CN117143997A

CN117143997A - Primer set, kit and detection method for PKD1 gene mutation detection

Info

Publication number: CN117143997A
Application number: CN202311423460.0A
Authority: CN
Inventors: 王姣姣; 冯韶华; 费嘉; 樊海洋; 汤曼
Original assignee: Beijing Jiabao Renhe Medical Technology Co ltd; Beijing Zhongyi Kangwei Medical Devices Co ltd
Current assignee: Beijing Jiabao Renhe Medical Technology Co ltd; Beijing Zhongyi Kangwei Medical Devices Co ltd
Priority date: 2023-10-31
Filing date: 2023-10-31
Publication date: 2023-12-01
Anticipated expiration: 2043-10-31
Also published as: CN117143997B

Abstract

The invention discloses a primer group, a kit and a detection method for PKD1 gene mutation detection, wherein the primer group comprises 6 pairs of primers for amplifying the whole region of a PKD1 gene and the total range of the upper and lower streams of the region is about 15kb, and the amplified products of each pair of primers have an overlapping region of more than 10 kb. According to the invention, a group of primers are designed to successfully amplify the whole PKD1 gene, PCR amplification is not required in the library construction process, the third generation sequencing technology is utilized to successfully construct the PKD1 gene haplotype and detect mutation in the PKD1 gene, and other family samples are not required in the carrier haplotype construction process; the detection result of the invention can be comparable to that of a sample for WGS sequencing, thereby greatly reducing the detection cost and the detection period and being more suitable for clinical detection service.

Description

Primer set, kit and detection method for PKD1 gene mutation detection

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group, a kit and a detection method for PKD1 gene mutation detection.

Background

Adult polycystic kidney disease (polycystic kidney disease, PKD) is a common hereditary kidney disease, mostly autosomal dominant inheritance. Patients usually develop diseases after adulthood, and the incidence rate is estimated to be 1/400-1/1000. Autosomal dominant hereditary polycystic kidney disease (autosomal dominant polycystic kidney disease, ADPKD) is the most common hereditary kidney disease, and genetic studies indicate that the occurrence of the disease is mainly caused by protein kinase D1 (PKD 1) or protein kinase D2 (PKD 2) mutation, wherein PKD1 is type i polycystic kidney disease, accounting for about 80% -85% of cases; PKD2 is type II polycystic kidney disease, accounting for about 10% -15% of cases. ADPKD is characterized by the onset of disease at any age, and is frequently occurring in 30-50 years old, has autosomal dominant inheritance characteristics, is developed instead of generation, and clinical abnormalities caused by gene defects are accompanied by symptoms such as liver cyst, pancreatic cyst, ovarian cyst, vascular system, malformation of heart valve structure and the like besides accumulated kidneys. About 1200 tens of thousands of patients worldwide are deeply affected by the disease, and about 50% of patients develop final-stage renal failure, requiring life support by allogeneic kidney transplantation or life-long hemodialysis treatment. While about 150 tens of thousands of people in our country are afflicted with the disease. ADPKD not only creates serious physical and mental distraction to the patient, but also places a heavy economic burden on the patient's home. Therefore, the gene detection of the patient can determine the cause of the disease, the prognosis is directly related, the prenatal diagnosis result is provided, and a guarantee is enhanced for the healthy infant.

PKD1 gene was located in 3 rd sub-band (16p13.3) of short arm 1 region of human chromosome 16, and the total length of the gene was about 47kb, which was 46 exons in total, encoding a transmembrane glycoprotein composed of 4302 amino acid residues, polycystin 1 (PC 1). 6 pseudogenes homologous to the PKD1 gene exist on chromosome 16, the homology of the pseudogenes with the 1-33 exons of the genes is up to 97.7%, it is reported that about 80% of pathogenic mutations occur in the region, and the GC content of partial DNA sequences in the region is up to 85%, so that a certain difficulty exists in gene amplification and verification of mutation sites is also difficult. In addition, PKD1 genes are more mutated and have no mutation hot spots, involving the whole gene, so specific detection of PKD1 genes is very challenging.

