CN116606931A - POCT system for directly detecting methylation genes and application thereof - Google Patents

Abstract

The application provides a POCT system for directly detecting a methylation gene, which is based on combination of an isothermal amplification method and a CRISPR/Cas method, and comprises methylation sensitive restriction enzyme, an isothermal amplification reagent and the CRISPR/Cas reagent, wherein double-stranded DNA obtained by the CRISPR/Cas reagent and Wen Kuozeng is specifically identified, so that the component of the single-stranded report DNA with a fluorescent group is cut by the trans-cleavage activity of the Cas enzyme is activated. The method provided by the application uses the genomic DNA which is more close to clinical application, and has the advantages of lower detection limit and wider detection range. Furthermore, the entire reaction can be completed within 1h, and is thus well suited for immediate detection.

Description

POCT system for directly detecting methylation genes and application thereof

Technical Field

The application relates to the field of biological detection, in particular to a POCT (point of care testing) system for directly detecting methylation genes and application thereof.

Background

DNA methylation is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent bonding of a methyl group at the cytosine 5' carbon of a genomic CpG dinucleotide under the action of a DNA methyltransferase. A large number of researches show that DNA methylation can cause changes of chromatin structure, DNA conformation, DNA stability and interaction modes of DNA and protein, thereby controlling gene expression and playing an important role in the generation and development of tumors. Thus, differentially expressed methylated DNA is an important biomarker for early diagnosis and prognostic monitoring of tumors. Sulfite-dependent detection methods (BS) are gold standards for clinical nucleic acid gene methylation detection. In this method, unmethylated cytosine (C) is converted to uracil (U) after sulfite treatment of DNA, and methylated cytosine is kept unchanged. The treated DNA is sequenced or PCR using specific primers to obtain very accurate DNA sequence methylation site information. However, the method has the defects of complicated operation steps, long time consumption, severe experimental conditions, easy degradation of target genes, incomplete transformation and low recovery rate, and the inherent defects result in inaccurate analysis of the result methylation (especially the gene with extremely low abundance) detection result and poor result repeatability, so that the clinical detection effect of the method is severely limited.

The Methylation Sensitive Restriction Enzyme (MSRE) just can overcome the defect of the BS, and the method utilizes the characteristic that the methylation sensitive restriction enzyme does not cut a methylation region to digest DNA into fragments with different sizes, and then carries out subsequent analysis. The reaction condition is mild, so that degradation of DNA can be greatly avoided. In addition, the cleaved products of the MSRE reaction generally do not require purification to allow subsequent detection reactions, as compared to BS requiring product recovery and purification. However, this detection method generally has a relatively low detection sensitivity and requires further amplification of the product, i.e. an expensive PCR instrument. PCR is the most widely used nucleic acid amplification technology, and is widely applied in terms of sensitivity and specificity, however, the PCR needs repeated thermal denaturation and cannot get rid of the limitation of depending on instruments and equipment, so that the application of the PCR in clinical field detection is limited.

Since the beginning of the 90 s of the 20 th century, many laboratories have developed isothermal amplification techniques that do not require thermal denaturation, such as Recombinant Polymerase Amplification (RPA), loop-mediated isothermal amplification (LAMP), strand-displacement isothermal amplification (SDA), rolling circle isothermal amplification (RCA), etc., have been developed. Among these, SDA, RCA and RPA have been successfully used for detection of methylated genes. SDA and RCA are long experiments that take several hours to peak the product of interest, resulting in lower sensitivity in methylation detection. However, RPA can peak the amplification amount of the target product within half an hour and has extremely high accuracy, and thus is widely used in disease detection.

In recent years, nucleic acid detection such as SHERLOCK, DETECTR, based on the regularly spaced clustered short palindromic repeats (CRISPR) technique, has received attention for its rapid and sensitive nature. In previous studies, CRISPR/Cas12 b-binding BS technology (called HOLMESv 2) has been used in DNA methylation detection. However, the complexity of the HOLMESv2 assay procedure limits its clinical application. Therefore, development of a rapid, sensitive, simple and easy-to-use methylation gene detection technique is very important for clinical disease detection.

Disclosure of Invention

In order to solve the above problems, the present application provides a POCT system for directly detecting methylated genes, which is based on isothermal amplification method and CRISPR/Cas method combination, comprising: component 1: it includes methylation sensitive restriction enzymes that digest genomic DNA where methylation modified sequence fragments of interest will remain intact and non-methylated sequence fragments will be specifically cleaved; component 2: the method comprises an isothermal amplification reagent, wherein the isothermal amplification reagent carries out isothermal amplification on the methylation modified target sequence fragment to obtain double-stranded DNA, and the unmethylated sequence fragment does not carry out isothermal amplification; and component 3: and (3) a CRISPR/Cas reagent, wherein the reagent specifically recognizes the double-stranded DNA, so that the Cas enzyme is activated to cleave the single-stranded reporter DNA with a fluorescent group in a trans-cleavage activity, and a fluorescent signal is generated to directly detect the methylation modified target sequence fragment.

In one embodiment, the methylation sensitive restriction enzyme is the endonuclease BstUI and the isothermal amplification reagent is an RPA isothermal amplification reagent.

In one embodiment, the CRISPR/Cas reagent is a CRISPR/Cas12a reagent.

In one embodiment, the CRISPR/Cas12a reagent comprises crRNA, cas12a enzyme and single-stranded reporter DNA with a fluorescent group, the crRNA guides Cas12a enzyme to recognize the double-stranded DNA, the methylation modified target sequence fragment is specifically cleaved, the double-stranded DNA comprises a nucleic acid fragment specifically combined with the crRNA, the nucleic acid fragment starting sequence is TTTN sequence, and N is any one base of A, C, G; the single-stranded reporter DNA having a fluorescent group is cleaved by the activated Cas12a enzyme to generate a fluorescent signal for specific detection.

