Method for removing escherichia coli DNA pollution in qPCR premix solution by using nuclease dsDNase
By adding dsDNase to the qPCR premix solution for digestion and inactivation, the false positive problem caused by E. coli DNA contamination in the qPCR premix solution was solved, and the accuracy and reliability of the detection results were achieved.
Patent Information
- Application Number
- CN202510295028.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-05-30
AI Technical Summary
There is E. coli DNA contamination in the existing qPCR premix solution, resulting in false positive problems when detecting residual DNA of E. coli.
E. coli DNA contamination was removed by adding nuclease dsDNase to the qPCR premix solution and undergoing digestion and inactivation treatment. The specific steps include digestion at 37°C for 5-15 minutes, followed by inactivation at 65-70°C for 5-10 minutes.
Effectively removes E. coli DNA contamination in qPCR premix solution, significantly reducing the false positive rate of the test results, making the test results more accurate and reliable.
Smart Images
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Abstract
Description
Technical Field
[0001] This application relates to the field of molecular detection, and more specifically, it relates to a method for removing Escherichia coli DNA contamination in qPCR premix using nuclease dsDNase. Background Art
[0002] Generally, a PCR amplification system includes five basic components: amplification primers, 4 dNTPs, DNA polymerase, amplification template, and PCR reaction buffer (buffer). A qPCR premix is generally a mixture of other components except for the template and primers mixed in the optimal ratio, and the template and primers are directly added during use.
[0003] Most of the common qPCR premixes on the market are contaminated with Escherichia coli DNA. When manufacturing a kit for detecting residual Escherichia coli DNA, the contamination of Escherichia coli DNA in the qPCR premix will have a great impact. How to remove the Escherichia coli DNA contamination in the qPCR premix is a key technology for manufacturing a kit for detecting residual Escherichia coli DNA. dsDNase is an endonuclease that can cleave the phosphodiester bond in DNA to generate oligonucleotides with 5'-phosphate and 3'-hydroxyl ends. dsDNase can specifically digest double-stranded DNA (dsDNA) without digesting single-stranded DNA, primers, probes, and RNA. dsDNase is heat-sensitive and can be rapidly inactivated at 55°C.
[0004] Currently, there is no reported method for removing Escherichia coli DNA contamination by adding nuclease dsDNase to qPCR premix. Summary of the Invention
[0005] The present invention proposes a method for removing Escherichia coli DNA contamination in qPCR premix using nuclease dsDNase. According to the characteristics that dsDNase can specifically digest double-stranded DNA without digesting single-stranded DNA, primers, probes, and RNA and is heat-sensitive, the qPCR premix is mixed and digested with dsDNase, inactivated, and then primers, probes, and template DNA are added for real-time fluorescence quantitative PCR detection, and the negative result can reach a state of no amplification. This solves the technical problem of false positives caused by Escherichia coli DNA contamination in the qPCR premix used in the kit for detecting residual Escherichia coli DNA.
[0006] To achieve the above-mentioned invention purpose, this application provides a method for removing Escherichia coli DNA contamination in qPCR premix using nuclease dsDNase, including the following steps: adding nuclease dsDNase to the qPCR premix, digesting, and inactivating.
[0007] Further, in a 10 μL qPCR system, the addition amount of nuclease dsDNase is 0.3 - 0.7 μL; or, in a 20 μL qPCR system, the addition amount of nuclease dsDNase is 1 - 2 μL.
[0008] Further, in a 20 μL qPCR system, the addition amount of nuclease dsDNase is 2 μL.
[0009] Further, the digestion refers to digestion at 37°C for 5 - 15 mins, and the inactivation refers to inactivation at 65 - 70°C for 5 - 10 mins.
[0010] Further, the digestion refers to digestion at 37°C for 10 mins, and the inactivation refers to inactivation at 70°C for 5 mins.
[0011] Further, the qPCR premix includes:
[0012] Primer 0.4 μL;
[0013] Probe 0.2 μL;
[0014] Reference primer 0.4 μL;
[0015] Reference probe 0.2 μL;
[0016] Probe qPCR Super qPCR premix / BioSmart U + All - Powerful Multiple Probe qPCR Pre qPCR premix 10 μL;
[0017] Nuclease dsDNase 2 μL;
[0018] ddH2O is added to make up to 20 μL.
[0019] Further, the method includes the following steps: The qPCR premix and the nuclease are mixed and reacted at 37°C for 10 minutes, inactivated at 70°C for 5 minutes, then Escherichia coli and the reference primer - probe are added, ddH2O is added to 20 μL, and mineral oil is added to cover; The reaction program is pre - denaturation at 95°C for 5 mins, denaturation at 95°C for 10 s, annealing and extension at 60°C for 40 s, for 45 cycles.
