CrRNA for detecting L858R single base mutation of EGFR gene and kit applying crRNA

Abstract

The invention provides crRNA for detecting L858R single base mutation of an EGFR (Epidermal Growth Factor Receptor) gene and a kit applying the crRNA. The nucleotide sequence of the crRNA for detecting the single base mutation of the EGFR gene L858R is as shown in SEQ ID NO: 11; the first basic group and the fifth basic group of the nucleotide sequence of the crRNA for detecting the single base mutation of the EGFR gene L858R are independently selected from one of A, U and C; the second base, the third base, the sixth base and the seventh base of the nucleotide sequence of the crRNA for detecting the single base mutation of the EGFR gene L858R are independently selected from one of A, U and G. According to the crRNA provided by the invention, the recognition efficiency of the crRNA on a wild type target can be reduced, the specificity and sensitivity of the crRNA on mutant target detection can be improved, the EGFR L858R mutant target and the wild type target can be effectively distinguished, and the crRNA has extremely important clinical guiding significance on the treatment strategy and prognosis of the non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a crRNA for detecting the L858R single-base mutation of the EGFR gene and a kit using the crRNA. Background Art

[0002] Primary lung cancer is the most common malignant tumor in my country, with the highest incidence and mortality rate, and it poses a serious threat to human health. Non-small cell lung cancer (NSCLC) is the main type of lung cancer, accounting for about 85% to 90% of all lung cancers. Most patients are already in the late stage or have distant metastasis when they seek medical treatment, and their prognosis is poor. Mutations in some genes can lead to lung cancer. For example, mutations in genes such as EGFR, BRAF, and KARS can be detected in some lung cancer patients. L858R is an important mutation form of the EGFR gene, in which the L858R mutation mainly occurs in the arginine mutation at position 858 of the 21st exon of the EGFR gene to leucine. Existing studies have shown that the EGFR gene L858R mutation makes patients with non-small cell lung cancer more likely to develop drug resistance or drug resistance-related mutations during clinical treatment, thereby affecting the therapeutic efficacy or prognosis of non-small cell lung cancer.

[0003] At present, the clinical diagnosis and treatment of non-small cell lung cancer patients can be performed by quantitative real-time polymerase chain reaction (Q-PCR), non-radioactive in situ hybridization (fluorescence in situ hybridization, Fish), multiplex PCR, etc. However, the specificity of the EGFR gene L858R mutant target is low when the above methods are used to detect the EGFR gene L858R mutant target, and it is difficult to distinguish the EGFR L858R mutant target from the wild-type target. Summary of the invention

[0004] In order to solve the problem that existing gene mutation detection methods are difficult to distinguish between EGFR L858R mutant targets and wild-type targets, and to improve the specificity and sensitivity of detection of EGFR L858R mutant targets, the present invention provides a crRNA for detecting the EGFR gene L858R single-base mutation and a kit using the crRNA.

[0005] According to the first aspect of the present invention, a crRNA for detecting a single base mutation of the EGFR gene L858R is provided, and the nucleotide sequence of the crRNA for detecting a single base mutation of the EGFR gene L858R is shown in SEQ ID NO: 11; the 1st and 5th bases of the nucleotide sequence of the crRNA for detecting a single base mutation of the EGFR gene L858R are independently selected from one of A, U, and C; the 2nd, 3rd, 6th, and 7th bases of the nucleotide sequence of the crRNA for detecting a single base mutation of the EGFR gene L858R are independently selected from one of A, U, and G.

[0006] Based on the PAM sequence of EGFR L858R, the inventors of the present application obtained crRNA-21C for detecting single-base mutations of EGFR gene L858R as shown in SEQ ID NO: 9 and crRNA-21G for detecting single-base mutations of EGFR gene L858R as shown in SEQ ID NO: 10 through conventional design. When the single-base mutation of EGFR gene L858R was detected using crRNA-21C and crRNA-21G, it was found that both the mutant and wild-type targets of EGFR gene L858R can activate the Cas12a protein bound to crRNA-21G and crRNA-21C. It can be seen that the crRNA screened by the traditional PAM sequence is difficult to achieve a good specific detection of single-base point mutation targets by Cas12a protein.

[0007] Based on the above findings, the inventors of the present application modified the crRNA-21C for detecting the single base mutation of the EGFR gene L858R as shown in SEQ ID NO: 9 by introducing mismatches, and introduced mismatch bases into the 1st, 2nd, 3rd, 5th, 6th and 7th bases of the crRNA-21C with the nucleotide sequence as shown in SEQ ID NO: 9, respectively, to obtain the crRNA for detecting the single base mutation of the EGFR gene L858R as claimed in the present invention, and the general formula of its nucleotide sequence is shown in SEQ ID NO: 11. When the crRNA with the nucleotide sequence as shown in SEQ ID NO: 11 provided by the present invention is used to detect the EGFR L858R mutant target and the wild-type target, the SNR values ​​of the two are significantly different compared with those of crRNA-21C. Therefore, the present invention is based on crRNA-21C, and introduces additional different types of mismatched bases at different sites near its mutation detection site according to the mismatch introduction method to obtain the modified crRNA (that is, the nucleotide sequence provided by the present invention is shown in SEQ ID NO: 11 as the crRNA for detecting the EGFR gene L858R single base mutation). It can effectively reduce the recognition efficiency of crRNA for the wild-type target EGFR gene, improve the specificity and sensitivity of crRNA for the detection of the mutant target EGFR gene, and can effectively distinguish between EGFR L858R mutant targets and wild-type targets, which has extremely important clinical guiding significance for the treatment strategy and prognosis of non-small cell lung cancer.

[0008] Preferably, the nucleotide sequence of crRNA used to detect the EGFR gene L858R single base mutation is selected from at least one of SEQ ID NOs: 12 to 29.

