A ROS1 gene fusion detection kit based on CRISPR-Dx technology

Abstract

The present invention discloses a ROS1 gene fusion detection kit based on CRISPR-Dx technology. The kit includes an RT-RPA amplification system and a CRISPR-Dx detection system, and has the advantages of short reaction time, simple equipment requirements and high detection sensitivity. The entire reaction process of the kit only takes 10 to 30 minutes, which has obvious detection advantages compared to traditional detection methods that take several hours. The kit only needs to react at a constant temperature of 35 to 45°C, has low requirements for reaction equipment, and has a wider range of applications. The detection sensitivity of existing similar products is 25 copies / μL. The kit first amplifies the sample to be tested by the RT-RPA amplification system, and then further uses the CRISPR-Dx detection system to detect the amplified product. The detection sensitivity can reach 10 copies / μL, which is far superior to existing similar products.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid detection technology, and in particular to a ROS1 gene fusion detection kit based on CRISPR-Dx technology. Background Art

[0002] Lung cancer caused by ROS1 gene mutation is a relatively rare type of lung cancer, mainly caused by the breakage and fusion of ROS1 gene with other genes, which usually occurs in non-small cell lung cancer, accounting for about 1-2% of the total number of lung cancers. Its clinical manifestations and treatment methods are similar to other types of non-small cell lung cancer, including surgery, radiotherapy, chemotherapy and targeted therapy. Among them, targeted therapy is a treatment method targeting specific gene mutations in tumor cells, which has the best therapeutic effect on ROS1 lung cancer, so it is necessary to perform typing detection for ROS1 gene fusion.

[0003] The existing detection methods for ROS1 mainly include immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time fluorescence quantitative PCR (PCR) and next-generation sequencing (NGS). These detection methods usually have poor detection sensitivity and specificity, resulting in low accuracy and reliability of the test results; the main disadvantages are long detection time, high cost and cumbersome operation. Therefore, it is urgent to develop a faster and more efficient method for detecting ROS1 gene fusion lung cancer.

[0004] Nucleic acid detection technology (CRISPR-based Diagnostics, CRISPR-Dx) is a detection technology developed based on the trans-cleavage characteristics of the CRISPR-Cas12 and CRISPR-Cas13 families.

[0005] For a long time, polymerase chain reaction (PCR) and its derivative technologies such as conventional PCR, fluorescent quantitative PCR, digital droplet PCR, etc. have been the gold standard for nucleic acid detection. However, the PCR reaction requires three steps: denaturation, annealing, and extension. It has high requirements for equipment and operators, requires high temperature conditions, is prone to nonspecific amplification, and has a long reaction time (1 to 2 hours), which limits its application to medical testing institutions. The recombinase polymerase isothermal amplification technology (RPA) has a faster detection reaction (20 minutes) and can react at room temperature, but its specificity is poor, nonspecific amplification is prone to occur, and it is easily affected by environmental nucleic acid contamination and false positive results. CRISPR-Dx can react quickly at room temperature and has good specificity, but it is not sensitive enough when used alone, and it is easy to miss the diagnosis when the nucleic acid concentration is low.

[0006] Since the sensitivity of CRISPR-Dx still needs to be optimized, a research team combined it with RPA to significantly improve the sensitivity. Unfortunately, RPA is prone to non-specific amplification, which reduces the accuracy of the combined detection technology.

[0007] The existing technology tends to adopt the method of cascade amplification, by adding a deoxyribonucleic acid (DNA) substrate with a similar structure to the template to the reaction system, to achieve the effect of extremely low concentration DNA template with extremely high sensitivity and specificity without amplification, for example: 1. The cascade amplification effect is achieved by adding DNA / RNA substrates, but the detection limit is at the pM level, the sensitivity is insufficient, and the reaction time takes 100 minutes, requiring two enzymes, Cas12 and Cas13, and the cost of DNA / RNA substrates is relatively high; 2. The Csm6 enzyme with signal amplification function is connected in series with the Cas13 enzyme, which can detect nucleic acids as low as 50 aM within 20 minutes, but the cost of connecting the enzymes in series is extremely high; 3. The cascade amplification effect is achieved by adding RNA substrates containing probes, with a detection limit as low as 5 aM, but the reaction time takes 4 hours, and the substrate structure is relatively complex, and the cost of RNA probe synthesis is relatively high. Summary of the invention

[0008] The purpose of the present invention is to disclose a ROS1 gene fusion detection kit based on CRISPR-Dx technology to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0009] To achieve the above object, the present invention is implemented through the following technical solutions:

[0010] The first aspect of the present invention is to provide a ROS1 gene fusion detection kit.

