CN111893166A - Reagent composition, kit and detection system for CCDC6-RET fusion gene detection - Google Patents
Reagent composition, kit and detection system for CCDC6-RET fusion gene detection Download PDFInfo
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Abstract
The present disclosure relates to a reagent composition for detecting a CCDC6-RET fusion gene, which comprises a crRNA, a Cas12a enzyme and a single-stranded DNA fluorescent probe, wherein the crRNA comprises a nucleotide sequence shown as SEQ ID NO. 1. By utilizing the reagent composition provided by the disclosure, the CCDC6-RET fusion gene in a sample to be detected can be detected with high sensitivity, the detection time is short, the required equipment is simple, the reaction condition is mild, and the quantitative detection and the visual detection of the CCDC6-RET fusion gene can be realized at normal temperature.
Description
Technical Field
The disclosure relates to the technical field of biology, in particular to a reagent composition, a kit and a system for detecting a CCDC6-RET fusion gene.
Background
The RET gene is located on the long arm of human chromosome 10, has a total length of about 60kb, and contains 21 protooncogene exons. The RET gene can code tyrosine kinase receptor RET protein, and the RET protein can be combined with ligand and stimulate intracellular phosphorylation to participate in regulating the growth and differentiation of body cells. The RET gene is easy to break at the coding region of the tyrosine kinase domain, and the 3 'end after the breakage is fused with the 5' end of the partner gene to form fusion mutation, which can cause the transformation of protooncogenes and the activation of kinases to induce malignant tumors. Fusion mutations of the RET gene occur in about 2% of solid tumors such as lung adenocarcinoma and 20% of thyroid papillary carcinoma. Fusion mutations of the RET gene the types of fusion in lung adenocarcinoma are mainly KIF5B-RET and CCDC 6-RET. The targeted drug aiming at the fusion mutation of the RET gene has good treatment effect on tumors. The detection of the fusion mutated RET fusion gene has great clinical value for clinical treatment and reasonable medication guidance.
At present, the detection method of the CCDC6-RET fusion gene mainly comprises FISH, IHC, RT-PCR and PCR/Sanger sequencing.
However, the detection method has low detection sensitivity to the CCDC6-RET fusion gene, the detection time is long, and the requirements of part of the methods on detection conditions are high.
Disclosure of Invention
In order to solve the problems of low sensitivity, long required time and high requirement on detection conditions in the conventional CCDC6-RET fusion gene detection method, the present disclosure provides a reagent composition, a kit and a system.
In order to achieve the above objects, in a first aspect, the present disclosure provides a reagent composition for CCDC6-RET fusion gene detection, the reagent composition comprising a crRNA, a Cas12a enzyme, and a single-stranded DNA fluorescent probe, wherein the crRNA comprises a nucleotide sequence shown as seq id No. 1.
Optionally, the reagent composition further comprises a primer pair, wherein the primer pair comprises nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.
Optionally, the CCDC6-RET fusion gene is the gene with CCDC6exon1-RETexon12 fusion.
Optionally, the single-stranded DNA fluorescent probe comprises a nucleotide sequence shown as seq id No.4 and a fluorophore, and the fluorophore comprises at least one of a FAM fluorophore, a HEX fluorophore, and a TAMRA fluorophore.
In a second aspect, the present disclosure provides a kit for detecting a CCDC6-RET fusion gene, which contains the reagent composition according to any one of the first aspect.
Optionally, the kit further comprises a reaction systemBuffer, RNase inhibitor, Mg2+And water.
Optionally, the kit contains the following components in parts by weight:
the 20 mu L reagent contains 0.3-0.4 mu M of primer pair, 300-500 nM crRNA, 14-15 mu L of Rehydrationbuffer, 150-200 nM single-stranded DNA fluorescent probe, 5-10U of RNase inhibitor, 2-3 mu L of NEBuffer2.1, 0.3-0.6 mu L of magnesium acetate, and 200-300 nM Cas12a enzyme.
In a third aspect, the present disclosure provides a use of the reagent composition of any one of the first aspect in the preparation of a kit for the detection of the CCDC6-RET fusion gene.