Due to the results of the PKD1 gene itself and the mutation type, detection methods for PKD1 genes are continuously innovated with the continuous development of new technologies. The existing methods for detecting PKD1 include LR-PCR, denaturing high performance liquid chromatography, LR-PCR combined high-throughput sequencing, exon capturing-high-throughput sequencing method and the like. The denaturing high performance liquid chromatography technology is used for a PKD1 gene mutation screening method, and although mutation detection rate can be improved to a certain extent, instability exists in detection results and accuracy of the results is also required to be improved. The current classical method for detecting PKD1 gene mutation is nested PCR-Sanger sequencing, the method needs to amplify target genes through LR-PCR, then target amplification sequencing is carried out again aiming at mutation sites, and then analysis results are obtained. Zhang Shuzhong et al amplify PKD1 full coding region by combining LR-PCR and nested PCR amplification to determine specific sites and types of mutation, and primers used in the research are up to dozens of groups, and although the result is accurate, the operation is very complicated, the denaturation high performance liquid chromatography technology is required to be combined, the detection cost is high, and the detection efficiency is required to be improved. Further TW201339309A discloses a primer set and a kit for detecting PKD1 gene mutation, which have similar problems as those of Zhang Shuzhong et al. Xiao Pan et al specifically amplified the 12, 14, 21 exon fragments of PKD1 gene by PCR, had limited detection regions, did not have variation in detecting the entire gene region of PKD1, and had certain limitations. CN104531883a discloses a kit and a detection method for detecting mutation of PKD1 gene, wherein long fragment amplicon is amplified by 5-8 to amplify exon 2-46 of PKD1 gene, and then NGS is used for detection, and although the method can detect samples rapidly and efficiently, the detection of high GC region of PKD1 gene still has defects due to GC preference of the technical method.

Disclosure of Invention

The invention aims to overcome the technical defects of the background technology and provides a primer group, a kit and a detection method for PKD1 gene mutation detection. According to the invention, a group of primers are designed to successfully amplify the whole PKD1 gene, PCR amplification is not required in the library construction process, the third generation sequencing technology is utilized to successfully construct the PKD1 gene haplotype and detect mutation in the PKD1 gene, and other family samples are not required in the carrier haplotype construction process; the detection result of the invention can be comparable to that of a sample for WGS sequencing, thereby greatly reducing the detection cost and the detection period and being more suitable for clinical detection service.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a primer set for detecting PKD1 gene mutation, wherein the primer set comprises 6 pairs of primers for amplifying the whole region of PKD1 gene, and the amplified products of each pair of primers have an overlapping region of more than 10 kb;

the sequences of the 6 pairs of primers are as follows (hg 19 reference genome):

for amplification of PKD1 gene chr16:2130793-2144241, the forward primer sequence is shown as SEQ ID NO. 1, and the reverse primer sequence is shown as SEQ ID NO. 2;

for amplification of PKD1 gene chr16:2134219-2154799, the forward primer sequence of which is shown as SEQ ID NO. 3, and the reverse primer sequence of which is shown as SEQ ID NO. 4;

for amplification of PKD1 gene chr16:2144661-2166375, the forward primer sequence of which is shown as SEQ ID NO. 5, and the reverse primer sequence of which is shown as SEQ ID NO. 6;

for amplification of PKD1 gene chr16:2151918-2173659, the forward primer sequence of which is shown as SEQ ID NO. 7, and the reverse primer sequence of which is shown as SEQ ID NO. 8;

for amplification of PKD1 gene chr16:2158065-2178648, the forward primer sequence of which is shown as SEQ ID NO. 9, and the reverse primer sequence of which is shown as SEQ ID NO. 10;

for amplification of PKD1 gene chr16:2171520-21918496, the forward primer sequence of which is shown as SEQ ID NO. 11, and the reverse primer sequence of which is shown as SEQ ID NO. 12.

The sequences of the 6 pairs of primers are specifically shown in table 1:

TABLE 1 primer sequences for amplifying PKD1 Gene by LR-PCR according to the present invention

Note that: in the table, F is a forward primer (upstream primer), and R is a reverse primer (downstream primer); the PCR primers in the table are in turn from top to bottom: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12.

Preferably, the primer set is used to amplify the entire region of the PKD1 gene, and is within about 15kb of the region upstream and downstream thereof.

