Liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology

Abstract

The invention relates to the technical field of nucleic acid detection, and discloses a liver cancer lncRNA HULC detection kit based on a CRISPR-Dx technology, and the liver cancer lncRNA HULC detection kit comprises an RT-RPA reaction system and a CRISPR / Dx reaction system; the CRISPR / Dx reaction system comprises crRNA, and the nucleotide sequence of the crRNA is as shown in SEQ ID NO. 1 or SEQ ID NO. 2; the RT-RPA reaction system comprises a forward primer F7-HULC-RPA of which the nucleotide sequence is as shown in SEQ ID NO.4, and a reverse primer R7-HULC-RPA of which the nucleotide sequence is as shown in SEQ ID NO.5, or a reverse primer R8-HULC-RPA of which the nucleotide sequence is as shown in SEQ ID NO.6. The RT-RPA reaction system comprises a forward primer F7-HULC-RPA of which the nucleotide sequence is as shown in SEQ ID NO.3. The kit provided by the invention has the characteristics of high efficiency and rapidness, and the reaction process for detection can be completed only in 10-35 minutes. Compared with a traditional detection means which needs reaction time of several hours, the method has the advantages that the time required by detection is greatly shortened, the detection efficiency is remarkably improved, a powerful guarantee is provided for rapidly obtaining a detection result, and the method has obvious advantages in time cost.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology. Background Art

[0002] Long non-coding RNA (lncRNA) plays a key role in the development, progression, metastasis, and prognosis of hepatocellular carcinoma (HCC), and therefore holds great potential as a diagnostic biomarker for HCC. Antisense oligonucleotides (ASOs) or small interfering RNA (siRNA) can be used therapeutically to silence oncogenic lncRNAs, reducing their expression in HCC cells and thereby inhibiting malignant behaviors such as proliferation, migration, and invasion. Monitoring lncRNA expression levels after surgery can help regulate the expression of these lncRNAs, helping to eliminate residual tumor cells and reduce recurrence rates. Furthermore, during chemotherapy, certain lncRNAs may affect the sensitivity of chemotherapeutic drugs. Modulating these lncRNAs can enhance the sensitivity of HCC cells to chemotherapeutic drugs and improve their efficacy.

[0003] Among them, abnormal expression of the highly up-regulated in liver cancer (HULC) transcript in liver cancer is closely related to the development of liver cancer. Its specific mechanism involves the regulation of multiple gene expressions and the effects on related signaling pathways, playing a key role in the occurrence and development of liver cancer. Therefore, HULC can be used as a target for liver cancer detection.

[0004] Existing methods for detecting HULC include qRT-PCR, gene chip technology, and in situ hybridization technology. Among them, qRT-PCR has high sensitivity, strong specificity, and accurate quantitative capabilities, but its throughput is low and it has high requirements for sample quality; gene chip technology can achieve high-throughput detection and can comprehensively analyze gene expression profiles, but its quantitative accuracy is poor and the cost is high; in situ hybridization technology can be intuitively located at the tissue and cell levels and has high value in pathological diagnosis, but its operation process is complicated and semi-quantitative analysis is quite difficult. Each of the above detection methods has its own advantages and disadvantages, and they all occupy an important position in the research and diagnosis of liver cancer. In view of this, there is an urgent need to develop a more convenient and efficient detection method. Summary of the Invention

[0005] The present invention aims to provide a liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology to solve the technical problems existing in the prior art.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology, comprising an RT-RPA reaction system and a CRISPR / Dx reaction system; the CRISPR / Dx reaction system comprises crRNA, and the nucleotide sequence of the crRNA is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the crRNA is as shown in SEQ ID NO.1.

[0009] Preferably, the CRISPR / Dx reaction system further comprises DEPC water, MgCl2, Buffer, RNase inhibitor, LbCas12a and a probe, and the nucleotide sequence of the probe is shown in SEQ ID NO.3.