[0009] Preferably, the nucleotide sequence of crRNA used to detect the EGFR gene L858R single base mutation is shown in SEQ ID NO: 25.

[0010] According to a second aspect of the present invention, a kit for detecting L858R and T790M single base mutations in the EGFR gene is provided, the kit comprising the above-mentioned crRNA for detecting the L858R single base mutation in the EGFR gene and the crRNA for detecting the T790M single base mutation in the EGFR gene; the nucleotide sequence of the crRNA for detecting the T790M single base mutation in the EGFR gene is shown in SEQ ID NO: 32.

[0011] Preferably, the above kit also includes a first primer, which is used to amplify EGFR gene L858R; the first primer includes an upstream primer A and a downstream primer B, the nucleotide sequence of the upstream primer A is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer B is shown in SEQ ID NO: 2.

[0012] Preferably, the above kit also includes a second primer, which is used to amplify EGFR gene T790M; the second primer includes an upstream primer C and a downstream primer D, the nucleotide sequence of the upstream primer C is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer D is shown in SEQ ID NO: 4.

[0013] Preferably, the above kit further comprises MgCl2 solution, KCl solution, and NaCl solution. 2+ The working concentration of KCl solution is 2-16 mM. + The working concentration is 0-1mM, and the Na in the NaCl solution + The working concentration is 0~2mM.

[0014] Preferably, the Mg in the MgCl2 solution 2+ The working concentration is 12 mM.

[0015] Preferably, the above kit further comprises Tri-HCl, the working concentration of Tri-HCl is 2 mM, and the pH is 7.4.

[0016] In the process of detecting the single-base mutations of EGFR gene L858R and T790M using the kit provided in this scheme, the Mg in the detection system was 2+ , K + and Na + The concentration (working concentration) of Mg 2+ , K + and Na + The working concentration is within the above range, which is beneficial to improve the accuracy of the test results.

[0017] Preferably, the above kit further comprises a reporter probe having a single-stranded DNA structure, one end of the reporter probe is labeled with a fluorescent group, and the other end is labeled with a quenching group, and the nucleotide sequence of the reporter probe is shown in SEQ ID NO:5. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of Example 1 for detecting two point mutation sites, L858R and T790M, in the EGFR gene of tissue and single tumor cell samples using PCR / CRISPR-Cas12a.

[0019] Figure 2 This is a schematic diagram of the principle of using PCR / CRISPR-Cas12a to detect the two point mutation sites of EGFR gene L858R and T790M in Example 1, in which the Cas12a protein specifically recognizes the crRNA to form a complex to recognize the target.

[0020] Figure 3 The detection pattern diagram of the crRNA provided in Example 1 for the fully matched target, the single base mismatch target and the double base mutation target.

[0021] Figure 4 This is a reference sequence diagram near the L858R mutation site of the EGFR gene provided in Example 2.

[0022] Figure 5 The graphs show the real-time fluorescence kinetic detection results of detecting the synthetic L858R mutant target and the wild-type target using crRNA-21C and crRNA-21G, respectively, as provided in Example 2.

[0023] Figure 6 The fluorescence intensity detection result diagram of the system when the synthetic L858R mutant target and wild-type target were detected using crRNA-21C and crRNA-21G respectively provided in Example 2.

[0024] Figure 7 The result diagram of the detection signal-to-noise ratio between the mutant target and the wild-type target at the same time by the crRNA with introduced mismatched bases provided in Example 3.

[0025] Figure 8 This is a schematic diagram of the detection principle of crRNA-21C-6U for L858R mutant target and wild-type target obtained by introducing mismatch on the basis of crRNA-21C in Example 3.

[0026] Fig. 9 Real-time fluorescence kinetic results of detection of L858R mutant target and wild-type target by Cas12a protein under the guidance of crRNA-21C-6U provided in Example 3.

[0027] Fig.10 The real-time fluorescence signal intensity difference result graph of the system when the CRISPR / Cas12a detection system detects L858R mutant target and wild-type target under the guidance of crRNA-21C-6U based on ultraviolet radiation observation provided in Example 3 is provided.

[0028] Fig.11This is a graph showing the results of Example 3 using urea-PAGE to detect the difference in cleavage of the L858R mutant target and the wild-type target by the Cas12a protein under the guidance of crRNA-21C.

[0029] Fig.12 This is a graph showing the results of differential detection of the cleavage of the L858R mutant target and the wild-type target by urea-PAGE using crRNA-21C-6U-guided Cas12a protein.

[0030] Fig.13 This is a graph comparing the sensitivity of detecting the EGFR gene L858R mutant target using crRNA-21C and crRNA-21C-6U in Example 3.

[0031] Fig.14 This is a reference sequence diagram near the T790M mutation site of the EGFR gene provided in Example 4.

[0032] Fig.15 Real-time fluorescence kinetic detection result graph of Cas12a protein detection of T790M mutant target and wild-type target under the guidance of crRNA-20U provided in Example 4.

[0033] Fig.16 The real-time fluorescence signal intensity difference result graph of detecting T790M mutant target and wild-type target based on ultraviolet radiation observation of CRISPR / Cas12a detection system under the guidance of crRNA-20U provided in Example 4.

[0034] Fig.17 This is a graph showing the results of differential detection of the cleavage of the T790M mutant target and the wild-type target by urea-PAGE using crRNA-20U-guided Cas12a protein.

[0035] Fig.18 This is the agarose gel electrophoresis result after amplifying the L858R and T790M mutation sites of the EGFR gene in Example 5.

[0036] Fig.19 This is a result graph showing the linear relationship between the fluorescence intensity of the detection system and the concentration of the mutant target when the amplified products of the EGFR gene L858R and T790M mutation sites are detected using crRNA-21C-6U and crRNA-20U as provided in Example 5.

