qPCR Primer Probes

Definition

1.qPCR (Quantitative Polymerase Chain Reaction) - Quantitative Polymerase Chain Reaction

*commonly known as real-time fluorescence quantitative PCR.

*It is a technique that uses fluorescent signals to monitor and quantify DNA/RNA templates in real time during the PCR amplification process. It not only determines the presence of the target gene (qualitative) but also accurately calculates its initial content (quantitative).

2.Primer

*A primer is a short single-stranded DNA fragment.

*It specifically recognizes the ends of the target DNA sequence to be amplified and serves as the starting point for DNA replication. Think of it as a "hook" that specifically latches onto the target fragment to be replicated.

3.Probe

*A probe is also a short single-stranded DNA fragment, typically designed between two primers.

*One end of the probe is linked to a reporter (such as FAM) and the other to a quencher.

*When the quencher and reporter groups are in close proximity (i.e., on an intact probe), the fluorescence emitted by the reporter group is absorbed by the quencher, and the instrument cannot detect the fluorescent signal.

*During PCR, DNA polymerase encounters the probe during DNA replication and uses its exonuclease activity to hydrolyze and cleave the probe. This separates the reporter and quencher groups, and the fluorescence emitted by the reporter group is no longer quenched, allowing it to be detected by the instrument.

In summary:

qPCR primer probes are a set of carefully validated primer pairs and fluorescently labeled probes designed for real-time quantitative PCR (qPCR) experiments. Working together, they ensure the specific, sensitive, and quantitative detection of extremely low levels of specific gene sequences in a sample.

The Role of qPCR Primers and Probes

1.Specificity: The sequences of primers and probes are designed based on unique regions of the target gene, ensuring that only the desired target (such as a viral gene) is amplified and detected, without misdetecting other unrelated genes, thus avoiding false positives.

2.Signal Generation: The probe is key to qPCR quantitative detection. With each amplified target DNA strand, a probe is hydrolyzed, releasing a fluorescent signal. Therefore, the intensity of the fluorescence is proportional to the amount of PCR product.

3.Precise Quantification: The instrument monitors changes in the fluorescence signal in real time throughout the PCR cycle. The greater the initial template amount in the sample, the smaller the number of cycles required for the fluorescence signal to reach the set threshold (Ct value). By comparing with a standard of known concentration, the copy number of the target gene in the original sample can be accurately calculated.

Disease-Related

qPCR primer-probe technology is relevant to nearly all diseases involving pathogen infection and gene expression, and has extremely wide applications.

1.Infectious Disease Testing (Most Well-Known Applications):

*Viral Diseases: COVID-19 (COVID-19), influenza, HIV/AIDS, hepatitis B (HBV), hepatitis C (HCV), Ebola, dengue fever, etc. "Nucleic Acid Testing" during the pandemic involves the use of qPCR primers and probes to detect SARS-CoV-2 RNA.

*Bacterial Diseases: Tuberculosis, gonorrhea, chlamydia, sepsis pathogen identification, etc.

*Other Pathogens: Malaria, fungal infections, etc.

2.Genetic Disease Diagnosis:

Used to detect gene mutations, deletions, or duplications associated with genetic diseases. Examples include thalassemia, hemophilia, Duchenne muscular dystrophy, and hereditary breast cancer (BRCA1/2 genes).

3.Cancer:

*Tumor Marker Testing: Detects overexpression of genes associated with specific cancers (e.g., AFP mRNA in liver cancer).

*Minimal Residual Disease Monitoring: Detects minimal residual cancer cells in the body after treatment to assess the risk of recurrence.

*Gene Mutation Testing: Detects mutations in cancer driver genes (e.g., EGFR, KRAS) to guide targeted drug therapy.

4.Autoimmune Diseases and Scientific Research:

Used to study changes in the expression levels of specific genes during disease progression. While not a direct diagnosis, this technology is crucial for understanding disease mechanisms and developing new drugs.

Application Scenarios

1.Clinical Diagnosis:

Hospital Laboratory Departments/Third-Party Testing Laboratories: Conduct pathogen nucleic acid testing, genetic disease screening, tumor genotyping, etc. This is currently the primary application scenario.

2.Disease Prevention and Control and Public Health:

CDC (Centers for Disease Control and Prevention): Conduct epidemic monitoring, source tracing, outbreak investigations, and strain typing for infectious diseases.

3.Scientific Research:

University/Institute Laboratories: Conduct gene expression analysis (to determine whether a gene is up- or down-regulated under specific conditions), genotyping, microRNA analysis, and epigenetic studies (such as DNA methylation).

4.Food Safety and Animal Quarantine:

*Detect pathogenic microorganisms (such as Salmonella and E. coli) in food.

*Detect genetically modified ingredients.

*Ports of entry conduct pathogen quarantine on animals and plants to prevent the introduction of foreign diseases.

5.Forensic Science:

DNA fingerprinting is used for individual identification and paternity testing.