[0010] Preferably, the CRISPR / Dx reaction system comprises 8-15 μL of DEPC water, 0-3 μL of 25 mM MgCl2, 0-2 μL of buffer, 0.5 μL of RNase inhibitor, 0.2-1.5 μM of Lb Cas12a, 1 μL of 10 μM probe, and 0.2-2 μL of 1 μM crRNA; the reaction temperature of the CRISPR / Dx reaction system is 38-42 ° C, and the reaction time is 20-25 min.

[0011] Preferably, the CRISPR / Dx reaction system is specifically DEPC water 16.5-(x+y) μL, 25mM MgCl2 1.5 μL, Buffer 2 μL, RNase inhibitor 0.5 μL, 1 μM Lb Cas12a x μL, 10 μM probe 1 μL and 1 μM crRNA y μL, wherein 0.4 μL≤x≤0.8 μL, x:y=1-2:1-4.

[0012] Preferably, the RT-RPA reaction system includes a forward primer F7-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.4 and a reverse primer R7-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.5 or a reverse primer R8-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.6.

[0013] Preferably, the RT-RPA reaction system includes a forward primer F7-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.4 and a reverse primer R7-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.5.

[0014] Preferably, the RT-RPA reaction system further comprises Buffer A, a template substrate, Buffer B and DEPC water; the RT-RPA reaction temperature is 38-42° C., and the reaction time is 15-20 min.

[0015] Preferably, the RT-RPA reaction system further comprises 10-29.5 μL of Buffer A, 1-2.5 μL of 10 μM forward primer, 1-2.5 μL of 10 μM reverse primer, 1-10 μL of template substrate, 0.5-2 μL of Buffer B, and 0-23 μL of DEPC water.

[0016] The present invention also provides an application of a liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology in detecting liver cancer lncRNA HULC.

[0017] In summary, compared with the prior art, the solution of the present invention has the following beneficial effects:

[0018] 1. Significantly shortened reaction time: The kit provided by the present invention is highly efficient and rapid for detection, with the entire reaction process taking only 10-35 minutes to complete. Compared to traditional detection methods that often take several hours to react, the present invention greatly shortens the detection time required, significantly improves detection efficiency, provides a strong guarantee for rapid acquisition of test results, and demonstrates a clear advantage in terms of time and cost.

[0019] 2. Simple reaction equipment requirements: The kit provided by the present invention has significant advantages in terms of reaction equipment for detection. The low requirements for reaction equipment make the detection method widely applicable, especially suitable for places with limited resources such as remote areas and economically underdeveloped areas with relatively backward technical conditions. It does not require complex and expensive temperature control equipment, which lowers the detection threshold and facilitates promotion and application.

[0020] 3. Higher detection sensitivity: The present invention adopts a unique combination of technologies. First, RT-RPA isothermal amplification technology is used to efficiently amplify the template, so that the target nucleic acid sequence can be enriched in large quantities. On this basis, the CRISPR / Dx reaction system is further introduced to accurately detect the amplified products. Through this innovative technical process, compared with traditional detection methods, the present invention can detect the target substance more keenly, effectively improve the detection sensitivity, and provide a reliable means for the detection of trace samples or low-abundance target molecules, thereby enhancing the accuracy and reliability of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of a detection kit for liver cancer lncRNA HULC based on CRISPR-Dx technology of the present invention;

[0022] Figure 2 is the expression level of HULC in different cell lines in Example 1;

[0023] Figure 3 It is a dotted line graph of the detection results of the HULC site detected by different crRNA and different primer combinations in Example 2;

[0024] Figure 4 The different Mg in Example 3 2+ Effect of concentration on HULC CRISPR / Dx assay results;

[0025] Figure 5 is the effect of different Cas12a and crRNA ratios on HULC CRISPR / Dx detection in Example 4;

[0026] Figure 6 The sensitivity and specificity of CRISPR / Dx in detecting HULC in Example 5. DETAILED DESCRIPTION

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] like Figure 1As shown, the present invention adopts CRISPR / Dx technology. Primers, crRNA and probe sequences are designed according to lncRNAs sequences. Template amplification and template detection are performed under fixed RT-RPA reaction conditions. After the template is detected, the probe will be cleaved by the enzyme activated in the CRISPR / Dx reaction system to produce a fluorescent signal, and finally the result can be interpreted.