[0037] Fig. 20 This is a diagram of the DNA detection pattern of tissue samples provided in Example 5.

[0038] Fig.21This is a mutation scatter plot of Example 5 for detecting L858R and T790M in tissue sample DNA.

[0039] Fig. 22 This is a graph showing the consistency analysis results of Example 5 using NGS gene sequencing technology and PCR / CRISPR-Cas12a technology to detect EGFR gene L858R and T790M mutations in tissue samples.

[0040] Fig.23 This is a heat map comparing the fluorescence intensity of detecting the L858R mutation rate using NGS and detecting the L858R mutation using PCR / CRISPR-Cas12a in Example 5.

[0041] Fig.24 For each ion (Mg) in the detection system in Example 7 2+ , K + 、Na + ) and the optimization results of Tris-HCl working concentration, PCR product addition volume, and PCR amplification cycle number.

[0042] Fig.25 This is a schematic diagram of Example 8 combining PCR and CRISPR-Cas12a to detect a single tumor cell.

[0043] Fig.26 This is a diagram of the fluorescence staining results of H1975 cells and A549 cells under a fluorescence microscope provided in Example 8.

[0044] Fig. 27 The result graph of the combined PCR and CRIPSR-Cas12a provided in Example 8 for detecting the EGFR L858R and T790M mutations of a single tumor cell, respectively. DETAILED DESCRIPTION

[0045] The following is a further clear and complete description of the technical features in the technical solution provided by the present invention in conjunction with the specific implementation methods. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0046] Example 1

[0047] In this example, PCR technology and CRISPR / Cas12a technology are combined to detect two point mutation sites, L858R and T790M, in the EGFR gene of tissue and single tumor cell samples. The PCR / CRISPR-Cas12a detection process is as follows: Figure 1As shown, specifically, genomic DNA extracted from tumor tissue and genomic DNA amplified from a single tumor cell enriched by a microfluidic chip are used as test samples, and PCR primers as shown in Table 1 are designed, and the genomic DNA obtained from the test sample is PCR amplified for the two point mutation sites of EGFR gene L858R and T790M, respectively, and then CRISPR / Cas12a technology is used to specifically identify the mutation site, and the specific target activates the trans-cleavage activity of Cas12a protein to efficiently cut the single-stranded DNA reporter probe labeled with a fluorescent group and a quenching group at both ends in the system (the nucleotide sequence of the single-stranded DNA reporter probe is shown in SEQ ID NO: 5, wherein one end is connected to a quenching group BHQ1, and the other end is connected to a fluorescent group HEX, specifically BHQ1-TTATT-HEX), and the single-stranded DNA reporter probe is cut and broken to release a detectable fluorescent signal to achieve detection.

[0048] Table 1 PCR primers used to amplify the two point mutation sites of EGFR gene L858R and T790M

[0049]

[0050] In the process of detecting the two point mutation sites of EGFR gene L858R and T790M by PCR / CRISPR-Cas12a, the principle of Cas12a protein specifically recognizing crRNA to form a complex to recognize the target is as follows Figure 2 As shown, Cas12a protein relies on programmable crRNA to recognize the target, that is, crRNA binds to Cas12a protein to form a Cas12a-crRNA complex, and the Cas12a protein in the Cas12a-crRNA complex recognizes the target under the guidance of crRNA. The target activates the trans-cleavage activity of Cas12a protein to cut the short-chain DNA reporter probes labeled with fluorescent groups and quenching groups at both ends, and the single-stranded DNA reporter probe is cut and broken to release the fluorescent group. The fluorescent signal in the detection system is used to detect the single-base mutation sites of EGFR gene L858R and T790M. This recognition method relying on base complementary pairing ensures its specificity, especially the seed region composed of the first few bases of crRNA binding to the target, which plays a major role in the specific recognition of crRNA.

[0051] However, in the process of using PCR / CRISPR-Cas12a to detect the two point mutation sites of EGFR gene L858R and T790M, the situation of activating Cas12a for single base mismatch targets still exists. This is mainly because the thermodynamic changes brought about by single base mutations are small, and crRNA has a certain tolerance for them. The detection mode of crRNA for fully matched targets, single base mismatch targets and double base mutation targets is shown in the figure Figure 3 As shown, the CRISPIR-Cas12a system specifically recognizes the target by crRNA and DNA base complementary pairing, and there is a certain fault tolerance. Both the fully matched target and the single mismatched base target can activate the trans-cutting activity of Cas12a protein, but the activation ability of the double base mutation target to Cas12a is significantly reduced. So when PCR / CRISPR-Cas12a technology is applied to the point mutation detection of a single base, the conventionally designed crRNA is sometimes difficult to realize the distinction between mutant and wild type. Therefore, in subsequent experiments, the present inventors refer to the primer design principle of cyclic amplification retardation PCR (ARMS-PCR), by introducing additional mismatches in the sequence near the crRNA recognition mutation site to reduce the recognition efficiency of crRNA to off-target sequences, and the present inventors find that compared to the situation where a single mismatch exists in the seed region of the target, when there are two mismatches in the seed region of the target, the recognition efficiency of crRNA to the target can be significantly reduced.

[0052] Based on the above findings, the inventors of the present application screened crRNA by introducing mismatches in the design of crRNA for detecting the single-base mutation L858R in the EGFR gene in subsequent experiments to screen out crRNA with higher detection specificity.

[0053] Example 2 crRNA for detecting the single base mutation of EGFR gene L858R

[0054] This embodiment aims to obtain crRNA for detecting the single-base mutation of EGFR gene L858R by conventional design, and use the designed crRNA to detect the mutant target and wild-type target of EGFR gene L858R.

