CN111876486A - BCR-ABL1 fusion gene three subtype typing detection kit - Google Patents
BCR-ABL1 fusion gene three subtype typing detection kit Download PDFInfo
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Abstract
The invention belongs to the field of genes, and particularly relates to a kit for detecting three subtype types of BCR-ABL1 fusion genes. Firstly, a primer probe composition for simultaneously detecting three subtype genes BCR-ABL1 is disclosed, wherein the three subtype genes BCR-ABL1 are P190, P210 and P230 respectively, and the detected nucleotide sequences are shown as SEQ ID NO: 1-9. The invention adopts the specific downstream primer of ABL1 gene to carry out reverse transcription, and the reverse transcription efficiency is higher than that of a random primer and oligo dT; the multiple system simultaneously qualitatively and quantitatively detects three subtypes and internally controls, thereby reducing pollution and waste possibly caused by multiple sample adding; the same downstream primer is adopted in the multiple amplification system, so that the consistency of the amplification efficiency is ensured as much as possible; the internal control probe is placed in the amplicon, and the abnormal condition that the fusion ratio exceeds 100 percent can not occur.
Description
Technical Field
The invention belongs to the field of genes, and particularly relates to a kit for detecting three subtype types of BCR-ABL1 fusion genes.
Background
Acute Lymphocytic Leukemia (ALL) is a common hematological malignancy, has high morbidity and mortality, and seriously harms physical and psychological health of people (especially teenagers). The etiology and pathogenesis of most ALL are not completely understood, and at present, it is widely believed to be the result of the interaction of genetic factors and environmental factors. Among genetic factors, the occurrence of a fusion gene is considered to be one of the most important risk factors. As early as 1960, Nowwell and hunterford discovered an abnormal "minichromosome" in the G-group chromosomes of 7 Chronic Myelogenous Leukemia (CML) cases, and this newly discovered abnormal chromosome was later named Philadelphia chromosome (Philadelphia chromosome). With the use of quinacrine fluorescence and giemsa staining methods, the philadelphia chromosome was found in 1973 to be indeed ectopic between chromosomes 9 and 22 ((t 9; 22) (q 34; q 11)). The human genome project started in the 80's of the 20 th century discovered, using somatic hybridization techniques, the ABL1(c-ABL, Abelson murine leukameria homolog) protooncogene located on chromosome 9. In the same year, De Klein et al have confirmed that Philadelphia chromosome is a chimeric gene formed by ectopic mapping of the ABL1 gene from chromosome 9 to chromosome 22, and the resulting fusion protein has tyrosine kinase activity. In the case of CML positive to Philadelphia chromosome, the site of the break ectopic to chromosome 22 of ABL1 is located in the 5.8-kb region, Groffen et al, which is designated BCR (Breakpoint Cluster region).
Although the BCR-ABL1 fusion gene has been discovered for over 30 years, other ABL1 related fusion genes related to ALL have not been discovered in succession until recently. The relationship of ABL 1-related fusion genes to ALL will be reviewed herein. Breakpoints of the BCR gene can occur in multiple regions, including mainly: (1) between exons 13 and 14 (e13a2, pro b2a 2); (2) between exons 14 and 15 (e14a2, pro b3a2), this region is also called the major breakpoint cluster region (M-BCR), and the BCR-ABL1 fusion gene formed at this position encodes the P210 protein; (3) between exons 1 and 2 (e1a2), this region is also called the minor breakpoint cluster region (m-BCR), and the BCR-ABL1 fusion gene formed at this position encodes the P190 protein. Of the philadelphia chromosome positive leukemia patients, approximately 50% of adult ALL and 80% of pediatric ALL patients were positive for P190 protein.
In addition, it has been reported in the literature that BCR breakpoints occur in other locations in a small number of cases. For example, a fusion gene (e19a2) formed by a BCR breakpoint in a micro breakpoint cluster region (mu-BCR) and an ABL1 breakpoint in exon 2 encodes P230 protein. The BCR-ABL1 fusion gene from e6a2 was found in individual T-ALL patients.
The coiled-coil domain (coiled-coil domain) and tyrosine residues of the BCR protein are contained in the BCR-ABL1 fusion protein. The BCR protein part can enhance the dimerization of BCR-ABL1 fusion protein, thereby promoting the tyrosine phosphorylation on the kinase activation loop (kinase activation loop) of the two tumor proteins. And the over-strong kinase activity of the protein can promote the cell over proliferation and inhibit the apoptosis.
The existing technology for detecting BCR-ABL1 fusion gene is usually used for detecting by a reverse transcription-PCR method, but the existing technology has the following defects: 1) reverse transcription efficiency of the random primer is not high; 2) amplification efficiencies of external internal control and detection subtypes are often inconsistent, resulting in detection ratios above 100%.
