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Frontiers of Science丨Demystifying Digital PCR

The novel coronavirus (SARS-CoV-2) pandemic has led to a public health crisis1. Delta and Lambda SARS-CoV-2 variants have appeared in more than 90 countries around the world2. How to quickly and accurately diagnose the virus, especially the variant strain, is of great significance to the treatment and epidemic prevention work. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most widely used COVID-19 detection technology worldwide3. In the epidemic prevention and control work, different nucleic acid detection technologies have also been rapidly developed, including digital PCR (dPCR), Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) and Transcription-Mediated Amplification (TMA). The dPCR is an emerging nucleic acid amplification technique that allows absolute quantification of nucleic acids4. It has the advantages of high sensitivity, high precision and strong anti-interference ability.

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01/What is Digital PCR?

Digital PCR technology, also known as the third-generation PCR technology, is a new method for highly sensitive detection and absolute quantification of nucleic acids5. Compared with traditional PCR, dPCR adds the operation of partitioning the reaction system, separating the reaction system (10 µl) into tens of thousands of tiny independent reaction systems. The nucleic acid template is sufficiently diluted during this separation process. Ideally, each droplet contains 1 molecule of the nucleic acid template. After the amplification is completed, the fluorescent signals of all droplets are identified and counted, and the number of negative and positive reactions is calculated. Finally, the concentration of the target molecule is calculated by the Poisson distribution principle, thereby realizing the absolute quantification of the target molecule. Digital PCR does not rely on standard curve quantification and is not affected by PCR amplification efficiency, with higher sensitivity and accuracy.

Accordingly, dPCR is particularly well suited for applications that require the detection of small amounts of input nucleic acid or the finer resolution of target amounts among samples. For example, rare sequence detection, copy number variation (CNV) analysis, and gene expression analysis of the rare targets.

02/The History of digital PCR

The dPCR rose out of an approach first published in 1988 by Cetus Corporation when researchers showed that a single copy of the β-globin gene could be detected and amplified by PCR6. This was achieved by diluting DNA samples from a normal human cell line with DNA from a mutant line having a homozygous deletion of the β-globin gene until it was no longer present in the reaction.

In 1999, Bert Vogelstein et al. first proposed the concept of digital PCR (dPCR) in PNAS, Proceedings of the National Academy of Sciences, and showed that the technique could be used to study rare cancer mutations.

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♦ 2003

Kinzler and Vogelstein continued to refine dPCR and created an improved method they called the BEAMing technique, an acronym for “beads, emulsions, amplification, and magnetism.” The BEAMing technology uses an emulsion to separate amplification reactions in a single tube. This change made it possible for scientists to scale the method to thousands of reactions in a single run. With the development of nanofabrication and microfluidics, digital PCR technology has encountered the best opportunity to break through the bottleneck.

♦ 2006

Fluidigm introduced the first commercially available chip-based digital PCR system. The IFC platform of Fluidigm uses a physical matrix strategy, and their qdPCR 37K system “integrated circuit” leverages the company’s microfluidics expertise to individually distribute 48 samples across 770 micro reaction units.

♦ 2013

Fluidigm Corporation introduced the first commercially available chip-based digital PCR system. The IFC platform of Fluidigm uses a physical matrix strategy, and their qdPCR 37K system “integrated circuit” leverages the company’s microfluidics expertise to combine 48 Each sample is distributed in 770 micro reaction units one by one.

♦ Now

The basic methods of dPCR have been established. Based on the different forms of reaction units, they can be mainly divided into three types of systems: microplate, microchamber and droplet. At present, the main digital PCR products on the market are droplet-based dPCR and chip-based dPCR, such as Bio-Rad’s QX200 droplet system and Thermo Fisher’s Quant Studio system.

03/Advantages of digital PCR

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Digital PCR is one of the most dramatic innovations in the life sciences. Compared with other traditional molecular diagnostic techniques, the advantages of digital PCR technology are:

• High sensitivity

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dPCR essentially turns a traditional PCR reaction into tens of thousands of PCR reactions, and the target sequences are independently detected in these tens of thousands of reaction units, thereby greatly improving the detection sensitivity.

• Unparalleled precision

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Accurately detecting low-level mutant genes in the presence of a large number of wild-type genes is one of the current research difficulties. Competitive reactions seriously affect the detection of mutant genes. The massive sample partitioning afforded by dPCR enables the reliable measurement of small fold differences in target DNA sequence copy numbers among samples. Therefore, the dPCR technology is particularly suitable for detecting rare mutations in complex backgrounds.

• Absolute quantification 

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The dPCR technology provides an absolute count of target DNA copies per input sample without the need for running standard curves, making this technique ideal for measurements of target DNA, viral load analysis, and microbial quantification. 

• High reproducibility

The data for the mean and CV values of FAM, HEX and Cy5 channel concentrations indicated that there was no statistical difference (p<0.05) between the three tested concentrations under the experimental conditions described.

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• Strong tolerance

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Blood, feces, food, soil and other samples contain a large number of inhibitors of PCR reaction, which greatly affects the efficiency of PCR reaction. In addition, in some assays, it is difficult to prepare the standard material required to determine the standard curve. The advantages of dPCR being unaffected by PCR inhibitors and independent of standard curves make it particularly suitable for accurate quantitative detection of gene expression in these complex samples.

04/Application field

The scientific community is increasingly demanding data accuracy and reliability of results. More laboratories are also beginning to pay attention to digital PCR technology with absolute quantification, high sensitivity and high reproducibility. As an emerging technology for the absolute quantification of target nucleic acids, dPCR is highly sensitive and specific to low-abundance DNA and highly resistant to amplification inhibitors. Therefore, dPCR can be used for the detection of low-copy viruses, the determination of viral load, the preparation of standard materials, the monitoring of virus concentration in the environment, the detection of virus mutation, and the evaluation of SARS-CoV-2 drugs.

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Vazyme offers more dPCR series products to meet different needs of customers:

NO.

Product Name

Cat.No.#

1.

Ace Taq DNA Polymerase

P401-MD1

2.

Cham Taq DNA Polymerase

P122-MD2

3.

Taq Pro HS DNA Polymerase for ddPCR

PN102

4.

HiScript II Reverse Transcriptase

R201

 

 

 

Reference

1. Choi H, Cho W, Kim M H, et al. Public health emergency and crisis management: case study of SARS-CoV-2 outbreak[J]. International journal of environmental research and public health, 2020, 17(11): 3984.

2. Liu H, Wei P, Zhang Q, et al. The Lambda variant of SARS-CoV-2 has a better chance than the Delta variant to escape vaccines[J]. BioRxiv, 2021.

3. Rauch J N, Valois E, Ponce-Rojas J C, et al. Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase–Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students[J]. JAMA network open, 2021, 4(2): e2037129-e2037129.

4. Tan C, Fan D, Wang N, et al. Applications of digital PCR in COVID‐19 pandemic[J]. View, 2021, 2(2): 20200082.

5. Morcia C, Ghizzoni R, Delogu C, et al. Digital PCR: what relevance to plant studies?[J]. Biology, 2020, 9(12): 433.

6. Saiki, Randall K., et al. “Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.” Science 239.4839 (1988): 487-491.

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