[0055] L858R is an important mutation form of EGFR, and the mutation mainly occurs in the thymine (T) to guanine (G) mutation at position 858 of exon 21. The sequences of the EGFR gene L858R mutant target and wild-type target are shown in Table 2.

[0056] Table 2 Nucleotide sequences of EGFR gene L858R mutant target and wild-type target

[0057]

[0058] The reference sequence near the L858R mutation site of the EGFR gene (Homo sapiens Gene ID: 1956) was obtained from the Gene database of NCBI. Figure 4 As shown, the base marked in red is the single base mutation site L858R of the EGFR gene, and the base marked in yellow is the PAM sequence (TTTG) located near the L858R mutation site and located in the forward and reverse chains respectively.

[0059] Table 3 Nucleotide sequences of crRNA-21C and crRNA-21G obtained by conventional design

[0060]

[0061] Note: In the sequences in Table 3, base N represents base U.

[0062] A crRNA with a length of 20 nt and completely complementary to the EGFR target was designed for the potential PAM sequence near the L858R single-base mutation site of the EGFR gene, wherein the fixed sequence of the crRNA is shown in SEQ ID NO: 8, and the designed crRNAs are composed of a fixed sequence plus a sequence with a length of 20 nt designed according to the PAM sequence (i.e., a detection sequence). The fixed sequence and detection sequence of the crRNA are shown in Table 3, wherein the crRNA detection sequence with a length of 20 nt designed according to the PAM sequences of the forward and reverse chains is as follows, the crRNA with a nucleotide sequence as shown in SEQ ID NO: 9 (named as crRNA-21C, designed according to the following PAM sequence) and the nucleotide sequence as shown in SEQ ID The crRNA shown in NO: 10 (named as crRNA-21G, designed according to the previous PAM sequence), the above crRNA-21C and crRNA-21G are used to detect the EGFR gene L858R single base mutation, and the synthetic L858R mutant target and wild-type target (synthetic sequence refer to Table 2) are detected by crRNA-21C and crRNA-21G, respectively. The real-time fluorescence kinetic detection results are shown in Figure 5 The fluorescence intensity detection results of the system are shown in Figure 6 shown.

[0063] Depend on Figure 5 It can be seen that although the detection efficiency of crRNA-21C designed for L858R is significantly higher than that of crRNA-21G under the same conditions, the detection signal shows that it is difficult to distinguish the mutant and wild-type targets; Figure 6It can be seen that the fluorescence intensity detection results of the system also showed that the fluorescence intensity of crRNA-21C-mediated detection was higher than that of crRNA-21G at the same time, but there was no obvious difference in the fluorescence intensity of crRNA-21C detection of wild-type targets and mutant targets.

[0064] It can be seen that both the L858R mutant and wild-type targets of the EGFR gene can activate the Cas12a protein bound to crRNA-21G and crRNA-21C. The crRNA screened by the traditional PAM sequence is difficult to achieve the specific detection of the Cas12a protein for single-base point mutation targets.

[0065] Example 3

[0066] This embodiment aims to obtain a crRNA for detecting the L858R single-base mutation of the EGFR gene based on Example 2 through conventional design, and to transform and screen the crRNA by introducing mismatches to screen out a crRNA with higher detection specificity, so as to reduce the recognition efficiency of crRNA for the wild-type target EGFR gene and improve the specificity of crRNA for detecting the mutant target EGFR gene.

[0067] In order to select a crRNA with higher detection efficiency for detecting the single base mutation of the EGFR gene L858R, the crRNA-21C detection sequence with a nucleotide sequence as shown in SEQ ID NO: 9 is used as the basis, and additional different types of mismatched bases are introduced at different sites near the mutation detection site according to the mismatch introduction method to obtain the modified crRNA, the general formula of which is shown in SEQ ID NO: 11. Specifically, mismatched bases are introduced at the 1st, 2nd, 3rd, 5th, 6th, and 7th bases of the crRNA-21C detection sequence with a nucleotide sequence as shown in SEQ ID NO: 9, and the base C bound to the mutation site is taken as coordinate 0, and its upstream (towards the 5' end of the crRNA) is recorded as coordinates -1, -2, and -3, respectively, and its downstream (towards the 3' end of the crRNA) is recorded as coordinates +1, +2, and +3, respectively. Different types of mismatched bases are introduced at each site to obtain the crRNA detection sequence shown in Table 4, and the crRNA fixed sequence is consistent with Example 2.

[0068] Table 4 crRNA detection sequences for detecting the EGFR gene L858R single base mutation

[0069]

[0070]

[0071] Note: In the sequences in Table 4, except for the bases N at positions 1 to 3 and positions 5 to 7 in SEQ ID NO: 11 which represent A, C, G or U, the other bases N all represent base U.

[0072] The nucleotide sequences obtained in Table 4 above, such as the crRNA shown in SEQ ID NOs: 12 to 29, were used to detect the wild-type target and mutant target of the EGFR gene, respectively, and the ratio of the detection fluorescence signal values ​​of the crRNA introducing the mismatched base for the mutant target and the wild-type target at the same time was compared, which was the signal-to-noise ratio SNR. The results are shown in FIG. Figure 7 As shown, **** indicates P < 0.0001 and * indicates P < 0.05.

[0073] Depend on Figure 7 It can be seen that compared with crRNA-21C whose nucleotide sequence is shown in SEQ ID NO: 9, when the crRNA with nucleotide sequences such as SEQ ID NOs: 12 to 29 is used to detect mutant targets and wild-type targets, the SNR values ​​of both are higher than crRNA-21C. Except for crRNA-21C-3A, the SNR values ​​of other crRNAs for detecting mutant targets and wild-type targets are significantly different from those of crRNA-21C. Moreover, among the crRNAs listed above, the signal-to-noise ratio of U / G mismatch at coordinates +2 and +3 is most significantly improved (P<0.0001), and the detection specificity of crRNA-21C-6U, in which cytosine (C) is mutated to uracil (U) at coordinate +2, is the strongest, and its detection SNR is 3.76 times that of the original crRNA-21C.

