PKD1 and PKD2 amplification primer group, kit and gene mutation type detection method

Abstract

The invention discloses a PKD1 and PKD2 amplification primer group, a kit and a gene mutation type detection method, by optimizing primer design sites, interference of pseudogenes in an amplification process can be avoided, a PKD1 gene and a PKD2 gene can be specifically amplified in a single tube, a long fragment is combined with a nanopore sequencing technology, the defect of structural variation detection by NGS sequencing can be overcome, and the detection accuracy is improved. The detection time and cost can be greatly reduced, and polycystic kidney disease detection can be effectively assisted.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnostic technologies, and particularly to PKD1 and PKD2 amplification primer sets, reagent kits, and methods for detecting gene mutation types. Background Art

[0002] Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease in humans, which is mainly caused by mutations in the polycystic kidney disease 1 gene (PKD1) located on chromosome 16 and the polycystic kidney disease 2 gene (PKD2) located on chromosome 4. This disease mostly occurs after the age of 30. The most obvious symptom is the formation of cysts in the kidneys. As cells abnormally proliferate and fluid is secreted, the cysts gradually expand, compressing the surrounding tissues and affecting kidney function, ultimately leading to the development of end-stage renal disease. In addition, this disease can also affect other organs, causing cysts to appear in multiple parts of the liver and pancreas, bringing a heavy burden to patients. Polycystic kidney disease caused by PKD1 gene mutations often has an earlier onset and usually develops into end-stage renal disease at around 55 years old on average. The remaining small part (15%) is caused by PKD2 mutations, and relatively speaking, the symptoms appear later.

[0003] The PKD1 gene is located in the 16p13.3 region of chromosome 16, with a total length of 47 kb and consisting of 46 exons in total. Studies have found that exons 1-33 of PKD1 have 6 extremely similar pseudogenes (PKD1P1-P6), with a similarity of up to 97.7%. However, statistics show that the mutation positions of the vast majority of patients also occur in these regions with pseudogenes. In addition, the GC content in some regions of PKD1 is as high as 80%, which makes it difficult to successfully amplify the high-GC regions in the PKD1 gene by conventional PCR amplification. In addition, the types of mutations that occur in the PKD1 gene are complex. In addition to point mutations and Indels, there are also large fragment deletions, duplications and other variations, which makes the detection of PKD1 not easy. In contrast, the PKD2 gene is not interfered by pseudogenes, and the main type of variation is point mutation. Therefore, the detection difficulty is lower than that of PKD1.

[0004] Currently, the detection methods for polycystic kidney disease gene mutations include LR-PCR plus Sanger sequencing. The main steps are to first perform long-fragment amplification on the gene, and then perform nested amplification on the locus to be detected followed by Sanger sequencing. This method has cumbersome steps, is time-consuming and laborious to operate, and due to the limited read length of first-generation sequencing, once there are large-fragment variations, it may lead to amplification failure and thus unable to be detected. For the detection of structural variations in polycystic kidney disease, the commonly used detection method is the MPLA method. Although this method can detect the structural variations of PKD1, it is also complex to operate and requires the synthesis of dozens of probes for detection. In addition, this method cannot detect single-base variations. With the development of NGS sequencing technology, there are also reports of combining LR-PCT with NGS sequencing to detect polycystic kidney disease. However, due to the GC bias and short read length of NGS sequencing, there are still certain defects when using NGS sequencing technology to detect the high-GC region of the PKD1 gene.

[0005] The existing detection methods also have the following problems: 1) It is impossible to complete the detection of different types of variations through a single experiment. Often, the results can only be obtained by the mutual cooperation of two experimental methods, which directly leads to too long a detection process cycle and complex experimental operation problems; 2) The pseudogenes of the PKD1 gene and the high-GC region will affect the binding of probes in the MLPA method and the binding of primers in the Sanger sequencing process; 3) The detection method of MLPA cannot obtain clear structural variation sites and can only determine the presence or absence of variations.

[0006] Nanopore sequencing technology determines the sequence of nucleic acid molecules based on the change in the current signal caused by single-stranded nucleic acid molecules passing through the biological protein nanopore channel. Therefore, for nanopore sequencing, its read length has no limit and can reach lengths of hundreds of Kb or even Mb levels. It is very suitable for gene detection with complex variations. Summary of the Invention

[0007] The present invention proposes PKD1 and PKD2 amplification primer sets, kits, and gene mutation type detection methods to avoid the interference of pseudogenes and be suitable for nanopore sequencing to improve detection efficiency.