In one embodiment, the present application provides the use of the POCT system for detecting methylated genes described above, comprising the steps of: step 1: digesting the genomic DNA with a methylation sensitive restriction enzyme, wherein the methylation modified target sequence fragment remains intact and the non-methylated gene fragment is specifically cleaved; step 2: digesting the genome DNA by using the methylation sensitive restriction enzyme in the step 1 as a template, and carrying out isothermal amplification, wherein the methylated genome DNA is amplified into a target sequence segment double-stranded DNA, and the non-methylated genome DNA is not subjected to isothermal amplification; step 3: the double-stranded DNA in the step 2 is used as a template to perform CRISPR/Cas activation, the double-stranded DNA activates the reverse cleavage activity of Cas enzyme, the non-specific cleavage is performed on the fluorescence modified single-stranded reporter DNA to generate a fluorescence signal, and the non-methylated sequence fragment cannot generate double-stranded DNA due to non-isothermal expansion, so that Cas12a enzyme cannot be activated, and no fluorescence signal is generated; and step 4: directly judging whether the methylation gene exists or not through a fluorescent signal.

In one embodiment, the methylation sensitive restriction enzyme is the endonuclease BstUI and the isothermal amplification is RPA isothermal amplification.

In one embodiment, the CRISPR/Cas activation is a CRISPR/Cas12a activation.

In one embodiment, the CRISPR/Cas12a activation comprises using crRNA, cas12a enzyme, and single stranded reporter DNA with a fluorescent group; the crRNA guides the Cas12a enzyme to recognize the double-stranded DNA, the methylation modified target sequence fragment is specifically cut, the double-stranded DNA comprises a nucleic acid fragment specifically combined with the crRNA, the initial sequence of the nucleic acid fragment is a TTTN sequence, and N is any one base of A, C, G; the single-stranded reporter DNA having a fluorescent group is cleaved by the activated Cas12a enzyme to generate a fluorescent signal for specific detection.

In one embodiment, the nucleic acid fragment initiation sequence is a TTTC sequence.

In one embodiment, the sequence of interest fragment is the SEPT9 gene promoter SEPT9:77373475-77373595 fragment.

Currently available methylation gene detection methods, such as Jin Biaozhun sulfite-dependent methods, require DNA degradation and detection failure caused by severe conditions such as strong acid and high temperature, and MSRE has mild reaction conditions to avoid DNA degradation, but incomplete digestion leads to detection accuracy and sensitivity to be improved.

Compared with the DNA methylation detection method in the prior art, the linear DNA detection difficulty is lower in all other technologies at present, and the genomic DNA with the supercoiled structure is not used, so that the method provided by the application has the advantages that the genomic DNA is more close to clinical application, the detection limit is lower, and the detection range is wider. Furthermore, the entire reaction can be completed within 1h, and is thus well suited for point of care testing (POCT).

Specifically, the application provides a DNA methylation detection system combining isothermal amplification (RPA) and a CRISPR/Cas12a (MeCRISPR) system based on a Methylation Sensitive Restriction Endonuclease (MSRE) technology, compared with the traditional methylation specific PCR, the RPA reaction condition based on an endonuclease BstUI is mild, and the detection speed is high (the whole reaction is lower than 1 h). Shows good performance in DNA methylation detection, and can accurately detect the methylation level of very low concentration (1 copy/. Mu.L) and content (0.01%). In addition, the sensitivity and specificity of MeCRISPR in clinical detection of cervical cancer can reach up to 100% and 92.3%, and the MeCRISPR has excellent application value and prospect.

The application utilizes the specificity of BstUI, can effectively distinguish methylated DNA and unmethylated DNA, and simultaneously, the efficient amplification of RPA to target DNA can generate a large amount of dsDNA (double-stranded DNA), then the dsDNA is specifically identified by Cas12a, PAM sites are needed in the identification process, thereby avoiding the defect of nonspecific amplification of RPA technology, and finally, the trans-cleavage activity of CRISPR/Cas12a is activated to perform nonspecific cleavage to the single-stranded reporter gene modified by fluorescent groups to generate fluorescent signals, so that the detection method has higher sensitivity and selectivity and good anti-interference capability and repeatability.

Since the resulting dsDNA is partially complementary to the crRNA, the trans-cleaving activity of Cas12a is activated. Cas12a may arbitrarily cleave a nearby fluorescent reporter probe (such as FAM-DNA-BHQ 1) to release a green fluorescent signal. In addition, the method can also be used for realizing real-time detection through experimental visualization by changing the detection mode and using ultraviolet lamp analysis. When the SEPT9 is subjected to methylation modification, the RPA reaction can be activated to amplify a large amount of dsDNA in a short time, and then the trans-cleavage activity of the Cas12a is activated, so that the FAM-DNA-BHQ1 reporter gene with single chain is not specifically cleaved. After cleavage of the reporter probe containing FAM group modification, an absorption peak was observed at an excitation wavelength of 480nm at a wavelength of 525nm, and a green fluorescent signal was detected under an ultraviolet lamp. In contrast, unmethylated SEPT9, because it fails to activate Cas12a trans-cleavage activity, has no significant green fluorescent signal at 480nm excitation wavelength or uv lamp. Based on the detection principle of the sensor, the methylation modification condition of SEPT9 can be successfully distinguished, and the detection method can be applied to an instant detection or high-flux detection platform.

