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Traditional qPCR instrument is combined with CMOS and a personal computer, and a photoelectric feedback automatic fluorescence detection system is designed to realize quantitative real-time PCR. The key to reaction efficiency lies in how to ensure that the temperature of the detection reagent completely matches the set temperature. However, for most traditional real-time fluorescent PCR systems, the temperature cycling is controlled by detecting the temperature of the heating well plate. It cannot directly measure the temperature in the reaction reagent PCR tube, which will cause the deviation in the actual temperature of the reagent to be as expected. Therefore, in this paper, we raise a method of directly detecting the temperature in the reaction tube of the reagent during the temperature cycling is adopted. According to the deviation from the expected value, the set temperature of the PCR instrument is adjusted to make the actual temperature of the reagent closer to the expected value. Through this method, we also realized the temperature calibration and optimization of the TEC circulation system we built. Experiments show that this low-cost, portable real-time quantitative PCR system can detect and analyze pathogens in situ.