Primer for rapidly detecting 11beta-OHD pathogenic chimeric gene and application thereof
By designing the primer group for 11β-OHD pathogenic chimeric genes, combined with QPCR and long clip PCR technology, the difficulty of detecting 11β-OHD pathogenic genes in existing technology has achieved rapid and accurate detection , Reduced the risk of clinical diagnosis errors.
Patent Information
- Application Number
- CN202411292737.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2025-01-28
AI Technical Summary
The prior art is difficult to quickly and accurately detect the chimeric genes of 11β-OHD pathogenic, resulting in clinical misdiagnosis, missed diagnosis or delayed diagnosis.
The primer group for fast detection of 11β-OHD pathogenic chimeric genes, combined with QPCR and long clips PCR technology, can directly screen patients and carriers carrying CYP11B2/CYP11B1 chimeric genes.
The rapid and accurate testing of the 11β-OHD pathogenic chimeric genes reduced the risk of misdiagnosis, missed diagnosis or delay diagnosis, and low cost, suitable for clinical applications.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular biology and genetic testing, and in particular relates to primers for rapidly detecting 11β-OHD pathogenic chimeric genes and applications thereof. Background Art
[0002] 11β-hydroxylase deficiency (11β-OHD) is a common congenital adrenal hyperplasia (CAH). 21-hydroxylase deficiency (21-OHD) is the most common cause of CAH, accounting for 90-99% of the total cases; in contrast, 11β-OHD is the second leading cause of CAH, accounting for about 5-8% of the total cases. 11β-OHD has been shown to be caused by mutations in the CYP11B1 gene, which encodes 11β-hydroxylase, is located on chromosome 8q21, contains 9 exons, and encodes 503 amino acids. Humans also have a gene CYP11B2 that is highly homologous to CYP11B1 and encodes aldosterone synthase. The CYP11B1 and CYP11B2 genes are arranged in tandem on the long arm of chromosome 8, with an interval of 45 kb. The two have 95% exon sequence homology and 90% intron sequence homology, and intron 5 is the region with the greatest difference.
[0003] Most CYP11B1 gene mutations are point mutations, with missense and nonsense mutations being the most common, followed by splice site mutations and small deletion or insertion mutations. In addition, CYP11B1 mutations may also be the result of abnormal recombination between this gene and the highly homologous aldosterone synthase encoding gene CYP11B2. Unequal allele exchange occurs between CYP11B2 and CYP11B1 genes during meiosis, resulting in gametes lacking normal copies of CYP11B1 and CYP11B2. These copies are deleted through unequal crossing over, ultimately forming CYP11B2 / CYP11B1 chimeric genes, see Appendix. Figure 1 . The CYP11B2 / CYP11B1 chimeric gene is composed of the 5' end sequence of the CYP11B2 gene and the 3' end sequence of the CYP11B1 gene, and the breakpoints are dispersed, making it difficult to determine the specific breakpoint positions. Cases of 11β-OHD caused by pathogenic CYP11B2 / CYP11B1 chimeric genes are relatively rare. The CYP11B2 / CYP11B1 chimeric gene complicates the molecular diagnosis of 11β-OHD, often leading to misdiagnosis, missed diagnosis, or delayed diagnosis of 11β-OHD in clinical practice.
[0004] Currently, with the widespread application of high-throughput sequencing (NGS) in clinical practice, copy number variation (CNV) detection algorithms extracted from NGS data have developed rapidly. However, their ability to detect CNVs containing homologous gene families or pseudogene families is not so reliable and the cost is high. Therefore, it is urgent to develop a simple and rapid molecular diagnostic method for detecting mosaic genes and other types of large fragment deletion genes that can be routinely used in the diagnosis of 11β-OHD. Summary of the invention
[0005] Based on the above problems, the present invention provides primers for rapid detection of 11β-OHD pathogenic chimeric genes and their applications. The primers designed by the present invention can be directly used for qPCR detection, which can preliminarily assist clinical practice in rapid molecular detection of 11β-OHD pathogenic chimeric genes. It is the first innovative application of long-fragment PCR technology to identify the rearrangement pattern of highly homologous genes.