[0008] The first aspect of the present invention is to propose PKD1 and PKD2 amplification primer sets, including a first primer set for amplifying the PKD1 gene and a second primer set for amplifying the PKD2 gene; the nucleotide sequences of the first primer set are shown as SEQ ID NO: 1-6, and the nucleotide sequences of the second primer set are shown as SEQ ID NO: 7-30.

[0009] The second aspect of the present invention lies in providing the use of the primer set in the preparation of products for detecting PKD1 and PKD2 gene mutations or products for diagnosing autosomal dominant polycystic kidney disease.

[0010] The third aspect of the present invention lies in providing a kit for detecting PKD1 and PKD2 gene mutations, which includes the primer set described in the first aspect.

[0011] Furthermore, the detection kit further includes at least one of nucleic acid extraction reagents, multiplex PCR reaction reagents, common sequences, barcode ligation PCR reaction reagents, and nanopore library construction reagents.

[0012] Furthermore, the multiplex PCR reaction reagents include DNA polymerase and buffer.

[0013] Preferably, the DNA polymerase is TaKaRa LA Taq DNA polymerase; and / or, the buffer includes Tris-HCl with a concentration of 10 - 100 mM, glycerol with a concentration of 5 - 20 wt%, DTT with a concentration of 1 - 5 mM, KCl with a concentration of 30 - 50 mM, MgCl2 with a concentration of 2 - 8 mM, dNTP with a concentration of 0.1 - 0.5 mM, DMSO with a concentration of 5 - 15 wt%, and betaine with a final concentration of 0.5 - 1.5 M. More preferably, the buffer composition is: Tris-HCl with a concentration of 50 mM, glycerol with a concentration of 10%, DTT with a concentration of 2.5 mM, KCl with a concentration of 40 mM, MgCl2 with a concentration of 4 mM, dNTP with a concentration of 0.3 mM, DMSO with a concentration of 10%, and betaine with a final concentration of 1 M.

[0014] The fourth aspect of the present invention lies in providing a method for amplifying all exons of PKD1 and PKD2 genes, which includes the step of performing PCR amplification using the primer set described in the first aspect.

[0015] The fifth aspect of the present invention lies in providing the use of the primer set described in the first aspect or the kit described in the third aspect in the detection of PKD1 and PKD2 gene mutations, and this application is not for diagnostic purposes.

[0016] The sixth aspect of the present invention lies in providing a method for detecting PKD1 and PKD2 gene mutations, which is not for diagnostic purposes, and includes amplifying the PKD1 and PKD2 genes of the sample to be tested using the primer set described in the first aspect, then sequencing the amplicons, and comparing the sequencing results with the reference genome to further determine the PKD1 and PKD2 gene mutation results.

[0017] Furthermore, the sequencing is based on nanopore sequencing, including but not limited to the ONT sequencing platform of Oxford Nanopore, all domestic nanopore sequencing platforms such as Puyi Biotechnology, BGI Genomics, and Jinshi Technology.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The primer set provided by the present invention can specifically amplify the PKD1 gene and the PKD2 gene in a single tube. By optimizing the primer design sites, interference from pseudogenes during the amplification process can be avoided. Through the amplification of long gene fragments combined with nanopore sequencing technology, the shortcomings of NGS sequencing in detecting structural variations can be made up for. The results of the present invention can greatly reduce the detection time and cost, and can effectively assist in the detection of polycystic kidney disease.

[0020] The primer set of the present invention is applied to the nanopore sequencing technology with a short detection process time. There is no need to assist other detection methods, and the PKD1 and PKD2 genes can be detected within 8 hours. Only one experiment is required to simultaneously complete the detection of different types of mutations. The operation is simple, the time is short, and the cost is low, which can effectively assist in the detection of polycystic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the gel image of the bands after single and multiplex amplification of the primer set described in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0023] In one embodiment, to solve the deficiencies of existing detection means, amplification primers for the PKD1 and PKD2 genes and a corresponding library construction and sequencing kit are provided. The primers used can cover all exon regions of PKD1 and PKD2. When designing the primers, the pseudogene regions are avoided by selecting the design sites, thus avoiding the amplification of pseudogenes and enabling the specific amplification of the PKD1 true gene. To address the time-consuming and laborious problems of existing detection schemes, all the amplification primers for PKD1 and PKD2 in this kit are mixed into one tube for amplification. In addition, by optimizing the amplification and library construction processes, the entire process can be completed within 8 hours. In addition, this amplification kit adopts the method of long fragment amplification for the PKD1 gene, which can directly detect the structural variations in the PKD1 gene, so that the detection of different mutation types of the PKD1 and PKD2 genes can be completed with only one sequencing. In addition, the library construction reagents provided in this kit can be paired with different platform nanopore sequencing platforms, not limited to the ONT sequencing platform of Oxford Nanopore, the Polyseq sequencing platform of Puyi Biotechnology, and domestic sequencing platforms such as Cycloneseq of MGI.