In a fourth aspect, the present disclosure provides a system for CCDC6-RET fusion gene detection, the system comprising a first reaction tube, a second reaction tube, a detector, and a computing device; wherein the content of the first and second substances,
an RPA reagent is arranged in the first reaction tube and is used for carrying out RPA amplification on cDNA of a sample to be detected to obtain an amplification product, wherein the RPA reagent contains an RPA primer, and the RPA primer comprises nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3;
the second reaction tube is internally provided with a Cas12a enzyme, a single-stranded DNA fluorescent probe and a crRNA, and is used for carrying out Cas12a enzyme digestion reaction to obtain an enzyme digestion product when the amplification product exists, wherein the crRNA comprises a nucleotide sequence shown as SEQIDNO.1, and the enzyme digestion reaction time is 5-15 minutes;
the detector is provided with an ultraviolet light-emitting device and a fluorescence detection device, the ultraviolet light-emitting device is used for carrying out ultraviolet light irradiation on the enzyme digestion product, and the fluorescence detection device is used for detecting the fluorescence value of the enzyme digestion product under the ultraviolet light irradiation;
the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
and when the fluorescence value of the enzyme digestion product under the ultraviolet irradiation is 500-3000 detected by the fluorescence detection device, judging that the CCDC6-RET fusion gene exists in the sample to be detected.
Optionally, the CCDC6-RET fusion gene is a CCDC6exon1-RETexon12 fusion gene.
By the technical scheme, the reagent composition provided by the disclosure can be used for carrying out high-sensitivity detection on the CCDC6-RET fusion gene in a sample to be detected, the detection time is short, required equipment is simple, the reaction condition is mild, and quantitative detection and visual detection on the CCDC6-RET fusion gene can be realized at normal temperature.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The first aspect of the present disclosure provides a reagent composition for CCDC6-RET fusion gene detection, which comprises a crRNA, a Cas12a enzyme and a single-stranded DNA fluorescent probe, wherein the crRNA comprises a nucleotide sequence shown as seq id No. 1.
By using the reagent composition, when the crRNA is specifically bound with the CCDC6-RET fusion gene, the detection activity of the Cas12a enzyme is activated, the Cas12a enzyme in the activated state can cut the single-stranded DNA fluorescent probe and release a fluorescent group in the fluorescent probe, and therefore, whether the CCDC6-RET fusion gene exists in the detection system can be judged according to whether the fluorescent group is detected. The reagent composition can be used for carrying out high-sensitivity detection on the CCDC6-RET fusion gene in a sample to be detected, has short detection time, simple required equipment and mild reaction conditions, and can realize quantitative detection and visual detection on the CCDC6-RET fusion gene at normal temperature.
According to the present disclosure, the reagent composition may further include a primer pair for RPA amplification of the CCDC6-RET fusion gene region, which may include nucleotide sequences shown in seq id No.2 and seq id No. 3.
By using the primer pair, the CCDC6-RET fusion gene region in a sample to be detected can be amplified, the abundance of the CCDC6-RET fusion gene in a detection system is improved, and crRNA can be more easily matched with the CCDC6-RET fusion gene, so that the detection sensitivity is improved, and high-sensitivity detection in a complex sample/heterozygote mutation sample is realized.
Optionally, the CCDC6-RET fusion gene is a CCDC6exon1-RETexon12 fusion gene. The CCDC6-RET fusion gene is typically present in tumor cells, such as lung cancer cells, thyroid cancer cells, leukemia cells, breast cancer cells, colorectal cancer cells, esophageal cancer cells, and melanoma cells.
In accordance with the present disclosure, the single-stranded DNA fluorescent probe may be selected from a wide range, and single-stranded DNA fluorescent probes that do not hybridize or react with other components of the detection system may be used in the present disclosure. Illustratively, the single-stranded DNA fluorescent probe may include a nucleotide sequence shown as seq id No.4 and a fluorophore including at least one of a FAM fluorophore, a HEX fluorophore, and a TAMRA fluorophore.
In a second aspect of the present disclosure, there is provided a kit for detecting a CCDC6-RET fusion gene, which contains the reagent composition according to any one of the first aspect.
Optionally, the kit further comprises a reaction system buffer solution, an RNase inhibitor and Mg2+And water.