[0011] The kit described in the first aspect of the present invention includes an RT-RPA amplification system, wherein the RT-RPA amplification system includes at least 14 forward primers and 4 reverse primers, wherein the forward primer is selected from the nucleotide sequences shown in SEQ ID No: 9 to 46, and the reverse primer is selected from the nucleotide sequences shown in SEQ ID No: 47 to 55.

[0012] The fusion types of ROS1 gene and other gene breakage fusions are shown in Table 1:

[0013]

[0014] The RT-RPA amplification system can achieve composite amplification. The upstream primer combinations designed thereby respectively target appropriate sites of the spliced ​​genes, and the downstream primer combinations respectively target the exons of the ROS1 gene, thereby achieving typing amplification of the fusion gene in the test sample, laying a solid foundation for subsequent further testing.

[0015] In some application embodiments of the first aspect of the present invention, the preferred forward primer consists of the nucleotide sequence shown in SEQ ID No:9, SEQ ID No:11, SEQ ID No:12, SEQ ID No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ ID No:22, SEQ ID No:23, SEQ ID No:28, SEQ ID No:30, SEQ ID No:34, SEQ ID No:35, SEQ ID No:38, SEQ ID No:42 and SEQ ID No:45, and the preferred reverse primer consists of the nucleotide sequence shown in SEQ ID No:47, SEQ ID No:49, SEQ ID No:51 and SEQ ID No:55.

[0016] In some application embodiments of the first aspect of the present invention, the RT-RPA amplification system further comprises buffer A and buffer B. The buffer A provides the buffer conditions for the RT-RPA reaction and contains reverse transcriptase; buffer B is magnesium acetate and is used to start the RT-RPA reaction.

[0017] In some application embodiments of the first aspect of the present invention, the reaction temperature of the RT-RPA amplification system is 35-45° C. Further, the reaction temperature is selected from any one of 35.0° C., 37.5° C., 38.0° C., 38.5° C., 39.0° C., 39.5° C., 40.0° C., 40.5° C., 41.0° C., 41.5° C., 42.0° C., 42.5° C., and 45.0° C.

[0018] In some application embodiments of the first aspect of the present invention, the kit further comprises a CRISPR-Dx detection system, the CRISPR-Dx detection system comprising at least 4 CrRNA fragments, the 4 CrRNA fragments respectively targeting exon 32, exon 34, exon 35 and exon 36 of the ROS1 gene. If there is a fusion gene in the sample to be detected, the corresponding amplification product can be obtained by the composite amplification of the RT-RPA amplification system, and on this basis, the target DNA can be accurately identified by cooperating with the CRISPR-Dx detection system, activating the Cas12a cutting probe, and obtaining a fluorescent signal.

[0019] In some application embodiments of the first aspect of the present invention, the crRNA fragment is selected from the nucleotide sequences shown in SEQ ID No: 1 to 8.

[0020] In some application embodiments of the first aspect of the present invention, the crRNA fragment is selected from the nucleotide sequences shown in SEQ ID No: 1, SEQ ID No: 4, SEQ ID No: 6 and SEQ ID No: 7.

[0021] In some application embodiments of the first aspect of the present invention, the CRISPR-Dx detection system further comprises a probe, the nucleotide sequence of the probe is 5'-TTATTATT-3', the 5' end of the probe is provided with a fluorescent group, and the 3' end of the probe is provided with a fluorescent quenching group. The probe is cut by the activated Cas12a, so that the fluorescent group at the 5' end and the fluorescent quenching group at the 3' end are separated, thereby emitting a fluorescent signal, indicating that the test result is positive.

[0022] In some application embodiments of the first aspect of the present invention, the CRISPR-Dx detection system further includes a magnesium chloride solution, a buffer, an RNase inhibitor and Lb Cas12a.

[0023] In some application embodiments of the first aspect of the present invention, the reaction temperature of the CRISPR-Dx detection system is 37-42°C, preferably 40°C.