Disclosure of Invention
The invention discloses a subtype detection kit for detecting three subtypes of BCR-ABL1 fusion genes, which solves the problems of low reverse transcription efficiency, inconsistent amplification efficiency of external internal control and subtype detection and the like in the prior art.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a primer probe composition for simultaneously detecting three subtype genes BCR-ABL1, wherein the three subtype genes BCR-ABL1 are P190, P210 and P230 respectively, and the primer probe composition comprises:
the sequences of the primer and the probe of the P190 are shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;
the sequences of the primer and the probe of the P210 are shown in SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;
the sequences of the primer and the probe of the P230 are shown in SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;
the sequence of the forward primer of the ABL1 gene is shown as SEQ ID NO: 7, the reverse primer sequence is shown as SEQ ID NO: 8, and the probe sequence is shown as SEQ ID NO: shown at 9.
In a second aspect, the invention discloses a kit, which comprises the primer probe composition.
Preferably, the kit further comprises 10 × PCR buffer, dntps, and Taq DNA polymerase.
It should be understood that the kit components disclosed in the present invention are not limited to the above components, and those skilled in the art can add or replace other components as needed and all fall within the scope of the present invention.
Preferably, the concentration of the upstream primer in the kit is 1000nM, the concentration of the probe is 250nM, and the concentration of the downstream primer is 4000 nM.
The third aspect of the invention discloses a method for simultaneously detecting three subtype genes of BCR-ABL1, which comprises the following steps: the above-described kit was used for detection.
Preferably, the test sample is derived from oral exfoliated cells and/or blood-extracted RNA.
In some embodiments of the invention, the test sample is from blood RNA.
Preferably, the method comprises the following steps: the specific primer is adopted for reverse transcription and then the amplification is carried out by the digital PCR technology.
The digital PCR technology is a variety of digital PCR technologies.
The term "multiplex digital PCR" refers to a digital PCR reaction in which two or more pairs of primers are added to the same digital PCR reaction system to simultaneously amplify multiple nucleic acid fragments. Preferably, the reverse transcription system comprises 5 × RT buffer, reverse transcriptase, reverse primer of ABL1 gene and RNA.
Preferably, the digital PCR amplification system comprises water, 10 XPCR buffer, dNTP and Taq DNA polymerase.
Preferably, the amplification procedure comprises: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 deg.C for 15 seconds, annealing at 60 deg.C for 1min, and circulation 40 times.
It should be understood that the amplification procedure is not limited to the above procedures, and one skilled in the art can select any suitable amplification procedure to complete the present invention as required and all within the scope of the present invention.
The fourth aspect of the invention discloses the application of the primer probe composition, the kit and the method in the field of gene detection. Preferably, the gene detection field is the BCR-ABL1 fusion gene detection field.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the invention has the following remarkable advantages and effects:
(1) reverse transcription is carried out by using a specific downstream primer of an ABL1 gene, and the reverse transcription efficiency is higher than that of a random primer and oligo dT;
(2) the multiple system simultaneously qualitatively and quantitatively detects three subtypes and internally controls, thereby reducing pollution and waste possibly caused by multiple sample adding;
(3) the same downstream primer is adopted in the multiple amplification system, so that the consistency of the amplification efficiency is ensured as much as possible;
(4) the internal control probe is placed in the amplicon, and the abnormal condition that the fusion ratio exceeds 100 percent can not occur.
Drawings
FIG. 1 is a design of an experiment in an embodiment of the present invention;
FIG. 2 is a dot diagram showing the results of detection in the NTC group (negative control group without template) according to the example of the present invention (the left panel is a ROX-VIC diagram, and the right panel is a FAM-CY5 diagram);
FIG. 3 is a dot diagram showing the results of tests conducted in the NC group (normal control group) (the left is ROX-VIC diagram, and the right is FAM-CY5 diagram) according to the example of the present invention;
FIG. 4 is a dot diagram showing the results of P190+ set tests in the example of the present invention (the left is ROX-VIC diagram, and the right is FAM-CY5 diagram);
FIG. 5 is a dot diagram showing the results of P210+ group tests in the example of the present invention (the left is ROX-VIC diagram, and the right is FAM-CY5 diagram);
FIG. 6 is a scattergram of the test results of the P230+ group in the example of the present invention (the left is ROX-VIC, and the right is FAM-CY 5).