[0074] Next, the detection effect of crRNA-21C-6U with a nucleotide sequence such as SEQ ID NO: 25 was further verified. The detection principle of crRNA-21C-6U obtained by mismatch introduction on the basis of crRNA-21C for L858R mutant target and wild-type target is as follows Figure 8 As shown in Figure 2, the real-time fluorescence kinetic results of Cas12a protein detection of L858R mutant target and wild-type target under the guidance of crRNA-21C-6U are as follows: Fig. 9 As shown, the real-time fluorescence signal intensity difference of the system when the CRISPR / Cas12a detection system detects L858R mutant targets and wild-type targets under the guidance of crRNA-21C-6U is observed based on ultraviolet radiation. Fig.10 As shown, the results of differential detection of the cleavage of L858R mutant target and wild-type target by Cas12a protein under the guidance of crRNA-21C and crRNA-21C-6U were shown in Figure 1. Fig.11 , Fig.12 As shown, Fig.10 From left to right: wild-type target, mutant target, ultrapure (RF) water. Fig.12 A is the result of urea-PAGE electrophoresis. Fig.12 B is the grayscale analysis result of each band in the urea-PAGE electrophoresis diagram, the ordinate Relative gray value represents the relative grayscale value, Wild type DNA represents the wild-type DNA target, and Mutant DNA represents the mutant DNA target.

[0075] Depend on Fig. 9 and Fig.10 It can be seen that using crRNA-21C-6U-mediated CRISPR-Cas12a detection of the EGFR gene L858R single base mutation, the 2-hour signal value and the 6-hour fluorescence intensity can clearly distinguish the mutant target from the wild-type target.

[0076] Depend on Fig.11 It can be seen that the products of crRNA-21C-mediated CRISPR reaction for 1 hour and 2 hours were silver stained after gel running, and it was found that at 1 hour, there were a large number of residual uncut targets for both wild-type and mutant targets, but at 2 hours, no obvious bands were seen for both mutant and wild-type targets and were completely cut.

[0077] Depend on Fig.12 It can be seen that after 2 hours of crRNA-21C-6U-mediated CRISPR reaction, the product was silver stained and it was found that the mutant target was almost completely cut, but there was still an obvious uncut band for the wild type.

[0078] We further studied the sensitivity of crRNA-21C and crRNA-21C-6U in detecting the EGFR gene L858R mutant target and compared the results. Fig.13 As shown, Fig.13 A represents the detection result of crRNA-21C, Fig.13 B represents the detection result of crRNA-21C-6U, NC represents 0% (only 50pM wild-type DNA target, no mutant target exists), and the percentage represents the mutant DNA concentration / wild-type DNA concentration, for example, 10% = 5pM mutant DNA / 50pM wild-type DNA.

[0079] Depend on Fig.13It can be seen that when using crRNA-21C and crRNA-21C-6U to detect the fully matched target (0%), the overall detection efficiency of crRNA-21C-6U is reduced compared with crRNA-21C, but the detection sensitivity has not changed significantly (the detection limit of crRNA-21C LOD = 562.47aM), and in the process of detecting targets containing 10% mutations, crRNA-21C is difficult to detect the presence of mutant genes, but crRNA-21C-6U can effectively detect mutant targets as low as 0.1%.

[0080] The above results show that this embodiment is based on crRNA-21C, and introduces additional different types of mismatch bases at different sites near its mutation detection site according to the mismatch introduction method to obtain the modified crRNA, which can effectively reduce the recognition efficiency of crRNA for the wild-type target EGFR gene and improve the specificity of crRNA for detecting the mutant target EGFR gene.

[0081] Example 4

[0082] This embodiment aims to design a crRNA for detecting the single-base mutation T790M in the EGFR gene, and use the designed crRNA to detect the mutant target and wild-type target of the EGFR gene T790M.

[0083] T790M is another major mutation form of EGFR, which mainly occurs in the mutation of cytosine (C) to thymine (T) at position 790 of exon 20. The sequences of the EGFR gene T790M mutant target and wild-type target are shown in Table 5.

[0084] Table 5 Nucleotide sequences of EGFR T790M mutant target and wild-type target

[0085]

[0086] The reference sequence of the mutation site near T790M of EGFR gene (Homo sapiens Gene ID: 1956) was obtained from the Gene database of NCBI. Fig.14 As shown, the base marked in red is the T790M single base mutation site of the EGFR gene.

[0087] Depend on Fig.14 It can be seen that there is no natural PAM sequence near the T790M single base mutation site of the EGFR gene. Therefore, in this embodiment, guanine (G) and cytosine (C) in the original sequence CAGCTC are replaced with two thymines (T) by PCR, so that the sequence becomes CATTTC to form a PAM sequence (TTTC).

[0088] SEQ ID NO: 32 (crRNA-20U)

[0089] ANCANGCAGCNCANGCCCNN

[0090] Note: In SEQ ID NO: 32, base N represents base U.

[0091] For the above-mentioned PAM sequence formed by artificially replacing 2 Ts near the T790M single-base mutation site of the EGFR gene, a crRNA detection sequence with a length of 20 nt and completely complementary to the EGFR target was designed, and its nucleotide sequence is shown in SEQ ID NO: 32, named crRNA-20U (detection sequence), and the crRNA-20U fixed sequence is consistent with Example 2 for detecting the T790M single-base mutation of the EGFR gene, and using crRNA-20U to detect the synthesized wild-type target and mutant target respectively, and the real-time fluorescence kinetic detection results of the detection of T790M mutant target and wild-type target by Cas12a protein under the guidance of crRNA-20U are as follows Fig.15 As shown, the real-time fluorescence signal intensity difference between the T790M mutant target and the wild-type target detected by the CRISPR / Cas12a detection system under the guidance of crRNA-20U is shown in FIG. Fig.16 As shown, the results of the differential detection of the cleavage of the T790M mutant target and the wild-type target by Cas12a protein under the guidance of crRNA-20U are shown in Figure 1. Fig.17 As shown, Fig.16 From left to right are wild-type target, mutant target, and ultrapure (RF) water.

