Amplification Method:
Isothermal Fluorescence PCR: In isothermal fluorescence PCR, DNA amplification occurs at a constant temperature. This means that the reaction takes place at a single, constant temperature throughout the entire process.
Real-time PCR: Real-time PCR, also known as quantitative PCR (qPCR), uses a thermal cycling process. It involves multiple temperature cycles, including denaturation, annealing, and extension steps. These temperature cycles are typically controlled by a thermal cycler machine.
Temperature Control:
Isothermal Fluorescence PCR: As the name suggests, isothermal fluorescence PCR maintains a constant temperature during the reaction, typically around 60-65℃. This simplifies the equipment needed for the reaction as compared to real-time PCR.
Real-time PCR: Real-time PCR involves thermal cycling, where the temperature is repeatedly changed between different levels (e.g., denaturation at around 95℃, annealing at around 55-65℃, and extension at around 72℃). This requires a thermal cycler with precise temperature control.
Detection Method:
Isothermal Fluorescence PCR: Fluorescence detection is used in both isothermal fluorescence PCR and real-time PCR. However, in isothermal fluorescence PCR, fluorescence signals are typically detected continuously without the need for cycling between different temperature steps.
Real-time PCR: In real-time PCR, fluorescence signals are measured after each cycle of temperature change (typically after the annealing/extension step). This allows for real-time monitoring of DNA amplification as it occurs during the thermal cycling process.
Applications:
Isothermal Fluorescence PCR: This technique is suitable for certain applications, such as rapid point-of-care testing, where simplicity and speed are essential. It can be used for qualitative detection of specific DNA sequences.
Real-time PCR: Real-time PCR is widely used for both qualitative and quantitative analysis of DNA. It is highly sensitive and allows for the measurement of the initial amount of DNA in a sample, making it useful for applications like gene expression analysis, viral load quantification, and genotyping.
In summary, the main difference between isothermal fluorescence PCR and real-time PCR is the temperature control and amplification method. Isothermal fluorescence PCR maintains a constant temperature throughout the reaction and is suitable for simpler, rapid applications, while real-time PCR involves thermal cycling and is used for a wide range of quantitative and qualitative DNA analysis applications.