CN118064565A - Competitive CRISPR-Dx detection system - Google Patents
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Abstract
The invention relates to a competitive CRISPR-Dx detection system. The detection system comprises a first substrate fragment, a second substrate fragment, general purpose CrRNA and Lbcas a, wherein the nucleotide sequence of the first substrate fragment is shown as SEQ ID No. 1 or SEQ ID No. 3, the nucleotide sequence of the second substrate fragment is shown as SEQ ID No. 2 or SEQ ID No. 4, and the nucleotide sequence of the general purpose CrRNA is shown as SEQ ID No. 5. After binding of the Lbcas a to the universal CrRNA, the target sequence is cis-cleaved, and the single-stranded regions of the first and second substrate fragments are trans-cleaved, resulting in a large number of short single-stranded DNA fragments rich in "T" bases. These short single-stranded DNA fragments rich in "T" bases resemble the probe sequence and therefore compete with the probe and reduce the fluorescent signal, thus distinguishing them from higher fluorescent signals in the absence of the target sequence.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a competitive CRISPR-Dx detection system.
Background
Nucleic acid detection techniques (CRISPR-based Diagnostics, CRISPR-Dx) are detection techniques developed based on the trans-cleavage properties of the CRISPR-Cas12 and CRISPR-Cas13 families.
Polymerase Chain Reaction (PCR) and its derivative techniques such as conventional PCR, fluorescent quantitative PCR, digital droplet PCR, etc. have long been gold standards for nucleic acid detection. However, the PCR reaction requires three steps of denaturation, annealing and extension, has high requirements on equipment and operators, requires high-temperature conditions, is easy to generate nonspecific amplification, and has long reaction time (1-2 hours), so that the application of the PCR reaction is limited to medical examination institutions. The recombinase polymerase isothermal amplification (RPA) detection reaction is faster (20 minutes), and can react at normal temperature, but the specificity is poor, nonspecific amplification is easy to occur, and false positive results are easy to occur due to the influence of environmental nucleic acid pollution. CRISPR-Dx can rapidly react at room temperature and has good specificity, but the sensitivity is insufficient when the CRISPR-Dx is used alone, and missed diagnosis is easy to occur when the concentration of nucleic acid is low.
Because the sensitivity of CRISPR-Dx is still to be optimized, a research team combines the CRISPR-Dx with RPA, and the aim of remarkably improving the sensitivity is fulfilled. Unfortunately, RPA is prone to non-specific amplification, resulting in reduced accuracy of the detection technique after binding.
For both sensitivity and accuracy, the prior art provides the following ideas: the reaction system is divided into droplets to increase local concentration, electrochemical biosensors are used, cas-based cascade amplification, crispr ribonucleic acid (CrRNA) is modified, a plurality of CrRNA, metal-enhanced fluorescence, and the like. The prior art has longer reaction time and higher common cost, and is not beneficial to the popularization of the technology; RNA substrates are difficult to preserve due to the widespread presence of RNases (RNases).
Disclosure of Invention
The invention aims to disclose a competitive CRISPR-Dx detection system, which solves one or more technical problems existing in the prior art and provides at least one beneficial selection or creation condition.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
the first aspect of the present invention is to provide a competitive CRISPR-Dx detection system.