In the present application, the SEPT9 gene promoter SEPT9:77373475-77373595 (121 bp) contains 3 cleavage sites of 5 '-CGCG-3'. Methylated and unmethylated DNA was first treated with BstUI methylation sensitive restriction enzyme that recognizes and digests the "5'-CGCG-3'" site. Therefore, unmethylated DNA will be cleaved by BstUI. Unmethylated modified SEPT9 was completely digested by BstUI. In contrast, methylated SEPT9 had no effect and the structure remained intact. The structurally intact methylated SEPT9 was used to activate the RPA amplification reaction. In the RPA system, a recombinase binds to a primer to locate homologous sequences in the duplex and exponentially amplify the target region. Thus, large amounts of dsDNA can be obtained by RPA amplification experiments. Meanwhile, the obtained dsDNA can be specifically identified by the Cas12a enzyme due to the existence of PAM sequence sites, so that the detection specificity of the dsDNA is increased. The high specificity and self-amplifying capability of the CRISPR/Cas12a system further improves the detection performance of the detection system.

In the present application we selected MSRE (BstUI) to be able to recognize and cleave 3 sequences containing 5'-CGCG-3' in this fragment, thus ensuring that unmethylated DNA is completely digested. In addition, the large amount of dsDNA amplified by the RPA reaction is specifically recognized by CRISPR/Cas12a with the aid of PAM sites, activating its trans-cleavage activity for fluorescent detection (visualization and high throughput). The MeCRISPR system has higher sensitivity and specificity in real clinical sample detection, and can be used for detecting cervical cancer in actual clinical diagnosis.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a basic schematic of the present application, wherein FIG. 1A is a schematic representation of the activation of methylated and unmethylated DNA by BstUI methylation sensitive restriction endonuclease treatment followed by isothermal amplification reactions, and FIG. 1B is a schematic representation of the reaction of the generated dsDNA to activate a CRISPR/Cas12a system cleavage reporter to generate a fluorescent signal;

FIG. 2 is a graph showing the result of dsDNA electrophoresis of target gene fragments amplified by RPA;

FIG. 3 is a CRISPR/Cas12a detection result graph, wherein FIG. 3A is a fluorescence spectrum detection result graph and FIG. 3B is a visualization detection result graph;

FIG. 4 is a graph showing the results of optimizing various experimental parameters according to the present application, wherein FIG. 4A is a graph showing the results of electrophoresis of amplification products at various substrate concentrations, FIG. 4B is a graph showing the results of electrophoresis of amplification products at various concentrations of BstUI, and FIG. 4C is a graph showing the results of electrophoresis of amplification products at various concentrations of Mg ²⁺ FIG. 4D is a graph showing electrophoresis results of amplification products of primers at different concentrations, FIG. 4E is a graph showing electrophoresis results of amplification products at different RAP isothermal amplification temperatures, FIG. 4F is a graph showing electrophoresis junctions of amplification products at different RAP isothermal amplification timesFig. 4G is a graph of fluorescence spectrum detection results of different CRISPR/Cas12a reaction temperatures, and fig. 4H is a graph of visualization detection results of different CRISPR/Cas12a reaction temperatures;

FIG. 5 is a graph of performance results of a methylation detection system of the present application, wherein FIG. 5A is a graph of the results of the mSEPT9 test at different concentrations, and FIG. 5B is a graph of the results of the mSEPT9 test at different levels;

FIG. 6 is a visual result diagram of the methylation detection system of the present application, wherein FIG. 6A is a schematic diagram of the detection principle of the methylation detection system of the present application, FIG. 6B is a visual result diagram of mSEPT9 at different concentrations, and FIG. 6C is a visual result diagram of mSEPT9 at different contents;

FIG. 7 is a graph showing the effect of the methylation detection system of the present application on clinical diagnosis of cervical cancer, wherein FIGS. 7A and 7B are a graph (7A) and a graph (7B) showing the results of fluorescent signal values of a sample of exfoliated cells from a cervical cancer patient and the results of signal values of a sample from a non-cervical cancer patient, respectively, under the same amount of genomic DNA.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.

Example one method of the application for detecting cervical cancer samples

Methylation of the SEPT9 promoter region is considered to be an effective biomarker for cervical cancer diagnosis, and detection is carried out by the following method. The principle of the detection method in this embodiment is shown in fig. 1. As can be seen from FIG. 1A, methylated and unmethylated DNA were first treated with BstUI methylation sensitive restriction enzyme that recognizes and digests the "5'-CGCG-3'" site. Therefore, unmethylated DNA will be cleaved by BstUI.

In the present application, the SEPT9 gene promoter SEPT9:77373475-77373595 (121 bp) contains 3 cleavage sites of 5 '-CGCG-3'. Thus, unmethylated modified SEPT9 was completely digested by BstUI. In contrast, methylated SEPT9 had no effect and the structure remained intact. The structurally intact methylated SEPT9 was used to activate the RPA amplification reaction. In the RPA system, a recombinase binds to a primer to locate homologous sequences in the duplex and exponentially amplify the target region. Thus, large amounts of dsDNA can be obtained by RPA amplification experiments.

Meanwhile, as shown in fig. 1B, since the generated dsDNA is partially complementary to the crRNA, the trans-cleavage activity of Cas12a is activated. Cas12a can arbitrarily cleave the nearby fluorescent reporter probe (FAM-DNA-BHQ 1) to release the green fluorescent signal. In addition, the method can also be used for realizing real-time detection through experimental visualization by changing the detection mode and using ultraviolet lamp analysis. When the SEPT9 is subjected to methylation modification, the RPA reaction can be activated to amplify a large amount of dsDNA in a short time, and then the trans-cleavage activity of the Cas12a is activated, so that the FAM-DNA-BHQ1 reporter gene with single chain is not specifically cleaved. After cleavage of the reporter probe containing FAM group modification, an absorption peak was observed at an excitation wavelength of 480nm at a wavelength of 525nm, and a green fluorescent signal was detected under an ultraviolet lamp. In contrast, unmethylated SEPT9, because it fails to activate Cas12a trans-cleavage activity, has no significant green fluorescent signal at 480nm excitation wavelength or uv lamp. Based on the detection principle of the sensor, the methylation modification condition of SEPT9 can be successfully distinguished, and the detection method can be applied to an instant detection or high-flux detection platform.