[0006] To solve the above technical problems, the present invention provides a primer set for rapid detection of 11β-OHD pathogenic chimeric gene, comprising the following two groups of primers: a primer set CYP11B2-Intron5-F primer and a CYP11B2-Intron5-R primer, and a primer set CYP11B1-Intron5-F primer and a CYP11B1-Intron5-R primer; the sequences of the CYP11B2-Intron5-F primer, the CYP11B2-Intron5-R primer, the CYP11B1-Intron5-F primer and the CYP11B1-Intron5-R primer are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively;
[0007] The primer set CYP11B2-Intron5-F primer and CYP11B2-Intron5-R primer are used to detect the gene fragment at the 5th intron of the CYP11B2 gene, and the primer set CYP11B1-Intron5-F primer and CYP11B1-Intron5-R primer are used to detect the gene fragment at the 5th intron of the CYP11B1 gene.
[0008] Furthermore, the primer set also includes L-CYP11B1-F primer, L-CYP11B1-R primer, L-CYP11B2-F primer and L-CYP11B2-R primer, and the sequences of the L-CYP11B1-F primer, L-CYP11B1-R primer, L-CYP11B2-F primer and L-CYP11B2-R primer are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.
[0009] In order to solve the above technical problems, the present invention also provides the use of a primer set in preparing a product for detecting 11β-OHD.
[0010] Furthermore, the product is used to detect CYP11B2 / CYP11B1 chimeric genes.
[0011] Furthermore, the product is a qPCR detection kit, and the qPCR reaction system of the qPCR detection kit is as follows: Hieff Universal Blue qPCR SYBR Green Master Mix 10 μL, Forward Primer (10 μM) 0.4 μL, Reverse Primer (10 μM) 0.4 μL, template DNA (5-50 ng) 2 μL, add sterile ultrapure water to make the reaction system 20 μL; the qPCR reaction program of the qPCR detection kit is as follows: pre-denaturation, 95°C, 2 min, cycle 1; denaturation, 95°C, 10 sec, annealing / extension, 60°C, 30 sec, cycle 40.
[0012] In order to solve the above technical problems, the present invention also provides the use of a primer set in preparing a product for detecting 11β-OHD.
[0013] Furthermore, the product is used to detect CYP11B2 / CYP11B1 chimeric genes.
[0014] Further, the product is a Long extension-PCR detection kit, and the Long extension-PCR reaction system is as follows: 10×LA PCR Buffer II (Mg 2+plus) 5μL, TaKaRa LA Taq HS (5U / μl) 0.5μL, dNTP Mixture (2.5mM each) 8μL, Forward Primer (10μM) 1μL, Reverse Primer (10μM) 1μL, template DNA (100-300ng) 5μL, sterile ultrapure water supplemented to 50μL;
[0015] The Long extension-PCR reaction procedure was as follows: pre-denaturation at 94°C for 1 min 1, denaturation at 98°C for 10 sec 30, annealing / extension at 72°C for 8 min, and 72°C for 10 min 1.
[0016] Further, the analysis method of the product test results is as follows:
[0017] (1) Chimeric gene heterozygotes:
[0018] A: The upstream of the chimeric gene carried includes the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R: the copy number of the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is equivalent to that of normal people, while the copy number of the target gene fragment amplified by the other primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is half that of normal people;
[0019] B: The upstream of the carried chimeric gene includes the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R: the copy number of the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is equivalent to that of normal people, while the copy number of the target gene fragment amplified by the other primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is half that of normal people;
[0020] (2) Homozygous chimeric gene:
[0021] A: The upstream of the chimeric gene carried includes the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R: The copy number of the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is equivalent to that of normal people, while the other primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R cannot detect the presence of the target gene fragment;
[0022] B: The upstream of the carried chimeric gene includes the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R: the copy number of the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is equivalent to that of normal people, while the other primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R cannot detect the presence of the target gene fragment;
[0023] (3) Other situations:
[0024] If the chimeric gene carried includes part of the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R and part of the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R, then the qPCR results of the DNA samples of heterozygous patients will show that the copy number of the target gene fragment amplified by the two pairs of primers is halved compared with that of normal people, while the two pairs of primers cannot detect the presence of the target gene fragment in homozygous patients.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] (1) The present invention designs two pairs of amplification primers based on the specific sequence differences between the CYP11B1 gene and the CYP11B2 gene at the 5th intron. The designed primers can be directly used for qPCR detection, and can preliminarily determine whether 11β-OHD patients carry CYP11B2 / CYP11B1 chimeric genes based on the relative quantitative analysis of the copy numbers of CYP11B1 and CYP11B2 genes in patients and carriers;