[0092] Depend on Fig.15 and Fig.16 It can be seen that when the crRNA-20U designed for T790M is used to detect the single-base mutation of the EGFR gene T790M, the detection signal shows that crRNA-20U can distinguish the mutant and wild-type targets very well. The fluorescence signal of the mutant target detected at 2 hours and the fluorescence intensity at 6 hours are significantly different from those of the wild-type target.

[0093] Depend on Fig.17 It can be seen that silver staining of the product after 0.5 hours of CRISPR reaction showed that crRNA-20U can completely cut the mutant target within half an hour, while for the wild-type target of the same concentration, there is still a significant residue within the same reaction time, which is not recognized and cut by crRNA-20U.

[0094] The above results show that the crRNA-20U designed in this embodiment for the EGFR gene T790M single base mutation site as shown in SEQ ID NO: 32 can bind to the Cas12a protein and specifically recognize the target, thereby activating the trans-cleavage activity of the Cas12a protein, thereby efficiently cutting the single-stranded DNA reporter probe (one end of the reporter probe is labeled with a fluorescent group and the other end is labeled with a quenching group, and the nucleotide sequence of the reporter probe is shown in SEQ ID NO: 5), releasing a detectable fluorescent signal, thereby achieving specific detection of the EGFR gene T790M single base point mutation target.

[0095] Example 5

[0096] This embodiment aims to combine PCR technology and CRISPR-Cas12a technology and use crRNA-21C-6U designed in Example 2 for detecting the single base mutation of EGFR gene L858R and crRNA-20U designed in Example 4 for detecting the single base mutation of EGFR gene T790M to detect the single base mutations of EGFR gene L858R and T790M.

[0097] First, the EGFR gene L858R and T790M mutation sites were amplified using the PCR primers shown in Table 1 and verified by agarose gel electrophoresis. Fig.18 As shown. Fig.18 It can be seen that the PCR primers shown in Table 1 can be used to amplify the L858R and T790M mutation sites of the EGFR gene.

[0098] Secondly, crRNA-21C-6U and crRNA-20U were used to detect the products obtained by amplifying the L858R and T790M mutation sites of the EGFR gene as described above. The linear relationship between the fluorescence intensity of the detection system and the concentration of the mutant target was as follows: Fig.19 As shown, Fig.19 A represents the linear relationship between fluorescence intensity and L858R mutant target concentration, Fig.19 B shows the linear relationship between fluorescence intensity and T790M mutant target concentration. Fig.19 It can be seen that when crRNA-21C-6U is used to detect the L858R mutation target of the EGFR gene, the detection limit LOD of the L858R mutation target is 315.26aM, and when crRNA-20U is used to detect the T790M mutation target of the EGFR gene, the detection limit LOD of the T790M mutation target is 1090.70aM.

[0099] Furthermore, the repeatability experiment of PCR-Cas12a detection using three crRNAs, crRNA-21C, crRNA-21C-6U and crRNA-20U, was also carried out. The results are shown in Table 6, where the relative standard deviation (RSD, relative standard deviation) is small, which proves that the detection using the three crRNAs, crRNA-21C, crRNA-21C-6U and crRNA-20U, has good repeatability.

[0100] Table 6 Repeatability experimental results of three crRNA detections

[0101]

[0102]

[0103] Furthermore, 43 patients with non-small cell carcinoma were recruited and divided into an EGFR gene L858R mutation group (n=20) and a wild-type group (n=23) according to their next-generation sequencing results. The samples of tumor tissue samples of the above patients with T790M mutation in the next-generation sequencing (n=6) were used as the EGFR gene T790M mutation group, and patients without T790M mutation in the next-generation sequencing results (n=12) were randomly selected as the wild-type group. Fig. 20 The tissue sample DNA detection pattern diagram shown in the figure, DNA was extracted from the tumor tissue samples of the above patients and diluted to a uniform concentration of 20ng / μL for detection of L858R and T790M. The detection mutation scatter plot of L858R and T790M in the tissue sample DNA is shown in Fig.21 As shown, **** indicates P < 0.0001, *** indicates P < 0.001. Fig.21 It is known that the detection fluorescence intensity of L858R and T790M mutant targets is significantly higher than that of L858R and T790M wild-type targets (P < 0.0001), which is consistent with the second-generation sequencing results, and illustrates that crRNA-21C-6U and crRNA-20U can be used to specifically detect EGFR gene L858R and T790M mutation sites, and L858R and T790M mutant targets and wild-type targets can be effectively distinguished well. It can be seen that compared with the second-generation sequencing results of clinical standards, the present invention uses PCR technology and CRISPR-Cas12a technology, and the crRNA for detecting EGFR gene L858R single base mutations provided by the present invention and the crRNA for detecting EGFR gene T790M single base mutations can achieve the same detection effect as the second-generation sequencing, and as a more convenient and low-cost detection means, it has more value for application in clinical detection.