[0024] Example 1: Design of PKD gene-specific primers and optimization of amplification buffer

[0025] 1. Primer design

[0026] Exons 1 to 33 of the PKD1 gene have 6 highly homologous pseudogenes, namely PKD1P1, PKD1P2, PKD1P3, PKD1P4, PKD1P5, and PKD1P6. The sequence similarity between these pseudogenes and exons 1 to 33 of PKD1 is as high as 97.7% - 95.5%. These pseudogenes are non-functional genes, that is, they do not express proteins. However, these pseudogenes will have an adverse effect on the detection of the PKD1 gene. First, if the primers are designed in the homologous regions with the pseudogenes, it will lead to the presence of pseudogene fragments in the amplification products, which will interfere with Sanger sequencing, NGS sequencing, and even third-generation sequencing.

[0027] To avoid the influence of pseudogenes, the following strategy is applied for primer design in this kit: First, select the 5' UTR and 3' end regions (exon34 - 46) of the PKD1 gene to design primers. These regions have no pseudogene copies and high specificity, so they can be used as the optional range for primer design; in addition, the long-fragment PCR amplification strategy is adopted. The long-fragment products can cover multiple exons, increasing specificity while reducing the probability of pseudogene amplification; furthermore, by using the specific bases of the PKD1 true gene at the 3' end of the primers, the specificity of amplifying the true gene is also improved. The specific primer design process of this kit is as follows: The PKD1 gene is divided into three long fragments for amplification, including fragment 1 (exon1 - exon2), fragment 2 (exon2 - 23), and fragment 3 (exon23 - 46) regions respectively. For the outer primers in fragments 1 and 3, select the 5' UTR and 3' end regions to design primers according to the above strategy. For the other primers in fragments 1, 2, and 3, the primer design scheme with a specific 3' end is adopted. 5 pairs of primers are designed for each fragment for amplification testing, and the designed primers are compared using Blast to ensure that the primers only amplify the PKD1 gene and do not amplify the PKD1P1 - P6 genes.

[0028] For the PKD2 gene, since there is no pseudogene interference and its mutation types are mainly point mutations, when designing primers, obtain the sequences of each exon of the PKD2 gene and its upstream and downstream regions, and use primer5 to design primers to ensure that the amplification range of the primers includes the entire exon and at least 50 bp of its upstream and downstream regions. 3 pairs of primers are designed for each fragment for amplification testing, and the designed primers are compared using Blast to ensure that the primers only amplify the PKD2 gene.

[0029] 2. Optimization of amplification buffer

[0030] Amplification Buffer Optimization: Since the GC content of some regions of both the PKD1 and PKD2 genes is higher than 80%, it is difficult for conventional PCR amplification buffers to amplify these regions. Therefore, this kit has optimized the PCR buffer. The optimized amplification buffer includes Tris-HCl at a concentration of 10 - 100 mM, glycerol at 5 - 20 wt%, DTT at 1 - 5 mM, KCl at 30 - 50 mM, MgCl2 at 2 - 8 mM, dNTP at 0.1 - 0.5 mM, DMSO at 5 - 15 wt%, betaine at a final concentration of 0.5 - 1.5 M, and Takara LA Taq. Preferably, the buffer components are: Tris-HCl at a concentration of 50 mM, glycerol at 10%, DTT at 2.5 mM, KCl at 40 mM; MgCl2 at 4 mM, dNTP at 0.3 mM, DMSO at 10%, betaine at a final concentration of 1 M, and Takara LA Taq. After testing, better amplification results can be obtained using the optimized buffer.

[0031] Experimental Verification: Ten different human nucleic acids were selected to verify the primers designed above. First, single amplification verification was performed on all the designed primers, and the primers that could amplify all samples in the single verification were used as standby primers. Subsequently, the primers were mixed for multiplex amplification. Further, the concentration ratio between different primers was adjusted according to the sequencing read depth. For primers with a high sequencing depth, the primer concentration ratio was decreased, and for primers with a low sequencing depth, the concentration was increased or they were replaced. Finally, the primer pool in Table 1 was selected. The primer pool contains a total of 14 pairs of primers. Experimental verification shows that there is little interference between these primers, and they can more uniformly complete the detection of the PKD1 and PKD2 genes. Figure 1 This is the amplification electrophoresis result diagram of this primer pool, including the results of single amplification of each primer and multiplex amplification of all primers.