[0027] (2) The primers designed by the present invention are combined with a DNA polymerase combination having both good polymerase activity and error correction ability, and the complete CYP11B1 and CYP11B2 genes can be amplified with the help of long-fragment PCR technology, thereby distinguishing these two highly homologous genes and analyzing their rearrangement more accurately and efficiently;
[0028] (3) Compared with the defects of the currently commonly used probe hybridization method, such as the difficulty in probe design, strict molecular hybridization conditions and high cost, and long detection time, the primer-combined long-fragment PCR detection method of the present invention can more quickly and conveniently directly screen out 11β-OHD patients and carriers carrying the CYP11B2 / CYP11B1 chimeric gene, and the cost is low. It can greatly improve the misdiagnosis, missed diagnosis or delayed diagnosis of 11β-OHD in clinical practice, and has great innovative significance for the rapid molecular detection of 11β-OHD in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 A schematic diagram of the formation of the CYP11B1 / CYP11B2 or CYP11B2 / CYP11B1 chimeric gene mentioned in the background technology of the present invention;
[0030] Figure 2 Schematic diagram of the specific amplified fragments of 171 bp on the human CYP11B1 genome and 182 bp on the CYP11B2 genome to be amplified in the embodiments of the present invention;
[0031] Figure 3 and Figure 4All of them are the research results of specific genomic sequences in which the CYP11B1 gene and the CYP11B2 gene of the embodiments of the present invention are different;
[0032] Figure 5 Schematic diagram of the copy number of intron 5 of CYP11B1 and CYP11B2 genes in patients and carriers in the embodiments of the present invention;
[0033] Figure 6 A diagram showing the test results of patient samples according to an embodiment of the present invention;
[0034] Figure 7 The present invention is an embodiment of the present invention, which uses long-range PCR and Sanger sequencing technology to clarify the Sanger sequencing chromatogram of the breakpoints of the CYP11B2 / CYP11B1 chimeric gene in 11β-OHD patients. DETAILED DESCRIPTION
[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.
[0036] Example:
[0037] Although the human CYP11B1 gene and CYP11B2 gene are highly homologous, CYP11B1 gene Intron 5 and CYP11B2 gene Intron 5 have their own specific genome sequences, see Appendix Figure 2 The specific genome sequences of the two are somewhat different, see Appendix Figure 3 and attached Figure 4 The inventors used SnapGene software to analyze the differences in the Intron5 sequences of the two genes and found that there were significant differences between the "......cacatttct......" sequence and the "......gggctctgt......" sequence of Intron 5 of the CYP11B1 and CYP11B2 genes ( Figure 4 The middle boxes mark the start and end sites of these differential sequences).
[0038] In addition, the inventors used UCSC's Genome Browser (http: / / genome.ucsc.edu / ) to analyze the genomic DNA sequences near intron5 of human CYP11B1 and CYP11B2, and copied the sequences of exon5, intron5 and exon6 of CYP11B1 and CYP11B2 to word for storage. The yellow bases show the specific sequences of intron5 of CYP11B1 and CYP11B2. In the specific sequences of intron5 of CYP11B1 and CYP11B2, the inventors designed forward primer sequences CYP11B1-intron 5F and CYP11B2-intron 5F for amplifying specific fragments of 171 bp on the CYP11B1 genome and 182 bp on the CYP11B2 genome, respectively (see Figure 3 The specific sequences of intron5 of CYP11B1 and CYP11B2 are marked and bolded).
[0039] According to the above differences, the inventors designed two pairs of amplification primers to amplify the specific fragments of 171 bp on the CYP11B1 genome and 182 bp on the CYP11B2 genome (see Figure 2 The 171 bp amplified fragment (B1 amplified fragment, B1-AF) on the CYP11B1 genome contains a specific sequence fragment of part of the CYP11B1 gene Intron 5 + a 110 bp fragment of the CYP11B1 gene Intron 5 + a partial fragment of part of the CYP11B1 gene Exon 5. The 182 bp amplified fragment (B2 amplified fragment, B2-AF) on the CYP11B2 genome contains a specific sequence fragment of the CYP11B2 gene Intron 5 + a 110 bp fragment of the CYP11B2 gene Intron 5 + a partial fragment of part of the CYP11B2 gene Exon 5 (see Figure 2 ).
