dPCR Primer Probes

Definition

dPCR (digital PCR)

①It is the third-generation PCR technology and a major innovation following conventional PCR and qPCR (real-time fluorescence quantitative PCR).

②A traditional PCR reaction system is evenly distributed into tens of thousands or even millions of tiny droplets or reaction wells, each unit acting as an independent "microreactor."

③Key Advantage: Through this "divide and conquer" strategy, dPCR can directly and absolutely quantify the number of target molecules (without the need for a standard curve) and has extremely high sensitivity, enabling the detection of extremely low amounts of nucleic acids (such as rare mutations and trace amounts of pathogens).

Primer Probe

This is a commonly used detection element in qPCR technology and is also widely used in dPCR. It is usually referred to as a TaqMan probe.

Composition:

1.Primers: A pair of short, single-stranded DNA fragments that specifically recognize the target DNA sequence and initiate amplification.

2.Probe: An even shorter, labeled DNA sequence. It is labeled at both ends with:

*A reporter (e.g., FAM): emits a fluorescent signal at one end.

*A quencher (quencher): suppresses the reporter's emission when the two come into contact.

*Working Principle: During PCR amplification, the Taq enzyme degrades the probe as it extends the DNA strand, separating the reporter and quencher groups and releasing a fluorescent signal. Each target molecule amplified generates a fluorescent signal.

In summary:

dPCR primer probes are a highly specific set of primers and probes designed for digital PCR (dPCR) experiments. Their purpose is to enable dPCR technology to accurately and specifically detect and absolutely quantify extremely low amounts of target nucleic acid (DNA or RNA) in a sample.

The Role of dPCR Primers and Probes

In a dPCR system, the core functions of primer probes include:

*Specific Recognition: Primers precisely bind to the ends of the target gene sequence being detected, ensuring that only the nucleic acid segment of interest (e.g., a gene in the novel coronavirus or a mutation site in cancer) is amplified.

*Signal Generation and Amplification: Probes convert the invisible nucleic acid amplification process into a detectable fluorescent signal. Each amplified target molecule emits a fluorescent signal.

*Achieving Absolute Quantification: This is the most powerful feature of dPCR. After the reaction is complete, the device scans each droplet/well. Units that emit fluorescence are labeled "positive," while those that do not are labeled "negative." By counting the proportion of positive droplets and applying the Poisson distribution statistical model, the absolute copy number of the target molecule in the original sample (e.g., the number of copies per milliliter) can be directly calculated, completely eliminating the need for a standard curve.

Disease-Related

DPCR primer-probe technology, due to its ultra-high sensitivity, plays a vital role in the research and diagnosis of a variety of diseases, particularly in the following situations::

1. Cancer

①Liquid biopsy: Detecting circulating tumor DNA (ctDNA) in the blood. ctDNA is typically present at very low levels, and dPCR can accurately detect rare mutations (such as EGFR, KRAS, and BRAF mutations) in ctDNA, enabling early cancer screening, medication guidance, efficacy monitoring, and drug resistance detection.

②Cancer genomics: Studying gene copy number variations (CNVs) and low-abundance fusion genes.

2. Infectious diseases

①Precise viral load quantification: Particularly suitable for scenarios requiring extremely high-precision monitoring of viral levels, such as therapeutic efficacy assessment for HIV, HBV (hepatitis B virus), and HCV (hepatitis C virus).

②Rare pathogen detection: Detecting extremely low levels of pathogens in the presence of high background nucleic acid, such as in early-stage or latent infections.

3.Genetic Diseases

①Prenatal Diagnosis: Non-invasive prenatal testing (NIPT) is performed by analyzing cell-free fetal DNA (cffDNA) in maternal peripheral blood to screen for chromosomal aneuploidies such as Down syndrome. This method is safer and more accurate than traditional methods.

②Monogenic Genetic Diseases: Detecting small differences in allele frequency for carrier screening and disease typing.

4.Other Areas

Organ Transplant Rejection Monitoring: Detecting donor-derived cell-free DNA (dd-cfDNA) provides early warning of transplant rejection.

Application Scenarios

dPCR primer probes have a wide range of applications, primarily in cutting-edge fields such as scientific research and clinical diagnostics:

1.Rare target detection

When the target sequence is present at extremely low levels (<0.1%) in a sample, making it difficult to detect using conventional qPCR, dPCR is the tool of choice. For example, monitoring trace amounts of cancer cell DNA remaining after cancer treatment (minimal residual disease, MRD).

2.Scenarios requiring absolute quantification

①The precise copy number, rather than relative changes, is required. For example:

②Viral load standard calibration：Developing precise standard curves and standards for qPCR experiments.

③Gene expression studies：Accurately measuring transcript levels of low-expressed genes.

④Next-generation sequencing (NGS) result validation: Validating and accurately quantifying rare mutations detected by NGS.

3.Precise measurement in complex backgrounds

When samples contain numerous similar sequences or inhibitors that could affect qPCR efficiency, dPCR offers greater robustness to interference, resulting in more reliable results. For example, detecting low-frequency mutations in a background of abundant wild-type genes.

4.Development of molecular diagnostic products

As the core technology of IVD (in vitro diagnostic) products, it is used to develop highly sensitive early cancer screening and infectious disease detection kits.