[0031] Example 1 qPCR detection of HULC expression in liver cancer cell lines and other cell lines

[0032] qPCR can accurately measure the expression of specific genes in cells. In this example, qPCR was used to verify the relative expression levels of HULC in HepG2 liver cancer cells, HCT116 colorectal cancer cells, and BEAS-2B bronchial epithelial cells. Using HULC as the detection target, the reaction system was configured using the primers and probes in Table 1: 10 μL of 2X PCR mix, 1 μL each of the target primer (10 μM), the forward primer, and the reverse primer, 1 μL of the target probe (10 μM), 5 ng of cDNA, and DEPC water to 20 μL. The reaction procedure was 95°C for 30 seconds, followed by 45 cycles of (95°C for 15 seconds, 55°C for 30 seconds).

[0033] Table 1 qPCR amplification primer and probe sequences

[0034]

[0035]

[0036] After the reaction, the results were processed. BEAS-2B cells were used as the control group. All experiments were repeated three times and the average values ​​were taken for plotting. Figure 2 .

[0037] Figure 2 The higher the bar, the higher the HULC expression level. This shows that the expression level of HULC in liver cancer cell lines is much higher than that in other cell lines (BEAS-2B, HepG2), so HULC lncRNAs are suitable for detecting liver cancer.

[0038] Example 2 Screening of HULC Primer Probes

[0039] Taking HULC as the detection target, this example sets up an experimental group (positive group) and a control group (blank group). The positive group uses HepG2 cell genomic RNA as a template, and the blank group uses DEPC water. Use the primers in Table 2 to configure according to Table 3. After RT-RPA amplification of the target, use the crRNA and probe in Table 4 to configure according to Table 5 for CRISPR detection. The primer and crRNA combination with the highest amplification efficiency are screened for subsequent experiments. The RT-RPA reaction system formula is as follows: 14μL Buffer A, 1μL forward primer (10μM), 1μL reverse primer (10μM), 5μL template substrate, and 1μL Buffer B. After mixing, the reaction is carried out at a reaction temperature of 40°C and a reaction time of 15min. The CRISPR / Dx system formula is as follows: DEPC water 1.5μL, MgCl2 (25mM) 2μL, Buffer 2μL, RNase inhibitor 0.5μL, Lb Cas12a (1μM) 1μL, probe (10μM) 1μL, crRNA (1μM) 2μL. Mix all components except the probe in advance and let it stand at room temperature for 5-15 minutes. Then add the probe and mix thoroughly. Then transfer 10μL of RT-RPA product to the CRISPR / Dx mixture. After mixing, react at a reaction temperature of 40°C and a reaction time of 20 minutes.

[0040] Table 2 RT-RPA amplification primer sequences

[0041]

[0042] Table 3 RT-RPA reaction system

[0043] Reagents Dosage (μL) Buffer A 10-29.5 Forward primer (10 μM) 1-2.5 Reverse primer (10 μM) 1-2.5 DEPC water 0-23 Template substrate 1-10 Buffer B 0.5-2

[0044] Table 4 crRNA and probe sequences

[0045]

[0046] Table 5 CRISPR / Dx reaction system

[0047]

[0048]

[0049] After the reaction, the results were interpreted as relative fluorescence units (RFU). All experiments were repeated three times and the average value was taken for plotting. Figure 3 .

[0050] The stronger the light signal, the more templates are recognized by crRNA in the reaction system, the more probes are cut by Lb Cas12a protein in the reaction solution, and the corresponding primer and crRNA combination is optimal. Figure 3 In the reaction system, the top three primer and crRNA combinations for the amount of template recognized by crRNA against HULC genomic RNA from HepG2 cells were F7-RPA+R7-RPA+crRNA1, F7-RPA+R7-RPA+crRNA2, and F7-RPA+R8-RPA+crRNA1. In this example, the F7-RPA and R7-RPA primers and crRNA1 were used for subsequent detection of the HULC locus.