Optionally, the kit contains the following components in parts by weight: the 20 mu L reagent contains 0.3-0.4 mu M of primer pair, 300-500 nM crRNA, 14-15 mu L of Rehydrationbuffer, 150-200 nM single-stranded DNA fluorescent probe, 5-10U of RNase inhibitor, 2-3 mu L of NEBuffer2.1, 0.3-0.6 mu L of magnesium acetate, and 200-300 nM Cas12a enzyme.
Optionally, the kit may further comprise a blank control, a negative control and a positive control.
The third aspect of the present disclosure provides a use of the reagent composition of any one of the first aspect in preparing a kit for detecting a CCDC6-RET fusion gene.
A fourth aspect of the present disclosure provides a system for CCDC6-RET fusion gene detection, the system comprising a first reaction tube, a second reaction tube, a detector, and a computing device; the first reaction tube is internally provided with an RPA reagent for carrying out RPA amplification on cDNA of a sample to be detected to obtain an amplification product, wherein the RPA reagent contains an RPA primer, and the RPA primer comprises nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3; the second reaction tube is internally provided with a Cas12a enzyme, a single-stranded DNA fluorescent probe and a crRNA, and is used for carrying out Cas12a enzyme digestion reaction to obtain an enzyme digestion product when the amplification product exists, wherein the crRNA comprises a nucleotide sequence shown as SEQIDNO.1, and the enzyme digestion reaction time is 5-15 minutes; the detector is provided with an ultraviolet light-emitting device and a fluorescence detection device, the ultraviolet light-emitting device is used for carrying out ultraviolet light irradiation on the enzyme digestion product, and the fluorescence detection device is used for detecting the fluorescence value of the enzyme digestion product under the ultraviolet light irradiation; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows: and when the fluorescence value of the enzyme digestion product under the ultraviolet irradiation is 500-3000 detected by the fluorescence detection device, judging that the CCDC6-RET fusion gene exists in the sample to be detected.
Optionally, the CCDC6-RET fusion gene is a CCDC6exon1-RETexon12 fusion gene.
The detector can be an ultraviolet gel imager, and the wavelength of ultraviolet light can be 420-460 nm. By using the system, the CCDC6-RET fusion gene in a sample to be detected can be sensitively, quickly and simply detected.
Optionally, the RPA reagent comprises the following components in parts by weight: the 20. mu.L reagent contains 0.3-0.4. mu.M of primer pair, 14-15. mu.L of Rehydrationbuffer, 5-10U of RNase inhibitor, 2-3. mu.L of NEBuffer2.1 and 0.3-0.6. mu.L of magnesium acetate.
A fifth aspect of the present disclosure provides a method for CCDC6-RET fusion gene detection, the method comprising: a. extracting RNA of a sample to be detected, and carrying out reverse transcription to obtain cDNA of the sample to be detected; b. mixing the cDNA of the sample to be detected with an RPA reagent, and carrying out RPA amplification to obtain an amplification product; wherein, the RPA reagent contains an RPA primer, and the RPA primer comprises nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3; c. mixing the amplification product with Cas12a enzyme, a single-stranded DNA fluorescent probe and crRNA, and reacting for 15-20 min to obtain a shearing product; wherein, the crRNA comprises a nucleotide sequence shown as SEQ ID NO.1, the single-stranded DNA fluorescent probe can comprise a nucleotide sequence shown as SEQ ID NO.3 and a fluorescent group, and the fluorescent group can comprise at least one of FAM fluorescent group, HEX fluorescent group and TAMRA fluorescent group; d. and placing the shearing product under ultraviolet light, detecting the fluorescence value of the shearing product, and judging that the CCDC6-RET fusion gene exists in the sample to be detected when the fluorescence value is 500-3000.
Wherein, the sample to be tested can be human whole blood, cell or tissue sample.
Preferably, the method of the present disclosure is not used for diagnosis, or there is no one-to-one correlation between the qualitative and quantitative results of the CCDC6-RET fusion gene and whether the disease occurs, which is not the diagnosis result, but the qualitative and quantitative detection results of the CCDC6-RET fusion gene can be used as intermediate information for the reference of the clinician.
The method disclosed by the invention can realize rapid and sensitive detection on tumors with the CCDC6-RET fusion gene within 40 minutes.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
Example 1 detection method and determination of detection result
1. Synthesis of crRNA and primers
Sequence synthesis was performed according to the crRNA sequence, primer sequence and single-stranded DNA fluorescent probe sequence shown in Table 1.
