Real-time quantitative PCR, the full name of Real-time Quantitative PCR (qPCR for short), is a molecular biology experimental technology that combines PCR amplification technology and fluorescence detection technology, and can perform real-time detection during the PCR amplification process. Monitor and quantify the copy number of specific DNA or RNA sequences.

1. Advantages and disadvantages of real-time quantitative PCR

advantage:

1. High sensitivity and specificity: qPCR can detect very low concentrations of nucleic acid molecules and accurately identify target sequences. This makes it particularly important in clinical diagnostics, such as detecting viral load.

2. Strong quantitative ability: Through the accumulation of fluorescence signals, the progress of the reaction can be monitored in real time, thereby achieving absolute or relative quantification.

3. Fast and efficient: Compared with traditional PCR, qPCR can complete the detection of a large number of samples in a shorter time, usually within one hour.

4. Intuitive data: Through real-time curve graphs, researchers can intuitively observe the amplification curve, which facilitates analysis and result interpretation.

shortcoming:

1. Higher cost: qPCR instruments and fluorescent dyes are relatively expensive, and the running costs are also high.

2. High technical requirements: operators need to have certain professional knowledge and skills to avoid experimental errors.

3. Susceptible to contamination: Due to its high sensitivity, sample contamination may lead to inaccurate results, so strict experimental condition control is required.

2. Equipment composition of real-time quantitative PCR

The real-time quantitative PCR system mainly consists of the following parts:

1. qPCR instrument: This is the core equipment, equipped with a fluorescence detection system, used to monitor changes in fluorescence signals during the PCR reaction.

2. Thermal cycler: Controls the temperature changes of the reaction system to achieve denaturation, annealing and extension of DNA.

3. Optical detection system: used to detect the fluorescence signal in the reaction tube and convert it into an electrical signal for analysis.

4. Software: used for data collection, processing and analysis, and generating results such as amplification curves and standard curves.

3. Operation method of real-time quantitative PCR

The operation method mainly includes the following steps:

1. Sample preparation: Extract the DNA or RNA in the sample, and convert the RNA into cDNA through reverse transcriptase.

2. Reaction system preparation: including the mixing of primers, probes or dyes, dNTPs, Taq enzymes, buffers and template nucleic acids.

3. Reaction condition setting: Set appropriate PCR reaction procedures according to specific experimental requirements, including pre-denaturation, cycle denaturation, annealing and extension steps.

4. Data collection and analysis: At the end of each cycle, the instrument will automatically record the fluorescence signal, and perform data analysis through the software to calculate the starting nucleic acid amount of the sample.

Real-time quantitative PCR has a wide range of applications in scientific research and clinical diagnosis. Although it has a certain cost and technical threshold, its unparalleled accuracy and efficiency make it an irreplaceable tool. For scientific researchers and medical workers, mastering the operating procedures and equipment usage of this technology is the key to improving experimental efficiency and data accuracy.