CN111455046A - Nucleic acid detection method for mutation of ADGRG6 enhancer and kit thereof - Google Patents
Nucleic acid detection method for mutation of ADGRG6 enhancer and kit thereof Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The invention discloses a nucleic acid detection method for mutation of an ADGRG6 enhancer and a kit thereof, wherein the method comprises the following steps: providing a DNA to be detected; amplifying the DNA to be detected by using a primer pair P to obtain an amplification product; mixing the Cas12a protein, a buffer solution, crRNA and a FAM-TTTTTT-BHQ probe to form a premixed solution; the crRNA is 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3'; and adding the amplification product into the premix solution, and performing fluorescence quantitative measurement. The nucleic acid detection method for the ADGRG6 enhancer mutation, which is designed aiming at the ADGRG6 enhancer mutation and simultaneously uses the Cas12a as a premix for preparing the ADGRG6 enhancer mutation, has high specificity and sensitivity on single base mutation.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a nucleic acid detection method for mutation of an ADGRG6 enhancer and a kit thereof.
Background
Bladder cancer is a common malignant tumor of the urinary system, the high-incidence age group is 50-70 years old, and the incidence rate of bladder cancer in men is 3-4 times that in women. The pathological types of bladder cancer in the histological classification of urinary tract system tumor in urinary tract system tumor pathology and genetics published by world health organization in 2004 are divided into urothelial carcinoma, bladder squamous cell carcinoma, bladder adenocarcinoma and the like. In fact, over 90% of patients with bladder cancer are urothelial carcinoma of the bladder, and therefore, the term bladder cancer is referred to as urothelial carcinoma of the bladder.
Bladder cancer has molecular heterogeneity, with genetic material containing multiple single nucleotide mutations, chromosomal translocations, and even genomic abnormalities. Many protooncogenes become oncogenes due to mutation, and the production of oncoprotein after transcription and translation promotes canceration of cells. Common mutant genes in bladder cancer include RAS, CCND, ERBB2, FGFR3 and the like. Therefore, the detection of the known mutation by the nucleic acid has important significance for judging the molecular type of the tumor, and assisting diagnosis and treatment.
The traditional nucleic acid detection method is mainly based on a fluorescent quantitative PCR method, and can efficiently and accurately identify whether the host contains exogenous DNA such as bacteria, viruses, fungi and the like; however, for the detection of single base mutation, the method still cannot maintain excellent specificity in practical application; and often rely on further judgment as to whether there is a mutation.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a method for detecting nucleic acid of ADGRG6 enhancer mutation and a kit thereof, which aims to solve the problem of low specificity of the existing bladder cancer mutation nucleic acid detection method to single base mutation.
The technical scheme of the invention is as follows:
a method for detecting nucleic acid with ADGRG6 enhancer mutation, which comprises the following steps:
providing a DNA to be detected;
amplifying the DNA to be detected by using a primer pair P to obtain an amplification product;
mixing the Cas12a protein, a buffer solution, crRNA and a FAM-TTTTTT-BHQ probe to form a premixed solution; the crRNA is 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3';
and adding the amplification product into the premix solution, and performing fluorescence quantitative measurement.
The kit for detecting the nucleic acid with the mutation of the ADGRG6 enhancer is characterized in that the detection marker of the kit is the mutation of the ADGRG6 enhancer, the kit comprises a primer pair P which is designed for the mutation of the ADGRG6 enhancer and is used for amplifying DNA to be detected, and a premixed solution which consists of Cas12a, crRNA-mismatched protein, a buffer solution, crRNA and a FAM-TTTTTT-BHQ probe; the crRNA was 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3'.
Has the advantages that: the invention designs crRNA aiming at the mutation of an ADGRG6 enhancer, and uses the crRNA and Cas12a to prepare a premixed solution for nucleic acid detection at the same time, thereby obtaining the nucleic acid detection method based on the mutation of the ADGRG6 enhancer of Cas12 a; the matching of the crRNA in the premix and the target DNA containing the PAM sequence can activate the activity of the Cas12a for non-specifically cutting the DNA, the activity can shear the FAM-TTTTTT-BHQ fluorescent probe, the FAM fluorescent group loses the limit of a BHQ quenching group, a fluorescent signal can be generated, the generation of the signal can indicate whether the target DNA matched with the crRNA is contained in the object to be detected, and the method can specifically distinguish single base mutation; in addition, the nucleic acid detection method has the characteristics of rapidness, accuracy and simplicity.