[0040] The inventors designed forward primers for qPCR (CYP11B1-Intron5-F and CYP11B2-Intron5-F) from the specific sequence fragments of Intron 5 of the CYP11B1 gene and the specific sequence fragments of Intron 5 of the CYP11B2 gene, respectively (see Figure 2 and Figure 3), reverse primers (CYP11B1-Intron5-R and CYP11B2-Intron5-R) were designed in Exon 5 of CYP11B1 gene and Exon 5 of CYP11B2 gene, respectively. The length of the two forward primers was between 18-19 bp, and the length of the reverse primer was 18 bp.
[0041] Using the genomic DNA of patients and carriers as a template, qPCR relative quantitative analysis was performed based on the copy numbers of CYP11B1 and CYP11B2 genes in patients and carriers, which could preliminarily determine the chimerism of three types of CYP11B2 / CYP11B1 chimeric gene carriers or three types of CYP11B2 / CYP11B1 chimeric genes in 11β-OHD patients.
[0042] Carriers of CYP11B2 / CYP11B1 chimeric genes will present in three situations, resulting in three different q PCR results for CYP11B2 and CYP11B1 genes:
[0043] (1) CYP11B1 halved carriers: If the subject is a heterozygote carrying a chimeric gene, and the upstream of the chimeric gene includes Intron5 of the CYP11B2 gene, then the qPCR results using the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R will theoretically show that the copy number is similar to that of a normal person. The relative quantitative results of another primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R will theoretically show that the copy number is halved compared to that of a normal person (see Figure 5 B) in;
[0044] (2) CYP11B2 half-carriers: If the upstream of the chimeric gene carried by the tested individual does not include the heterozygote of CYP11B2 gene Intron 5, then theoretically, using the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R for qPCR, the copy number of CYP11B2 is halved compared to that of a normal person; using the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R for qPCR, the copy number of CYP11B1 is similar to that of a normal person (see Figure 5 B) in;
[0045] (3) Carriers of both CYP11B1 and CYP11B2: If the chimeric gene carried by the person being tested is a heterozygote that includes neither CYP11B1 gene Intron 5 nor CYP11B2 gene Intron 5, then theoretically, using the two pairs of primers CYP11B2-Intron5-F / CYP11B2-Intron5-R or CYP11B1-Intron5-F / CYP11B1-Intron5-R for qPCR, the copy numbers of both CYP11B2 and CYP11B2 are halved compared to normal people (see Figure 5 B).
[0046] 11β-OHD patients carrying CYP11B2 / CYP11B1 chimeric genes will also appear in three situations, resulting in three different q PCR results of CYP11B2 and CYP11B1 genes:
[0047] (1) Patients with CYP11B1 cannot be detected: If the person being tested is a homozygote carrying a chimeric gene that includes the CYP11B2 gene Intron5 but does not include the CYP11B1 gene Intron5, then theoretically the results will show that when the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is used for qPCR, the copy number is similar to that of a normal person. When the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is used for qPCR, the presence of the target gene fragment will not be detected (see Figure 5 C in
[0048] (2) Patients with CYP11B2 cannot be detected: If the person being tested is a homozygote carrying a chimeric gene with only the CYP11B1 gene Intron5 upstream, then theoretically the results will show that the presence of the CYP11B2 gene cannot be detected by qPCR using the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R, while the copy number of the CYP11B2 gene by qPCR using the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is similar to that of normal people (see Figure 5 C in
[0049] (3) Patients with neither CYP11B1 nor CYP11B2 can be detected: If the chimeric gene carried by the person being tested is a heterozygote that includes neither CYP11B1 gene Intron 5 nor CYP11B2 gene Intron 5, then theoretically, the copy number of CYP11B2 and CYP11B2 cannot be detected using the two primer pairs CYP11B2-Intron5-F / CYP11B2-Intron5-R or CYP11B1-Intron5-F / CYP11B1-Intron5-R for qPCR (see Figure 5 C).
