rad also provides online documents and articles related to dPCR in their Learning Center such as the Planning Droplet Digital™ PCR Experiments.

Digital PCR: RainDance

RainDance Technologies offers an ultra-sensitive droplet-based system, the RainDrop Plus™ dPCR (Digital PCR) System that can generate millions of droplets from source sample DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). The system features increased sensitivity capabilities for use in critical applications, including expression analysis, copy number variation, and in detecting rare mutants that demand high sensitivity and have small amounts of sample available. Included below are elaborate findings regarding the typical workflow process in dCPRs, time taken to detect targets, and training resources offered.

RAINDDANCE’s dPCR WORKFLOW PROCESS FOR DETECTING LEUKEMIA

  • The first step involves generating droplets from a sample source, i.e., RNA or DNA samples. Then, the samples are partitioned into millions of Picoliter droplets to enable a highly accurate and sensitive nucleic acid detection. The process generates millions of droplets allowing single PCR (polymerase chain reactions).
  • The next phase involves thermal cycling the reaction to break the emulsion and using a next-generation sequencing detector to sequence the sample. Alternatively, labs can maintain the sample and use RainDance’s sense instrument, a fluorescence detector capable of identifying wild type mutants and empty droplets.
  • The third stage involves analyzing the droplets and grouping them accordingly using RainDance’s RainDrop Analyst II Software. In this stage, the exact number of copies of a DNA (deoxyribonucleic acid) molecule in the droplet is then calculated based on the presence or absence of fluorescence in each partition.
  • The dPCR platform uses Poisson statistics to establish the distribution of target DNA molecules among the PCRs. At limiting dilution, most reactions would contain one or zero target DNA molecules. Therefore, increasing the number of partitions elevates the likelihood of augmenting sensitivity for the detection of minimal amounts of targets.
  • RainDance’s RainDrop Plus Digital PCR System is capable of generating up to 10,000,000 droplets with the application of Poisson statistics only possible when the target molecules exceed 1,000,000.
  • In cases where large amounts of target molecules are needed, such as when quantitating small levels of virus in a high host background, the RainDrop Analyst II Software provides the option of Poisson statistics.
  • Overall, digital PCR technology is highly suitable for examining rare events and analyzing large samples efficiently and cost-effectively, because the data is reproducibly available across different industries’ end-users, instruments, and laboratories.

TIME TAKEN TO DETECT TARGETS

STAFF TRAINING REGARDING PRODUCTS

Digital PCR Sensitivity and Resolution

dPCR’s sensitivity impacts quantification of leukemia cells by improving the sensitive techniques to measure residual CML cells in low levels of patient samples providing information about when the leukemia patients can suspend their TKI-therapy without CML recurrence. We could not find information on the benefits of higher or lower sensitivity and how sensitivity can be maximized with dPCR and the comparison of dPCR’s sensitivity to its resolution and the pros and cons of each. Below are our helpful findings and detailed methodology.

dPCR’s impact on the quantification of leukemia cells.

  • According to Chinmay Seth, the marketing manager at Clinical Diagnostics at Bio-Rad Laboratories, the quantitative PCR or qPCR method is the current standard of measurement, but it is not accurate enough with low levels of BCR-ABL.
  • Due to this, the quantitative PCR or qPCR method for CML patients with very low or undetectable levels of BCR-ABL1 does not provide information about when to stop the administration of TKI-therapy without CML recurrence.
  • This has increased the need for more sensitive and specific techniques to measure residual CML cells such as the ddPCR method.
  • Research efforts like these will help pave the way for digital PCR (dPCR) to become a new standard for monitoring chronic myelogenous leukemia (CML).

HELPFUL FINDINGS

Benefits of higher or lower sensitivity

  • The qPCR method is not accurate below-down to one copy in 10,000 normal copies from the patient samples, lower sensitivity presents some disadvantages such as poor sensitivity, low sensitivity, bone marrow only, does not detect other clonal events, and poor standardization among others.
  • The above disadvantages can be prevented/corrected by the use of ddPCR method to quantify concentration low as 1 copy in 100,000 ref copies.
  • In contrast, there are also some advantages which consist of being a method widely available, standardized, fast, and very sensitive, at higher levels.
  • Recently, a new method qPCR-based is under the FDA review, the digital PCR or dPCR method is sensitive enough to detect rare DNA sequences.
  • But unlike qPCR, this will enable a pathologist to quantify a target sequence absolutely, without the need for a reference curve, meaning that ddPCR can quantify BCR-ABL concentrations as low as one copy in 100,000 reference copies, which means that this method is more sensitive and accurate than qPCR.

Compare dPCR’s sensitivity to its resolution and the pros and cons of each.

  • The dPCR does not need a standard curve to provide an absolute quantification.
  • On the other hand, when the sensitivity increases along with the number of partitions and the volume of the sample, it can reduce the precision of measurement.

Digital PCR and Next Generation Sequencing

Next-generation sequencing and dPCR both reduce the errors caused by PCR. However, the two technologies are designed to work together rather than compete. It does not appear that next-generation sequencing technology will disrupt the dPCR market.

DIGITAL PCR AND NEXT GENERATION SEQUENCING

  • One of the first methods of sequencing was the Sanger method developed in the 1970s. Sanger can be inaccurate, and the technique will miss certain sequences under a specific threshold.
  • The Human Genome Project developed a new sequencing method in the 1990s and 2000s that sequenced strands in parallel, which increased efficiency. This technique, using newer and more powerful computers, could sequence extremely quickly.
  • The next generation of sequencing techniques started in earnest with companies like Solexa that improved on the Human Genome Project’s work.
  • Single molecule sequencing” (SMS) is the latest sequencing technique. SMS examines single molecules rather than an entire strand at once.

IMPLICATIONS FOR PCR

  • PCR is the general term for amplifying (i.e., copying) DNA sequences. PCR techniques introduce artifacts during amplification, which improvements in both sequencing and PCR correct.
  • Quantitative PCR (qPCR) and Digital PCR (dPCR) are two modern PCR techniques. QPCR performs PCR in realtime while dPCR breaks PCR into smaller steps; both increase the accuracy of PCR.
  • SMS accurately sequences a single molecule, which reduces the effects of PCR amplification. SMS is thus an alternative way to increase the accuracy of PCR without requiring new PCR techniques.
  • However, dPCR and next-generation sequencing techniques are not competitors. Workflows often try to integrate both technologies to increase accuracy. SMS can accurately sequence DNA while dPCR amplifies sequences with fewer artifacts.
  • The two systems work together. The technologies are not intended to disrupt each other’s market.
TDM

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