A kit for detecting PKD1 gene mutation, the kit being used for PKD1 gene mutation detection and PKD1 gene haplotype construction, comprising the primer set for PKD1 gene mutation detection as described above, wherein the concentration of each primer in the primer set is preferably 10 μm.

Preferably, the kit comprises PCR reaction reagents for amplifying a long fragment sequence of PKD1 gene, and/or reagents for extracting genomic DNA from a sample and reagents for third generation Nanopore sequencing of the treated PCR products.

Preferably, the PCR reaction reagents for amplifying the long fragment sequence of the PKD1 gene include 2 x Phanta Flash Master Mix, which includes DNA polymerase, buffer, and dntps; the DNA polymerase is preferably a mixture of DNA polymerases with high fidelity, high yield, rapid extensibility, and suitability for use in high GC systems.

Preferably, the total PCR reaction system is 50. Mu.L, including 2X Phanta Flash Master Mix. Mu.L, 2. Mu.L of each of the upstream and downstream primers (concentration: 10. Mu. Mol/L), 2. Mu.L of the DNA template (DNA amount: 50 to 60 ng), 10. Mu.L of the PCR Enhancer, and the ultra-pure water to 50. Mu.L.

The above reagents are commercially available or formulated, and the commercial route and formulation methods are well known to those skilled in the art.

Preferably, the kit amplifies the entire region of the PKD1 gene and its upstream and downstream ranges of about 15kb by optimizing the reaction time and reaction temperature.

A method for in vitro detection of PKD1 gene mutations and construction of PKD1 gene haplotypes using the primer set or kit as described above for non-disease diagnostic purposes, comprising the steps of:

(1) Extracting genomic DNA from a peripheral blood sample of a subject;

(2) Using genome DNA as a template, setting optimized reaction time and reaction temperature by using the primer group for PKD1 gene mutation detection, wherein each pair of primers independently amplify the whole region of PKD1 gene and the upstream and downstream of the region of PKD1 gene by about 15 kb;

(3) Purifying and recycling the 6 PCR products obtained in the step (2);

(4) Measuring the concentration of the purified PCR product, and mixing the purified PCR products according to a certain proportion to obtain a PCR product mixture of 1 subject;

(5) Directly constructing a Nanopore library from the PCR product mixture of the subject, and performing ONT sequencing on a MinION chip to obtain the sequence of a long fragment PCR product;

(6) And analyzing the obtained PCR product data by Clair3 software, comparing the obtained sequence fragments with a reference PKD1 gene, extracting SNVs and Indels, and determining SNVs and haplotype conditions of the sample PKD1 gene.

Preferably, in the step (2), the reaction time and the reaction temperature are:

PCR cycle parameter setting: 98-30 s, and cycle number 1 cycle; denaturation at 98 ℃ for 10s, tm-20s, 72-6 min, cycle number of 20-60 cycles, preferably 35 cycles, extension at 72 ℃ for 5min; wherein the Tm of the amplicon primers 1, 3 and 5 is 60-70 ℃, preferably 64 ℃; 2. the Tm of the amplicon primers 4 and 6 is 60-70℃and preferably 66 ℃.

Compared with the prior art, the invention has the beneficial effects that:

(1) Aiming at the result characteristics and mutation types of PKD1 genes, the invention provides a primer group, a kit and a detection method for PKD1 gene mutation detection; the invention uses 6 pairs of long fragment amplicon primers to amplify the whole region of PKD1 gene, and each pair of primers has more than 10kb overlapping region between amplified products; the whole PKD1 gene and the upstream and downstream of the PKD1 gene are subjected to targeted amplification by using a long-fragment PCR technology, the purified PCR products are mixed according to a certain proportion and then are directly constructed into an ONT library, a Nanopore sequencer mk-1C is used for sequencing, and PKD1 gene haplotype information is obtained through bioinformatics analysis or PKD1 gene mutation information is directly detected;

(2) According to the invention, a group of primers are designed to successfully amplify the whole PKD1 gene, PCR amplification is not required in the library construction process, the third generation sequencing technology is utilized to successfully construct the PKD1 gene haplotype and detect mutation in the PKD1 gene, and other family samples are not required in the carrier haplotype construction process;

(3) The detection result of the invention can be comparable to that of a sample for WGS sequencing, thereby greatly reducing the detection cost and the detection period and being more suitable for clinical detection service.

