Real-time fluorescent quantitative PCR (qPCR) technology is widely used in the field of pet detection and other areas, possessing numerous advantages, but it also has some disadvantages and limitations. The details are as follows:

Advantages

• High Sensitivity: It is capable of detecting an extremely low number of nucleic acid copies. Even if there is only a small amount of pathogen nucleic acid or target gene in the sample, it can be accurately detected. This is helpful for identifying infections at the early stage of a disease when the number of pathogens is still very small, providing a basis for early intervention and treatment.
• High Specificity: By designing specific primers and probes, qPCR can detect specific pathogen or gene sequences. It can accurately distinguish between similar pathogens or genes, effectively avoiding false positive results and improving the accuracy of detection.
• Accurate Quantification: It can not only qualitatively determine whether the target nucleic acid exists in the sample but also precisely measure its content. It can accurately quantify the pathogen load, gene expression level, etc., providing important quantitative indicators for disease diagnosis, treatment monitoring, and prognosis assessment.
• Fast and Efficient: The entire detection process is relatively quick, usually completed within 2 to 4 hours. Compared with traditional detection methods such as the culture method, it greatly shortens the detection time, which is beneficial for rapid diagnosis and timely treatment.
• High Throughput: It can detect multiple samples simultaneously and process a large number of samples in a short time. It is suitable for large-scale pet disease screening, vaccine efficacy evaluation, and other tasks, improving detection efficiency and reducing costs.

Disadvantages

• High Requirements for Samples: The collection, preservation, and transportation of samples must be strictly standardized. Otherwise, it may lead to nucleic acid degradation, affecting the accuracy of the detection results. For example, if a blood sample is not separated into serum in a timely manner or a fecal sample is not properly preserved, the quality of the nucleic acid may decline.
• High Cost of Instruments and Reagents: qPCR requires the use of specialized real-time fluorescent quantitative PCR instruments, and the price of these instruments is expensive, usually ranging from hundreds of thousands to millions of yuan. At the same time, the price of supporting reagents is also relatively high, and different detection items require the use of different reagents, increasing the detection cost.
• Existence of Technical Errors: Errors in sample addition during the experimental operation process, slight differences in the reaction system, etc., can all affect the repeatability and accuracy of the detection results. In addition, errors may also be introduced in links such as the design of primers and probes and the detection of fluorescent signals.
• Requires Professional Personnel to Operate: This technology involves professional knowledge in multiple aspects such as molecular biology and instrument operation. Operators need to undergo specialized training and be familiar with the experimental process and instrument operation. Otherwise, operational errors are likely to occur, affecting the detection results.

Limitations

• Can Only Detect Known Sequences: qPCR can only design primers and probes for detection based on known pathogen or gene sequences. If encountering new and unknown pathogens or gene mutations, it may not be able to carry out effective detection. It is necessary to redesign the primers and probes and establish and verify the methodology.
• Difficulty in Detecting Mixed Infections: When a pet has a mixed infection of multiple pathogens, qPCR may not be able to accurately distinguish and quantify each pathogen. Because the detection of different pathogens may interfere with each other, affecting the accuracy of the detection results.
• Cannot Completely Replace Other Detection Methods: Although qPCR has unique advantages in pathogen detection, genetic analysis, and other aspects, for the diagnosis of some diseases, it is still necessary to combine other methods such as clinical symptoms, pathological examination, and serological testing for comprehensive judgment. The diagnosis cannot be made solely based on the qPCR results.