[0024] Compared with the prior art, the above technical solution of the present invention has at least the following advantages or beneficial effects:

[0025] 1) Short reaction time: The entire reaction process of the kit only takes 10 to 30 minutes, which has obvious detection advantages compared to traditional detection methods that take several hours;

[0026] 2) Simple reaction equipment: The kit only needs to be kept at a constant temperature of 35-45°C, which has low requirements for reaction equipment and a wider range of applications;

[0027] 3) Higher detection sensitivity: The detection sensitivity of existing similar products is 25 copies / μL. The kit first amplifies the sample to be tested using the RT-RPA amplification system, and then further uses the CRISPR-Dx detection system to detect the amplified product. The detection sensitivity can reach 10 copies / μL, which is far higher than that of existing similar products.

[0028] 4) Gene fusion typing: The CRISPR-Dx detector contained in the kit uses CrRNA that targets different exon regions of the ROS1 gene, which can realize the typing detection of ROS1 gene fusion and meet the needs of subsequent ROS1 lung cancer targeted treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1It is a dot-line graph of the detection results of C1 crRNA detecting ROS1 gene fusion in Example 1;

[0030] Figure 2 It is a dotted line graph of the detection results of C2 crRNA detecting ROS1 gene fusion in Example 1;

[0031] Figure 3 It is a dot-line graph of the detection results of C3 crRNA detecting ROS1 gene fusion in Example 1;

[0032] Figure 4 It is a dotted line graph of the detection results of C4 crRNA detecting ROS1 gene fusion in Example 1;

[0033] Figure 5 It is a dot-line graph of the detection results of C5 crRNA detecting ROS1 gene fusion in Example 1;

[0034] Figure 6 It is a dot-line graph of the detection results of C6 crRNA detecting ROS1 gene fusion in Example 1;

[0035] Figure 7 It is a dot-line graph of the detection results of C7 crRNA detecting ROS1 gene fusion in Example 1;

[0036] Figure 8 It is a dot-line graph of the detection results of C8 crRNA detecting ROS1 gene fusion in Example 1;

[0037] Fig. 9 is a bar graph of the amplification results of primer combinations 1 to 3 in Example 2;

[0038] Fig.10 is a bar graph of the amplification results of primer combinations 4 to 6 in Example 2;

[0039] Fig.11 is a bar graph of the amplification results of primer combinations 7 to 9 in Example 2;

[0040] Fig.12 is a bar graph of the amplification results of primer combinations 10 to 12 in Example 2;

[0041] Fig.13 is an electrophoresis gel image of the amplification temperature of the optimized RT-RPA amplification system in Example 3;

[0042] Fig.14 This is an electrophoresis gel image of the amplification time of the optimized RT-RPA amplification system in Example 4;

[0043] Fig.15 The detection of C6R34 in Example 5 was carried out for Mg 2+ Concentration optimized histogram;

[0044] Fig.16 It is the detection of S2R32 in Example 5 for Mg 2+ Concentration optimized histogram;

[0045] Fig.17 is a bar graph of the optimization of the types of buffer solutions for detecting C6R34 in Example 6;

[0046] Fig.18 is a bar graph of the optimization of the types of buffer solutions for detecting S2R32 in Example 6;

[0047] Fig.19 It is a bar graph of the optimization of the Cas12a addition ratio by detecting C6R34 in Example 7;

[0048] Fig. 20 It is a bar graph of the optimization of the Cas12a addition ratio by detecting S2R32 in Example 7;

[0049] Fig.21 This is a heat map for verifying the sensitivity of the kit in Example 8. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0051] Example 1. Construction of the kit

[0052] (1) According to the ROS1 gene fusion type shown in Table 1, the corresponding crRNA is designed to target the fusion gene. The details are shown in Table 2.

[0053]

[0054] The probe design is FAM-TTATTATT-BHQ1. The corresponding CRISPR-Dx reaction system design is shown in Table 3.

[0055]

[0056] (2) According to the ROS1 gene fusion type shown in Table 1, design the corresponding RT-RPA amplification primers, as shown in Table 4.

