Background-free DNA amplification method based on CRISPR-Cas12a pretreatment and application
By combining CRISPR-Cas12a pretreatment with magnetic separation and SERS detection technology, the sensitivity and specificity issues of the CRISPR-Cas12a system in nucleic acid detection have been solved, achieving efficient transduction and low background noise detection of double-stranded DNA, making it suitable for rapid and accurate detection of complex samples.
Patent Information
- Application Number
- CN202511208654.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-27
- Publication Date
- 2025-11-18
AI Technical Summary
Existing CRISPR-Cas12a systems suffer from limited sensitivity and background introduced during off-target amplification in nucleic acid detection, leading to poor accuracy and reliability of detection results, especially for low-concentration targets.
A background-free DNA amplification method based on CRISPR-Cas12a pretreatment was adopted. Non-target nucleic acids were removed by pre-cutting and magnetic separation technology, and SERS detection technology was combined to achieve efficient and specific nucleic acid amplification.
It achieves efficient transduction of double-stranded DNA, reduces background noise, and improves detection sensitivity and specificity, making it suitable for rapid and accurate detection of complex samples.
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Figure CN120967068A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of biological detection, and specifically discloses a background-free DNA amplification method based on CRISPR-Cas12a pretreatment and application. BACKGROUND
[0002] Viral nucleic acid molecule diagnostics are extremely powerful tools in human and animal disease control, food safety testing, and environmental monitoring. This urgency is most apparent during repeated outbreaks and epidemics of various infectious agents, including DNA-based pathogens such as African swine fever virus (ASFV) and monkeypox virus (MPXV), and RNA-based pathogens. As an important disease detection technology, molecular diagnostics has become an ideal solution with specificity, speed, and the ability to detect large-scale epidemics. Quantitative polymerase chain reaction (qPCR) is generally considered the gold standard, but due to the complexity of the process equipment, it is not suitable for on-site detection. Isothermal amplification techniques, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), are on the rise due to the need for only a simple temperature control module. However, the high operating cost, complex primer design, or false positive problems due to the multi-enzyme system or the use of four specific primers targeting six regions of the target hinder their application in low-resource environments. Fortunately, rolling circle amplification (RCA) has the characteristics of affordable operating costs and simple primer design, providing another complementary or alternative technology. To our knowledge, the application of RCA in most DNA virus detection is limited, mainly due to the inherent challenge of using RCA to directly recognize double-stranded targets, while RCA lacks the ability to effectively unwind and replicate double-stranded DNA (dsDNA) without prior modification.
[0003] Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein (Cas12a) is a endonuclease of V-A type CRISPR system, which can guide single-stranded DNA (ssDNA) to target dsDNA with a single CRISPR RNA (crRNA), thus cutting the single-stranded DNA to become a tool for detecting dsDNA in the next generation. This is mainly due to their excellent properties such as high fidelity and programmability. In addition, these systems show high versatility as they can be seamlessly integrated with a variety of readout methods. These include but are not limited to electrochemistry, colorimetry, fluorescence detection and surface-enhanced Raman scattering techniques. However, the CRISPR-Cas12a system itself is challenged by its limited sensitivity. To compensate for these limitations, the mainstream strategy is to first use isothermal amplification methods such as LAMP, RPA or RCA to pre-amplify nucleic acid molecules. Although these methods significantly improve the sensitivity of CRISPR-Cas12a, this mandatory pre-amplification of CRISPR-Cas12a can introduce off-target amplification, which needs to be balanced in terms of sensitivity and specificity. In addition, the selection scheme after amplification with CRISPR-Cas12a specific targeting function, although it temporarily shields off-target amplification, these intact and underutilized non-specific amplification products have not been effectively disposed of and have the potential to cause sample cross-contamination through aerosol transmission. This consequence not only artificially reduces the efficiency of nucleic acid amplification, but also causes distortion of the detection signal, thereby affecting the accuracy and reliability of the diagnostic results, especially for low concentration targets.
[0004] Therefore, in view of the above challenges, it is of great significance to develop a method of pre-cleaning oligonucleotides before amplification for robust and sensitive nucleic acid detection. SUMMARY
[0005] The present application aims to overcome the defects existing in the prior art and provide a background DNA-free amplification method based on CRISPR-Cas12a pretreatment and application.