Drawings

FIG. 1 is a schematic diagram showing the result of LR-PCR amplification of PKD1 gene long fragment amplicon in example 1 of the present invention; the agarose gel electrophoresis result shows that 6 amplicon products of PKD1 gene have single band, the fragment size 1 is 13449bp, and the other 5 amplicon fragments have sizes of 20.3-21.7 kb; wherein the 23kb Marker is a DNA Marker (lambda DNA/HindIII, solabio); the 15kb Marker is a DNA Marker (Trans 15K DNA Marker, full-size gold organism), lanes 1-6 are long fragment amplicons of LR-PCR amplified PKD1 genes 1-6 respectively;

FIG. 2 is a visual image of IGV sequencing of the 1-case clinical sample PKD1 gene Oxford Nanopore in example 1 of the present invention; the figure shows that the coverage of 6 amplicons (primer design positions: 2130793-2196637, hg 19) in all introns and exons of the PKD1 gene reaches 100%;

FIG. 3 is an IGV view showing the detection result of mutation sites of PKD 1-positive samples in example 1 of the present invention.

Detailed Description

For a better understanding of the present invention, reference will be made to the following description of specific embodiments and accompanying drawings. It is to be understood that these examples are provided only for further illustration of the present invention and are not intended to limit the scope of the present invention. It should be further understood that after reading the description of the present invention, those skilled in the art make some insubstantial changes or modifications to the present invention, which still fall within the scope of the present invention.

The following is a detailed description of 1 sample of three-generation whole genome sequencing performed in the early stage and 1 PKD1 positive patient. In specific embodiments, the invention uses long fragment PCR technology to target and amplify the whole PKD1 gene and the upstream and downstream of the PKD1 gene to about 15kb, mixes the purified PCR products according to a certain proportion, directly constructs ONT library, uses a Nanopore sequencer to sequence, obtains PKD1 gene haplotype information through bioinformatics analysis or directly detects PKD1 gene mutation information. The invention successfully amplifies the whole PKD1 gene by designing a group of primers, does not need to carry out PCR amplification in the library construction process, successfully constructs PKD1 gene haplotypes by utilizing the third-generation sequencing technology and detects mutation in the PKD1 gene, and does not need other family samples in the carrier haplotype construction process.

Example 1

1. Sample genomic DNA extraction

A sample of 0.2ml of peripheral blood from the subject was taken, and DNA was extracted according to the instructions of the blood genomic DNA extraction kit. The extracted DNA sample, which is the template for the next PCR reaction, was assayed with the Qubit reagent. In this example, 1 sample with three-generation whole genome sequencing performed earlier and 1 PKD 1-positive patient were combined for detection, and the pathogenic site of PKD 1-positive patient was of known mutation type Chr16:2160724 c.4444C>T.

2. LR-PCR amplification of PKD1 Gene

6 pairs of PKD1 gene amplification primers are designed and synthesized according to the PKD1 gene sequence. LR-PCR amplification was performed using Phanta Flash Master Mix to amplify 6 large fragment sequences, named amplicon numbers 1-6, respectively, with primer sequences and amplified product sizes shown in Table 1. The total PCR reaction system was 50. Mu.L, including 2X Phanta Flash Master Mix. Mu.L, 2. Mu.L of each of the upstream and downstream primers (concentration: 10. Mu. Mol/L), 2. Mu.L of the DNA template (DNA amount: 50 to 60 ng), 10. Mu.L of PCR Enhancer (Beijing nuozhen Biotech Co., ltd.) and ultrapure water to a 50. Mu.L system. PCR cycle parameter setting: 98-30 s, and cycle number 1 cycle; denaturation at 98℃for 10s, tm-20s,72℃to 6min, cycle number of 35 cycles, extension at 72℃for 5min. Wherein the Tm of amplicon primer nos. 1, 3 and 5 is 64 ℃; 2. the Tm of the amplicon primers 4 and 6 was 66 ℃.

After the LR-PCR reaction was completed, 5. Mu.L of the sample was removed and subjected to 1% agarose gel electrophoresis. The results showed that the fragment sizes of the 6 amplicons were consistent with the theoretical values of the design and that the bands were clear (see FIG. 1).