Materials and reagents

HeLa genomic DNA was purchased from Semer Feishmania technology (China). RPA primers, PAGE related reagents (DNA marker (25-500 bp), gelRed nucleic acid dye, TE buffer and loading buffer) were purchased from Shanghai Biotechnology Inc., crRNA and fluorescent group-labeled single-stranded DNA (ssDNA-FQ reporter) were purchased from Guangzhou Laibo Biotechnology Inc.Basic RPA kit was purchased from TwitDx corporation. Jurkat genomic DNA, lbaCas12a (Cpf 1), bstUI methylation sensitive restriction enzymes were purchased from NEB (beijing) limited. DNA/RNase free water, phenol, chloroform and DNA markers (100-1500 bp) were all from Tiangen Biochemical technologies (Beijing) Inc. Genomic DNA extraction kits were purchased from Jingshan biotechnology (Jiangsu) Inc.

TABLE 1 DNA and RNA sequences used in the examples of the present application

Note that: the red sequence is BstUI enzyme digestion recognition site; yellow sequence is CRISPR/Cas12a recognition site; the blue sequence is the spacer (spacer) on crRNA responsible for the complementarity to 21 bases downstream of the motif (protospacer adjacent motif, PAM, typically TTTN sequence, N A, C, G bases) on double-stranded DNA named primordial spacer.

Second, extraction of cervical exfoliated cell genome DNA

Genomic DNA of cervical exfoliated cells is extracted using a commercial kit, for example, a nucleic acid extraction kit (magnetic bead method) from Jingshan biotechnology (Jiangsu) limited. The concentration and quality of the extracted genome DNA are detected by Nanodrop, and the concentration of a DNA sample (OD 260/280=1.6-2.0; OD260/230 is more than or equal to 3.0; DNA concentration is more than or equal to 20 ng/. Mu.L, recommended as 100 ng/. Mu.L) with qualified quality is adjusted to 20 ng/. Mu.L, and the nucleic acid sample with adjusted concentration is ready for use. Then, the obtained genomic DNA was used as a target DNA, and the practical applicability of the method was verified.

Digestion of target DNA with BstUI methylation sensitive restriction endonucleases

The target DNA (mSEPT 9 and unsePT 9) was digested with BstUI endonuclease, and the total volume of the reaction was 20. Mu.L. Specifically, 2. Mu.L of the target DNA solution, 2. Mu.L (10X) of the buffer solution, 2. Mu.L (20U) of BstUI and 14. Mu.L of DNA/RNase-free water were added and reacted at 60℃for 30 minutes to obtain the cleavage product of the target DNA.

Fourth RPA amplification

UsingThe cleavage product obtained in the above step was amplified by the Basic RPA kit (Twistdx Co.) to obtain a target DNA fragment, and double-stranded DNA (dsDNA) was amplified. Wherein the reaction steps are as follows: according to the specification, taking 29.5 mu L of RPA reaction buffer solution to resuspend RPA freeze-dried powder to obtain uniform and transparent RPA reaction solution; then, 2. Mu.L of the digested product obtained in the above step (without purification), 2. Mu.L of the reverse primer (10. Mu.M), 2. Mu.L of the forward primer (10. Mu.M) and 12.85. Mu.L of DNA/RNase-free water were added to the RPA reaction solution and thoroughly mixed. Finally, 1.65. Mu.L of magnesium acetate (280 mM) was added, and the total reaction volume was 50. Mu.L. The reaction was carried out at 42℃for 15 minutes to give a reaction product (dsDNA).

Fifth, electrophoretic analysis

The amplification product obtained in the above amplification step was verified by using a non-denaturing 3% polyacrylamide gel. Because the directly synthesized double-stranded target DNA fragments or plasmid samples containing target gene fragments are linear DNA, the real detection environment cannot be simulated. Since the genomic DNA extracted from a real clinical sample is of supercoiled structure, which is far more complex than linear DNA, we used HeLa and Jurkat as methylation positive and negative samples, and dsDNA of RPA amplified target gene fragments as targets for electrophoretic analysis. Since substances in the RPA reaction interfere with agarose electrophoresis and if no product recovery is performed, tailing of the run-out band occurs, we used 20. Mu.L of phenol/chloroform (1:1) solution to mix well with the RPA product, and centrifuged to take the supernatant. After the supernatant was mixed with 6×DNA loading buffer, PAGE electrophoresis was performed in 1×TAE buffer at 130V for 40 minutes. Finally, taking a photograph of the gel after electrophoresis by using a Tanon UV image, and collecting an image. Both methylated SEPT9 (mSEPT 9) and unmethylated SEPT9 (unsePT 9) have 121bp bases, unsePT9 contains 3 BstUI "5'-CGCG-3'" sites, and therefore, after the unsePT9 reacts with BstUI endonuclease, the unsePT9 will be cut off at the cleavage site. As can be seen from FIG. 2, the band amplified specifically by the RPA reaction was observed in lane 3 (mSEPT 9 group), whereas no apparent target gene fragment was found in both lane 2 (unsePT 9 group) and lane 1 (negative control group). Since mSEPT9 thus methyl modification cannot be kept intact by restriction enzyme digestion, a large amount of dsDNA can be obtained for the RPA amplification template. These results indicate that both msepat 9 and unSEPT9 can be specifically recognized by BstUI.

