Fluorescence quantitative PCR instrument (Real-time Fluorescence Quantitative PCR

Fluorescence quantitative PCR instrument (Real-time Fluorescence Quantitative PCR Instrument) is an instrument widely used in the field of molecular biology. The following will provide a detailed introduction from several aspects including its principle, specific applications, characteristics, and precautions:

Principle

This instrument adds fluorescent groups to the PCR reaction system and uses the accumulation of fluorescent signals to monitor the entire PCR process in real time. Finally, quantitative analysis of unknown templates is performed through a standard curve. During the PCR amplification process, after each cycle, the fluorescent signal will increase correspondingly. By detecting the change of fluorescent signal, the amplification of DNA can be determined, thus realizing the quantification of the initial template.

Specific applications

• Pathogen detection: It can quickly and accurately detect the nucleic acids of pathogens such as viruses and bacteria. For example, the detection of the new coronavirus provides strong support for the early diagnosis and prevention and control of diseases.
• Gene expression analysis: Study the expression levels of specific genes in different tissues and cells under different physiological states or treatment conditions, which helps to deeply understand the function and regulatory mechanism of genes.
• Genetic disease diagnosis: Detect and quantitatively analyze mutations in genes related to genetic diseases to assist in clinical diagnosis and genetic counseling.
• Drug research and development: It can be used to study the influence of drugs on gene expression, evaluate drug efficacy and mechanism of action, and provide a basis for new drug research and development.

Characteristics

• High sensitivity and specificity: It can detect nucleic acid templates with extremely low copy numbers and has high specificity for the target sequence, reducing false positive and false negative results.
• Accurate quantification: Through real-time monitoring of fluorescent signals, accurate quantification of nucleic acid templates can be achieved, and the results are accurate and reliable.
• High throughput: Multiple samples can be detected simultaneously at one time, greatly improving detection efficiency and saving time and cost.
• High degree of automation: It has functions such as automatic sampling, reaction program setting, data acquisition and analysis. The operation is simple and reduces human errors.

Precautions

• Sample quality: The sample collection, preservation and processing processes must be carried out in strict accordance with the requirements to avoid sample degradation or contamination and affect the accuracy of the detection results.
• Reagent selection: High-quality and stable PCR reagents and fluorescent dyes should be selected to ensure the specificity and sensitivity of the reaction.
• Instrument maintenance: Regularly clean, calibrate and maintain the instrument, and check the performance of components such as the optical path system and temperature control system to ensure the normal operation of the instrument.
• Data analysis: Correctly set the threshold and baseline, and use appropriate data analysis methods to scientifically and reasonably interpret the experimental results and avoid misjudgment.