The following is a detailed introduction:

As an important molecular biology detection method, Quantitative Real – time Polymerase Chain Reaction (qPCR) is widely used in various fields. It has advantages such as high sensitivity and strong specificity, but also has drawbacks like expensive instruments and high requirements for experimental personnel.

Advantages

• High Sensitivity
  This technology can detect nucleic acid templates with extremely low copy numbers. Even if there is only a small amount of target DNA or RNA in the sample, through the exponential amplification effect of PCR and the precise detection of fluorescent signals, quantitative analysis of trace nucleic acids can be achieved. For example, in the early stage of virus infection, when the virus nucleic acid content in blood or tissue samples is extremely low, qPCR can still accurately detect it.
• Strong Specificity
  By designing specific primers and fluorescent probes, it can precisely identify the target nucleic acid sequence. Only the specific gene fragment is amplified and detected, effectively avoiding interference from non – specific amplification and ensuring the accuracy of the test results. Taking the detection of the SARS – CoV – 2 virus as an example, primers and probes can be designed targeting its unique gene sequence to accurately distinguish this virus from other similar viruses.
• Accurate Quantification
  During the PCR amplification process, the change of fluorescent signals is monitored in real – time. Based on the cycle number (Ct value) when the fluorescent signal reaches the set threshold, the initial nucleic acid template content in the sample can be accurately calculated. It can achieve accurate quantification of nucleic acids within a wide dynamic range, precisely measuring from a few copies to millions of copies.
• Fast and Efficient
  The entire experimental process can usually be completed within 2 – 3 hours. There is no need for cumbersome subsequent operations such as electrophoresis detection as in traditional PCR, which greatly shortens the detection time and improves work efficiency. It is very suitable for scenarios such as clinical diagnosis and epidemic monitoring that require rapid results.
• Good Repeatability
  Experimental results are relatively less affected by human and environmental factors. As long as the experimental conditions are properly controlled, the experimental results among different laboratories and operators have high consistency and repeatability, providing reliable data support for scientific research and clinical diagnosis.
• High – throughput
  Combined with automated instruments and software, multiple samples can be detected and analyzed simultaneously. One experiment can detect dozens or even hundreds of samples, meeting the needs of large – scale screening and detection. It has great advantages in disease census, genotyping and other research.

Disadvantages

• Expensive Instrumentation
  The qPCR instrument is a sophisticated high – tech device, usually priced from several hundred thousand to over a million yuan. Moreover, the supporting reagents and consumables are relatively expensive, resulting in high detection costs. For some small laboratories or units with limited economic conditions, it may be difficult to afford.
• High Requirements for Sample Quality
  The nucleic acids in the sample need to be intact and of high purity. If the sample is contaminated, degraded, etc. during collection, transportation, or processing, it will seriously affect the accuracy and reliability of the test results. For example, if RNA is degraded during extraction due to improper operation, accurate test results cannot be obtained.
• Influence of Inhibitors
  There may be some substances in the sample, such as hemoglobin, heparin, polysaccharides, etc., which can inhibit the activity of Taq enzyme, thus affecting the PCR amplification efficiency and causing deviations in the test results. When processing complex samples, strict sample pretreatment is required to remove inhibitors.
• High Technical Difficulty
  The experiment involves multiple aspects such as primer and probe design, reaction system optimization, instrument operation, and data analysis. Any problem in any link may affect the experimental results. Therefore, experimental personnel are required to have high professional knowledge and rich operating experience.
• Limited Detection Range
  Although qPCR has a relatively wide dynamic detection range, for some samples with extremely high or low nucleic acid content, it may exceed its linear detection range. Multiple dilution or concentration operations on the sample are required, increasing the complexity of the experiment and errors.
• Difficulty in Detecting Unknown Sequences
  This technology relies on known nucleic acid sequences to design primers and probes. For nucleic acids with unknown sequences or newly emerging pathogens, sequence determination and analysis need to be carried out first before corresponding detection methods can be established. This somewhat limits its ability to quickly detect unknown pathogens.