[0057]

[0058]

[0059] The corresponding reaction system is designed as shown in Table 5:

[0060]

[0061] (3) Taking the 14 ROS1 fusion types in Table 1 as the detection targets, select the corresponding upstream primers and downstream primers from the primer combinations shown in Table 4 for RT-RPA amplification, and screen the primer combination with the highest amplification efficiency for subsequent experiments. Each group of experiments has an experimental group and a control group. The RT-RPA amplification system is initially configured as follows: 14 μL of Buffer A, 1 μL of a forward primer mixture with a concentration of 10 μM, 1 μL of a reverse primer mixture with a concentration of 10 μM, 5 μL of a template substrate, and 1 μL of Buffer B. After mixing, the reaction was carried out at a reaction temperature of 40°C and a reaction time of 15 minutes.

[0062] The formula of CRISPR-Dx detection system is as shown in Table 3: DEPC water 11.5 μL, MgCl2 with a concentration of 25 mM 2 μL, buffer solution 2 μL, RNase inhibitor 0.5 μL, Lb Cas12a with a concentration of 1 μM 1 μL, probe with a concentration of 10 μM 1 μL, CrRNA with a concentration of 1 μM 2 μL. Mix all ingredients except the probe in advance, let stand at room temperature for 5-15 minutes, then add the probe and mix thoroughly, then transfer 10 μL of the amplified product to the CRISPR-Dx detection system mixture, mix well and react at 40°C for 20 minutes.

[0063] After the reaction is completed, the results are interpreted. Figures 1 to 8 As shown. The substrate of the NTC group is DPEC water, and the other components are exactly the same as the positive experimental group. The stronger the fluorescence signal, the more templates are recognized by crRNA in the reaction system, and the more probes are cut by Lb Cas12a protein in the reaction solution. Figure 1 , 4 , 6, and 7 show that the crRNA detection groups all have strong corresponding fluorescence signals, while the control group has almost no fluorescence signal changes; Figure 2 , 3 The fluorescence signals of some experimental groups of the crRNA groups shown in Figures 5, 8 were low, which could easily lead to false negative interpretations. Therefore, the crRNAs shown in C1, C4, C6, and C7 were selected into the CRISPR-Dx detection system.

[0064] Example 2: Screening of optimal primer combinations for fusion sites

[0065] The primers designed according to Table 4 were integrated into multiple primer combinations to detect their amplification efficiency.

[0066] Primer combinations 1 to 3 for amplification of S2R32, S4R32, S13R32, SL4R32, and C6R32.

[0067] Primer combination 1: select 6 upstream primers including F2-RT-RPA, F7-RT-RPA, F10-RT-RPA, F16-RT-RPA, F21-RT-RPA and F33-RT-RPA, and 1 downstream primer R4-RT-RPA;

[0068] Primer combination 2: 7 upstream primers including F1-RT-RPA, F3-RT-RPA, F10-RT-RPA, F13-RT-RPA, F20-RT-RPA, F26-RT-RPA and F34-RT-RPA, and 1 downstream primer R3-RT-RPA were selected;

[0069] Primer combination 3: Select 6 upstream primers, including F6-RT-RPA, F10-RT-RPA, F17-RT-RPA, F21-RT-RPA, F25-RT-RPA and F35-RT-RPA, and 1 downstream primer R7-RT-RPA.

[0070] Primer combinations 4 to 6 amplified C6R34, S4R34, S13R34, SL4R34, and E10R34.

[0071] Primer combination 4: select 6 upstream primers including F2-RT-RPA, F10-RT-RPA, F16-RT-RPA, F18-RT-RPA, F21-RT-RPA and F28-RT-RPA, and 1 downstream primer R2-RT-RPA;

[0072] Primer combination 5: 6 upstream primers including F1-RT-RPA, F9-RT-RPA, F10-RT-RPA, F11-RT-RPA, F20-RT-RPA and F27-RT-RPA, and 1 downstream primer R1-RT-RPA were selected;

[0073] Primer combination 6: Select 6 upstream primers including F6-RT-RPA, F10-RT-RPA, F17-RT-RPA, F19-RT-RPA, F21-RT-RPA and F29-RT-RPA, and 1 downstream primer R6-RT-RPA.

[0074] Primer combinations 7 to 9 amplified T8R35, G8R35, and L16R35.