[0050] Two DNA polymerases are used in long-extension PCR to react. One is a heat-resistant DNA polymerase (commonly used Taq enzyme) used in conventional PCR reactions. It has a strong extension ability and relies on this enzyme for chain extension. The other is a heat-resistant DNA polymerase (commonly used Pfu enzyme) with 3′→5 exonuclease activity. It has a good proofreading function and can cut off the mismatched bases and reuse the first enzyme for chain extension. The two enzymes have their own advantages and disadvantages. The extension ability of the first enzyme and the proofreading function of the second enzyme are fully utilized, so that the two enzymes complement each other in the reaction to complete the amplification of long-fragment DNA. By using a combination of DNA polymerases with good polymerase activity or error correction ability, the length of the amplifiable DNA fragment can be extended. Using this feature, the inventors applied the Long-extension PCR technology to amplify the complete CYP11B1 and CYP11B2 genes to further identify these two highly homologous genes and more accurately analyze their rearrangements.
[0051] The inventors first used real-time fluorescence quantitative PCR (qPCR) to quantitatively analyze the patient's genes to preliminarily determine whether the 11β-OHD patient carries the CYP11B2 / CYP11B1 chimeric gene.
[0052] Template DNA was extracted using the blood DNA extraction kit of Tiangen Biochemical Technology Co., Ltd., and two sets of primers were designed by the above method. In this example, human telomerase reverse transcriptase (TERT, NM_198253.3) and human actin (ACTB, NM_001101.5) were used as internal references for genomic DNA. Among them, the primer sequences of CYP11B1-Intron5-F, CYP11B1-Intron5-R, CYP11B2-Intron5-F, CYP11B2-Intron5-R, and the sequences of TERT and ACTB are shown in Table 1. Using the genomic DNA of patients and carriers as templates, qPCR relative quantitative analysis was performed according to the copy number of CYP11B1 and CYP11B2 genes in patients and carriers, and it can be preliminarily determined whether 11β-OHD patients carry CYP11B2 / CYP11B1 chimeric genes.
[0053] Table 1 Primer sequences for genomic DNA qPCR
[0054]
[0055] Next, the inventors prepared a qPCR amplification reaction kit, primers, template DNA, a tip without nuclease residue, a reaction tube, etc., and prepared a 20uL reaction system according to the kit in an ultra-clean workbench, as shown in Table 2.
[0056] Table 2 qPCR reaction system
[0057]
[0058] Add the above components to eight rows of tubes respectively, and be sure to shake gently to mix thoroughly, avoid vigorous shaking to produce too many bubbles, and centrifuge briefly to empty the small bubbles in the tubes. Set the qPCR reaction program according to Table 3.
[0059] Table 3 qPCR reaction procedure
[0060]
[0061]
[0062] After the reaction, the amplification curve and melting curve were analyzed, and TERT and ACTB were used as internal reference genes. -ΔΔCt Relative quantitative analysis was performed.
[0063] Next, the inventors used Long-extension PCR+electrophoresis to further determine whether homologous recombination occurred between the CYP11B2 gene and the CYP11B1 gene to form a fusion gene and to determine the type of the chimeric gene.
[0064] Four pairs of primer combinations (CYP11B1F / B1R, CYP11B2F / B2R, CYP11B1F / B2R, CYP11B2F / B1R) were designed to amplify the long fragments of the samples. The primer sequences are shown in Table 5. Gel electrophoresis was used to determine whether homologous recombination occurred between the CYP11B2 gene and the CYP11B1 gene to form a fusion gene and to determine the type of chimeric gene. Gel electrophoresis was used to determine whether homologous recombination occurred between the CYP11B2 gene and the CYP11B1 gene to form a fusion gene and to determine the type of chimeric gene.
[0065] Prepare the Long extension-PCR amplification reaction kit (using TaKaRaLAT aq StartVersion), primers, template DNA, pipette tips, reaction tubes, etc., were prepared into a 50uL reaction system according to the kit in an ultra-clean workbench (see Table 4); the primer sequences used in Long extension-PCR are shown in Table 5, and the Long extension-PCR reaction procedure is shown in Table 6.
[0066] Table 4 Long extension-PCR reaction system
[0067]
[0068]
[0069] Table 5 Primer sequences for long extension-PCR amplification of genomic DNA long fragment PCR
[0070]
[0071] Table 6 Long extension-PCR reaction program
[0072]
[0073] After the DNA samples of 11β-OHD patients were amplified by the above-mentioned Long-extension PCR, the amplified chimeric gene products were purified by gel excision recovery, and their concentrations were adjusted to a range suitable for sequencing (50-100 ng / μL), followed by Sanger sequencing.