The competitive CRISPR-Dx detection system provided by the first aspect of the invention comprises a first substrate fragment, a second substrate fragment, general purpose CrRNA and Lbcas a, wherein the nucleotide sequence of the first substrate fragment is shown as SEQ ID No. 1 or SEQ ID No. 3, the nucleotide sequence of the second substrate fragment is shown as SEQ ID No. 2 or SEQ ID No. 4, and the nucleotide sequence of the general purpose CrRNA is shown as SEQ ID No. 5. The first substrate fragment and the second substrate fragment are sequentially composed of a self-complementary A region, a single-chain A region, a pairing region, a single-chain B region and a self-complementary B region, and the self-complementary A region and the self-complementary B region of the same substrate fragment are complementarily paired. The Lbcas a combines with the generic CrRNA to form a generic CrRNA-Lbcas a complex. In the absence of target sequence in the reaction system, the generic CrRNA-Lbcas a complex cleaves the pairing region of the first and second substrate fragments in cis and cleaves the probe in trans, yielding a stronger detection signal. And in the presence of the target sequence in the reaction system, after cis-cleavage of the target sequence by the universal CrRNA-Lbcas a complex, trans-cleaving the single-stranded A or single-stranded B regions of the first and second substrate fragments to produce a plurality of short single-stranded DNA fragments enriched in "T" bases. These short single-stranded DNA fragments rich in "T" bases are similar to the probes and therefore compete with the probes, reducing the signal from the probes and thus distinguishing them from the higher signal intensity in the absence of target sequence.
The nucleotide sequence of the first substrate fragment is:
5'-CCCCCCTTTTTATTTTTATTTTTATTTTTTTTGTGTCCCAGTCATATCCGTTGCTTTTTATTTTTATTTTTATTTTTGGGGGG-3'(SEQ ID No:1) Or alternatively, the first and second heat exchangers may be,
5'-CCCCCCTTTTTATTTTTATTTTTATTTTTTTTGGTTGTCCCAGTCATATCCGTTGCTTTTTATTTTTATTTTTATTTTTGGGGGG-3'(SEQ ID No:3);
The nucleotide sequence of the second substrate fragment is:
5'-GGGGGGTTTTTATTTTTATTTTTATTTTTGCAACGGATATGACTGGGACACAAATTTTTATTTTTATTTTTATTTTTCCCCCC-3'(SEQ ID No:2) Or alternatively, the first and second heat exchangers may be,
5'-GGGGGGTTTTTATTTTTATTTTTATTTTTGCAACGGATATGACTGGGACAACCAAATTTTTATTTTTATTTTTATTTTTCCCCCC-3'(SEQ ID No:4).
The nucleotide sequence of the universal CrRNA is as follows:
5’-UAAUUUCUACUAAGUGUAGAUGUUGUCCCAGUCAUAUCCGUUGCTATTATT-3’(SEQ ID No:5)。
In some embodiments of the first aspect of the present invention, the first substrate fragment and the second substrate fragment form a double-loop structure after annealing, the double-loop structure includes a first loop structure and a second loop structure, the first substrate fragment forms the first loop structure, the second substrate fragment forms the second loop structure, and the pairing region of the first substrate fragment and the pairing region of the second substrate fragment are complementarily paired through hydrogen bonds to form the double-loop structure. The double-loop substrate is capable of generating more short single-stranded DNA fragments rich in "T" bases when subjected to Lbcas a trans-cleavage, helping to further amplify the signal changes.
In some embodiments of the first aspect of the invention, the reaction system of the annealing treatment comprises enzyme-free water, a DNA annealing buffer, the first substrate fragment and the second substrate fragment. The DNA return buffer may be selected from commercially available products such as D0251 from bi-cloud.
In some embodiments of the first aspect of the present invention, the annealing process is performed by: 98℃for 1 minute; reducing to 25 ℃ at a rate of 1 ℃ per minute; preserving at 25 ℃. The hydrogen bond is opened in the annealing treatment process through heating, and the pairing area of the first annular structure can be mutually identified and paired with the pairing area of the second annular structure by cooling at a constant speed, so that the double annular structure is formed.
In some embodiments of the first aspect of the invention, the CRISPR-Dx detection system further comprises enzyme-free water, buffer, magnesium chloride solution, rnase inhibitor, assay CrRNA, and probe. The CRISPR-Dx detection system has a plurality of expansion possibilities, and can be prepared into a fluorescent detection kit or a colloidal gold test strip according to different use scenes and use requirements, but is not limited to the preparation method. And (3) performing a control experiment on the experimental group containing the sample to be detected and the blank group without adding the sample to be detected, and judging through the change of the fluorescence values detected by the two groups, so as to obtain whether the sample to be detected contains the template fragment.
