When interpreting the results of a fluorescent RT-PCR kit, the most straightforward way to assess the results is to check whether the amplification curve appears normal. Many researchers have been troubled by abnormal amplification curves. Let’s take a look at several typical examples of abnormal amplification curves and analyze them together. Now, let’s dive into some key knowledge about the detection using the swine fever virus real-time fluorescent RT-PCR kit!

　　 1. Normal amplification curve for fluorescent RT-PCR

　　As the experiment progresses, the amplified product accumulates continuously, and the fluorescence signal increases accordingly. The fluorescence signal is collected at each cycle; after a certain number of cycles (e.g., 40 cycles), the following amplification curve is obtained. The horizontal axis represents the number of amplification cycles, and the vertical axis represents the fluorescence intensity.

　　This also requires clarifying the following basic concepts.

　　Baseline: Refers to the period during the initial few cycles of a PCR amplification reaction when the fluorescence signal changes only slightly—approaching a nearly straight line. This straight line is called the baseline. Just understanding this concept is sufficient; it doesn't significantly affect experimental procedures.

　　Threshold Line: Typically, the fluorescence signal from the preceding 15 cycles is used as the background fluorescence signal. The threshold is set at 10 times the standard deviation of the fluorescence signal over 3 to 15 cycles, which corresponds to the exponential phase of PCR amplification (as indicated by the arrow in the figure).

　　 2. Standard Curve

　　What is the purpose of creating a standard curve? A standard curve can be used to determine the initial quantity of an unknown sample. When absolute quantification is required, a standard curve is essential.

　　The CT values of each template exhibit a linear relationship with the logarithm of the initial copy number of that template; the greater the initial copy number, the lower the CT value. The standard samples are diluted by a fixed multiple to yield approximately 5–6 concentrations, and then subjected to RT-PCR under the appropriate reaction conditions. A linear regression curve is plotted, with the logarithm of the standard sample copy number as the x-coordinate and the CT value as the y-coordinate. The copy number of unknown samples can be calculated based on the equation of this curve.

　　 3. Dissolution Curve

　　In short, it’s about examining the specificity of the dye-labeling method’s results. To put it even more simply, it’s about confirming whether the primers are effective in terms of minutes.

　　I won’t go into the details of the complex underlying principles. The key lies in how you interpret and assess the results. If the reaction produces a single product, the melting curve will show a sharp peak. However, if there’s dimer formation or nonspecific amplification, you’ll see at least two peaks—or even more pronounced deviations. When using SYBR Green dye, it’s essential to generate a melting curve; after all, this dye is inherently nonspecific.

　　In the experiment, everyone will still encounter many problems. Let me list a few common ones:

　　(1) After 40 cycles, does the amplification curve show no plateau phase? Or does the amplification curve fail to exhibit exponential growth? In this case, your initial template concentration is likely too low—most probably, the curve hasn't yet reached a plateau after 40 cycles. You can verify whether this is the issue by increasing the number of cycles.

　　(2) Regarding the melting curve, for example, are there multiple peaks, or does a sharp peak have smaller sub-peaks, or is there no peak at all? Since the melting curve is closely related to the primer and the reaction system’s temperature, if there’s no peak at all, it’s likely that the primers themselves are problematic—hence, I recommend redesigning the primers. In cases where there are small sub-peaks, you can eliminate this effect by optimizing reaction conditions, such as fine-tuning the annealing temperature.

　　The above is an introduction to the knowledge related to the detection of swine fever virus using the real-time fluorescent RT-PCR kit. If you’d like to learn more, feel free to contact us anytime!