3. PCR product purification treatment

The PCR product obtained was purified by means of Agencourt XP magnetic beads (Beckman Coulter Co., U.S.A.), the amount of magnetic beads and the PCR system were mixed in equal amounts of 1:1, and the mixture was left at room temperature for 3min after shaking. And (3) placing the solution on a magnetic rack for adsorption after instantaneous centrifugation, and sucking and discarding the supernatant after the solution is clarified. Washing the magnetic beads with 80% ethanol for 2 times, absorbing and removing excessive ethanol, drying the magnetic beads, adding nuclease-free water for eluting, and measuring the concentration of the purified product with a Qubit 2.0 nucleic acid protein quantitative instrument.

Mixing the purified 6 amplicons according to a certain proportion, wherein the amount of DNA after mixing is not less than 1000ng.

4. Library construction

Library construction was performed using Ligation Sequencing Kit of Oxford Nanopore Technologies (SQK-LSK 110) as follows:

1. NDA end repair reaction

1) Dissolving the reagent on ice;

2) The reagents were added in Eppendorf tubes as follows:

3) Lightly mixing by using a pipetting gun, and lightly flicking and uniformly mixing by fingers if the integrity of the long fragments is required to be maintained;

4) Amplification was performed using a PCR apparatus at 20℃for 5min and 65℃for 5min;

5) Preparing magnetic beads, shaking and uniformly mixing, and balancing at room temperature for 30min;

6) Transferring the amplified samples into 1.5mL centrifuge tubes respectively;

7) Adding 60 mu L of resuspended magnetic beads into a sample, and mixing by a flick test tube;

8) Incubating for 5min at room temperature on a four-dimensional rotator;

9) 500 μl of 70% absolute ethanol solution was prepared for each sample;

10 Placing the sample on a magnetic rack, standing, sucking the supernatant, holding the sample on the magnetic rack, adding 200 mu L of 70% absolute ethanol solution, standing for 30s, discarding the ethanol, and repeating for one time (adding 200 mu L of 70% absolute ethanol solution, standing for 30s, discarding the ethanol);

11 Using a pipette to absorb the ethanol, and drying for 30s, so as to avoid drying until the magnetic beads are cracked;

12 Taking off the test tube from the magnetic rack, re-suspending the magnetic beads in 27 mu L NF water, and incubating for 2min at room temperature;

13 Placing the test tube on a magnetic rack until the eluent is clear;

14 27 μl of eluate was transferred to a new 1.5mL centrifuge tube;

15 1. Mu.L was taken out for quantification, and the target amount of the obtained sample was > 700ng (Qubit concentration 20 to 60 ng/. Mu.L).

2. barcode ligation reaction

1) Thawing Native barcode at room temperature, one for each sample;

2) 500ng of the samples after the end repair were dissolved in 22.5. Mu.L NF water;

3) Sequentially adding the reagents in the following table, flicking and uniformly mixing;

4) Lightly mixing by using a pipetting gun, and lightly flicking and uniformly mixing by fingers if the integrity of the long fragments is required to be maintained;

5) Incubating for 10min at room temperature on a four-dimensional rotator;

6) Adding 50 mu L of resuspended magnetic beads into a sample, and mixing by a flick test tube;

7) Incubating for 5min at room temperature on a four-dimensional rotator;

8) 500 μl of 70% absolute ethanol solution was prepared for each sample;

9) Placing the sample on a magnetic rack, standing, sucking the supernatant, holding the sample on the magnetic rack, adding 200 μL of 70% absolute ethanol solution, standing for 30S, discarding ethanol, and repeating for one time (adding 200 μL of 70% absolute ethanol solution, standing for 30S, discarding ethanol);

10 Using a pipette to absorb the ethanol, and drying for 30s, so as to avoid drying until the magnetic beads are cracked;

11 Taking off the test tube from the magnetic rack, re-suspending the magnetic beads in 28 mu L NF water, and incubating for 2min at room temperature;

12 Placing the test tube on a magnetic rack until the eluent is clear;

13 28. Mu.L of eluate was transferred to a new 1.5mL centrifuge tube;

14 Taking out 1 mu L for quantification (the Qubit concentration is 5-15 ng/. Mu.L);

15 Mixing equimolar amounts (technical support recommendations) of samples added with different barcode into a 1.5mL centrifuge tube to make the combined sample amount reach 700ng;

16 700ng of sample was diluted into 65 μl NF water;

17 If the mixed sample volume of 700ng exceeds 65. Mu.L, the mixed sample is purified with 2.5 times the magnetic beads and the product is eluted with 65. Mu.L NF water.