In some embodiments of the first aspect of the invention, the probe has a fluorescent group attached to the 5 'end and a fluorescence quenching group attached to the 3' end.
In some embodiments of the first aspect of the invention, the nucleotide sequence of the probe is: 5'-TTATTATT-3'. The short single-stranded DNA fragment rich in "A" and "T" bases released by the Lbcas a for trans-cleavage of the substrate with the double-loop structure is highly similar to the nucleotide sequence of the probe, so that the Lbcas a can be prevented from trans-cleaving the probe, and the fluorescence value of the detection result is reduced.
In some embodiments of the first aspect of the invention, the nucleotide sequence of the assay CrRNA is set forth in SEQ ID Nos. 6 to 8. The assay CrRNA shown in SEQ ID No. 6 is used to detect the ERBB2 gene, the assay CrRNA shown in SEQ ID No. 7 is used to detect the EGFR gene, and the assay CrRNA shown in SEQ ID No. 8 is used to detect the DYS gene.
In some embodiments of the first aspect of the invention, the buffer is a 10 x HOLMES buffer.
In some embodiments of the first aspect of the invention, the detection system has a volume of 20. Mu.L, comprising 2. Mu.L of 10 XHOLMES buffer, 1.5. Mu.L of 25mM magnesium chloride solution, 0.25. Mu.L of 40U/. Mu.L of RNase inhibitor, 1. Mu.M of the universal CrRNA 0.25.25. Mu.L, 1. Mu.M of the assay CrRNA 0.25.25. Mu.L, 10 pmol/. Mu.L of Lbcas a 0.5. Mu.L, 10mM of the probe 1. Mu.L, 1. Mu.L of substrate solution containing the first and second substrate fragments diluted 50-fold, 12.25. Mu.L of non-enzymatic water, and 1. Mu.L of sample solution to be tested.
The invention has the advantages that:
(1) By adding the first substrate fragment, the second substrate fragment and the common CrRNA into a CRISPR-Dx reaction system, a large number of short single-stranded DNA fragments rich in 'T' bases can be released to inhibit the cas12 enzyme from cutting the probe, so that the sensitivity is higher than that of the conventional CRISPR-Dx method.
(2) Because the sensitivity is higher than that of the conventional CRISPR-Dx method, the RPA amplification is not needed, so that the non-specific amplification of the RPA is avoided, and the specificity is improved.
(3) The two substrate fragments added in the invention are DNA, and the theoretical stability is higher than that of RNA substrate.
(4) Since both substrate fragments are DNA, the reaction requires only the addition of cas12 enzyme and no cas13 enzyme, resulting in lower costs.
(5) As the CRISPR-Dx detection system is used as an interpretation standard by taking the ratio of the fluorescent value of the experimental group to the fluorescent value of the blank group when being applied to the detection by a fluorescence method, the ratio is stable within the range of 10-30 minutes, the reaction time of 20 minutes can meet the diagnosis requirement, and the problem of long reaction time of the CRISPR-Dx in the prior art is solved.