[0104] In addition, NGS gene sequencing technology was used to detect EGFR gene L858R and T790M mutations in tissue samples. Fig.21 The PCR / CRISPR-Cas12a detection results were analyzed for consistency of L858R and T790M mutations. The results are as follows Fig. 22 As shown in the figure, the fluorescence intensity comparison heat map of L858R mutation rate detected by NGS and L858R mutation detected by PCR / CRISPR-Cas12a is shown in the figure. Fig.23 As shown, Fig.23 The top one is the heat map of mutation rate detected by NGS, and the bottom one is the heat map of fluorescence signal intensity when the PCR / CRISPR-Cas12a method was used to detect L858R mutation for 90 minutes. Fig.21 , Fig. 22 and Fig.23 It can be seen that the Kappa test k for L858R mutation detection is 0.9059, and the Kappa test k for T790M mutation detection is 1.0000. The PCR / CRISPR-Cas12a test results are almost completely consistent with the NGS test results, with good consistency. The high and low detection fluorescence signal values ​​have a certain correlation with the mutation rate of sequencing, which further proves the accuracy of the specific detection results of crRNA-21C-6U and crRNA-20U for the L858R and T790M mutation sites of the EGFR gene.

[0105] Example 6

[0106] A kit for detecting single-base mutations of EGFR gene L858R and T790M, the kit comprising: crRNA for detecting single-base mutation of EGFR gene L858R, crRNA for detecting single-base mutation of EGFR gene T790M, a first primer, a second primer, MgCl2 solution, KCl solution, NaCl solution, Tri-HCl, and a reporter probe;

[0107] The nucleotide sequence of the crRNA for detecting the EGFR gene L858R single base mutation is selected from at least one of SEQ ID NOs: 12 to 29, and the nucleotide sequence of the crRNA for detecting the EGFR gene T790M single base mutation is shown in SEQ ID NO: 31;

[0108] The first primer is used to amplify EGFR gene L858R, and the first primer includes an upstream primer A and a downstream primer B, the nucleotide sequence of the upstream primer A is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer B is shown in SEQ ID NO: 2;

[0109] The second primer is used to amplify EGFR gene T790M, and the second primer includes an upstream primer C and a downstream primer D, the nucleotide sequence of the upstream primer C is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer D is shown in SEQ ID NO: 4;

[0110] Mg in MgCl2 solution 2+ The working concentration is 2~16mM,

[0111] K in KCl solution + The working concentration is 0~1mM,

[0112] Na in NaCl solution + The working concentration is 0-2mM;

[0113] The working concentration of Tri-HCl is 2 mM, pH 7.4;

[0114] The reporter probe has a single-stranded DNA structure, one end of which is labeled with a fluorescent group HEX, and the other end is labeled with a quenching group BHQ1. The nucleotide sequence of the reporter probe is shown in SEQ ID NO: 32, and its specific nucleotide sequence and structure are as follows: BHQ1-TTATT-HEX.

[0115] Example 7

[0116] In order to maximize the detection efficiency of crRNA-21C-6U for detecting the single base mutation of EGFR gene L858R designed in Example 3 and crRNA-20U for detecting the single base mutation of EGFR gene T790M designed in Example 4 in the CRISPR / Cas12a system, this example aims to optimize the MgCl2 solution in the kit provided in Example 6. 2+ 、K of KCl solution + 、Na in NaCl solution + and the working concentration of Tris-HCl during the detection process (i.e., the actual concentration in the detection system), the volume of PCR product added, the number of PCR amplification cycles, and other conditions. 2+ , K + 、Na + ) and the optimization results of the working concentration of Tris-HCl, the volume of PCR product added, and the number of PCR amplification cycles (cycle) are as follows Fig.24 As shown, Fig.24 A represents different Mg 2+ The signal-to-noise ratio (SNR) test results under concentration, Fig.24 B indicates different K + The signal-to-noise ratio (SNR) test results under concentration, Fig.24 C represents different Na + The signal-to-noise ratio (SNR) test results under concentration, Fig.24 D represents the signal-to-noise ratio (SNR) test results at different Tris-HCl concentrations. Fig.24 E represents the signal-to-noise ratio (SNR) test results at different PCR product addition volumes. Fig.24 F represents the signal-to-noise ratio (SNR) test results at different PCR cycle numbers. Fig.24 In F, target indicates mutant target, control indicates wild-type target, and Fluorescence intensity (AU) indicates fluorescence intensity.

[0117] Depend on Fig.24 It can be seen that Na in the CRISPR / Cas12a detection system + and K + The presence of Mg2+ will affect the activity of Cas12a to a certain extent and thus affect the final SNR detection results. 2+ The detection effect of CRISPR / Cas12a in 2mM and pH7.4 Tri-HCl is the best. Then PCR is linked to the CRISPR / Cas12a system. After PCR amplification, the product is added to the CRISPR / Cas12a system for reaction. It is found that as the number of PCR cycles increases, false positive results will occur. The optimal number of PCR cycles is determined to be 35 cycles through experiments. In addition, adding the product to the CRISPR-Cas12a system at a ratio of 1:5 can achieve the best detection effect.

[0118] Example 8

[0119] Circulating tumor cells (CTCs) contain a wealth of tumor-related information. In order to achieve high-sensitivity detection of CTCs, this example refers to Fig.25 The schematic diagram of combined PCR and CRISPR-Cas12a detection of single tumor cells shown in the figure uses microfluidic enrichment combined with PCR-Cas12a to detect EGFR gene T790M and L858R mutations in single tumor cells in circulating tumor cells. Specifically, 5 to 10 mL of blood from patients with non-small cell lung cancer is collected and passed through a microfluidic chip. At this time, the tumor cells are retained on the microfluidic chip, and the residual white blood cells and tumor cells are distinguished by fluorescent staining. Then, the tumor cells with confirmed identity are picked up with a cell needle and the whole genome amplified, and then PCR and CRIPSR-Cas12a are combined to detect them.