[0075] Primer combination 7: 5 upstream primers, F4-RT-RPA, F12-RT-RPA, F15-RT-RPA, F22-RT-RPA and F30-RT-RPA, and 1 downstream primer, R5-RT-RPA, were selected;

[0076] Primer combination 8: 5 upstream primers, F5-RT-RPA, F12-RT-RPA, F15-RT-RPA, F23-RT-RPA and F31-RT-RPA, and 1 downstream primer, R8-RT-RPA, were selected;

[0077] Primer combination 9: Select 5 upstream primers including F8-RT-RPA, F12-RT-RPA, F15-RT-RPA, F24-RT-RPA and F32-RT-RPA, and 1 downstream primer R5-RT-RPA.

[0078] Primer combinations 10 to 12 for amplification of G4R36.

[0079] Primer combination 10: 2 upstream primers F14-RT-RPA and F37-RT-RPA, and 1 downstream primer R2-RT-RPA were selected;

[0080] Primer combination 11: select 2 upstream primers F14-RT-RPA and F36-RT-RPA, and 1 downstream primer R9-RT-RPA;

[0081] Primer combination 12: Select 2 upstream primers, F14-RT-RPA and F38-RT-RPA, and 1 downstream primer, R9-RT-RPA.

[0082] Since some fusion genes have poor specificity and need to be detected using multiple upstream primers, the number of upstream primers in each primer combination is greater than the number of spliced ​​genes in Table 1.

[0083] The RT-RPA amplification system formula is as follows: 14 μL Buffer A, 1 μL forward primer at a concentration of 10 μM, 1 μL reverse primer at a concentration of 10 μM, 5 μL template substrate, and 1 μL Buffer B. After mixing, the reaction temperature is 40°C and the reaction time is 15 minutes. The CRISPR-Dx detection system formula is as follows: 11.5 μL DEPC water, 2 μL MgCl2 at a concentration of 25 mM, 2 μL buffer solution, 0.5 μL RNase inhibitor, 1 μL Lb Cas12a at a concentration of 1 μM, 1 μL probe at a concentration of 10 μM, and 2 μL CrRNA at a concentration of 1 μM. Mix all the ingredients except the probe in advance, let it stand at room temperature for 5 to 15 minutes, then add the probe and mix thoroughly, then transfer 10 μL of the amplified product to the CRISPR-Dx mixture, mix it, and react at a temperature of 40°C for 20 minutes. The test results are as follows Figures 9 to 12 As shown in the figure, each primer combination can successfully amplify the target fragment, among which primer combinations 2, 5, 7, and 10 have the best amplification effect. Since ROS1 gene fusion has multiple types, even if ROS1 is fused with the same gene, the fusion region will be inconsistent (such as S2R32, S4R32, and S4R34). Therefore, the RT-RPA composite amplification of 4 tubes can complete the typing screening of the ROS1 gene before the test, so that the subsequent CRISPR-Dx test can be carried out smoothly.

[0084] Example 3: Optimization of amplification temperature for RT-RPA amplification system

[0085] In order to determine the optimal reaction temperature of the RT-RPA reaction system, this example sets 5 gradient temperatures in the range of 35~45°C to amplify the target. The RT-RPA system formula is configured as follows: 14 μL of Buffer A, 1 μL of forward primer with a concentration of 10 μM, 1 μL of reverse primer with a concentration of 10 μM, 5 μL of template substrate, and 1 μL of Buffer B. After mixing the 5 groups of reaction systems, amplification was performed at temperatures of 35°C, 37.5°C, 40°C, 42.5°C, and 45°C, respectively, with a reaction time of 15 minutes, and then verified by 1.5% agarose gel electrophoresis. In this example, C6R34, S2R32, and T8R35, which appear most frequently in three different ROS1 exons, are selected as representatives for verification. The results are as follows Fig.12 The darker the color of each lane in the electrophoresis gel image, the more products are obtained by RT-RPA amplification, indicating that the optimal reaction temperature of the RT-RPA amplification system is 40°C, and the subsequent examples are all reacted at 40°C.