[0020] In summary, the present application has the following beneficial effects:
[0021] The present invention uses nuclease to treat the Escherichia coli DNA contamination in the qPCR premix, thereby establishing a detection system for residual Escherichia coli DNA. The false positive of the detection result is greatly reduced, solving the problem of false positive caused by Escherichia coli DNA contamination in the qPCR premix, making the detection result more accurate and reliable. Description of the Drawings
[0022] Figure 1 : Negative amplification result of real-time fluorescence quantitative PCR for Example 1( Figure 1 -A: Without nuclease - negative - 10 uL; Figure 1 -B: Nuclease 0.3 uL - 10 uL; Figure 1 -C: Nuclease 0.5 uL - 10 uL; Figure 1 -D: Nuclease 0.7 uL - 10 uL; Figure 1 -E: Without nuclease - negative - 20 uL; Figure 1 -F: Nuclease 1 uL - 20 uL; Figure 1 -G: Nuclease 1.5 uL - 20 uL; Figure 1 -H: Nuclease 2 uL - 20 uL);
[0023] Figure 2 : Negative amplification result of real-time fluorescence quantitative PCR for Example 2( Figure 2 -A: Without nuclease - 10 uL; Figure 2 -B: Digested for 5 mins - 10 uL; Figure 2 -C: Digested for 10 mins - 10 uL; Figure 2 -D: Digested for 15 mins - 10 uL)
[0024] Figure 3 : Negative amplification result of real-time fluorescence quantitative PCR for Example 3 (nuclease 2 uL - 20 uL - digested for 10 mins). Detailed Implementation Manner
[0025] The technical solutions and effects of the present application will be further described in detail below in conjunction with examples and drawings. It can be understood that the specific examples described herein are only for explaining the invention, rather than limiting the invention.
[0026] I. Experimental Materials
[0027] The following primers and probes (Table 1) were synthesized by Shanghai Diwin Biotech Co., Ltd. and Shanghai Bioliggen Biotech Co., Ltd.;
[0028] Table 1 Primers and Probes
[0029]
[0030] Probe qPCR SuperqPCR Premix (UDG) (E106) was purchased from Shanghai Jinan Protein Technology Co., Ltd.;
[0031] The dsDNase nuclease was purchased from Jiangsu Bristol-Myers Squibb Biotechnology Co., Ltd.;
[0032] RNase free Water was purchased from Sangon Biotech (Shanghai) Co., Ltd.;
[0033] BioSmart U+AllPowerful Multiple Probe qPCR PreqPCR Premix (ONE TUBE) was purchased from Nanjing Novizan Biotech Co., Ltd.
[0034] II. Experimental methods
[0035] 1. Detection system (20 μL):
[0036] For each of the two sets of primers for Escherichia coli, 0.4 μL was added, and 0.2 μL of the probe was added. The final concentration of the primers and probe was 0.2 μM;
[0037] For the reference gene primers, 0.4 μL was added, and 0.2 μL of the probe was added. The final concentration of the primers and probe was 0.2 μM;
[0038] Probe qPCR SuperqPCR Premix (UDG) / BioSmart U+All-Powerful Multiple Probe qPCR PreqPCR Premix (ONE TUBE) was added at 10 μL;
[0039] dsDNase nuclease was added at 2 μL;
[0040] Finally, ddH 2 O was added to 20 μL.
[0041] (The input volume was reduced proportionally for the 10 μL system)
[0042] 2. Reaction conditions
[0043] The qPCR premix and the nuclease were mixed and reacted at 37 °C for 10 minutes. After inactivating at 70 °C for 5 minutes, the first set of Escherichia coli and the reference gene primers and probe were added to one tube, and the second set of Escherichia coli and the reference gene primers and probe were added to the other tube. ddH 2 O was added to 20 μL, and mineral oil was added to cover;
[0044] The reaction program was pre-denaturation at 95 °C for 5 mins, denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 40 s, for 45 cycles.
[0045] It should be noted that the above system can be appropriately adjusted according to the nuclease reaction, such as the temperature and time of digestion and inactivation, so as to achieve a state of negative without amplification.
[0046] III. Implementation Examples
[0047] Example 1
[0048] Verify the effect of different amounts of nuclease input on removing Escherichia coli DNA contamination in qPCR premix.
[0049] (1) Setting of the addition amount of dsDNase nuclease:
[0050] 10 μL qPCR system: Add 0, 0.3, 0.5, 0.7 μL of dsDNase nuclease respectively;
[0051] 20 μL qPCR system: Add 1, 1.5, 2 μL of dsDNase nuclease respectively;
[0052] (2) Reaction conditions:
[0053] Digest at 37 °C for 5 mins and inactivate at 65 °C for 5 mins;
[0054] (3) Detection of negative amplification results by real-time fluorescence quantitative PCR:
[0055] As Figure 1 shown, Figure 1 -A shows the real-time fluorescence quantitative PCR detection results of the 10 μL qPCR system without adding nuclease; Figure 1 -B shows the real-time fluorescence quantitative PCR detection results of the 10 μL qPCR system with 0.3 μL of nuclease; Figure 1 -C shows the real-time fluorescence quantitative PCR detection results of the 10 μL qPCR system with 0.5 μL of nuclease; Figure 1 -D shows the real-time fluorescence quantitative PCR detection results of the 10 μL qPCR system with 0.7 μL of nuclease; Figure 1 -E shows the real-time fluorescence quantitative PCR detection results of the 20 μL qPCR system without adding nuclease; Figure 1 -F shows the real-time fluorescence quantitative PCR detection results of the 20 μL qPCR system with 1 μL of nuclease; Figure 1 -G shows the real-time fluorescence quantitative PCR detection results of the 20 μL qPCR system with 1.5 μL of nuclease; Figure 1 -H shows the real-time fluorescence quantitative PCR detection results of the 20 μL qPCR system with 2 μL of nuclease.
