The “New Coronary Virus Pneumonia Diagnosis and Treatment Program (Trial Ninth Edition)” uses nucleic acid detection Ct value ≥35 as one of the important basis for release of isolation management or discharge. So, what does the Ct value of the nucleic acid detection kit represent? Are the Ct values of different kits comparable? Does the lower the Ct value, the better the performance of the kit?
Ct value (Threshold Cycle, Ct) is the number of PCR cycles when the intensity of real-time fluorescence quantitative fluorescence signal exceeds the set threshold. Nucleic acid detection kits take the new crown as an example. For the same reaction of the same kit to detect two virus-containing samples, A and B, the size of the Ct value represents, to a certain extent, the number of copies of the viral gene, that is, the viral load. The lower the Ct of sample B, the higher the viral load. The Ct value is inversely proportional to the viral load and its infectivity. The pros and cons of different real-time fluorescence quantitative kits cannot be judged solely by the size of the Ct value, because the Ct values of different kits are not comparable. The performance evaluation of the kit needs to be measured from the aspects of sensitivity (minimum detection limit), specificity, precision, stability, and diagnostic application. Each reagent manufacturer has its own eighteen kinds of martial arts, and they have their own magical powers in application, so I won’t go into details here.
For conventional real-time quantitative PCR (QPCR) analysis instruments/systems, the acquisition of Ct values and their widespread use in pathogen detection applications are familiar to everyone. With the development of the industry and the advancement of technology, QPCR equipment and reagents have encountered opportunities and challenges. Yiou Think Tank (2021 Research Report on China’s Genetic Testing Industry: Technology) analyzed that most companies in my country’s QPCR industry are facing common problems based on technology. In response to these problems, the solutions are mainly divided into two types: accelerating technology improvement and deploying new technologies.
POCT for QPCR molecular diagnosis is one of the development directions of the industry, and the improvement of performance is an important factor to accelerate the application of the technology. For POCT equipment with single-sample throughput of QPCR, how to optimize the Ct value of the key parameter of a single reaction in R&D? The same equipment, the same reaction system, and the same template concentration, theoretically the larger the Ct value of a single reaction, the lower the efficiency of the enzyme in the reaction.
In the process of improving the efficiency of the enzyme reaction and optimizing the Ct value, a typical 40 PCR cycle fluorescence quantitative amplification curve can be analyzed first. The amplification curve in the figure below is divided into fluorescence background phase, exponential amplification phase, linear phase, and plateau phase. In order to achieve a lower fluorescence background, as far as possible to distinguish it from the fluorescence value of early exponential amplification, it is necessary to test from the reaction consumables carrier, primer probe design screening, and reaction buffer system. The exponential amplification period is the most direct reaction of enzyme activity, and it is also the proof of the perfect coordination of POCT equipment, reaction carriers and reagents. At this time, equipment temperature control, optical signal acquisition and analysis, carrier biocompatibility, and system performance of reagents need to be adjusted and tested in all aspects. Finally, the entire amplification is completed, and the results must be presented by a rigorous data algorithm to obtain a reasonable Ct value.

A small step in the optimization of the Ct value is the countless steps of the R&D personnel. In the anxiety and excitement of the day and night, every time “the mountains and rivers are nowhere to be seen, the willows are dark and the flowers are bright and there is another village”, which gives us the courage and passion to move forward again.