[0086] Example 4: Optimization of amplification time of RT-RPA amplification system

[0087] In order to determine the optimal reaction temperature of the RT-RPA reaction system, this example sets 5 gradient temperatures in the range of 0~15°C to amplify the target. The RT-RPA system formula is configured as follows: 14 μL of Buffer A, 1 μL of forward primer with a concentration of 10 μM, 1 μL of reverse primer with a concentration of 10 μM, 5 μL of template substrate, and 1 μL of Buffer B. After mixing, the 5 groups of reaction systems were amplified at 40°C, and the reaction times were set to 0 minutes, 1 minute, 5 minutes, 10 minutes and 15 minutes, respectively, and then verified by 1.5% agarose gel electrophoresis. In this example, C6R34, S2R32, and T8R35, which appear most frequently in three different ROS1 exons, were selected as representatives for verification. The results are as follows Fig.13 As shown. The darker the color of each lane in the electrophoresis gel, the more products amplified by RT-RPA are obtained, indicating that the optimal reaction temperature of the RT-RPA amplification system of the present invention is 40°C. The subsequent embodiments are all carried out at 40°C. The darker the color of each lane in the electrophoresis gel, the more products amplified by RT-RPA are obtained, which indicates that the optimal reaction temperature of the RT-RPA amplification system of the present invention is 40°C. The subsequent embodiments are all carried out at 40°C. Fig.13 It can be seen that the amount of product in the reaction time of 0 to 15 minutes generally shows an increasing trend. Considering the time cost, the product obtained by amplification for 10 minutes can meet the needs of subsequent CRISPR-Dx detection. Therefore, the RT-RPA amplification system selects 10 minutes as the amplification time.

[0088] Example 5: Additional addition of Mg 2+ Impact on CRISPR-Dx detection system

[0089] To determine the additional addition of Mg 2+ Impact on the CRISPR-Dx detection system. In this embodiment, 7 gradient concentrations are set in the range of 0~75 mM to detect the target. The total amount of the CRISPR-Dx detection system is 20 μL, including: 2 μL of buffer solution, 0.5 μL of RNA enzyme inhibitor, 1 μL of Lb Cas12a with a concentration of 1 μM, 1 μL of probe with a concentration of 10 μM, 2 μL of CrRNA with a concentration of 1 μM, and MgCl2 with a concentration of 25mM are added in the amounts of 0 μL, 12.5 μL, 25 μL, 37.5 μL, 50 μL, 62.5 μL and 70 μL respectively, and the balance is supplemented by DEPC water. Mix the other components except the probe in advance, let it stand at room temperature for 5~15 minutes, then add the probe and mix it thoroughly, then transfer 10 μL of the amplified product to the CRISPR-Dx mixture, set the reaction temperature to 40°C after mixing, and react for 20 minutes.

[0090] In this example, C6R34 and S2R32, which appear most frequently in two different ROS1 exons, are selected as representatives for verification. After the reaction is completed, the results are interpreted. Fig.14 and Fig.15 The higher the column, the stronger the fluorescence signal generated by the CRISPR-Dx reaction. Fig.14 and Fig.15 It can be seen that with the increase of Mg 2+ As the concentration increases, the fluorescence signal detected by NTC becomes stronger, and excessive background signals may lead to false positive results. 2+ The lower the additional amount, the lower the background signal and the lower the probability of false positive results. 2+ An additional amount of 0 mM was added for the reaction.

[0091] Example 6: Effect of buffer solution on CRISPR-Dx detection system

[0092] In order to determine the effect of different buffers on the CRISPR-Dx detection system, this example selected four buffer solutions commonly used in CRISPR-Dx reactions for comparative testing, including HolmesBuffer 1, HolmesBuffer 2, NEBufferr2.1 and Tris-HCl. The CRISPR-Dx detection system formula is as follows: 13.5 μL of DEPC water, 2 μL of buffer solution, 0.5 μL of RNase inhibitor, 1 μL of Lb Cas12a at a concentration of 1 μM, 1 μL of probe at a concentration of 10 μM, and 2 μL of CrRNA at a concentration of 1 μM. Mix all ingredients except the probe in advance, let stand at room temperature for 5 to 15 minutes, then add the probe and mix thoroughly, then transfer 10 μL of the amplified product to the CRISPR-Dx mixture, set the reaction temperature to 40°C after mixing, and the reaction time is 20 minutes.

[0093] In this example, C6R34 and S2R32, which appear most frequently in two different ROS1 exons, are selected as representatives for verification. After the reaction is completed, the results are interpreted. Fig.16 and Fig.17 The higher the column, the stronger the fluorescence signal generated by the CRISPR-Dx reaction. Fig.16 and Fig.17 It can be seen that the fluorescence signal obtained by using HolmesBuffer 1 for detection is the strongest and the result is the most stable, which can ensure that the error of the result obtained in each detection is small.