Drawings
FIG. 1 is a flow chart of a method for detecting a nucleic acid having an ADGRG6 enhancer mutation according to an embodiment of the present invention.
FIG. 2 is a schematic diagram of the detection principle of the nucleic acid detection method for the ADGRG6 enhancer mutation in the embodiment of the invention.
FIG. 3 is a graph showing the response of the nucleic acid detection method for the ADGRG6 enhancer mutation to the magnitude of the sample DNA concentration in example 1 of the present invention.
FIG. 4 is a diagram showing the evaluation of the specificity of the nucleic acid detecting method for the ADGRG6 enhancer mutation in example 1 of the present invention.
Detailed Description
The present invention provides a nucleic acid detection method for ADGRG6 enhancer mutation and a kit thereof, and the present invention is further described in detail below in order to make the object, technical scheme and effect of the present invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1, the embodiment of the present invention provides a method for detecting nucleic acid of ADGRG6 enhancer mutation, comprising the steps of:
s100, providing DNA to be detected;
s200, amplifying the DNA to be detected by using a primer pair P to obtain an amplification product;
s300, mixing the Cas12a protein, a buffer solution, crRNA and a FAM-TTTTTT-BHQ probe to form a premixed solution; the crRNA is 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3';
s400, adding the amplification product into the premix solution, and performing fluorescence quantitative measurement.
In this example, for purposes other than diagnosis and diagnosis, the nucleic acid detection method for the ADGRG6 enhancer mutation based on Cas12a, which designs crRNA for the ADGRG6 enhancer mutation and uses it together with Cas12a as a preparation premix, has specificity and high sensitivity to single base mutation; has the characteristics of rapidness, accuracy and simplicity.
Referring to fig. 2, this example specifically designs crRNA by targeting ADGRG6 enhancer mutation and uses it simultaneously with Cas12a to prepare a premix for nucleic acid detection; matching of the crRNA in the premix with target DNA (namely amplification product) containing a PAM sequence (TTTN) can activate the activity of the Cas12a for nonspecific DNA cleavage, the activity can shear the FAM-TTTTTT-BHQ fluorescent probe, the FAM fluorescent group loses the limit of a BHQ quenching group, a fluorescent signal can be generated, and the generation of the signal can indicate whether the target DNA matched with the crRNA is contained in the object to be detected; otherwise, if the object to be detected does not contain the target DNA capable of matching with the crRNA, the object to be detected is mismatched, and the mismatched intermediate cannot activate the activity of Cas12a for non-specific cleavage of DNA, so that a fluorescent signal is not generated.
In one embodiment, the ADGRG6 enhancer mutation is a G > a at position chr.6:142,706,206 of ADGRG 6. Further in one embodiment, the ADGRG6 enhancer mutation may be, but is not limited to, a bladder cancer ADGRG6 enhancer mutation.
In one embodiment, in step S200, the primer pair P is F:5' -TTC CAG CAA CCC CCA AGAAA, R:5'-CAG CTC CAC TCC ACC AAG TC-3'.
In one embodiment, the mode of amplification is PCR amplification or isothermal amplification. Further in one embodiment, the mode of amplification is PCR amplification; the PCR amplification comprises the following steps:
s201, placing the DNA to be detected in a PCR amplification system, and carrying out amplification for 5min at 95 ℃;
s202, sequentially carrying out treatment at 95 ℃ for 15S; at 58 ℃ for 15 s; amplifying at 72 ℃ for 15s, and repeating for 35-45 times;
s203, finally carrying out amplification at 72 ℃ for 2 min.
Specifically, step S201 may be performed to pre-denature the ADGRG6 enhancer mutation; step S203 is performed to sufficiently amplify the DNA to be tested.