Drawings
FIG. 1 is a schematic view of a double annular structure according to example 1;
FIG. 2 is a line graph of detection times for the optimized fluorescence CRISPR-Dx detection system of example 2;
FIG. 3 is a bar graph of dilution of DNA substrate combinations for the optimized fluorescence CRISPR-Dx detection system of example 2;
FIG. 4 is a bar graph of the amount of MgCl 2 used in the optimized fluorescence CRISPR-Dx detection system of example 2;
FIG. 5 is a bar graph of the amount of optimized fluorescence CRISPR-Dx detection system Lbcas a in example 2;
FIG. 6 is a bar graph of probe usage for the optimized fluorescence CRISPR-Dx detection system of example 2;
FIG. 7 is a graph of a control line of detection of ASFV gDNA using the conventional CRISPR-Dx detection system and the fluorescence CRISPR-Dx detection system of example 2;
FIG. 8 is a graph of the specificity verification of the fluorescence CRISPR-Dx detection system of example 2;
FIG. 9 is a bar graph of the specificity verification of the fluorescence CRISPR-Dx detection system of example 2;
FIG. 10 is a graph of fluorescence values for different concentrations of gDNA added in example 2;
FIG. 11 is a histogram of fluorescence values multiplied by the addition of gDNA at different concentrations in example 2;
FIG. 12 is a linear regression plot of fluorescence values at various gDNA concentrations in example 2;
FIG. 13 is a graph showing the results of the test strip method of example 3 in which gDNA of different concentrations was added;
FIG. 14 is a control line graph of the fluorescence CRISPR-Dx detection system of example 4 for detecting ERBB2 gene;
FIG. 15 is a photograph showing the detection of ERBB2 gene by CRISPR-Dx detection system of the test strip method in example 4;
FIG. 16 is a control line graph of the fluorescence CRISPR-Dx detection system of example 5 for EGFR gene detection;
FIG. 17 is a photograph showing the detection of DYS gene by the CRISPR-Dx detection system of the test strip method in example 6.
Detailed Description
Molecular biological assay methods not specifically described in the examples below are all described in reference to the guidelines for molecular cloning experiments (third edition) or according to the methods and product specifications; the method biological materials, unless otherwise specified, are commercially available.
Example 1: DNA substrate combinations were constructed to aid in the detection of African Swine Fever Virus (ASFV).
In the embodiment, ASFV is taken as a detection object, a DNA substrate combination is designed according to the T8 locus of the P72 gene of the ASFV, two groups of total 4 substrate fragments are obtained, the substrate fragments are 4 nucleotide single chains, the nucleotide sequences of the substrate fragments are shown in the table 1, CH8a and CH8b are matched for use, and CH9a and CH9b are matched for use.
TABLE 1 nucleic acid sequences of the substrate fragments of the DNA substrate combinations
The DNA substrate combination needs to be heated to open hydrogen bonds, and then annealing treatment is carried out, so that the pairing region of the first annular structure can be mutually identified and paired with the pairing region of the second annular structure, and then a double annular structure in an "infinity" shape shown in figure 1 is formed. The annealing system is shown in table 2.
TABLE 2 annealing System for DNA substrate combinations
| Component (A) | Volume (mu L) |
| Enzyme-free water | 4 |
| 5 Xannealing buffer liquid | 4 |
| 10 Mu mol/. Mu.L CH8a or CH9a | 6 |
| 10 Mu mol/mu L CH8b or CH9b | 6 |
| Totalizing | 20 |
The reaction procedure of the annealing system is as follows: 98℃for 1 minute; reducing to 25 ℃ at a rate of 1 ℃ per minute; preserving at 25 ℃.
Example 2: a fluorescence CRISPR-Dx detection system is constructed by utilizing DNA substrate combination.
A fluorescence CRISPR-Dx detection system was constructed using CH8a as the first substrate fragment and CH8b as the second substrate fragment as described in example 1 to obtain a kit with satisfactory specificity and sensitivity. The main reagent components of the kit comprise the DNA substrate combination, enzyme-free water, buffer solution, magnesium chloride solution, RNase inhibitor, lbcas a, general CrRNA and probe.