[0094] Example 7, the effect of the ratio of Cas12a to CrRNA on the CRISPR-Dx detection system

[0095] In order to determine the impact that different ratios of Cas12a to CrRNA may have on the CRISPR-Dx detection system, this embodiment selected 9 different concentration ratios of Cas12a:CrRNA for detection, as shown in Table 6.

[0096]

[0097] The RT-RPA amplification system and the CRISPR-Dx detection system were configured according to the parameters optimized in the previous example. In this example, C6R34 and S2R32, which appear most frequently in two different ROS1 exons, were selected as representatives for verification. After the reaction was completed, the results were interpreted. Fig.18 and Fig.19 As shown. The results show that different Cas12a:CrRNA ratios have little effect on the detection effect of the reaction system, indicating that Cas12a:CrRNA can meet the detection requirements of the CRISPR-Dx of the present invention for ROS1 gene fusion under the conditions of 20 nM:20 nM (1:1). Therefore, considering the comprehensive detection cost and detection effect, the CRISPR-Dx reaction of the present invention uses a Cas12a:CrRNA ratio of 1:1.

[0098] Example 8: Sensitivity test of the kit

[0099] The RT-RPA amplification system and the CRISPR-Dx detection system were configured according to the parameters optimized in the previous example. A step-concentration sample containing 14 ROS1 gene fusion templates was configured, with concentrations of 1 copy / μL, 5 copies / μL, 10 copies / μL, 20 copies / μL, and 50 copies / μL, respectively. The heat map of the detection results is shown in Fig. 20 As shown, it is intuitively shown that all fusion sites have good detection stability at 10 copies, and the 10-copy group of 14 ROS1 fusion sites is statistically analyzed with the control group, all p<0.01 (**), indicating that all 10-copy groups are significantly different from the control group, that is, the minimum copy number of all ROS1 gene fusions that can be detected by the kit is 10 copies.

[0100] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A ROS1 gene fusion detection kit, characterized in that: The invention comprises an RT-RPA amplification system and a CRISPR-Dx detection system, wherein the RT-RPA amplification system comprises 4 groups of primer combinations selected from the following: the first group of primer combinations consists of primers with nucleotide sequences such as SEQ ID No: 9, SEQ ID No: 11, SEQ ID No: 18, SEQ ID No: 21, SEQ ID No: 28, SEQ ID No: 34, SEQ ID No: 42 and SEQ ID No: 49; the second group of primer combinations consists of primers with nucleotide sequences such as SEQ ID No: 9, SEQ ID No: 17, SEQ ID No: 18, SEQ ID No: 19, SEQ ID No: 28, SEQ ID No: 35 and SEQ ID No: 47; the third group of primer combinations consists of primers with nucleotide sequences such as SEQ ID No: 12, SEQ ID No: 20, SEQ ID No: 23, SEQ ID No: 30, SEQ ID No: 38 and SEQ ID No: 51; the fourth group of primer combinations consists of primers with nucleotide sequences such as SEQ ID No: 22, SEQ ID No: 45 and SEQ ID The primer composition is shown in No:48; the CRISPR-Dx detection system includes 4 CrRNA fragments, and the CrRNA fragments are selected from the nucleotide sequences shown in SEQ ID No:1, SEQ ID No:4, SEQ ID No:6 and SEQ ID No:

7.

2. The kit according to claim 1, characterized in that The RT-RPA amplification system also includes buffer A and buffer B, wherein buffer A provides buffer conditions for the RT-RPA reaction and contains reverse transcriptase, and buffer B is magnesium acetate.

3. The kit according to claim 2, characterized in that: The reaction temperature of the RT-RPA amplification system is 35-45°C.

4. The kit according to claim 1, characterized in that The CRISPR-Dx detection system also includes a probe, the nucleotide sequence of the probe is 5'-TTATTATT-3', the 5' end of the probe is provided with a fluorescent group, and the 3' end of the probe is provided with a fluorescent quenching group.

5. The kit according to claim 4, characterized in that: The CRISPR-Dx detection system also includes a magnesium chloride solution, a buffer, an RNase inhibitor and Lb Cas12a.

6. The kit according to claim 5, characterized in that The reaction temperature of the CRISPR-Dx detection system is 37~42°C.

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