An Optimized Thermal Feedback Methodology for Accurate Temperature Control and High Amplification Efficiency during Fluorescent qPCR.

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.

Construction of dPCR and qPCR integrated system based on commercially available low-cost hardware.

Fluorescent quantitative PCR (qPCR) and digital PCR (dPCR) are two mainstream nucleic acid quantification technologies. However, commercial dPCR and qPCR instruments have a low integration, a high price, and a large footprint. To solve these shortcomings, we introduce a compound PCR system with both qPCR and dPCR functions. All the hardware used in this compound PCR system is commercially available and low-cost, and free software was used to realize the absolute quantification of nucleic acids. The compound PCR provides two working modes. In the qPCR mode, thermal cycling is realized by controlling the reciprocating motion of the x axis. The heating rate is 1.25 °C s-1 and the cooling rate is 1.75 °C s-1. We performed amplification experiments of the PGEM-3zf (+)1 gene. The performance level was similar to commercial qPCR instruments. In the dPCR mode, the heating rate is 0.5 °C s-1 and the cooling rate is 0.6 °C s-1. We performed the UPE-Q gene amplification and used the sequential actions of the two-dimensional mechanical sliders to scan the reaction products and used the method of regional statistics and back-inference threshold to get test results. The result we got was 1208 copies per µL-1, which was similar to expectations.