CRISPR/Cas12a detection

The high-throughput detection based on fluorescence spectrum and the visual detection based on ultraviolet lamp are based on trans-cleavage activity of Cas12a, so Cas12a reaction proceeds as follows, total reaction volume is 20 μl: mu.L of DNA/RNase free water, 2. Mu.L of Cas12 enzyme reaction buffer, 4. Mu.L of crRNA (1. Mu.M), 2. Mu.L of Cas12a (1. Mu.M), 2. Mu.L of fluorescent probe (5. Mu.MFAM-DNA-BHQ 1) and 2. Mu.L of reaction product were incubated at 45℃for 15 min. For high throughput detection, 80. Mu.L of DNA/RNase-free water was added and its fluorescent signal intensity at 525nm was measured with a multifunctional microplate reader (SpectraMax M5) at 480nm excitation. For visual detection, CRISPR/Cas12a cleavage product (without dilution) is placed under an ultraviolet lamp and photographed with a smartphone or digital camera.

As shown in fig. 3, the fluorescence spectrum and the visual detection result are shown in fig. 3A and 3B, respectively. As a result, it was found that only mSEPT9 produced strong fluorescence, whereas the fluorescence of unsePT9 and the negative control group was negligible. The above results indicate that msepat 9 can avoid BstUI site-specific digestion through 5mC modification, thereby triggering RPA reaction to generate a large amount of dsDNA, thereby specifically activating trans-cleavage activity of Cas12a to cleave the fluorescently labeled single-stranded reporter gene, releasing fluorescent signal. Meanwhile, unSEPT9 cannot activate subsequent RPA reactions and CRISPR/Cas12a reactions because it can be recognized by BstUI to be digested. In high-throughput detection (fig. 3A), only the msepat 9 group exhibited significant signal intensity at the 525nm emission wavelength, whereas the unSEPT9 and NC signal values were comparable, since in the msepat 9 group, the nonspecific cleavage property of Cas12a was activated, resulting in the cleavage of FAM-DNA-BHQ1, the FAM group separated from the quencher group, and the FAM group had a deliberate absorbance peak at the 525nm excitation wavelength. In the visual detection (fig. 3B), only the msepat 9 group showed significant green fluorescence under uv light, due to the cleavage of FAM-DNA-BHQ1, separation of the FAM group from the quencher group, releasing fluorescence. The above results indicate that the merspr system is capable of successfully detecting DNA methylation with low background interference and high sensitivity and accuracy.

Example two optimization experiments of the method of the application

In order to improve the sensitivity of the method, MSRE experiments (BstUI cleavage substrate concentration, bstUI concentration), RPA experiments (Mg ²⁺ Concentration, primer concentration, reaction time, reaction temperature) and CRISPR/Cas12a experiments (CRISPR/Cas 12a cleavage time and CRISPR/Cas12a reaction temperature) were optimized, see fig. 4 for specific results.

MSRE experimental parameter optimization

BstUI cleavage substrate concentration optimization: to achieve POCT (point of care detection) of the merspr technique, we first set the cleavage reaction time to 30min to optimize the concentration of cleaved substrate. MSRE (methylation sensitive restriction endonuclease) experimental condition optimization: mu.L of Jurkat gDNA (0/25/50/100/250/500 ng, i.e., 0, 1.25, 2.5, 5, 12.5, 25 ng/. Mu.L), 2. Mu.L (10X) of buffer, 2. Mu.L (20U) of BstUI and 14. Mu.L of DNA/RNase-free water were reacted at 60℃for 30 minutes to obtain the digested products of the target DNA. The digested product of the above step was amplified using an RPA kit to obtain a double-stranded DNA (dsDNA) amplification product, wherein the amplification step and the RPA amplification system were as described in reference example one. Finally, referring to the electrophoresis analysis in the first embodiment, the result of the RPA reaction is subjected to PAGE analysis to obtain the target gDNA digestion effect. As shown in FIG. 4A, the results of the PAGE experiments showed that specific amplification of target DNA occurred when the substrate amount was 100-500ng (5/12.5/25 ng/. Mu.L), indicating that Jurkat gDNA was incompletely digested under this condition, whereas no band of interest appeared in the 50ng (2.5 ng/. Mu.L) group, indicating that Jurkat gDNA was completely digested under this condition, and thus 2.5 ng/. Mu.L Jurkat gDNA (50 ng) was selected as the optimal cleavage substrate concentration for the MSRE experiment.

BstUI enzyme concentration optimization: we first set the cleavage reaction time to 30min and the substrate concentration to 2.5 ng/. Mu.L to optimize the optimal BstUI enzyme concentration. MSRE (methylation sensitive restriction enzyme) experiment BstUI enzyme concentration condition optimization: bstUI enzyme (i.e., 0, 0.1, 0.25, 0.5, 0.75, 1U/. Mu.L) at different concentrations, 2. Mu.L (10X) of buffer, 2. Mu.L (20U) of BstUI and 14. Mu.L of DNA/RNase free water were reacted at 60℃for 30 minutes to obtain the cleavage products of the target DNA. The digested product of the above step was amplified using an RPA kit to obtain a double-stranded DNA (dsDNA) amplification product, wherein the amplification step and the RPA amplification system were as described in reference example one. Finally, the RPA reaction product is processed in the fifth step, and is subjected to PAGE analysis, so that the target gDNA enzyme digestion effect is obtained. As shown in FIG. 4B, the results of the PAGE experiments showed that specific amplification of target DNA occurred at BstUI enzyme concentrations of 0, 0.1, 0.25, 0.5U/. Mu.L), indicating incomplete digestion of Jurkat gDNA under these conditions. Whereas no target band appeared at 0.75, 1U/. Mu.L, indicating that Jurkat gDNA was completely digested under this condition, and that incomplete digestion resulted in a high background signal in view of BstUI cleavage capacity associated with background signal, 1U/. Mu.L BstUI enzyme was chosen as the optimal enzyme concentration for MSRE experiments.