[0006] In order to achieve the above purpose, one of the technical solutions of the present application is: a background DNA-free amplification method based on CRISPR-Cas12a pretreatment, comprising the following steps:
[0007] S1: Interfering oligonucleotide and pre-primer pre-cleavage with double-stranded target viral nucleic acid in the sample as CRISPR-Cas12 primer: adding double-stranded target viral nucleic acid ASFV DNA into a PCR tube containing buffer, magnetic bead DNA / RNA pre-primer and Cas12a-crRNA for incubation;
[0008] S2: Magnetic separation of preprimers and amplification: The precipitate is collected by separating the magnetic beads with the help of a magnetic stand; after the precipitate is transferred to another PCR tube containing a round padlock template, T4 DNA ligase, phi29 DNA polymerase, and SERS labels, the solution is removed for enzyme inactivation treatment;
[0009] S3: Signal detection of amplification products: After adding an equal volume of NaCl solution to the enzyme-inactivated solution obtained in step S2, Raman detection is performed.
[0010] In a preferred embodiment of the present application, the molar ratio of magnetic bead DNA / RNA preprimers and Cas12a-crRNA in step S1 is 1:4-1:2.
[0011] In a preferred embodiment of the present application, the volume ratio of double-stranded target viral nucleic acid, buffer, and both magnetic bead DNA / RNA preprimers and Cas12a-crRNA in step S1 is (15-25):(5-15):(15-25).
[0012] In a preferred embodiment of the present application, the method of standing incubation is used in step S1, the incubation temperature is 35-42℃, and the incubation time is 10-30 min.
[0013] In a preferred embodiment of the present application, the molar ratio of T4 DNA ligase to phi29 DNA polymerase in step S2 is (3-3.5):1.
[0014] In a preferred embodiment of the present application, the SERS label used in step S2 is an Au nanoparticle modified Cy5 labeled DNA sequence.
[0015] In a preferred embodiment of the present application, the concentration of the round padlock template used in step S2 is 100-200 nM, and the round padlock template is a 3' and 5' end-to-end connected DNA circular sequence.
[0016] In a preferred embodiment of the present application, the method of standing incubation is used in step S2, the incubation temperature is 35-42℃, and the incubation time is 40-60 min.
[0017] In a preferred embodiment of the present application, the enzyme inactivation treatment in step S2 is performed at 70-90℃ for 5-20 min.
[0018] In a preferred embodiment of the present application, the concentration of the NaCl solution added in step S3 is 1-5 M.
[0019] In a preferred embodiment of the present application, the laser used in the SERS detection in step S3 can effectively excite the hot spots between the nanoparticle surface and the particles, and the Raman test conditions are as follows: the laser wavelength is 785 nm, the power is 5-15 mW, and the exposure time is 0.5-1.5 s.
[0020] In order to achieve the above object, the second technical scheme of the present application is: application of a background-free DNA amplification method based on CRISPR-Cas12a pretreatment in SERS detection of pathogen prevention and control in pig farming.
[0021] In a preferred embodiment of the present application, the SERS detection method comprises the following steps: dropping the background-free DNA amplification product based on CRISPR-Cas12a pretreatment on an aluminum film for Raman testing by SERS.
[0022] Compared with the prior art, the present application has the following beneficial effects:
[0023] 1. The present application uses a pre-cutting strategy to improve the amplification efficiency of target nucleic acids, randomly eliminates non-target nucleic acids while retaining specific targets, thereby realizing highly specific downstream nucleic acid amplification; therefore, the nucleic acid pretreatment scheme provided can be used as an ideal nucleic acid processing platform for efficient and highly specific amplification of target nucleic acids.
[0024] 2. The present application realizes the transduction of double-stranded DNA to single-stranded RNA, realizes low-background RCA amplification compatible with dsDNA, provides an efficient and economical solution for most DNA pathogen detection, has important public safety significance, and its method and performance characteristics may provide a reference and potential application for other DNA virus detection;
[0025] 3. The present application discloses the application of background-free nucleic acid amplification technology in SERS nucleic acid detection, which can analyze the trace amount of B646L gene in complex pig serum samples, and minimize the cross contamination of samples; by combining this innovative strategy with emerging portable Raman spectrometers, the method is expected to provide a rapid, direct and high-sensitivity solution for on-site ASFV detection. BRIEF DESCRIPTION OF DRAWINGS
[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, the preferred detailed description of the present application will be given below with reference to the drawings.