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1
As shown in FIG. 1, this example discloses a method for simultaneously detecting three subtype types of BCR-ABL1 fusion genes, which comprises:
firstly, designing a primer probe by using primer express software according to the sequence of a BCR-ABL1 fusion gene. The specific information is shown in the following table 1:
TABLE 1
Second, sample
(1) P190/P210 RNA sample: RNA was extracted from clinical samples using an RNA extraction kit.
(2) P230 DNA: biometrics synthesized DNA fragments.
(3) Wild-type sample of ABL1 gene: and extracting RNA of the oral exfoliative cells.
Thirdly, the reverse transcription system is shown in the following table 2:
TABLE 2
IV, reverse transcription conditions
42℃1h，70℃15min。
Fifth, the cDNA digital PCR amplification system is shown in the following Table 3:
TABLE 3
| Reagent | Volume (μ L) | Final concentration |
| Water (W) | 10.55 | |
| 10×PCR Buffer | 1.5 | 1X |
| dNTP | 0.3 | |
| Taq DNA polymerase | 0.3 | |
| P190-F(100μM) | 0.15 | 1000nM |
| P210-F(100μM) | 0.15 | 1000nM |
| P230-F(100μM) | 0.15 | 1000nM |
| ABL1-F(100μM) | 0.15 | 1000nM |
| ABL1-R(100μM) | 0.6 | 4000nM |
| P190-P(100μM) | 0.0375 | 250nM |
| P210-P(100μM) | 0.0375 | 250nM |
| P230-P(100μM) | 0.0375 | 250nM |
| ABL1-P(100μM) | 0.0375 | 250nM |
| cDNA or synthetic DNA | 1 | |
| Total up to | 15 |
Sixth, amplification procedure
98℃5min【98℃15s 60℃1min】*40。
Seventhly, the detection results are shown in the following table 4:
TABLE 4
And eighthly, the scatter diagram examples are shown in figures 2-6.
As can be seen from FIGS. 2 to 6, the primer probes of the present invention were normal in specificity, and no non-specific amplification was detected in the NTC group; detecting normal RNA, only detecting ROX signal; the detection of the P190 positive sample can simultaneously detect FAM and ROX signals, and the ratio is stable; the detection of the P210 positive sample can simultaneously detect VIC and ROX signals, and the ratio is stable; the double-stranded DNA synthesized by P230 can be detected to simultaneously detect CY5 and ROX signals, and the ratio is stable.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (10)
1. A primer probe composition for simultaneously detecting three subtype genes of BCR-ABL1, wherein the three subtype genes of BCR-ABL1 are P190, P210 and P230 respectively, and the primer probe composition comprises:
the sequences of the primer and the probe of the P190 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the specification;
the sequences of the primer and the probe of the P210 are respectively shown as SEQ ID NO: 3 and SEQ ID NO: 4 is shown in the specification;
the sequences of the primer and the probe of the P230 are respectively shown as SEQ ID NO: 5 and SEQ ID NO: 6 is shown in the specification;
the sequence of the forward primer of the ABL1 gene is shown as SEQ ID NO: 7, the reverse primer sequence is shown as SEQ ID NO: 8, and the probe sequence is shown as SEQ ID NO: shown at 9.
2. A kit comprising the primer probe composition of claim 1.
3. The kit of claim 2, further comprising 10 x PCR buffer, dntps, and Taq DNA polymerase.
4. The kit according to claim 2, wherein the concentration of the primer in the kit is 1000-5000nM, and the sequence concentration of the probe is 100-1000 nM; preferably, the concentration of the upstream primer is 1000nM, the concentration of the downstream primer is 4000nM, and the concentration of the probe is 250 nM.
5. A method for simultaneously detecting three subtype genes of BCR-ABL1, which comprises the following steps: detection using the kit of claims 2-4.
6. The method of claim 5, wherein the test sample is derived from oral exfoliated cells and/or blood-extracted RNA.
7. The method of claim 5, comprising: the primer is adopted for reverse transcription and then the amplification is carried out by the digital PCR technology.
8. The method of claim 7, wherein the reverse transcription system comprises 5 xrt buffer, reverse transcriptase, reverse primer for ABL1 gene, and RNA.
9. The method of claim 7, wherein the digital PCR amplification system comprises water, 10 XPCR buffer, dNTPs and Taq DNA polymerase.
10. Use of the primer probe composition according to claim 1, the kit according to claims 2 to 4 and the method according to claims 5 to 9 in the field of gene detection; preferably, the gene detection field is the BCR-ABL1 fusion gene detection field.
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| CN113718021A (en) * | 2021-07-30 | 2021-11-30 | 广州达安基因股份有限公司 | Primer, probe and kit for quantitatively detecting BCR-ABL1 fusion gene |
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