Since this reaction system principle reduces the fluorescence value by competing with the probe for downstream products generated after the addition of the target DNA, it is in principle desirable that the ratio (i.e. "fold") of the fluorescence value of the experimental group (to which the target DNA is added) to the blank group (to which the water is added) is as low as possible. The reaction time of a fluorescence CRISPR-Dx detection system and the dosage of each reagent need to be optimized: the reaction time was optimized as shown in FIG. 2, and the detection time was chosen to be 20 minutes, since the multiple was too high at 10 minutes; the DNA substrate combinatorial dilution factor optimization as described in fig. 3, "20×" is equivalent to "50×" dilution factor, significantly better than "100×" and "200×", with substrate dilution factor 50×beingultimately selected from a cost perspective; the amount of magnesium chloride solution (MgCl 2) shown in FIG. 4 was optimized, and 1.5. Mu.L was selected in consideration of factors, cost and experimental error; as shown in FIG. 5, the dosage of Lbcas a is optimized, and the multiple and the pipetting accuracy (cas 12 protein solution has viscosity and is easy to be adsorbed on a gun head, and the pipetting inaccuracy is caused by too small sample adding amount) are comprehensively considered to select 0.5 mu L; the probe amount was optimized as shown in FIG. 6, and 1. Mu.L was selected in consideration of the fold and cost. Since the universal CrRNA was designed based on the T8 site, the universal CrRNA could be used directly instead of the assay CrRNA when performing an ASFV-targeting assay, and thus the specific assay system is shown in table 3.
TABLE 3 fluorescence CRISPR-Dx detection System
The nucleotide sequence of the universal CrRNA is as follows:
5'-UAAUUUCUACUAAGUGUAGAUGUUGUCCCAGUCAUAUCCGUUGCTAT TATT-3' (SEQ ID No: 5). The sequence of the probe is as follows: 5'-TTATTATT-3', FAM is connected to the 5 'end of the probe, and BHQ1 is connected to the 3' end. The first substrate fragment contained in the DNA substrate combination solution is CH8a described in example 1, and the second substrate fragment is CH8b described in example 1, and both are annealed to form a substrate combination solution CH8.
Because the CRISPR-Dx detection system of the fluorescence method needs to take the ratio of the fluorescent value of the experimental group to the fluorescent value of the blank group as an interpretation basis, when the CRISPR-Dx detection system is used, the blank group is arranged by using enzyme-free water to replace a sample solution to be detected, and the fluorescent value of the blank group is taken as a background to be subtracted.
As shown in FIG. 7, the fluorescence curve of the traditional CRISPR-Dx detection system "498fMASFV gDNA" does not rise, the detection result is negative, and the gDNA with the concentration of 498fM is not detected by directly using the traditional CRISPR-Dx detection system; in the fluorescence CRISPR-Dx detection system provided by the invention, the substrate and ASFV gDNA are simultaneously added into the experimental group 'CH 8+498fM ASFV gDNA', and the fluorescence curve is lower than that of a blank group 'CH 8' with only the substrate, so that the added target DNA and the added water are separated and become detectable, and the detection limit is lower than that of the traditional CRISPR-Dx method.
After adding substrates to each of the three experimental systems, water, 498fM ASFV gDNA (target DNA), and plasmid DNA (hybrid DNA) containing Enhanced Green Fluorescent Protein (EGFP) gene were added to 498fM, and fold detection was performed. As shown in FIG. 8, the fluorescence curve of the mixed DNA added is basically as high as that of the water added, and the target DNA added is obviously reduced compared with that of the water added, so that the specificity of the detection method is good. The three groups had average fluorescence values of 1525, 1681, 832, respectively, at 20 min. When the threshold of the fold is 0.8 (the threshold is determined as follows), the target DNA fold=832/1525=0.55 <0.8, and the hybrid DNA fold=1681/1525=1.10 >0.8, so that the target DNA and the hybrid DNA can be distinguished, and the specificity of the constructed fluorescence CRISPR-Dx detection system is good.