(II) RPA optimization experiments

1.Mg ²⁺ Concentration optimization: we first formulated Mg at different concentrations ²⁺ (0, 10, 15, 20, 25, 30 mM) to optimize Mg ^{2 +} Is a concentration of (3). The method comprises the following specific steps: according to the specification, taking 29.5 mu L of RPA reaction buffer solution to resuspend RPA freeze-dried powder to obtain uniform and transparent RPA reaction solution; then, 2. Mu.L of the digested product obtained in the above step (without purification), 2. Mu.L of the reverse primer (10. Mu.M), 2. Mu.L of the forward primer (10. Mu.M) and 12.85. Mu.L of DNA/RNase-free water were added to the RPA reaction solution and thoroughly mixed. Finally, 2. Mu.L of magnesium acetate (different concentrations) was added, the total reaction volume being 50. Mu.L. The reaction was carried out at 42℃for 30 minutes to give a reaction product (dsDNA). Wherein the substrate digestion step and the system reference step three. Finally, the RPA reaction product is processed in the fifth step, and PAGE analysis is carried out to obtain the target gDNA amplification effect. As shown in FIG. 4C, the results of the PAGE experiments indicate that when Mg ²⁺ At concentrations of 10, 15, 20, 25mM, specific amplification of target DNA occurred. Whereas at 0 and 30mM no band of interest appears. In addition, the brightness of the target DNA band was maximized under 10mM conditions, indicating that the target DNA amplification efficiency was maximized under these conditions, and thus 10mM Mg was selected ²⁺ Optimal Mg as RPA experiment ²⁺ Concentration.

2. Primer concentration optimization: we first formulated primers at different concentrations (0, 100, 200, 400, 500 and 600 nM) to optimize primer concentration. The method comprises the following specific steps: according to the specification, taking 29.5 mu L of RPA reaction buffer solution to resuspend RPA freeze-dried powder to obtain uniform and transparent RPA reaction solution; then, 2. Mu.L of the digested product obtained in the above step (without purification), 2. Mu.L of the reverse primer (different concentrations), 2. Mu.L of the forward primer (different concentrations) and 12.85. Mu.L of DNA/RNase-free water were added to the RPA reaction solution and thoroughly mixed. Finally, 2. Mu.L of magnesium acetate (final concentration 10 mM) was added, and the total reaction volume was 50. Mu.L. The reaction was carried out at 42℃for 30 minutes to give a reaction product (dsDNA). Wherein the substrate digestion step and the system reference step three. Finally, the RPA reaction product is processed in the fifth step, and PAGE analysis is carried out to obtain the target gDNA amplification effect. As shown in FIG. 4D, the results of the PAGE experiments showed that specific amplification of target DNA occurred at primer concentrations of 100, 200, 400, 500 and 600 nM. Whereas at 0nM, no band of interest appears, indicating the specificity of the amplification. Furthermore, it was found that the brightness of the target DNA band was maximal under 400nM conditions, indicating that the target DNA amplification efficiency was maximal under this condition, and 400nM was therefore chosen as the optimal primer concentration for the RPA experiment.

3. And (3) temperature optimization: we first set different temperatures (38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃ and 43 ℃) to optimize the temperature of the reaction. The method comprises the following specific steps: according to the specification, taking 29.5 mu L of RPA reaction buffer solution to resuspend RPA freeze-dried powder to obtain uniform and transparent RPA reaction solution; then, 2. Mu.L of the digested product obtained in the above step (without purification), 2. Mu.L of the reverse primer (final concentration: 400 nM), 2. Mu.L of the forward primer (final concentration: 400 nM) and 12.85. Mu.L of DNA/RNase-free water were added to the RPA reaction solution and thoroughly mixed. Finally, 2. Mu.L of magnesium acetate (final concentration 10 mM) was added, and the total reaction volume was 50. Mu.L. The reaction was carried out at various temperatures for 30 minutes to give a reaction product (dsDNA). Wherein the substrate digestion step and the system reference step three. Finally, the RPA reaction product is processed in the fifth step, and PAGE analysis is carried out to obtain the target gDNA amplification effect. As shown in FIG. 4E, the result of the PAGE experiment shows that the target DNA was specifically amplified at 38℃at 39℃at 40℃at 41℃at 42℃and at 43 ℃. The brightness of the target DNA band was maximized at 42℃which indicates the highest target DNA amplification efficiency, and 42℃was selected as the optimal reaction temperature for the RPA experiment.

4. And (3) time optimization: in addition, RPA amplification time is also an important reaction parameter, and is closely related to the sensitivity of methylation gene detection. We first set different reaction times (0, 5, 10, 15, 20 and 30 min) to optimize the reaction time. The method comprises the following specific steps: according to the specification, taking 29.5 mu L of RPA reaction buffer solution to resuspend RPA freeze-dried powder to obtain uniform and transparent RPA reaction solution; then, 2. Mu.L of the digested product obtained in the above step (without purification), 2. Mu.L of the reverse primer (final concentration: 400 nM), 2. Mu.L of the forward primer (final concentration: 400 nM) and 12.85. Mu.L of DNA/RNase-free water were added to the RPA reaction solution and thoroughly mixed. Finally, 2. Mu.L of magnesium acetate (final concentration 10 mM) was added, and the total reaction volume was 50. Mu.L. The reaction was carried out at 42℃for various times to give the reaction product (dsDNA). Wherein the substrate digestion step and the system reference step three. Finally, the RPA reaction product is processed in the fifth step, and PAGE analysis is carried out to obtain the target gDNA amplification effect. As shown in FIG. 4F, the results of the PAGE experiments showed that no band of interest appeared at 0min and 5min, indicating the specificity of the amplification. Specific amplification of target DNA occurs at times of 5min, 10min, 15min and 30 min. In contrast, the brightness of the target DNA band reached the peak at 15min, which indicates that the target DNA amplification efficiency was highest under this condition, and 15min was chosen as the optimal reaction time for the RPA experiment.