[0027] Figure 1 The relative standard deviation of the Raman intensity of the 30 random point characteristic peaks in Example 1;
[0028] Figure 2 The performance of the method of the present application in real sample detection in Example 2;
[0029] Figure 3 Anti-interference of different double-stranded target viral nucleic acids in Example 1 and Comparative Example 1 and mixing them together. DETAILED DESCRIPTION
[0030] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be described in more detail below with reference to the drawings and specific embodiments, but the protection scope of the present application is not limited to these embodiments.
[0031] A background DNA amplification method based on CRISPR-Cas12a pretreatment, comprising the following steps:
[0032] S1: Pretreatment of interference oligonucleotide and preprimer with double-stranded target viral nucleic acid in sample as CRISPR-Cas12 primer: add double-stranded target viral nucleic acid ASFV DNA into a PCR tube containing buffer, magnetic bead DNA / RNA preprimer and Cas12a-crRNA for incubation;
[0033] S2: Magnetic separation and amplification of preprimer: collect the precipitate by separating the magnetic beads with the help of a magnetic stand; after transferring the precipitate to another PCR tube containing a round lock template, T4 DNA ligase, phi29 DNA polymerase and SERS label for incubation, remove the solution for enzyme inactivation treatment;
[0034] S3: Signal detection of amplification product: add an equal volume of NaCl solution to the enzyme inactivated solution obtained in step S2, and then perform Raman detection.
[0035] The molar ratio of magnetic bead DNA / RNA preprimer and Cas12a-crRNA in step S1 is 1:4-1:2.
[0036] The volume ratio of double-stranded target viral nucleic acid, buffer, magnetic bead DNA / RNA preprimer and Cas12a-crRNA in step S1 is (15-25):(5-15):(15-25).
[0037] The method of standing incubation is used in step S1, the incubation temperature is 35-42℃, and the incubation time is 10-30min.
[0038] The molar ratio of T4 DNA ligase and phi29 DNA polymerase in step S2 is (3-3.5):1.
[0039] The SERS label in step S2 is Au nanoparticle modified Cy5 labeled DNA sequence.
[0040] The concentration of the circular padlock template in step S2 is 100-200 nM, and the circular padlock template is a 3' and 5' end-to-end DNA circular sequence.
[0041] In step S2, the method of static incubation is used, the incubation temperature is 35-42℃, and the time is 40-60 min.
[0042] The enzyme inactivation treatment condition in step S2 is to keep 70-90℃ for 5-20 min.
[0043] The concentration of NaCl solution added in step S3 is 1-5M.
[0044] The laser used in SERS detection in step S3 can effectively excite the hot spots between the surface of the nanoparticles and the particles, and the Raman test conditions are laser wavelength 785 nm, power 5-15 mW, and exposure time 0.5-1.5 s.
[0045] A background-free DNA amplification method based on CRISPR-Cas12a pretreatment is applied to the SERS detection of pathogens in the prevention and control of pig industry.
[0046] Example 1
[0047] A background-free DNA amplification method based on CRISPR-Cas12a pretreatment, comprising the following steps:
[0048] S1: 35 μL of African swine fever nucleic acid ASFV DNA is added to a 200 μL PCR tube containing 16 μL of 10×Cas12a reaction buffer, 14 μL of 100 nM magnetic bead@DNA / RNA pre-primer, and 35 μL of 100 nM Cas12a-crRNA complex, and the final reaction volume reaches 100 μL; then the mixture is incubated at 37℃ for 20 min.
[0049] S2: The mixture after incubation in step S1 is collected by separating the magnetic beads under the help of the magnetic stand; then, the precipitate is transferred to another PCR tube containing 200 nM of circular padlock template, 3.5 U of T4 DNA ligase, 1 U of phi29 DNA polymerase, and 24 μL of Au nanoparticle modified Cy5 labeled DNA sequence SERS label, and finally 30 μL of mixture solution is obtained. After the mixture solution is incubated at 37℃ for 50 min, the solution is taken out and heated to 80℃ for 20 min to inactivate the enzyme.