[0074] The Sanger sequencing PCR reaction system is as follows:
[0075]
[0076] The Sanger sequencing PCR reaction procedure is as follows:
[0077]
[0078]
[0079] The above sequencing products were purified and then subjected to capillary electrophoresis. The obtained products were added to the Applied Biosystems 3500Dx Gene Analyzer for detection, and the first-generation sequencing results were read using Chrosome software.
[0080] In 2023, the inventors, in collaboration with the Department of Endocrinology and Metabolism of Guizhou Provincial People's Hospital, also collected a peripheral blood sample from a patient with CYP11B2 / CYP11B1 chimeric gene 11β-hydroxylase deficiency (11β-OHD) in Taijiang County, Qiandongnan Prefecture, Guizhou Province. This patient and the consanguineous marriage family reported by the inventors in 2023 were residents of the same township. The inventors extracted their DNA genes according to the aforementioned steps, and at the same time found the DNA of a normal person whose 11β-hydroxylase deficiency had been excluded as a control. Using ACTB and TERT as internal references, qPCR was performed using the CYP11B1-Intron5-F and CYP11B1-Intron5-R primer sets and the CYP11B2-Intron5-F and CYP11B2-Intron5-R primer sets, respectively, to amplify the peripheral blood DNA templates CYP11B1 and CYP11B2. The qPCR results are attached. Figure 6 , where: Figure A, with ACTB as the internal reference, qPCR could not detect the expression of CYP11B1 in this patient, while the expression of CYP11B2 was not significantly different from that of normal people; Figure B, with ACTB as the internal reference, CYP11B1 in this patient could not be detected, while the expression of CYP11B2 was not significantly different from that of normal people. It can be seen that the qPCR results show that CYP11B1 in this 11β-OHD patient cannot be detected, which means that there may be a lack of intron5 and exon6 of CYP11B1, while intron5 and exon6 of CYP11B2 gene are expressed normally. In other words, it is possible to screen out the attached Figure 5 The CYP11B2 / CYP11B1 chimeric gene fusion form has a normal copy number of CYP11B2 but undetectable CYP11B1.
[0081] Next, the inventors further used the aforementioned long-extension PCR and first-generation sequencing technology to clarify the specific sequence of the patient's chimeric gene.
[0082] See attached Figure 7 ,in, Figure 7 Figure A in the figure is the Sanger sequencing chromatogram of the CYP11B2 / CYP11B1 chimeric gene breakpoints of the above-mentioned 11β-OHD patient; Figure 7Figure B is a schematic diagram of the CYP11B2 / CYP11B1 chimeric gene of the above-mentioned 11β-OHD patient.
[0083] The base sequence of the CYP11B2 / CYP11B1 chimera highlighted in red is identical to that of CYP11B2, the base sequence highlighted in green is identical to that of CYP11B1, and the base sequence highlighted in purple is not found in either CYP11B1 or CYP11B2. CYP11B1 rs6391T>C SNP, CYP11B1rs6392 A>G SNP, a CYP11B1rss6395 T>G SNP, an IVS6+179C>T mutation, and an IVS-75C>T variant were found in intron 6 of the chimera. A c0.1086G>c (p.Leu.362=) synonymous mutation was detected in exon 6, and a c0.1157C>G (p.A386V) missense mutation was detected in exon 7. It can be seen that the detailed DNA sequence at the breakpoint of the CYP11B2 / CYP11B1 chimera includes exon 6, intron 6 and exon 7 of the CYP11B2 / CYP11B1 chimera gene.
[0084] The above is an embodiment of the present invention. The above embodiments and the specific parameters in the embodiments are only for the purpose of clearly describing the invention verification process, and are not intended to limit the patent protection scope of the present invention. The patent protection scope of the present invention is still subject to its claims. Any equivalent structural changes made by using the contents of the description and drawings of the present invention should be included in the protection scope of the present invention.