[0032] Table 1: Primer Sequences of the Finally Screened PKD Genes (F is the forward primer, R is the reverse primer)

[0033]

[0034]

[0035] Example 2 PKD1 and PKD2 Gene Detection Kit

[0036] The cost of single library construction for nanopore sequencing is relatively high. Multiple samples can be detected simultaneously in one sequencing run to share the cost, significantly reducing the detection cost per sample. This kit is designed with 96 barcode sequence tags, which are connected to the specific products of one-round amplification through common sequences. 96 samples can be detected simultaneously in one sequencing run, but it is not limited to detecting 96 samples. If there is a detection need, it can be extended to 384 samples. The present invention provides a kit for detecting PKD1 and PKD2 genes based on nanopore sequencing, including multiplex PCR reaction reagents, barcode ligation PCR reaction reagents, and nanopore library construction reagents. The components of the kit are shown in Table 2.

[0037] Table 2:

[0038]

[0039] Sample Detection Process of Example 3

[0040] 1) Nucleic Acid Extraction from Samples

[0041] Take 400 μL of the blood sample to be tested. It is recommended to use the magnetic bead method blood genomic DNA extraction kit (DP329) from Tiangen for nucleic acid extraction. The specific extraction steps refer to the kit instructions.

[0042] 2) Amplification of PKD1 and PKD2 Genes

[0043] The nucleic acids extracted from different samples are uniformly diluted to 50 ng / μL and then subjected to one-tube amplification of PKD1 and PKD2 genes according to the following table.

[0044] Reagent Volume μL Amplification buffer 15 Multiplex amplification primer 4 Sample DNA template 1

[0045] On a PCR instrument, amplify according to the following program:

[0046]

[0047] 3) Second-round Amplification

[0048] Prepare the following system for second-round amplification, and add different Barcode sequences to different samples for pooled sequencing:

[0049] Reagent Volume μL Amplification buffer 15 Barcode(1μM) 4 Product of the first round 1

[0050] On a PCR instrument, amplify according to the following program:

[0051]

[0052] 4) Product Purification

[0053] 4.1 Take a new 1.5 ml EP tube and evenly mix the PCR products ligated with barcodes in equal volume.

[0054] 4.2 Resuspend the AMPure XP magnetic beads by vortexing.

[0055] 4.3 Take 200 μl of the mixed PCR product, add 140 μl of AMPure XP magnetic beads, mix by flicking the EP tube, and incubate at room temperature for 5 minutes.

[0056] 4.4 Place the EP tube on the magnetic stand until the eluate is clear and colorless, then remove the supernatant.

[0057] 4.5 Place the EP tube on the magnetic stand, wash the magnetic beads with 200 μl of freshly prepared 80% ethanol, remove the ethanol and discard it.

[0058] 4.6 Repeat the previous step.

[0059] 4.7 Centrifuge briefly and place the EP tube back on the magnetic stand to remove all residual ethanol. Dry for about 30 seconds, but do not dry to the extent that the particles crack.

[0060] 4.8 Remove the EP tube from the magnetic stand, resuspend the magnetic beads in 52 μl of EP, and incubate at room temperature for 2 minutes.

[0061] 4.9 Place the EP tube on the magnetic stand until the eluate is clear and colorless.

[0062] 4.10 Transfer all the supernatant to a new 1.5 ml EB tube.

[0063] 4.11 Take 1 μl of the purified product and measure the concentration using the Qubit dsDNA HS Assay Kit.

[0064] 5) Adapter Ligation and Purification

[0065] 5.1 Prepare the adapter ligation system. This kit's optimized ligation system can be used for adapter ligation on different nanopore sequencing platforms. Only the Adapter Mix for different nanopore sequencing platforms needs to be replaced during the ligation process.

[0066] Component Dosage per person Purified DNA from the previous step 35μl Ligation buffer 50μl DNA Ligase 10μl Adapter Mix 5μl Total 100μl

[0067] 5.2 Place the PCR reaction tube on the PCR instrument; Program: 24°C for 10 min (reaction time: 10 min - 30 min. To shorten the detection time, 10 min is sufficient to meet the reaction requirements. If you want to improve the reaction effect, the reaction time can be appropriately extended).