Further in one embodiment, the PCR amplification system (primestar (TAKARA) amplification system) is:
in one embodiment, in step S300, the crRNA is prepared by chemical synthesis. Furthermore, the crRNA is designed and obtained aiming at the mutation site chr.6:142,706,206 of the mutation site of ADGRG6 enhancer, namely G > A, and is prepared by a chemical synthesis method. Compared with the method for preparing the crRNA (the concentration is generally less than 100nM and is low, and impurities which are difficult to remove, such as template DNA, are easy to introduce) by adopting in vitro transcription and purification, the crRNA prepared by the chemical synthesis method has high purity and extremely high concentration and is powdery, so that the premixed solution for nucleic acid detection with high content of the crRNA can be obtained, and the possibility of improving the detection sensitivity of the nucleic acid is provided. In one embodiment, in step S400, the fluorescence quantitative measurement is performed by a fluorescence quantitative PCR method. Specifically, the kit is placed into a fluorescent quantitative PCR instrument, a certain temperature (such as 25 ℃ and 37 ℃) is set for detection, and FAM fluorescent signals are collected every 1-10 min (such as 1min, 5min and 10min) of detection. The detection time and the collection times (such as 24 times) are set according to the needs.
The invention also provides a kit for detecting nucleic acid with mutation of the ADGRG6 enhancer, wherein the detection marker of the kit is mutation of the ADGRG6 enhancer, the kit comprises a primer pair P which is designed aiming at the mutation of the ADGRG6 enhancer and is used for amplifying DNA to be detected, and a premixed solution which is composed of Cas12a protein, buffer solution, crRNA and FAM-TTTTTT-BHQ probe; the crRNA was 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3'.
In the embodiment, the kit containing the Cas12a protein and the crRNA has high and rapid sensitivity on the detection of the nucleic acid with the mutation of the ADGRG6 enhancer; the kit can specifically distinguish single base mutation and is convenient to carry.
In one embodiment, the ADGRG6 enhancer mutation is a G > a at position chr.6:142,706,206 of ADGRG 6; and/or
The primer pair P is F:5'-TTC CAG CAA CCC CCA AGA AA-3' and R is 5'-CAG CTC CAC TCCACC AAG TC-3'.
In one embodiment, the Buffer is Buffer, 10X. Specifically, Buffer, 10X comprises 1M NaCl, 500mM Tris-HCl, 100mM MgCl2，10mM DTT，pH 7.9@25℃。
In one embodiment, the concentration of the crRNA is greater than 150 nM.
The present invention will be described in detail below with reference to specific examples.
Example 1
(1) Extracting sample DNA by using a TIANAmp FFPE DNA kit (Tiangen Biotech) to be used as DNA to be detected;
(2) PCR amplification of sample DNA with primer pair P (F:5'-TTC CAG CAA CCC CCA AGA AA-3', R:5'-CAG CTC CAC TCCACC AAG TC-3');
the amplification enzyme was a primestar amplification system (from TAKARA) as follows:
the PCR amplification step is as follows:
(2.1) placing the sample DNA in a PCR amplification system, and carrying out amplification for 5min at 95 ℃ to pre-denature the ADGRG6 enhancer mutation;
(2.2) amplifying at 95 ℃ for 15s, at 58 ℃ for 15s and at 72 ℃ for 15s in sequence, and circulating for 35 times;
(2.3) carrying out extension amplification for 2min at 72 ℃ to obtain an amplification product.
(3) Preparation of Cas12a reaction system (i.e., premix):
(4) adding the amplification product into the premixed solution, putting the premixed solution into a fluorescent quantitative PCR instrument, setting the constant temperature of 37 ℃, carrying out nucleic acid detection, and collecting an FAM signal once in 1 min.
(5) According to the above-mentioned detection procedures, sample DNAs (DNAs containing the ADGRG6 enhancer mutation) were detected at different concentrations (1nM, 2nM, 5nM, 10nM, 20nM, 50nM, and 100nM), and the change in FAM signal with time of detection of the sample DNAs at different concentrations is shown in FIG. 3, which shows that the nucleic acid detection method of this example has a high sensitivity to DNA containing the ADGRG6 mutation with a detection limit of 2 nM.
(6) According to the above-described detection procedure, different types of sample DNAs (mutant type: sample DNA containing mutation of the ADGRG6 enhancer; wild type: sample DNA in which mutation of the ADGRG6 enhancer did not occur) were detected at the same concentration (10nM or 100nM), and the change of FAM signals with the detection time side of different types of sample DNA at the same concentration was shown in FIG. 4, which revealed that the nucleic acid detection method of this example had excellent specificity for single-base mutation.