[0056] Example 2
[0057] Verify the effect of different nuclease digestion times on removing Escherichia coli DNA contamination in qPCR premix.
[0058] (1) Setting of the addition amount of dsDNase nuclease:
[0059] For a 10 μL qPCR system: Add 0.7 μL of dsDNase nuclease;
[0060] (2) Reaction conditions:
[0061] Digest at 37°C for 5, 10, and 15 mins respectively, and inactivate at 70°C for 5 mins;
[0062] (3) Detection of negative amplification results by real-time fluorescence quantitative PCR:
[0063] As Figure 2 shown, Figure 2 -A shows the real-time fluorescence quantitative PCR detection results of a 10 μL qPCR system without adding nuclease; Figure 2 -B shows the real-time fluorescence quantitative PCR detection results of a 10 μL qPCR system with a digestion condition of 5 mins; Figure 2 -C shows the real-time fluorescence quantitative PCR detection results of a 10 μL qPCR system with a digestion condition of 10 mins; Figure 2 -D shows the real-time fluorescence quantitative PCR detection results of a 10 μL qPCR system with a digestion condition of 15 mins.
[0064] Example 3
[0065] Adjust the qPCR system, the input amount of nuclease, and the nuclease digestion time to achieve the purpose of completely removing the contamination of Escherichia coli DNA in the qPCR premix.
[0066] (1) Setting of the addition amount of dsDNase nuclease:
[0067] For a 20 μL qPCR system: Add 2 μL of dsDNase nuclease;
[0068] (2) Reaction conditions:
[0069] Digest at 37°C for 10 mins, and inactivate at 70°C for 5 mins;
[0070] (3) Detection of negative amplification results by real-time fluorescence quantitative PCR:
[0071] As Figure 3 shown, the contamination of Escherichia coli DNA in the qPCR premix can be completely removed under the above conditions.
[0072] The beneficial effects of the present invention are as follows: By using nuclease to treat the Escherichia coli DNA contamination in the qPCR premix, an Escherichia coli residual DNA detection system is established, and the false positives of the detection results are greatly reduced, solving the problem of false positives caused by Escherichia coli DNA contamination in the qPCR premix, making the detection results more accurate and reliable.
[0073] This specific embodiment is only an interpretation of the present application and does not limit the present application. After reading this specification, those skilled in the art can make modifications to this embodiment without creative contributions as needed, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.
Claims
1. A method for removing Escherichia coli DNA contamination in qPCR premix using nuclease dsDNase, characterized in that: The following steps are involved: Add nuclease dsDNase to qPCR master mix, digest and inactivate.
2. The method according to claim 1, characterized in that In a 10uL qPCR system, the amount of the nuclease dsDNase added is 0.3-0.7uL; or, in a 20uL qPCR system, the amount of the nuclease dsDNase added is 1-2uL.
3. The method according to claim 2, characterized in that In a 20uL qPCR system, the amount of nuclease dsDNase added is 2uL.
4. The method according to claim 1, characterized in that: The digestion refers to digestion at 37°C for 5-15 mins, and the inactivation refers to inactivation at 65-70°C for 5-10 mins.
5. The method according to claim 4, characterized in that The digestion refers to digestion at 37°C for 10 mins, and the inactivation refers to inactivation at 70°C for 5 mins.
6. The method according to claim 1, characterized in that The qPCR premix includes: Primer 0.4uL; Probe 0.2uL; Internal reference primer 0.4uL; Internal reference probe 0.2uL; Probe qPCR SuperqPCR premix / BioSmart U+All-Powerful MultipleProbe qPCR PreqPCR premix 10uL; Nuclease dsDNase 2uL; Add ddH2O to make up to 20uL.
7. The method according to claim 6, characterized in that The following steps are involved: The qPCR premix was mixed with nuclease and reacted at 37°C for 10 minutes. After inactivation at 70°C for 5 minutes, Escherichia coli and internal reference primer probe were added, ddH2O was added to 20uL, and mineral oil was added to cover. The reaction procedure was 95°C pre-denaturation for 5mins, 95°C denaturation for 10s, 60°C annealing and extension for 40s, and 45 cycles.