[0050] S3: The solution obtained in step S2 was added with an equal volume of 1M NaCl solution, and then the sample was detected by SERS using a portable SHINS-p785 Raman spectrometer equipped with a 785 nm laser. The power was 10 mW, and the exposure time was 1 s.
[0051] Figure 1 The relative standard deviation of the Raman intensity of the 30 random point characteristic peaks was calculated. As shown in the figure, the background-free DNA amplification method based on CRISPR-Cas12a pretreatment has excellent stability and can be used for reliable quantitative detection of target nucleic acids.
[0052] Example 2
[0053] The background-free DNA amplification method based on CRISPR-Cas12a pretreatment in Example 1 was used for low detection background and high-efficiency African swine fever nucleic acid SERS detection.
[0054] The background-free DNA amplification product based on CRISPR-Cas12a pretreatment in Example 1 was dropped on an aluminum film and tested by SERS.
[0055] Figure 2 The quantitative analysis ability of the method of the present application in real sample detection was calculated. As shown in the figure, the background-free DNA amplification method based on CRISPR-Cas12a pretreatment can detect trace amounts of African swine fever nucleic acid in real samples.
[0056] Comparative Example 1
[0057] The method is the same as Example 1, except that the double-stranded target viral nucleic acid is PRV DNA, PCV2 DNA, and single-base mismatch ASFV (Mis-1), respectively.
[0058] The anti-interference of different double-stranded target viral nucleic acids PRV DNA, PCV2 DNA, single-base mismatch ASFV (Mis-1), ASFV DNA, and the mixture of the above four double-stranded target viral nucleic acids (denoted as Mix) was tested, and the results are shown in Figure 3 As shown in the figure, the background-free DNA amplification method based on CRISPR-Cas12a pretreatment has strong anti-interference ability and can specifically detect African swine fever nucleic acid ASFV DNA.
[0059] The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.
Claims
1. A background-free DNA amplification method based on CRISPR-Cas12a pretreatment, characterized in that, Includes the following steps: S1: Pre-cutting of interfering oligonucleotides and pre-primers using double-stranded target viral nucleic acid in the sample as CRISPR-Cas12 primers: Add double-stranded target viral nucleic acid ASFV DNA to a PCR tube containing buffer, magnetic beads@DNA / RNA pre-primers and Cas12a-crRNA and incubate. S2: Magnetic separation and amplification of preprimes: The precipitate was collected by separating magnetic beads with the help of a magnetic rack; the precipitate was transferred to another PCR tube containing a circular padlock template, T4 DNA ligase, phi29 DNA polymerase, and SERS tag and incubated. The solution was then removed and subjected to enzyme inactivation treatment. S3: Signal detection of amplified products: Add an equal volume of NaCl solution to the enzyme-inactivated solution obtained in step S2, and then perform Raman detection.
2. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S1, the molar ratio of magnetic beads@DNA / RNA preprime to Cas12a-crRNA is 1:4 to 1:2, and the volume ratio of double-stranded target viral nucleic acid, buffer, and magnetic beads@DNA / RNA preprime to Cas12a-crRNA is (15 to 25):(5 to 15):(15 to 25).
3. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S1, a static incubation method is used, with an incubation temperature of 35–42°C and an incubation time of 10–30 min.
4. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S2, the molar ratio of T4 DNA ligase to phi29 DNA polymerase is (3-3.5):
1.
5. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S2, the SERS tag is a DNA sequence modified with Au nanoparticles and labeled with Cy5.
6. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S2, the circular padlock template is a DNA circular sequence with 3' and 5' ends linked together, and the concentration of the circular padlock template is 100-200 nM.
7. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S2, a static incubation method is used, with an incubation temperature of 35-42℃ and a time of 40-60 min. The enzyme inactivation treatment after incubation is carried out at 70-90℃ for 5-20 min.
8. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, The concentration of the NaCl solution added in step S3 is 1-5M.
9. The background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in claim 1, characterized in that, In step S3, the Raman test conditions are: laser wavelength of 785nm, power of 5-15mW, and exposure time of 0.5-1.5s.
10. The application of a background-free DNA amplification method based on CRISPR-Cas12a pretreatment as described in any one of claims 1-9 in the detection of SERS pathogens in the swine industry.