(III) CRISPR/Cas12a optimization experiments

1. And (3) temperature optimization: we first set different temperatures (35 ℃, 37 ℃, 39 ℃, 41 ℃, 43 ℃ and 45 ℃) to optimize the concentration of the reaction. The method comprises the following specific steps: the total reaction volume was 20. Mu.L: 8. Mu.L of DNA/RNase free water, 2. Mu.L of the enzyme reaction buffer for LCas12a, 4. Mu.L of crRNA (1. Mu.M), 2. Mu.L of Cas12a (1. Mu.M), 2. Mu.L of fluorescent probe (5. Mu.M FAM-DNA-BHQ 1) and 2. Mu.L of RPA reaction product were incubated for 15 minutes at different temperatures. Wherein the RPA test procedure and system are described in reference to example one. For high throughput detection, 80. Mu.L of DNA/RNase-free water was added and its fluorescent signal intensity at 525nm was measured with a multifunctional microplate reader (SpectraMax M5) at 480nm excitation. For visual detection, CRISPR/Cas12a cleavage product (without dilution) is placed under an ultraviolet lamp and photographed with a smartphone or digital camera. As shown in FIGS. 4G-4H, the fluorescence signal gradually increased from 35℃to 45℃with increasing temperature. And the fluorescence signal intensity was highest when the reaction temperature was 45 ℃ (fig. 4G). The result of visualization (FIG. 4H) also revealed that the green fluorescence signal was strongest at a reaction temperature of 45 ℃. The next experimental Cas12a reaction temperature was therefore 45 minutes.

Example three MeCRISPR System Performance study to detect DNA methylation

Compared with the DNA methylation detection method in the prior art, the linear DNA detection difficulty is lower in all other technologies at present, and the genomic DNA with the supercoiled structure is not used, so that the method provided by the application has the advantages that the genomic DNA is more close to clinical application, the detection limit is lower, and the detection range is wider. Furthermore, the entire reaction can be completed within 1h, and is thus well suited for point of care testing (POCT). These excellent properties may benefit from the following reasons: (1) The specific recognition of BstUI enzyme and the 3 restriction sites in selected fragments of the application allow for efficient discrimination between methylated and unmethylated DNA. (2) The efficient amplification capability of RPA can produce large amounts of dsDNA in a short time. (3) The obtained dsDNA can be specifically identified by Cas12a due to the existence of PAM sequence sites, so that the detection specificity of the dsDNA is increased. The high specificity and self-amplifying capability of the CRISPR/Cas12a system further improves the detection performance of the detection system.

To study the performance of the methylation detection system, we formulated different concentrations of mSEPT9 (0, 1, 2.5, 5, 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ cobies/. Mu.L), the performance of the MeCRISPR system on mSEPT9 detection was investigated under optimal experimental conditions. As shown in FIG. 5A, under 480nm laser stimulation, 1, 2.5, 5, 10 ² 、10 ³ 、10 ⁴ 、10 ⁵ The fluorescence signal value of copies/. Mu.L mSEPT9 at 525nm was gradually increased and significantly higher than that of the control group (0 copy/. Mu.L mSEPT 9).

Prior studies have shown that the proportion of methylation modifications in mammalian genomes is typically less than 0.1%, most being in the unmethylated modified state. Therefore, in a large number of unmethylated samples, it is important to detect a very low amount of methylated genes. To solve the above problems, we first set the amount of unmethylated target DNA (umSEPT 9) to 50ng, then add different amounts of methylated target DNA (mSEPT 9) to the reaction system of unmethylated SEPT9, prepare a series of concentration gradient mSEPT9 solutions (0%, 0.01%,0.05%,0.1%,1%,10%, and 100%), and further study the performance of the merspr system on mSEPT9 detection under optimal experimental conditions. As shown in FIG. 5B, the fluorescence signal values at 525nm of 0.01%,0.05%,0.1%,1%,10%, and 100% mSEPT9 were gradually increased under 480nm excitation, and were significantly higher than those of the control group (0% mSEPT9). The above results show that the MeCRISPR system adopted by the application has higher accuracy and sensitivity in methylation gene detection.

Example visual study of DNA methylation detection by the tetramerspr system

The visual detection of the tumor has the advantages of intuitiveness, convenience and the like, so that the visual detection method has great application value in the tumor detection for establishing a visual methylation gene detection platform. Thus, the present application combines the merspr system with a portable hand-held uv lamp to enable visualization of the detection. According to the detection principle of the merspr visualization (fig. 6A), when methylated target DNA is present (msepat 9), the tube appears green under uv light. While no significant green fluorescence appears in unmethylated DNA (msepat 9). Next, the present application investigated the sensitivity of the merspr system based on the methylation gene visualization assay. As shown in FIG. 6B, under UV light, the mSEPT9 concentration is from 0 to 10 ⁵ The gradient between copies/. Mu.L increases and the green fluorescence in the reaction tube shows a tendency to fade from colorless to dark. Notably, mSEPT9 as low as1 copy/. Mu.L could be identified visually by the MeCRISPR experiment. Furthermore, as can be seen from fig. 6C, the green fluorescence in the reaction tube showed a tendency to fade from colorless to dark as the msepat 9/unSEPT9 ratio was increased from 0 to 100% in a gradient under the uv lamp. Notably, as low as 0.01% mSEPT9 can be MeCRIThe SPR system recognizes. These results show that the MeCRISPR-based system adopted by the application has higher sensitivity to the visual detection of the methylation genes, and does not need complex instruments and equipment, thereby having better application prospect in the areas with limited resources.