TABLE 1
| Sequence name | Sequence of | SEQIDNO |
| CCDC6-RET-crRNA | tttgtaggtctccagctctatctt | 1 |
| CCDC6-RET-F | gtgctgaagatagagctggagacctacaaactg | 2 |
| CCDC6-RET-R | gacctgcttcaggacgttgaactctgacagcag | 3 |
| Single-stranded DNA fluorescent probe | FAM-ttatt-BHQ1 | 4 |
2. Sample processing
Taking a sample to be detected, extracting RNA of the sample to be detected from the sample to be detected according to a using method of an easy PurerRNAKit kit, wherein the extracted RNA needs to meet the condition that the ratio of OD260/OD280 is 1.7-2.0, and the concentration is more than 50 ng/muL. Then carrying out reverse transcription on the sample RNA to be detected which meets the requirement by using TranscriptionFirstStrandcDNAsynthesothers (Roche) to obtain sample cDNA to be detected, wherein the ratio of OD260/OD280 of the cDNA obtained by the reverse transcription is 1.8-2.0, and the concentration is more than 30 ng/muL. And taking the cDNA of the sample to be detected which meets the requirements as a template.
3. Construction of a detection System
(1) The RPA reaction system was prepared as in table 2.
TABLE 2
(2) 5 μ of LPrimer FreeRehydroregenerative buffer was added to the upper layer of an adsorption column containing Cas12a enzyme to form a Cas12a enzyme reagent with a final concentration of 250 nM.
(3) After configuring the RPA reaction system according to Table 2, carrying out the RPA amplification reaction for 15min to obtain the amplification product. And then centrifuging at 12000rpm for 5min to uniformly mix the Cas12a enzyme reagent in the adsorption column with the amplification product, and reacting for 5min to obtain a shearing product. And performing fluorescence detection on the shearing product by using a fluorescence microplate reader, and judging that the CCDC6-RET fusion gene exists in the sample to be detected when the detected fluorescence value is 500-3000.
Example 2 minimum detection Limit validation
Test samples for evaluation: the cDNA verified as CCDC6-RET fusion gene by next generation sequencing was diluted to 10 concentration by gradient4，103，102，101，100Template for evaluation of aM.
The evaluation templates were examined for each concentration according to the examination method of example 1, 3 times for each concentration gradient, and fluorescence was measured using a fluorescence microplate reader, and the fluorescence values (data presentation form mean ± standard deviation, i.e., "M ± SD") obtained by the examination are shown in table 3.
TABLE 3
As can be seen from Table 3, when the concentration of the template for evaluation was 101at aM, the fluorescence values detected by the method of example 1 were significantly increased. Illustrating that the method of example 1 has a minimum detection limit of 10 for CCDC6-RET fusion gene1aM. Therefore, the reagent composition provided by the disclosure has high sensitivity for detecting the CCDC6-RET fusion gene.
As can be seen from the example, the sensitivity of the method disclosed by the invention for detecting the CCDC6-RET fusion gene is high.
Example 3 accuracy verification
Samples 5 which were verified to be wild-type RET genes by the second-generation sequencing and 10 which were verified to be CCDC6-RET fusion genes by the second-generation sequencing were selected as samples for evaluation.
The samples for evaluation were subjected to blind tests according to the test method of example 1, and the corresponding number of test cases was counted, and the test was repeated 3 times for each sample, and the test results are shown in table 4.
TABLE 4
As can be seen from Table 4, the accuracy of detection of the CCDC6-RET fusion gene by the method of example 1 is 100% when the reagent composition is selected.
As can be seen from the example, the method disclosed by the invention has high accuracy in detecting the CCDC6-RET fusion gene.
Comparative example
Sequence synthesis was performed according to the crRNA sequences and primer sequences shown in Table 5.
TABLE 5
| Sequence code | Sequence of | SEQIDNO |
| CCDC6-RET-crRNA-D | tttgcgcaggtcgcggttctcctc | 5 |
| CCDC6-RET-F-D | gggggcattgtcatctcgccgttccgcctggag | 6 |
| CCDC6-RET-R-D | cacggccaccgtggtgtaccctgctctgccttt | 7 |
| Single-stranded DNA fluorescent probe-D | FAM-ttatt-BHQ1 | 4 |
1. Minimum detection limit verification
The lowest detection limit was performed according to the method of example 2, 3 times for each concentration gradient, and fluorescence was detected using a fluorescence microplate reader, and the fluorescence values obtained by the detection (data presentation form mean ± standard deviation, i.e., "M ± SD") are shown in table 6.