[0051] Example 3 Additional addition of Mg 2+ Impact on HULC CRISPR / Dx Assays

[0052] To determine the additional addition of Mg 2+ Influence on HULC CRISPR / Dx detection, this embodiment sets 6 gradient concentrations in the range of 0-62.5mM to detect the target. The RT-RPA system formula, reaction temperature and time in this embodiment are exactly the same as those in Example 2. The CRISPR / Dx system formula is as follows: DEPC water 13.5-xμL, MgCl2 (25mM) xμL, Buffer 2μL, RNase inhibitor 0.5μL, Lb Cas12a (1μM) 1μL, probe (10μM) 1μL, crRNA (1μM) 2μL. Mix all ingredients except the probe in advance, let it stand at room temperature for 5-15min, then add the probe and mix thoroughly, then transfer 10μL of RT-RPA product to the CRISPR / Dx mixture, mix it and react at a reaction temperature of 40°C and a reaction time of 20min. After the reaction is completed, the results are interpreted, and the results are shown in Figure 4 .

[0053] Figure 4 The higher the column, the stronger the fluorescence signal generated by the CRISPR / Dx reaction. 2+ The dosage has little effect on the test results, so there is no need to add additional Mg 2+ ; However, the Mg corresponding to the fluorescence peak value during HULC site detection 2+ The added amount was 37.5 mM (corresponding to 1.5 μL MgCl2 (25 mM)) and was used for subsequent experiments.

[0054] Example 4 Effect of different Cas12a:crRNA on HULC CRISPR / Dx detection

[0055] In order to determine the effect of different Cas12a:crRNA on HULC CRISPR / Dx detection, this example selected 6 different concentration ratios of Cas12a:crRNA for detection (see Table 6 for specific ratios). The RT-RPA system formula, reaction temperature and time in this example are exactly the same as those in Example 2. The CRISPR / Dx system formula is as follows: DEPC water 16.5-(x+y)μL, MgCl2 (25mM) 1.5μL, Buffer 2μL, RNase inhibitor 0.5μL, Lb Cas12a (1μM) xμL, probe (10μM) 1μL, crRNA (1μM) yμL. Mix all ingredients except the probe in advance, let it stand at room temperature for 5-15min, then add the probe and mix thoroughly, then transfer 10μL of RT-RPA product to the CRISPR / Dx mixture, mix it, and react at a reaction temperature of 40°C and a reaction time of 20min. After the reaction is completed, the results are interpreted, and the results are shown in the table. Figure 5 .

[0056] Table 6 Cas12a:crRNA ratio table

[0057] 1 2 3 4 5 6 Cas12a (μL) 0.4 0.4 0.4 0.8 0.8 0.8 crRNA (μL) 0.4 0.8 1.6 0.4 0.8 1.6 Proportion 1：1 1:2 1:4 2:1 2:2 2:4

[0058] Figure 5 The higher the column, the stronger the fluorescence signal generated by the CRISPR / Dx reaction. Figure 5 It can be seen that different Cas12a:crRNA ratios at the HULC site have little effect on the detection effect of the reaction system, indicating that Cas12a:crRNA can meet the detection effect of HULC by CRISPR / Dx in this method under the conditions of 400nM:400nM (1:1). Therefore, this embodiment uses Cas12a:crRNA as 1:1 (400nM:400nM) for detecting the HULC site in subsequent experiments.