[0120] In order to verify the feasibility of the above method, human lung cancer cells NCl-H1975 (EGFR L858R and T790M mutant) and A549 (EGFR L858R and T790M wild type) were added to the whole blood of healthy subjects, and the microfluidic chip and fluorescent antibodies CD45 and EPCAM were used for staining. The fluorescence staining results of H1975 cells (upper) and A549 cells (lower) under a fluorescence microscope are shown in Figure 2. Fig.26 As shown in the figure, the blue fluorescence is DIPA, the green fluorescence is EpCAM protein, and the red fluorescence is CD45. H1975 cells, A549 cells (green), and white blood cells (red) in the blood can be seen under a fluorescence microscope. At the same time, 1, 3, and 5 H1975 cells were picked out from the tumor cells with a cell needle, and 5 A549 cells were picked out as negative controls. After whole genome amplification using a single cell amplification kit, the product was diluted to a uniform concentration of 20 ng / μL and then combined with PCR and CRIPSR-Cas12a to detect EGFR L858R and T790M mutations, respectively. The results are shown in the figure. Fig. 27 As shown, Fig. 27 A. Fig. 27 B represents the fluorescence signal bar graph of single cell T790M and L858R mutation detection, respectively. H1-01, H1-02, H1-03 represent the results of 3 samples detected by picking 1 H1975 cell, respectively. H3-01, H3-02, H3-03 represent the results of 3 samples detected by picking 3 H1975 cells, respectively. H5-01, H5-02, H5-03 represent the results of 3 samples detected by picking 5 H1975 cells, respectively. A5 represents the test result of 5 A549 cells, ns represents no significant difference, **** represents P<0.0001, *** represents P<0.001, ** represents P<0.01, and * represents P<0.05.

[0121] Depend on Fig. 27 It can be seen that the EGFR L858R and T790M mutation signals were observed in 1, 3 and 5 H1975 cell groups, which were significantly higher than the negative control group of 5 A549 cells. In particular, the three groups of H1975 cells with 5 cells were significantly higher than the A549 cell group (P<0.00001). In addition, during the detection, it was found that some single cell H1975 cell groups had no detected mutations, and some mutation signals of 3 H1975 cell groups were low. This may be related to the mutation rate of EGFR L858R and T790M contained in the cell line.

[0122] The above results show that the purpose of detecting EGFR L858R and T790M mutations can be achieved by capturing or extracting single tumor cells from circulating tumor cells in clinical samples, and then using the crRNA-21C-6U for detecting the single base mutation of EGFR gene L858R and the crRNA-20U for detecting the single base mutation of EGFR gene T790M obtained by the invention, combined with PCR and CRISPR-Cas12a to detect single tumor cells.

[0123] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A crRNA for detecting the single-base mutation of EGFR gene L858R, characterized in that: The nucleotide sequence of the crRNA for detecting the EGFR gene L858R single base mutation is shown in SEQ ID NO: 11; The first and fifth bases of the nucleotide sequence of the crRNA for detecting the EGFR gene L858R single base mutation are independently selected from one of A, U, and C; The second, third, sixth and seventh bases of the nucleotide sequence of the crRNA for detecting the EGFR gene L858R single base mutation are independently selected from one of A, U and G.

2. The crRNA for detecting the EGFR gene L858R single base mutation according to claim 1, characterized in that: The nucleotide sequence of the crRNA used to detect the EGFR gene L858R single base mutation is selected from at least one of SEQ ID NOs: 12 to 29.

3. The crRNA for detecting the EGFR gene L858R single base mutation according to claim 1, characterized in that: The nucleotide sequence of the crRNA used to detect the EGFR gene L858R single base mutation is shown in SEQ ID NO:

25.

4. A kit for detecting single-base mutations L858R and T790M in EGFR gene, characterized in that: The kit comprises the crRNA for detecting the EGFR gene L858R single base mutation and the crRNA for detecting the EGFR gene T790M single base mutation as claimed in any one of claims 1 to 3; The nucleotide sequence of the crRNA for detecting the EGFR gene T790M single base mutation is shown in SEQ ID NO:

32.

5. The kit for detecting L858R and T790M single base mutations of EGFR gene according to claim 4, characterized in that: The kit also includes a first primer, which is used to amplify EGFR gene L858R; The first primer includes an upstream primer A and a downstream primer B. The nucleotide sequence of the upstream primer A is shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer B is shown in SEQ ID NO:

2.

6. The kit for detecting L858R and T790M single base mutations of EGFR gene according to claim 4, characterized in that: The kit also includes a second primer, which is used to amplify EGFR gene T790M; The second primer includes an upstream primer C and a downstream primer D. The nucleotide sequence of the upstream primer C is shown in SEQ ID NO:3, and the nucleotide sequence of the downstream primer D is shown in SEQ ID NO:

4.

7. The kit for detecting L858R and T790M single base mutations of EGFR gene according to claim 4, characterized in that: The kit also includes MgCl2 solution, KCl solution, and NaCl solution. The Mg in the MgCl2 solution 2+ The working concentration of KCl is 2-16 mM. + The working concentration of NaCl is 0-1 mM. + The working concentration is 0~2mM.

8. The kit for detecting L858R and T790M mutations of EGFR gene according to claim 7, characterized in that: The Mg in the MgCl2 solution 2+ The working concentration is 12 mM.

9. The kit for detecting L858R and T790M single base mutations of EGFR gene according to claim 4, characterized in that: The kit also includes Tri-HCl, the working concentration of which is 2 mM and the pH is 7.

4.

10. The kit for detecting L858R and T790M single base mutations of EGFR gene according to claim 4, characterized in that: The kit also includes a reporter probe having a single-stranded DNA structure, one end of which is labeled with a fluorescent group, and the other end of which is labeled with a quenching group. The nucleotide sequence of the reporter probe is shown in SEQ ID NO:5.

Images