| Real-time quantitative PCR (qPCR) technology is to add fluorescent reporter groups and fluorescent quencher groups to the PCR reaction system to achieve quantitative detection of nucleic acid molecules through fluorescent signals. During the reaction, PCR products are continuously generated as the amplification reaction proceeds, and the fluorescence signal continues to increase. The entire PCR process can be monitored in real time through the changes in the fluorescence signal, and the original template can be quantitatively analyzed through the standard curve. This article will provide a detailed introduction from five aspects: the reaction stages of real-time fluorescence quantitative PCR, common parameters, quantitative analysis principles, common classifications, and the advantages and disadvantages of different methods. 1. Reaction stage The entire reaction process of real-time fluorescence quantitative PCR can be divided into Baseline, index, and plateau phases 3 stages:
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| 2. Common parameters |
In order to facilitate quantitative analysis, two concepts, fluorescence threshold and cycle threshold (Cyclethreshold, Ct), are introduced in qPCR reactions:
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| 3. Principle of quantitative analysis |
| In the reaction system, if a certain fluorescence threshold is designed, the larger the amount of the starting template, the smaller the number of cycles required to reach the fluorescence threshold, and the smaller the Ct value. During the experiment, a standard curve is drawn using standard samples with known starting copy numbers. Since the number of PCR amplification products of the sample to be tested corresponds to the fluorescence intensity emitted by the fluorescent group, as long as the Ct value of the sample to be tested is obtained through qPCR, the starting copy number of the sample to be tested can be calculated through the standard curve. |
| 4. Common categories |
| At present, qPCR can be divided into two categories according to the chemical principles of the fluorescent substances used and the specificity of PCR detection: |
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| DNA dye method |
| Fluorescent dyes can quickly incorporate into double-stranded DNA and bind to it, emitting fluorescent signals, while fluorescent dye molecules that are not incorporated into double-stranded DNA will not emit any fluorescent signals. They can be used to detect specific and non-specific PCR amplification products, such as SYBR Green I and Eva Green. |
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| Fluorescence probe method |
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It can be divided into Taqman probe method and molecular beacon probe method. ① The Taqman probe method is to add a specific fluorescent probe at the same time as a pair of primers during PCR amplification. The two ends of the probe are labeled with a reporter fluorescent group and a quencher fluorescent group respectively. When the probe is intact, the fluorescent signal emitted by the reporter group is just absorbed by the quencher group, and there is no fluorescent signal in the system; as the reaction proceeds, the probe is degraded by the 5'-3' exonuclease of the Taq enzyme, separating the reporter group from the quencher group, and the signal detection system receives the fluorescent signal. |
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②Molecular beacon is an oligonucleotide with a stem-loop structure formed by base complementarity. Because the fluorescent group and the quenching group are very close, the fluorescence is quenched. During the PCR amplification process, as the DNA double helix melts, the stem-loop of the beacon hybridizes with the exposed DNA target sequence, the complementary region is pulled apart, causing the distance between the fluorescent group and the quenching group to increase, and the fluorescence is restored. |
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5. Advantages and disadvantages of three qPCR methods |
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Qihengxing PCR/RT-PCR/qPCR product list |
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