Molecular diagnosis is the fastest developing sub field in the field of IVD. It has the advantages of short detection time, high sensitivity and strong specificity. It is widely used in tumor concomitant diagnosis and screening of infectious diseases and genetic diseases. In the field of molecular diagnosis, PCR is not only the most mature technology platform with the largest market share, but also the "gold standard" technology of clinical diagnosis. In the traditional PCR reaction, the problem of nonspecific amplification often occurs. Nonspecific amplification directly affects the interpretation of detection results, and will lead to the decline of amplification sensitivity and even the yield of target fragments. How does nonspecific amplification occur and how can it be effectively avoided?
The poor sequence specificity of primers is the direct cause of nonspecific amplification. As the promoter of conventional DNA polymerase, its exonuclease activity can truncate the DNA primer sequence during the preparation of PCR system, so as to reduce the specificity of primers.
In addition, conventional DNA polymerase not only has exonuclease activity, but also has polymerase activity. Although the optimal extension temperature of DNA polymerase is 72℃, the polymerase is still active at room temperature. Therefore, during the preparation of PCR reaction and the initial heating process, primers may form nonspecific binding with some single strand templates and extend under the action of Taq DNA polymerase, resulting in the amplification of non-target sequences and affecting the specificity of the reaction.
Therefore, PCR systems are often configured on ice to inhibit the activity of DNA polymerase. Although this method is simple and cheap, it can not completely inhibit the enzyme activity, so it can not completely eliminate the amplification of nonspecific products.
Hot start polymerase is the core component of hot start PCR. At room temperature, the active site of DNA polymerase is blocked by enzyme modifier. Only after PCR denaturation at 95℃, the enzyme modifier falls off and the enzyme activity is started, avoiding non-specific amplification caused by enzyme.
At present, the commonly used hot start modification methods of DNA polymerase in the market mainly include chemical method, ligand method and antibody method. Among them, the blocking effect of antibody modified hot start polymerase is the best, and the release speed of enzyme activity is fast, which can greatly reduce the reaction time of PCR. It is a hot start enzyme modification method widely used in IVD market.
However, it should be noted that the traditional antibody hot start method only blocks the 5'→3' polymerase activity of Taq DNA polymerase, which can only prevent non-specific amplification caused by mismatch or primer dimer at low temperature. However, as mentioned above, Taq DNA polymerase not only has 5'→3' polymerase activity, but also has 5'→ 3' exonuclease activity. This exonuclease activity may lead to the degradation of mismatched probes or other materials at low temperature, resulting in some nonspecific signals.
As an innovative leader in the domestic molecular enzyme industry, Yeasen Biotechnology (Shanghai) Co., Ltd. (hereafter called Yeasen) focuses on the R & D and production of molecular enzyme raw materials and has the courage to innovate. Relying on its own mature antibody screening platform, it screens the blocking antibody with Taq DNA polymerase as the target molecule. After years of painstaking research and system optimization, it has developed the double-block anti-Taq DNA polymerase antibody, It can not only block the polymerase activity of Taq DNA polymerase, but also block the exonuclease activity of Taq DNA polymerase. In a two pronged approach, it can not only effectively prevent non-specific amplification caused by mismatch or primer dimer, but also prevent material degradation from generating non-specific signals, and double improve the stability of the reagent.
After blocking Taq polymerase with Yeasen’s double-block antibody and T company’s antibody, the positive samples were detected by ARMS-PCR.
Figure 1. ARMS-PCR amplification curve; Blue: T company’s blocking antibody; Red: Yeasen’s double-block antibody
It can be seen from Figure 1 that the Taq polymerase blocked by Yeasen’s double-block antibody has accurate typing and higher sensitivity in ARMS-PCR amplification.
The synthesized primer probes were matched with the reaction solution of Taq DNA polymerase blocked by unclosed and double blocked antibodies respectively, and reacted at 40℃ to detect the fluorescence signals of them.
Figure 2. Detection of blocking efficiency of double-block antibody for exonuclease activity; Yellow: unclosed Taq DNA polymerase group; Purple: Taq DNA polymerase group blocked by double-block antibody
It can be seen from Figure 2 that the double-block antibody can effectively block the exonuclease activity of Taq DNA polymerase.
Taq DNA polymerase was blocked with traditional blocking antibody and double-block antibody respectively, and configured into a full premix containing primer probe. 10000 copies of ASF plasmid were amplified at 4℃ for 10 days or at 37℃ for 1, 3 and 5 days. The amplification curve and Ct value changes were observed, and the effects of traditional blocking antibody and double-block antibody on the stability of full premix reaction solution were compared.
Figure 3. Amplification curve of 10000 copies of ASF plasmid amplified by traditional blocking antibody (a) and double-block antibody (b) blocking reaction solution; Blue: store at 4℃ for 10 days; Red: placed at 4℃ for 0 days (control)
It can be seen from Figure 3 that the double-block antibody can maintain the stability of the reaction solution and effectively improve the stability of the detection reagent than the traditional blocking antibody. The amplification curve and Ct value did not change significantly after the whole premix containing primer probe was blocked by double-block antibody and placed at 4℃ for 10 days.
Figure 4. Amplification curve of 10000 copies of ASF plasmid amplified by traditional blocking antibody (a) and double-block antibody (b) blocking reaction solution; Blue: store at 37℃ for 5 days; Green: 37℃ for 3 days; Yellow: 37℃ for 1 day; Red: placed at 37℃ for 0 days (control)
It can be seen from Figure 4 that the double-block antibody can maintain the stability of the reaction solution and effectively improve the stability of the detection reagent than the traditional blocking antibody. Double-block antibody blocks the whole premix containing primer probe, and the difference of Ct value is less than 0.2 after being placed at 37℃ for 1, 3 and 5 days.
When some primers cooperate with specific buffer and instrument, there will be baseline drift. Yeasen tried many methods to improve the baseline problem, and found that double-block antibody was more conducive to stable baseline.
Figure 5. Amplification curve of N gene (a) and ACT gene (b); Red: traditional blocking antibody; Green: double-block antibody
As can be seen from Figure 5, the use of double-block antibody under specific primers and buffer can help solve the problem of baseline drift.
100000, 10000, 1000 and 100 copies of ASF/ACT double plasmids were amplified with the reaction solution blocked by double-block antibody, and the standard curve was made.
Figure 6. Standard curves of ASF gene (a) and ACT gene (b) produced by double-block reaction solution
As can be seen from Figure 6, the standard curve R2 of ASF gene is 0.998, and the amplification efficiency is 98.848%; The standard curve R2 of ACT gene was 0.998 and the amplification efficiency was 98.113%.
Taq polymerase was blocked with imported T company’s antibody and Yeasen’s double-block antibody (cat#31303) respectively, and the enzyme activity was detected after heat shock at 95℃ for 20 s. It can be seen that 31303 can release more than 95% of the enzyme activity after heat shock at 95 ℃ for 20 s, which is equivalent to T company’s antibody.
Three types of Taq enzymes (wild type and 2 mutants) were blocked by Yeasen’s double-block antibody (cat#31303), and the blocking ratio was 1 μ g: 5 U, measure the fluorescence value and sealing efficiency. It can be seen that the blocking effect of Yeasen’s double-block antibody (cat#31303) is better for different types of Taq enzymes.
Taking water as the template as the negative control and mouse genome as the template as the positive control, 31303 of different batches were amplified. It was found that the target fragment was not amplified in the sample, indicating that there was no mouse genome residue in the antibody.
Figure 7. Mouse genome residue detection diagram
Note: - is the negative control, + is the positive control, and 1-5 are 31303 samples of different batches