According to the data of fig. 9, the ratio of added template target DNA was significantly different from the ratio of added other interfering DNA of the same concentration, and the threshold value = mean-3 x standard deviation = 1.104-3 x 0.099 = 0.807≡0.8, so the threshold value was set to 0.8-fold, and less than or equal to this value was defined as positive. It can be seen that the ratio of the fluorescence value of the added hybrid DNA to the fluorescence value of the added water at 20 minutes is greater than 0.8 times, while the ratio of the fluorescence value of the added target DNA to the fluorescence value of the added water is less than 0.8 times.
The result judging method comprises the following steps:
the ratio of the fluorescent value of the experimental group to the fluorescent value of the blank group is less than or equal to 0.8, and the experimental group should be judged to be positive.
The ratio of the fluorescent value of the experimental group to the fluorescent value of the blank group is more than 0.8, and the result is negative.
As shown in FIG. 10, it can be seen that overall, the higher the concentration of added DNA, the more pronounced the competition phenomenon and the lower the curve. As shown in fig. 11, the detection limit is 249fM on the premise of 0.8 times the threshold, and therefore, the detection limit of the fluorescence CRISPR-Dx detection system is 249fM. When the concentration of the template gDNA is 83-332 fM, the linear relation exists between the ratio and the concentration of the T8 sequence template (see FIG. 12), namely, the linear negative correlation exists between the fluorescence intensity multiple and the target DNA concentration, and the detection limit of the method can be estimated to be 207.41fM theoretically according to an equation. And even T8 sequence template concentrations as high as 2491fM will be diagnosed negative without the addition of DNA substrate combinations. Therefore, the detection limit is reduced by more than 10 times by adding the CRISPR-Dx detection system of the fluorescent method.
Example 3: and constructing a CRISPR-Dx detection system of the colloidal gold test strip by utilizing the DNA substrate combination.
Similarly, the DNA substrate provided in example 1 can be applied to a colloidal gold test strip.
The side-flow chromatography colloidal gold test strip rule does not need to set a blank group. Because of the difference of the carriers of the reaction system, the dilution factor of the DNA substrate solution needs to be reduced, and the concentration of the substrate fragment diluted by 15 times is better through the optimization test. The specific reaction system and reaction temperature conditions of the test strip method are shown in Table 4. And then the result can be interpreted.
TABLE 4 CRISPR-Dx detection System of colloidal gold test strip
| Component (A) | Volume (mu L) |
| Enzyme-free water | 12.25 |
| 10 XHOLMES buffer | 2 |
| 25MM magnesium chloride solution | 1.5 |
| 40U/. Mu.L RNase inhibitor | 0.25 |
| Universal CrRNA with 1 mu M target as T8 site | 0.5 |
| 10pmol/μL Lbcas12a | 0.5 |
| 10MM probe (P8 a-Cas 12-FAM-biotin) | 1 |
| 15-Fold diluted annealed DNA substrate combination solution | 1 |
| ASFV sample solution | 1 |
| Totalizing | 20 |
The result judging method comprises the following steps:
the control line (C line) develops color, the detection line (T line) develops color, and the judgment is negative.
The control line (C line) develops color, the detection line (T line) does not develop color, and the judgment should be positive.
Neither the C line nor the T line is colored, and the judgment is invalid.
The colloidal gold test strip shown in FIG. 13 sequentially measures the sample concentrations of 0, 83, 166, 249, 415, 498fM gDNA from left to right, and in general, the higher the DNA concentration is added, the more the competition phenomenon is obvious, the lighter the color of the detection line is, and the detection limit of the embodiment is 498fM. And even T8 sequence template concentrations as high as 2491fM will be diagnosed negative without the addition of DNA substrate combinations. Therefore, the detection limit is reduced by more than 5 times by adding the CRISPR-Dx detection system of the colloidal gold test strip.
Example 4: ERBB2 gene was detected using a competitive CRISPR-Dx detection system.
A CRISPR-Dx detection system targeting the ERBB2 gene of the MKN-45 cell line was constructed as provided in example 2, and is specifically shown in Table 5.
