Its detection principle mainly involves the following aspects:

I. Basic Principles of PCR

The PCR technique is the basis of qPCR. Its basic principle is similar to the natural replicationprocess of DNA, mainly consisting of three basic reaction steps: denaturation, annealing, and extension.

• Denaturation: Under high – temperature conditions (usually 90 – 95°C), the hydrogen bonds of the double – stranded DNA template break, dissociating into two single – stranded DNA molecules, providing conditions for the subsequent binding of primers to the template.
• Annealing: The reaction temperature is lowered (generally between 50 – 65°C), and the primers specifically bind to the complementary sequences of the single – stranded DNA template. Primers are short single – stranded DNA fragments synthesized artificially, which determine the starting position and specificity of PCR amplification.
• Extension: At an appropriate temperature (usually 70 – 75°C), DNA polymerase uses the single – stranded DNA as a template and dNTPs (deoxyribonucleoside triphosphates) as raw materials to synthesize a new DNA strand starting from the 3′ end of the primer according to the principle of base – pairing.

These three steps are continuously cycled. Each cycle approximately doubles the number of target DNA fragments. After n cycles, the number of target DNA fragments can theoretically be amplified to 2ⁿ times.

II. Principles of Fluorescent Signal Generation

In qPCR, the generation of fluorescent signals is the key to achieving quantitative detection. There are two common ways to generate fluorescent signals:

(I) Fluorescent Dye Method

• SYBR Green I: This is a commonly used fluorescent dye that can bind to the minor groove of double – stranded DNA. In the free state, SYBR Green I emits almost no fluorescence. However, when it binds to double – stranded DNA, the fluorescent signal is significantly enhanced. During the extension stage of the PCR reaction, as new double – stranded DNA is synthesized, SYBR Green I continuously binds to the double – stranded DNA, and the fluorescent signal increases accordingly. By detecting the intensity of the fluorescent signal, the accumulation of PCR products can be monitored in real – time. The advantages of this method are simple operation and low cost. It can bind to any double – stranded DNA and is suitable for the detection of multiple target genes. The disadvantage is its lack of specificity. It can bind not only to the target PCR products but also to non – specific amplification products, which may lead to false – positive results.

(II) Fluorescent Probe Method

• TaqMan Probe: The TaqMan probe is an oligonucleotide probe. Its 5′ end is labeled with a fluorescent reporter group (such as FAM), and its 3′ end is labeled with a fluorescent quencher group (such as TAMRA). When the probe is intact, the fluorescence emitted by the reporter group is absorbed by the quencher group, and no fluorescent signal can be detected. When the PCR reaction proceeds to the extension stage, the 5′ – 3′ exonuclease activity of Taq DNA polymerase hydrolyzes the TaqMan probe bound to the template, separating the reporter group from the quencher group, thus emitting a fluorescent signal. The intensity of the fluorescent signal is proportional to the number of PCR products. By detecting the change in the fluorescent signal, quantitative detection of the target gene can be achieved. The advantage of the TaqMan probe method is its high specificity. It can effectively distinguish between target and non – target genes, reducing false – positive results. The disadvantage is that the design and synthesis of the probe are costly, and it can only be used to detect specific target genes.

III. Quantitative Principles

In qPCR, the commonly used quantitative methods are relative quantification and absolute quantification:

(I) Relative Quantification

Relative quantification is to determine the relative expression level of the target gene in different samples by comparing the expression levels of the target gene and the reference gene (such as β – actin, GAPDH, etc., whose expressions are relatively stable in different samples) in different samples. The 2⁻ΔΔCt method is usually used for calculation, where ΔCt = Ct(target gene) – Ct(reference gene), and ΔΔCt = ΔCt(experimental group) – ΔCt(control group). Through this method, the expression differences of genes under different treatment conditions, in different tissues, or in different cells can be analyzed.

(II) Absolute Quantification

Absolute quantification is to determine the initial copy number of the target gene in the sample by constructing a standard curve. First, prepare standard products with known copy numbers, perform qPCR amplification on them, and obtain the Ct values (the number of cycles when the fluorescent signal reaches the set threshold) corresponding to different concentrations of the standard products. Use the logarithm of the copy number of the standard product as the abscissa and the corresponding Ct value as the ordinate to draw a standard curve. Then, perform qPCR amplification on the unknown sample, and find the corresponding copy number on the standard curve according to its Ct value, thus achieving absolute quantification of the target gene in the unknown sample.