TABLE 6
As can be seen from Table 6, the reagent composition and method of the present comparative example had a minimum detection limit of 10 for the CCDC6-RET fusion gene2aM. The reagent composition and the method disclosed by the invention have higher detection sensitivity and stronger detection capability on the CCDC6-RET fusion gene.
2. Accuracy verification
According to the method of the embodiment 3, the detection accuracy is verified, the corresponding detection examples are counted, each sample is repeatedly detected for 3 times, and the detection results are shown in table 7.
TABLE 7
As can be seen from Table 7, the reagent composition and method of the present comparative example showed a positive detection rate of 20% for the CCDC6-RET fusion gene. It is shown that the reagent composition and method of the present disclosure are more accurate in detecting the CCDC6-RET fusion gene.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
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Claims (10)
1. The reagent composition for detecting the CCDC6-RET fusion gene is characterized by comprising crRNA, a Cas12a enzyme and a single-stranded DNA fluorescent probe, wherein the crRNA comprises a nucleotide sequence shown as SEQ ID NO. 1.
2. The reagent composition of claim 1, further comprising a primer pair comprising the nucleotide sequences shown as SEQ ID No.2 and SEQ ID No. 3.
3. The reagent composition of claim 1, wherein the CCDC6-RET fusion gene is a CCDC6exon1-RETexon12 fusion gene.
4. The reagent composition of claim 1, wherein the single-stranded DNA fluorescent probe comprises the nucleotide sequence shown as seq id No.4 and a fluorophore comprising at least one of FAM fluorophore, HEX fluorophore and TAMRA fluorophore.
5. A kit for detecting a CCDC6-RET fusion gene, which comprises the reagent composition of any one of claims 1 to 4.
6. The kit according to claim 5, wherein the kit further comprises a reaction system buffer, an RNase inhibitor and Mg2+And water.
7. The kit according to claim 6, wherein the kit comprises the following components in parts by weight:
the 20 mu L reagent contains 0.3-0.4 mu M of primer pair, 300-500 nM crRNA, 14-15 mu L of Rehydrationbuffer, 150-200 nM single-stranded DNA fluorescent probe, 5-10U of RNase inhibitor, 2-3 mu L of NEBuffer2.1, 0.3-0.6 mu L of magnesium acetate, and 200-300 nM Cas12a enzyme.
8. Use of the reagent composition of any one of claims 1 to 4 in the preparation of a kit for the detection of a CCDC6-RET fusion gene.
9. A system for detecting a CCDC6-RET fusion gene is characterized by comprising a first reaction tube, a second reaction tube, a detector and a calculating device; wherein the content of the first and second substances,
an RPA reagent is arranged in the first reaction tube and is used for carrying out RPA amplification on cDNA of a sample to be detected to obtain an amplification product, wherein the RPA reagent contains an RPA primer, and the RPA primer comprises nucleotide sequences shown in SEQ ID NO.2 and SEQ ID NO. 3;
the second reaction tube is internally provided with a Cas12a enzyme, a single-stranded DNA fluorescent probe and crRNA, and is used for carrying out Cas12a enzyme digestion reaction to obtain an enzyme digestion product when the amplification product exists, wherein the crRNA comprises a nucleotide sequence shown in SEQ ID NO.1, and the enzyme digestion reaction time is 5-15 minutes;
the detector is provided with an ultraviolet light-emitting device and a fluorescence detection device, the ultraviolet light-emitting device is used for carrying out ultraviolet light irradiation on the enzyme digestion product, and the fluorescence detection device is used for detecting the fluorescence value of the enzyme digestion product under the ultraviolet light irradiation;
the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the discrimination as follows:
and when the fluorescence value of the enzyme digestion product under the ultraviolet irradiation is 500-3000 detected by the fluorescence detection device, judging that the CCDC6-RET fusion gene exists in the sample to be detected.
10. The system of claim 9, wherein the CCDC6-RET fusion gene is a CCDC6exon1-RETexon12 fusion gene.
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