The fluorescent PCR method, as the gold standard for nucleic acid detection of COVID-19, still faces problems such as inaccurate test results, false positives and false negatives. Except manual operation factors, the quality of assay kit which mainly depend on Hot-Start Taq DNA polymerase should be considered in further studies. Due to differences of enzyme raw materials from different manufacturers in the actual sample detection, it will be very difficult to screen out the perfect antibody-based hot-start enzyme.
We organize the evaluation indicators of the antibody-based hot-start enzyme:
In addition to the 5'-3' polymerase activity of Taq polymerase, TaqMan probe method qPCR also needs to detect the 5'-3' exonuclease activity of Taq polymerase, which is the defect of conventional Taq polymerase activity assay.
The amplification efficiency of PCR reaction is closely related to the performance of DNA polymerase. The template was diluted into a series of concentration gradients for qPCR reaction, the log value of the initial amount of template was used to plot the standard curve, and the amplification efficiency was calculated.
The sensitivity of Taq polymerase has a great relationship with many aspects, such as the purity of enzyme and its affinity with the substrate.
Hot-start Taq polymerase is one of the most important factors to improve the sensitivity and specificity of PCR amplification. The non-hot-start Taq polymerase will have a certain activity during the reagent preparation process, which is easy to perform non-specific amplification. And it will eventually lead to a decrease in sensitivity. An active and effectively blocked Taq polymerase is essential for efficient amplification of qPCR.
Considering the above indicators, the best choice for the one-step RT-qPCR detection reagent used for SARS-Cov-2 is the antibody-based hot-start polymerase. The antibody-modified hot-start enzyme can achieve a good effect of blocking enzyme activity, which has the best corresponding blocking effect and a high enzyme activity release speed. It is currently the most widely used DNA polymerase hot-start transformation method in the IVD market. The production of qualified IVD reagents requires strict control in raw material control, production management, quality inspection control and storage and transportation.
Hieff UNICON™ Hotstart E-Taq DNA Polymerase is a hot start DNA polymerase with double blocking by double antibodies independently developed by the company. This product not only blocks the 5'→3' polymerase activity of Taq DNA polymerase, but also blocks the 5'→3' exonuclease activity. Heating for 30 seconds at the pre denaturation temperature can completely inactivate the antibody and release DNA polymerase activity and exonuclease activity. The double blocking characteristic can not only effectively prevent the nonspecific amplification caused by mismatch or primer dimer, but also effectively inhibit the decline of fluorescence signal caused by probe degradation, so as to make the in vitro detection reagent more stable during transportation or use at room temperature.