TABLE 5 CRISPR-Dx detection System targeting ERBB2 Gene
The nucleotide sequence of the assay CrRNA is as follows:
5'-UAAUUUCUACUAAGUGUAGAUCCUCAACACUUUGAUGGCCA-3' (SEQ ID No: 6). The first substrate fragment contained in the DNA substrate combination solution is CH9a described in example 1, and the second substrate fragment is CH9b described in example 1, and both are annealed to form a substrate combination solution CH9. The fluorescent probe was the same as in example 2.
A conventional CRISPR-Dx assay system was used as a control. As a result of the detection, as shown in FIG. 14, the label of "94 ng/. Mu.L MKN-45gDNA" represents a control group, "CH9" represents a blank group to which only the DNA substrate combination solution was added, and the label of "CH9+94 ng/. Mu.L MKN-45gDNA" represents an experimental group. The experimental group fluorescence value/blank group fluorescence value= 1829.33/4623.67 =0.40 <0.8, and the detection system is proved to be applicable to the detection of ERBB2 genes, and the detection limit is lower than that of the traditional CRISPR-Dx method.
The same detection system is used for the colloidal gold test strip, the fluorescent probe is replaced by the colloidal gold test strip probe as described in the embodiment 3, the detection result is shown in fig. 15, and the control line (C line) and the detection line (T line) of the left blank test strip are developed; the control line (C line) of the test strip of the right test group is developed, and the detection line (T line) is not developed, so that the positive detection result is indicated.
Example 5: EGFR genes were detected using a competitive CRISPR-Dx detection system.
A CRISPR-Dx detection system targeting the EGFR gene of the NCI-H1975 cell line was constructed as provided in example 2, and is specifically shown in Table 6.
TABLE 6 CRISPR-Dx detection System targeting EGFR Gene
The nucleotide sequence of the assay CrRNA is as follows:
5'-UAAUUUCUACUAAGUGUAGAUGGCUGGCCAAACUGCUGGGUG-3' (SEQ ID No: 7). The first substrate fragment contained in the DNA substrate combination solution is CH9a described in example 1, and the second substrate fragment is CH9b described in example 1, and both are annealed to form a substrate combination solution CH9. The fluorescent probe was the same as in example 2.
A conventional CRISPR-Dx assay system was used as a control. The results of the detection are shown in FIG. 16, in which the label of "NCI-H1975 gDNA" represents the control group, "CH9" represents the blank group to which only the DNA substrate combination solution was added, and the label of "CH9+NCI-H1975 gDNA" represents the experimental group. The experimental group fluorescence value/blank group fluorescence value=2683/4260=0.63 <0.8, and the detection system is proved to be applicable to EGFR gene detection, and the detection limit is lower than that of the traditional CRISPR-Dx method.
Example 6: the DYS gene is detected by using a competitive CRISPR-Dx detection system.
A CRISPR-Dx detection system for the DYS gene targeting RD139 (EX 50 del) cell line (Dystrophin (DYS) gene with the 50 th exon deleted) and RD139 cell line (DYS gene normal) gDNA was constructed as provided in example 3, and is specifically shown in Table 7.
TABLE 7 CRISPR-Dx detection system targeting DYS gene
| Component (A) | Volume (mu L) |
| Enzyme-free water | 12.25 |
| 10 XHOLMES buffer | 2 |
| 25MM magnesium chloride solution | 1.5 |
| 40U/. Mu.L RNase inhibitor | 0.25 |
| 1 Mu M general CrRNA | 0.25 |
| 1 Mu M assay CrRNA | 0.25 |
| 10pmol/μL Lbcas12a | 0.5 |
| 10MM probe (P8 a-Cas 12-FAM-biotin) | 1 |
| 15-Fold diluted annealed DNA substrate combination solution | 1 |
| DYS sample solution | 1 |
| Totalizing | 20 |
The result judging method comprises the following steps:
the control line (C line) develops color and the detection line (T line) develops color, which indicates that the DYS gene is missing and should be judged as positive.