[0059] Example 5: Exploring the Sensitivity and Specificity of CRISPR / Dx in Detecting HULC and MALAT1

[0060] In this embodiment, experimental groups (positive and negative groups) and control groups (blank groups) were set, wherein the positive group used HepG2 cell genomic RNA as a template, the negative group used HCT116 cells and BEAS-2B cell genomic RNA as a template, and the blank group used DEPC water. In this embodiment, the RT-RPA system formula, reaction temperature and time are exactly the same as those in Example 2. The CRISPR / Dx system formula is as follows: DEPC water 15.7 μL, MgCl2 (25 mM) 1.5 μL, Buffer 2 μL, RNase inhibitor 0.5 μL, Lb Cas12a (1 μM) 0.4 μL, probe (10 μM) 1 μL, crRNA (1 μM) 0.4 μL, 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 thoroughly, then transfer 10 μL of RT-RPA product to the CRISPR / Dx mixture, and after mixing, react at a reaction temperature of 40 ° C and a reaction time of 20 minutes. After the reaction is completed, the results are interpreted. Figure 6 .

[0061] according to Figure 6 It can be seen that HULC can be detected in the RNA concentration range of 0.01-50 ng in HepG2 cells (positive group), but it cannot be detected at a concentration of 0.1 ng in HCT116 and BEAS-2B cells (negative group). This shows that under the technical solution of the present invention, the LOD of HULC can be measured below 0.01 ng / μL.

[0062] The above-described embodiments merely represent preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and such modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A CRISPR-Dx technology-based detection kit for liver cancer lncRNA HULC, characterized in that: It includes an RT-RPA reaction system and a CRISPR / Dx reaction system; the CRISPR / Dx reaction system includes crRNA, and the nucleotide sequence of the crRNA is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 1, characterized in that: The nucleotide sequence of the crRNA is shown in SEQ ID NO.

1.

3. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 1, characterized in that: The CRISPR / Dx reaction system also includes DEPC water, MgCl2, Buffer, RNase inhibitor, Lb Cas12a and a probe, and the nucleotide sequence of the probe is shown in SEQ ID NO.

3.

4. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 1, characterized in that: The CRISPR / Dx reaction system specifically includes 8-15 μL of DEPC water, 0-3 μL of 25 mM MgCl2, 0-2 μL of buffer, 0.5 μL of RNase inhibitor, 0.2-1.5 μM of Lb Cas12a, 1 μL of 10 μM probe, and 0.2-2 μL of 1 μM crRNA; the reaction temperature of the CRISPR / Dx reaction system is 38-42 ° C, and the reaction time is 20-25 min.

5. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 4, characterized in that: The CRISPR / Dx reaction system specifically includes 16.5-(x+y) μL of DEPC water, 1.5 μL of 25 mM MgCl2, 2 μL of buffer, 0.5 μL of RNase inhibitor, 1 μM Lb Cas12a x μL, 10 μM probe 1 μL and 1 μM crRNA y μL, wherein 0.4 μL≤x≤0.8 μL, x:y=1-2:1-4.

6. A liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology according to claim 1 or 2, characterized in that: The RT-RPA reaction system includes a forward primer F7-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.4 and a reverse primer R7-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.5 or a reverse primer R8-HULC-RPA having a nucleotide sequence as shown in SEQ ID NO.

6.

7. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 6, characterized in that: The RT-RPA reaction system includes a forward primer F7-HULC-RPA whose nucleotide sequence is shown in SEQ ID NO.4 and a reverse primer R7-HULC-RPA whose nucleotide sequence is shown in SEQ ID NO.

5.

8. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 6, characterized in that: The RT-RPA reaction system further includes Buffer A, a template substrate, Buffer B, and DEPC water; the RT-RPA reaction temperature is 38-42° C., and the reaction time is 15-20 min.

9. The CRISPR-Dx-based liver cancer lncRNA HULC detection kit according to claim 6, characterized in that: The RT-RPA reaction system further includes 10-29.5 μL of Buffer A, 1-2.5 μL of a 10 μM forward primer, 1-2.5 μL of a 10 μM reverse primer, 1-10 μL of a template substrate, 0.5-2 μL of Buffer B, and 0-23 μL of DEPC water.

10. Use of the liver cancer lncRNA HULC detection kit based on CRISPR-Dx technology according to any one of claims 1-5, 7-9 in detecting liver cancer lncRNA HULC.

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