[0068] 5.3 Transfer the PCR product to a new 1.5 ml EP tube, add 80 μl of AMPure XP magnetic beads, perform magnetic bead purification, replace 80% ethanol with PH for washing, and elute with 20 μl of EB to obtain purified DNA.

[0069] 5.4 Take 1 μl of the purified product and detect the concentration using the Qubit dsDNA HS Assay Kit.

[0070] 6) Sequencing on the machine

[0071] This detection kit can be used for sequencing with different nanopore platforms, including the ON T sequencing platform of Oxford Nanopore, Polyseq of Puyi Biotechnology, and domestic sequencing platforms such as CycloneSeq of MGI. Perform the operation on the machine according to the operation manual of the nanopore sequencer.

[0072] 7) Bioinformatics analysis

[0073] Perform bioinformatics analysis on the data downloaded from the machine, use the polycystic kidney detection software to compare the database, and analyze the detection results.

[0074] Example 4 Detection cases

[0075] Using the detection primer sets, kits and detection methods provided in the above examples, more than 2000 mutation sites on the PKD1 and PKD2 genes, SNVs and indels on the exons of the PKD1 and PKD2 genes; deletions and duplications within the PKD1 and PKD2 genes can be detected simultaneously.

[0076] In one example, 20 positive samples were detected using the detection kit and detection method provided in the above examples, and the detection results are shown in Table 3. By comparing the analysis results, all primer amplification fragments were completely detected, and each fragment obtained more than 100×. Comparing with the second-generation NGS detection results, the mutation sites were detected completely consistently, but some structural variations were not detected by the second generation, while this scheme detected them. Later, it was verified by sanger sequencing and MLPA, and the results were consistent with this scheme. In the current conventional polycystic kidney disease detection, a comprehensive detection result cannot be obtained by one method, and often multiple methods such as LR-PCR + sanger sequencing, MLPA, and second-generation NGS need to be combined for detection, which is complex in operation, time-consuming, and costly. However, the detection method of the kit in the present invention only requires one experiment to simultaneously complete the detection of different types of mutations, is simple in operation, time-saving, low in cost, and does not require sample pooling.

[0077] Table 3:

[0078]

[0079]

[0080] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A primer set for amplifying PKD1 and PKD2, characterized in that, It includes a first primer set for amplifying the PKD1 gene and a second primer set for amplifying the PKD2 gene; the nucleotide sequences of the first primer set are shown as SEQ ID NO: 1-6, and the nucleotide sequences of the second primer set are shown as SEQ ID NO: 7-30.

2. Use of the primer set according to claim 1 in the preparation of a PKD1 and PKD2 gene mutation detection product or an autosomal dominant polycystic kidney disease diagnosis product.

3. A kit for detecting PKD1 and PKD2 gene mutations, characterized in that, It includes the primer set according to claim 1.

4. The kit according to claim 3, characterized in that, It further includes at least one of a nucleic acid extraction reagent, a multiplex PCR reaction reagent, a common sequence, a barcode ligation PCR reaction reagent, and a nanopore library construction reagent.

5. The kit according to claim 4, wherein The multiplex PCR reaction reagent includes a DNA polymerase and a buffer.

6. The kit according to claim 5, wherein, The DNA polymerase is TaKaRa LA Taq polymerase; and / or, the buffer includes Tris-HCl with a concentration of 10-100 mM, glycerol with a content of 5-20 wt%, DTT with a concentration of 1-5 mM, KCl with a concentration of 30-50 mM, MgCl2 with a concentration of 2-8 mM, dNTP with a concentration of 0.1-0.5 mM, DMSO with a content of 5-15 wt%, and betaine with a final concentration of 0.5-1.5 M.

7. A method for amplifying all exons of PKD1 and PKD2 genes, characterized in that, The method includes the step of performing PCR amplification using the primer set according to claim 1.

8. Use of the primer set according to claim 1 or the kit according to any one of claims 3-6 in the detection of PKD1 and PKD2 gene mutations, and the use is for non-diagnostic purposes.

9. A method for detecting PKD1 and PKD2 gene mutations, for non-diagnostic purposes, characterized in that, It includes amplifying the PKD1 and PKD2 genes of a sample to be tested using the primer set according to claim 1, then sequencing the amplicons, and comparing the sequencing results with a reference genome to further determine the PKD1 and PKD2 gene mutation results.

10. The method according to claim 9, wherein The sequencing is based on the nanopore sequencing method.

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