In conclusion, the invention provides a nucleic acid detection method for mutation of an ADGRG6 enhancer and a kit thereof, and the nucleic acid detection method for mutation of the ADGRG6 enhancer based on Cas12a is obtained by designing crRNA aiming at mutation of the ADGRG6 enhancer and using the crRNA and Cas12a for preparing a premixed solution for nucleic acid detection; the matching of the crRNA in the premix and the target DNA containing the PAM sequence can activate the activity of the Cas12a for non-specifically cutting the DNA, the activity can shear the FAM-TTTTTT-BHQ fluorescent probe, the FAM fluorescent group loses the limit of a BHQ quenching group, a fluorescent signal can be generated, the generation of the signal can indicate whether the target DNA matched with the crRNA is contained in the object to be detected, and the method can specifically detect single base mutation; in addition, the nucleic acid detection method has the characteristics of rapidness, accuracy and simplicity.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (10)
1. A method for detecting a nucleic acid having an ADGRG6 enhancer mutation, comprising the steps of:
providing a DNA to be detected;
amplifying the DNA to be detected by using a primer pair P to obtain an amplification product;
mixing the Cas12a protein, a buffer solution, crRNA and a FAM-TTTTTT-BHQ probe to form a premixed solution; the crRNA is 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3';
and adding the amplification product into the premix solution, and performing fluorescence quantitative measurement.
2. The assay of claim 1 wherein the ADGRG6 enhancer mutation is at position chr.6:142,706,206 of ADGRG6, G > A; and/or
The primer pair P is F:5'-TTC CAG CAA CCC CCA AGA AA-3' and R is 5'-CAG CTC CAC TCC ACCAAG TC-3'.
3. The detection method according to claim 1, wherein the amplification means is PCR amplification or isothermal amplification.
4. The detection method according to claim 3, wherein the PCR amplification comprises the steps of:
placing the DNA to be detected in a PCR amplification system, and carrying out amplification for 5min at 95 ℃;
then sequentially keeping the temperature at 95 ℃ for 15 s; at 58 ℃ for 15 s; amplifying at 72 ℃ for 15s, and repeating for 35-45 times;
finally, amplification was performed at 72 ℃ for 2 min.
5. The method of claim 1, wherein the crRNA is prepared by chemical synthesis.
6. The detection method according to claim 1, wherein the quantitative fluorescence measurement is performed by a quantitative fluorescence PCR method.
7. The kit for detecting the nucleic acid with the mutation of the ADGRG6 enhancer is characterized in that a detection marker of the kit is the mutation of the ADGRG6 enhancer, the kit comprises a primer pair P which is designed aiming at the mutation of the ADGRG6 enhancer and is used for amplifying DNA to be detected, and a premixed solution which consists of Cas12a protein, a buffer solution, crRNA and a FAM-TTTTTT-BHQ probe; the crRNA was 5'-AAU UUC UAC UGU UGU AGA UUU GUA UGU UUA UAC AAA-3'.
8. The kit for detecting the nucleic acid of the ADGRG6 enhancer mutation according to claim 7, wherein the ADGRG6 enhancer mutation is G > A at position chr.6:142,706,206 of ADGRG 6; and/or
The primer pair P is F:5'-TTC CAG CAA CCC CCA AGA AA-3' and R is 5'-CAG CTC CAC TCC ACCAAG TC-3'.
9. The kit for detecting nucleic acid encoding an ADGRG6 enhancer mutation according to claim 7, wherein the Buffer is Buffer, 10X.
10. The kit for detecting the nucleic acid of the ADGRG6 enhancer mutation according to claim 9, wherein the concentration of the crRNA is greater than 150 nM.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112322739A (en) * | 2020-11-24 | 2021-02-05 | 深圳市罗湖区人民医院 | Method and kit for detecting ADGRG6 high-frequency mutation site |
| CN112980933A (en) * | 2020-12-30 | 2021-06-18 | 南方科技大学 | SNP (Single nucleotide polymorphism) typing detection method based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system and application thereof |
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