Real-time Quantitative Polymerase Chain Reaction Instrument

The real – time quantitative polymerase chain reaction (qPCR) instrument, also known as the real – time PCR instrument, is an advanced device for nucleic acid quantitative analysis. It is widely used in fields such as life science research, clinical diagnosis, and disease monitoring. Here is a detailed introduction:

Working Principle

• Fluorescent Chemistry Principle
  The real – time quantitative PCR instrument uses fluorescent dyes or fluorescent – labeled probes to indicate the amount of PCR amplification products. Commonly used fluorescent dyes such as SYBR Green I can bind to double – stranded DNA. During the PCR reaction, as double – stranded DNA is continuously synthesized, the fluorescence signal intensity is proportional to the amount of PCR products. Fluorescent – labeled probes, such as TaqMan probes, have higher specificity. They bind specifically to the target DNA sequence. During PCR amplification, the 5’→3′ exonuclease activity of Taq enzyme cleaves the probe, separating the fluorescent reporter group from the quencher group, thus generating a fluorescence signal.
• Real – time Monitoring Principle
  In each cycle of the PCR reaction, the instrument continuously monitors the fluorescence signal. As the PCR reaction progresses, the fluorescence signal gradually increases. By detecting and analyzing the fluorescence signal, the progress of the PCR reaction can be understood in real – time, and the copy number of the initial template can be accurately determined.

Instrument Structure

• Optical System
  It includes an excitation light source and a fluorescence detection system. The excitation light source usually uses a halogen – tungsten lamp, light – emitting diode (LED), etc., to provide excitation light for the fluorescent dyes or probes. The fluorescence detection system consists of components such as a photomultiplier tube (PMT) and a charge – coupled device (CCD), which are used to capture and measure the fluorescence signal.
• Thermal Cycling System
  It is composed of a heating module, a refrigeration module, and a temperature control system. It can rapidly and accurately achieve the different temperature conditions required for the PCR reaction, ensuring the specificity and high – efficiency of the PCR reaction.
• Sample Module
  It is used to place PCR reaction tubes or microplates. Usually, it has good heat – conduction performance to ensure uniform heating of the samples.

Technical Features

• High Sensitivity
  It can detect nucleic acid templates with extremely low copy numbers, enabling accurate detection of trace amounts of pathogen nucleic acids or genes with low expression levels.
• High Specificity
  Through the specific recognition of fluorescent – labeled probes and strict control of reaction conditions, it can effectively distinguish the target nucleic acid from other similar sequences, reducing false – positive results.
• Precise Quantification
  It can perform absolute or relative quantification analysis of nucleic acid templates, providing accurate data support for disease diagnosis, drug research and development, etc.
• High – throughput
  It can detect multiple samples simultaneously at one time, greatly improving the detection efficiency and being suitable for large – scale clinical tests and scientific research experiments.

Application Fields

• Clinical Diagnosis
  It is used for pathogen detection, such as the detection of SARS – CoV – 2, hepatitis B virus, human immunodeficiency virus, etc. It can also be used for the detection of tumor – related genes, assisting in the early diagnosis, prognosis assessment, and treatment monitoring of tumors.
• Disease Monitoring
  During the disease treatment process, by monitoring the changes in pathogen load or gene expression levels, the treatment effect can be evaluated, and clinical medication can be guided.
• Genetic Analysis
  It is used for genotyping, gene mutation detection, single – nucleotide polymorphism (SNP) analysis, etc., playing an important role in the diagnosis of genetic diseases, prenatal diagnosis, etc.
• Drug Research and Development
  During the drug research and development process, it can be used to evaluate the impact of drugs on gene expression, screen drug targets, and monitor gene changes during drug treatment.

Brands and Models

• Roche LightCycler 480
  It has an excellent optical system and thermal cycling performance, high detection sensitivity, and accurate and reliable results. It is suitable for a variety of application scenarios.
• ABI 7500
  It is widely used, easy to operate, and has a powerful software function. It can conduct various real – time quantitative PCR experiments and is highly recognized in both clinical and research fields.
• Bio – Rad CFX96
  It has the characteristics of high – throughput and high sensitivity, supports multiple fluorescence detection modes, and has real – time monitoring and data analysis functions, meeting the needs of different users.