The advantages and disadvantages of real-time fluorescence quantitative PCR technology are as follows:

Advantages

• High Sensitivity: This technology can detect an extremely low copy number of nucleic acids. For example, in the early screening of tumors, it can detect the DNA released by a very small number of tumor cells into the blood, which is helpful for the early detection of tumors.
• Strong Specificity: By designing specific primers and probes, it can precisely target and amplify the target nucleic acid sequence. When detecting the novel coronavirus, for instance, it can specifically identify the specific gene sequence of the novel coronavirus, avoiding confusion with other viruses or the human body’s own genes.
• Accurate Quantification: It is capable of accurately determining the initial amount or relative expression level of the target nucleic acid in the sample. In gene therapy research, it can precisely measure the changes in the expression level of the target gene before and after treatment, evaluating the therapeutic effect.
• Fast and Efficient: The entire detection process can usually be completed within 2 to 4 hours, and there is no need for cumbersome subsequent operations from sample processing to obtaining the results. In large-scale nucleic acid screening for the novel coronavirus, it can quickly process a large number of samples and identify infected individuals in a timely manner.
• Real-time Monitoring: During the PCR amplification process, it can monitor the changes in the fluorescence signal in real time, presenting the reaction process intuitively. Researchers can thus understand the dynamic changes of gene amplification, providing a basis for optimizing experimental conditions.
• Wide Application Range: It can be applied in multiple fields such as gene expression analysis, pathogen detection, tumor marker detection, and genetic disease diagnosis. In the agricultural field, it can also be used to detect genetically modified components in crops and plant pathogens.

Disadvantages

• High Instrument Requirements: Professional real-time fluorescence quantitative PCR instruments are required. These instruments are expensive, usually ranging from hundreds of thousands to millions of yuan. Moreover, subsequent maintenance and calibration also require professional personnel and costs, which is a significant burden for some small laboratories or institutions with limited funds.
• High Detection Cost: In addition to the cost of the instrument, the consumables such as fluorescently labeled primers, probes, and special PCR reagents are relatively expensive. The cost increases significantly during large-scale detection, which may limit its application in some regions or projects.
• Susceptible to Interference: Impurities and inhibitors in the sample may affect the efficiency of the PCR reaction, and the quality and purity of the sample are required to be high. For example, substances like humic acid contained in soil samples may inhibit the PCR reaction, resulting in inaccurate results.
• Difficulty in Primer and Probe Design: To ensure specificity and accuracy, primers and probes need to be carefully designed for specific target sequences. For some highly variable viruses, such as the influenza virus, continuous optimization of primer and probe design is required to ensure the accuracy of detection.
• Platform Differences Exist: Real-time fluorescence quantitative PCR instruments of different brands and models have differences in detection principles, fluorescence signal collection, and analysis methods. The detection results are difficult to compare directly between different platforms. In multi-center studies, a large amount of calibration and verification work is needed to ensure the consistency of the results.