The control line (C line) developed and the detection line (T line) did not develop, indicating that the DYS gene was present and negative.
Neither the C line nor the T line is colored, and the judgment is invalid.
The nucleotide sequence of the assay CrRNA is as follows:
5'-UAAUUUCUACUAAGUGUAGAUTTTACCGCCTTCCACTCAGAGCTCA-3' (SEQ ID No: 8). The first substrate fragment contained in the DNA substrate combination solution is CH9a described in example 1, and the second substrate fragment is CH9b described in example 1, and both are annealed to form a substrate combination solution CH9.
The detection CrRNA targets the 50 th exon (ex 50) of the DYS gene, the detection result is shown in figure 17, the control line (C line) of the DYS gene deletion group (DYS-) test strip on the left side develops color, the detection line (T line) develops color, and the detection result is positive; the control line (C line) of the right DYS gene normal group (DYS+) test strip is developed, the detection line (T line) is not developed, and the detection result is negative. The result shows that the detection system can be used for detecting DYS gene deletion. The method can realize early screening and home self-test of Du's Muscular Dystrophy (DMD) (the cause is DYS gene deletion).
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. A competitive CRISPR-Dx detection system is characterized by comprising a first substrate fragment, a second substrate fragment, a general purpose CrRNA and Lbcas a, wherein the nucleotide sequence of the first substrate fragment is shown as SEQ ID No. 1 or SEQ ID No. 3, the nucleotide sequence of the second substrate fragment is shown as SEQ ID No.2 or SEQ ID No. 4, and the nucleotide sequence of the general purpose CrRNA is shown as SEQ ID No. 5.
2. The detection system according to claim 1, wherein the first substrate fragment and the second substrate fragment are annealed to form a double-loop substrate, the double-loop substrate comprises a first loop structure and a second loop structure, the first substrate fragment forms the first loop structure, the second substrate fragment forms the second loop structure, and the pairing region of the first substrate fragment and the pairing region of the second substrate fragment are complementarily paired through hydrogen bonds to form the double-loop substrate.
3. The detection system of claim 2, wherein the reaction system of the annealing treatment comprises an annealing buffer, the first substrate fragment, and the second substrate fragment.
4. A detection system according to claim 3, wherein the annealing process is performed by the following reaction sequence: 98℃for 1 minute; reducing to 25 ℃ at a rate of 1 ℃ per minute; preserving at 25 ℃.
5. The detection system of any one of claims 1 to 4, further comprising a buffer, a magnesium chloride solution, an rnase inhibitor, a detection CrRNA, and a probe.
6. The detection system of claim 5, wherein the probe has a fluorescent group attached to the 5 'end and a fluorescence quenching group attached to the 3' end.
7. The detection system according to claim 5, wherein the probe has a nucleotide sequence of 5'-TTATTATT-3'.
8. The detection system according to claim 5, wherein the nucleotide sequence of the detection CrRNA is shown in any one of SEQ ID No. 6 to SEQ ID No. 8.
9. The detection system of claim 5, wherein the buffer is a 10 x HOLMES buffer.
10. The detection system according to claim 9, wherein the detection system has a volume of 20. Mu.L, comprising 2. Mu.L of 10 XHOLMES buffer, 1.5. Mu.L of 25mM magnesium chloride solution, 0.25. Mu.L of 40U/. Mu.L of RNase inhibitor, 1. Mu.M of the universal CrRNA 0.25.25. Mu.L, 1. Mu.M of the assay CrRNA 0.25.25. Mu.L, 10 pmol/. Mu.L of Lbcas a 0.5. Mu.L, 10mM of the probe 1. Mu.L, 1. Mu.L of substrate solution containing the first substrate fragment and the second substrate fragment diluted 50-fold, 12.25. Mu.L of enzyme-free water, and 1. Mu.L of sample solution to be tested.
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