Claims
1. A primer set for rapid detection of 11β-OHD pathogenic chimeric gene, characterized in that: The invention comprises the following two groups of primers: a primer group CYP11B2-Intron5-F primer and a CYP11B2-Intron5-R primer, and a primer group CYP11B1-Intron5-F primer and a CYP11B1-Intron5-R primer; the sequences of the CYP11B2-Intron5-F primer, the CYP11B2-Intron5-R primer, the CYP11B1-Intron5-F primer and the CYP11B1-Intron5-R primer are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively; The primer set CYP11B2-Intron5-F primer and CYP11B2-Intron5-R primer are used to detect the gene fragment at the 5th intron of the CYP11B2 gene, and the primer set CYP11B1-Intron5-F primer and CYP11B1-Intron5-R primer are used to detect the gene fragment at the 5th intron of the CYP11B1 gene.
2. The primer set for rapid detection of 11β-OHD pathogenic chimeric gene according to claim 1, characterized in that: The primer set also includes L-CYP11B1-F primer, L-CYP11B1-R primer, L-CYP11B2-F primer and L-CYP11B2-R primer. The sequences of the L-CYP11B1-F primer, L-CYP11B1-R primer, L-CYP11B2-F primer and L-CYP11B2-R primer are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.
3. Use of the primer set according to claim 1 in preparing a product for detecting 11β-OHD.
4. The use according to claim 3, characterized in that: The product is used to detect CYP11B2 / CYP11B1 chimeric genes.
5. The use according to claim 4, characterized in that: The product is a qPCR detection kit, and the qPCR reaction system of the qPCR detection kit is as follows: Hieff Universal Blue qPCR SYBR Green Master Mix 10 μL, Forward Primer (10 μM) 0.4 μL, Reverse Primer (10 μM) 0.4 μL, template DNA (5-50 ng) 2 μL, add sterile ultrapure water to make the reaction system 20 μL; the qPCR reaction program of the qPCR detection kit is as follows: pre-denaturation, 95°C, 2 min, cycle 1; denaturation, 95°C, 10 sec, annealing / extension, 60°C, 30 sec, cycle 40.
6. Use of the primer set according to claim 2 in preparing a product for detecting 11β-OHD.
7. The use according to claim 6, characterized in that: The product is used to detect CYP11B2 / CYP11B1 chimeric genes.
8. The use according to claim 7, characterized in that: The product is a Long extension-PCR detection kit, and the Long extension-PCR reaction system is as follows: 10×LA PCR Buffer II (Mg 2+ plus) 5 μL, TaKaRa LATaq HS (5U / μl) 0.5 μL, dNTP Mixture (2.5 mM each) 8 μL, Forward Primer (10 μM) 1 μL, Reverse Primer (10 μM) 1 μL, Template DNA (100-300 ng) 5 μL, sterile ultrapure water to 50 μL; The Long extension-PCR reaction procedure was as follows: pre-denaturation at 94°C for 1 min 1, denaturation at 98°C for 10 sec 30, annealing / extension at 72°C for 8 min, and 72°C for 10 min 1.
9. The use according to any one of claims 3 to 5, characterized in that The analysis method of the test results of the product is as follows: (1) Chimeric gene heterozygotes: A: The upstream of the chimeric gene carried includes the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R: the copy number of the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is equivalent to that of normal people, while the copy number of the target gene fragment amplified by the other primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is half that of normal people; B: The upstream of the carried chimeric gene includes the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R: the copy number of the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is equivalent to that of normal people, while the copy number of the target gene fragment amplified by the other primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is half that of normal people; (2) Homozygous chimeric gene: A: The upstream of the chimeric gene carried includes the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R: The copy number of the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R is equivalent to that of normal people, while the other primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R cannot detect the presence of the target gene fragment; B: The upstream of the carried chimeric gene includes the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R: the copy number of the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R is equivalent to that of normal people, while the other primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R cannot detect the presence of the target gene fragment; (3) Other situations: If the chimeric gene carried includes part of the target gene fragment amplified by the primer pair CYP11B2-Intron5-F / CYP11B2-Intron5-R and part of the target gene fragment amplified by the primer pair CYP11B1-Intron5-F / CYP11B1-Intron5-R, then the qPCR results of the DNA samples of heterozygous patients will show that the copy number of the target gene fragment amplified by the two pairs of primers is halved compared with that of normal people, while the two pairs of primers cannot detect the presence of the target gene fragment in homozygous patients.