PCR (Polymerase Chain Reaction) is a molecular biology technique used to amplify specific DNA fragments. With the development of biological and photoelectric signal technology, PCR has technically realized real-time monitoring of the amplification reaction process of DNA fragments, that is, real-time fluorescence quantitative PCR technology. It refers to adding fluorescent substances to the PCR reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and quantitatively analyze unknown templates.

01 The concept of quantitative PCR technology

Quantitative PCR technology has a broad concept and a narrow concept. Quantitative PCR technology in a broad sense refers to the quantification of the amount of PCR initial template by analyzing the final product of PCR or monitoring the PCR process as the standard with external or internal reference.

Quantitative PCR technology under the broad concept can be divided into five types:

1）External reference method + final product analysis. The so-called "external reference method" refers to the reaction of the sample and the positive reference in two reaction vessels. This type of sample has no quality control monitoring, which is prone to false negative and false positive results, which does not monitor the amplification efficiency, will resulting in inaccurate quantification.

2）Internal reference method + final product analysis. The so-called "internal reference method" refers to the reaction between the sample and the positive reference in a reaction vessel. This type of quality control monitoring of samples to exclude false negative results, but the quantification is not accurate.

3）External standard method + process monitoring. This type of monitoring of amplification efficiency allows accurate quantification of positive samples, but false negative results cannot be ruled out.

4）Internal reference method + process monitoring. Since the sample and the positive reference are reacted in one container, and the same Taq enzyme and reaction participants are used, there is competitive inhibition. The reaction with a high concentration of starting template will inhibit the reaction with a low concentration of starting template, so the quantification is not accurate.

5）External standard method + process monitoring + internal control. This type monitors amplification efficiency, quantifies positive samples accurately, and excludes false negative results. This type should be advocated, and it is also a commonly used technology in various scientific research and medical laboratories. However, there are also various detection modes for monitoring the PCR process.

02

Detection mode for PCR process monitoring

There are three detection modes most commonly used:

1）SYBR Green I detection mode. The temperature cycle is a three-step method of 94-55-72°C, with only primers, no probes, and fluorescent dyes embedded in the middle of the double-stranded helix. The signal is obtained by strong fluorescence detection in a specific direction. This reagent detection mode is prone to generate non-specific signals and has a large background light.

2）Probe mode (Taqman) Hydrolysis Probe. The temperature cycle is a two-step method of 94-60°C, and there are not only primers, but also another probe specific for the amplification template between the primer pairs. Two fluorescent dyes are bound to two adjacent bases of the probe, one dye receives the excitation light and the energy is transferred to the second dye, the second dye that receives the energy returns to the stable state by emitting characteristic photons. When the Taq enzyme extends the amplified chain at 60°C, it encounters the probe, and uses the 5'-3' exonuclease activity of the Taq enzyme to hydrolyze the probe into a single base, the distance between the single bases is relatively long. The energy of one dye cannot be transferred to the second dye, so it has to return to a stable state by emitting characteristic photons, and obtain a signal by fluorescence detection of the first dye in solution. This reagent detection mode increases the specificity of the detection signal, but due to the use of the 5'-3' exonuclease activity of Taq enzyme, the general reagent manufacturer only calibrates the polymerase activity of Taq enzyme, and does not give Taq enzyme 5' at the same time. - 3' exonuclease activity calibration, different batches of reagents will bring differences in quantification. In addition, the melting point temperature (Tm) of the probe is only required to be higher than 60 °C, which makes the specificity of different kits uneven, and cannot be used for quality control detection.

3）Hybridization Probes mode. The temperature cycle is a three-step method of 94-55-72°C, with primers, two adjacent probes specific for the amplification template are between the primer pairs, and a fluorescent dye is combined on the 3' base of one probe. A second fluorescent dye is attached to the 5' base of the other probe. At 55°C, both probes are just bound to the template, the energy from the excitation light of the first dye is transferred to the second dye, and the second dye that accepts the energy returns to the stable state by emitting characteristic photons, the signal is obtained by fluorescent detection of the second dye in the dual probe bound to the amplification template. In this reagent detection mode, the fluorescent signal is related to a specific hybridization temperature, and the concentration of the probe remains unchanged, so the melting curve can be detected after amplification as a specific quality control for the signal. In addition, this reagent detection mode can be used for point mutation detection.

# spacegen

K-ras Gene Mutations Detection Kit（Real Time PCR）

Detection Significance

1. Inoperable NSCLC patients should be tested for KRAS gene mutation before systemic treatment, and treatment should be guided according to molecular typing. If tissue samples are not available, circulating tumor DNA can be considered.

2. KRAS gene mutation detection was performed before neoadjuvant chemotherapy in the potentially resectable group of metastatic colorectal cancer or before systemic treatment in the palliative care group. The patients with KRAS mutation were resistant to cetuximab, panitumumab, and anti-HER2 therapy.

Performance Parameter