Example five real sample detection

In order to evaluate the effect of the MeCRISPR system in clinical diagnosis of cervical cancer, the MeCRISPR experiment is carried out to detect the mSEPT9 by taking a clinical sample commonly used in noninvasive diagnosis of cervical cancer, namely cervical exfoliated cells, as a material, and the detection effect is explored by comparing the MeCRISPR experiment with the gold standard-pathological detection of cervical cancer diagnosis. First, the genomic DNA sample of the extracted cervical exfoliated cells was used as a material, the concentration thereof was adjusted to 25 ng/. Mu.L, and the target DNA (mSEPT 9) was detected by the MeCRISPR method. As shown in FIG. 7A, the fluorescent signal value of the exfoliated cell sample of cervical cancer patient was significantly higher than that of the non-cervical cancer patient sample under the same amount of genomic DNA (25 ng/. Mu.L). Furthermore, the fluorescent intensity of the test tube under the ultraviolet lamp was visually observed under the same amount of genomic DNA (25 ng/. Mu.L), and it was found that the fluorescent intensity in the genomic DNA of the cervical cancer patient was significantly higher than that in the sample of the non-cancer patient (shown in FIG. 7B). Finally, 53 real samples are tested, the sensitivity of the application in cervical cancer detection is 100%, the specificity is 92.3%, the accuracy is 96.2%, and the excellent detection performance can be suitable for detection of complex real samples. The results show that the method has extremely high sensitivity and specificity in cervical cancer diagnosis for detecting the methylation of the cell DNA.

It is to be understood that this application is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are also encompassed by the appended claims.

Claims (10)

1. POCT system for direct detection of methylated genes, characterized in that the system is based on isothermal amplification method and CRISPR/Cas method combination, comprising:

component 1: it includes methylation sensitive restriction enzymes that digest genomic DNA where methylation modified sequence fragments of interest will remain intact and non-methylated sequence fragments will be specifically cleaved;

component 2: the method comprises an isothermal amplification reagent, wherein the isothermal amplification reagent carries out isothermal amplification on the methylation modified target sequence fragment to obtain double-stranded DNA, and the unmethylated sequence fragment does not carry out isothermal amplification;

and (3) a component 3: and (3) a CRISPR/Cas reagent, wherein the reagent specifically recognizes the double-stranded DNA, so that the Cas enzyme is activated to cleave the single-stranded reporter DNA with a fluorescent group in a trans-cleavage activity, and a fluorescent signal is generated to directly detect the methylation modified target sequence fragment.

2. The POCT system of claim 1, wherein the methylation sensitive restriction enzyme is endonuclease BstUI and the isothermal amplification reagent is RPA isothermal amplification reagent.

3. The POCT system of claim 1, wherein the CRISPR/Cas reagent is a CRISPR/Cas12a reagent.

4. The POCT system of claim 3, wherein the CRISPR/Cas12a reagent comprises crRNA, cas12a enzyme and single-stranded reporter DNA having a fluorescent group, the crRNA guides Cas12a enzyme to recognize the double-stranded DNA, the methylation-modified sequence fragment of interest is specifically cleaved, the double-stranded DNA comprises a nucleic acid fragment specifically binding to crRNA, the nucleic acid fragment initiation sequence is TTTN sequence, and N is A, C, G base; the single-stranded reporter DNA having a fluorescent group is cleaved by the activated Cas12a enzyme to generate a fluorescent signal for specific detection.

5. Use of the POCT system for detecting methylated genes according to claims 1-4, comprising the steps of:

step 1: digesting the genomic DNA with a methylation sensitive restriction enzyme, wherein the methylation modified target sequence fragment remains intact and the non-methylated gene fragment is specifically cleaved;

step 2: digesting the genome DNA by using the methylation sensitive restriction enzyme in the step 1 as a template, and carrying out isothermal amplification, wherein the methylated genome DNA is amplified into a target sequence segment double-stranded DNA, and the non-methylated genome DNA is not subjected to isothermal amplification;

step 3: the double-stranded DNA in the step 2 is used as a template to perform CRISPR/Cas activation, the double-stranded DNA activates the reverse cleavage activity of Cas enzyme, the non-specific cleavage is performed on the fluorescence modified single-stranded reporter DNA to generate a fluorescence signal, and the non-methylated sequence fragment cannot generate double-stranded DNA due to non-isothermal expansion, so that Cas12a enzyme cannot be activated, and no fluorescence signal is generated;

step 4: directly judging whether the methylation gene exists or not through a fluorescent signal.

6. The use according to claim 5, wherein the methylation sensitive restriction enzyme is the endonuclease BstUI and the isothermal amplification is RPA isothermal amplification.

7. The use of claim 5, wherein the CRISPR/Cas activation is a CRISPR/Cas12a activation.

8. The use of claim 5, wherein the CRISPR/Cas12a activation comprises using crRNA, cas12a enzyme, and single-stranded reporter DNA with a fluorescent group; the crRNA guides the Cas12a enzyme to recognize the double-stranded DNA, the methylation modified target sequence fragment is specifically cut, the double-stranded DNA comprises a nucleic acid fragment specifically combined with the crRNA, the initial sequence of the nucleic acid fragment is a TTTN sequence, and N is any one base of A, C, G; the single-stranded reporter DNA having a fluorescent group is cleaved by the activated Cas12a enzyme to generate a fluorescent signal for specific detection.

9. The use according to claim 8, wherein the nucleic acid fragment initiation sequence is a TTTC sequence.

10. The use according to claim 5, wherein the sequence fragment of interest is the SEPT9 gene promoter SEPT9:77373475-77373595 fragment.