3. Adapter ligation reaction

1) Vortex melting EB, nebnet Quick Ligation Reaction Buffer (5×), vortex T4 livese and AM ii at room temperature, and place on ice for use;

2) The reagents were added according to the following table system:

3) Flick test tube and vortex, incubate for 10min at room temperature on a four-dimensional rotator;

4) Preparing magnetic beads, shaking and uniformly mixing, and balancing at room temperature for 30min;

5) Adding 50 mu L of resuspended magnetic beads into a sample, and mixing by a flick test tube;

6) Incubating for 5min at room temperature on a four-dimensional rotator;

7) Placing a sample on a magnetic rack, standing, sucking out supernatant, keeping the sample on the magnetic rack, and cleaning by using 250 mu L of LFB;

8) Removing the supernatant by using a pipetting gun, taking the sample out of a magnetic rack, adding 250 mu L of LFB, placing the sample in the magnetic rack after the magnetic beads are resuspended by flicking, and sucking the supernatant after standing;

9) Repeating step 8) once;

10 Drying for 30s, taking the sample out of the magnetic rack, re-suspending by using 15 mu L of EB, incubating for 10min at room temperature, and culturing for 10min at 37 ℃ in a metal bath for high molecular weight DNA to improve the recovery rate of long fragments;

11 Placing the sample on a magnetic rack, standing until the eluent is clear, and transferring 15 mu L of the eluent into a 1.5mL centrifuge tube;

12 1. Mu.L was taken out and quantified, and the target amount of the sample was about 430ng (Qubit concentration 5 to 30 ng/. Mu.L).

5. Oxford Nanopore sequencing

The following reagents were added to a new 1.5mL centrifuge tube and vortexed to mix well, i.e., the on-machine sample:

the on-machine operation performed sequencing operations according to the Oxford Nanopore sequencer instructions. The sequencer adopts a Oxford Nanopore MinION Mk C single-molecule real-time sequencing system, and the Minion cell contains 512 nanopore channels, so that each DNA sample can be subjected to single-molecule real-time sequencing, and the DNA sequence is converted into an electric signal.

6. Analysis of results

The nano ultra-long read length original machine-down data is in a fast5 format, the fast5 format is converted into a fastq format after being subjected to baserolling, then the fastq data is compared to a GRCh37/hg19 reference genome by using minimum 2 software, and the variation detection and typing are carried out on the bam generated by the minimum 2 by using Clair3 software. Finally, IGV software is used to view the results and map the correlations.

1 sample LR-PCR tested for WGS data SNP and Indels results were detected and haplotype construction results were compared to WGS analysis results. The clinical PKD1 sample obtains SNP, indels locus and haplotype conditions on the PKD1 gene of the patient through data analysis, then the accuracy of SNP detection of the patient is verified through nested PCR, and the accuracy of haplotype construction is verified by detecting parents and parents of the patient.

Analysis of results:

the PKD1 gene was amplified by LR-PCR protocol, and FIG. 1 shows the single case of amplified fragment size and band for each amplicon. As can be seen from FIG. 1, the band of each amplicon is single and the fragment size is consistent with the set range. According to the invention, 1 positive PKD1 patient samples are detected, and as shown in FIG. 2, the designed long fragment amplicon can completely cover the whole PKD1 gene region through IGV observation, and the sequencing depth of the lowest region is also more than 30X. In addition, the pathogenic site of the patient (as shown in fig. 3) can be successfully detected by the LR-PCR method, the PKD1 haplotype of the patient is constructed, and the accuracy of haplotype construction is verified by detecting both parents of the patient by nested PCR.

Table 2 shows the haplotype information table of the patient and the SNP detection at the corresponding sites of the parents through nested PCR verification. In addition, as shown in Table 3, the haplotype construction results and LR-PCR results of the non-PKD 1 positive sample data of 1 example of WGS were compared, and as a result, it was found that LR-PCR had false positive Indels detection compared with WGS, mainly because the existence of PolyA and PolyT structures in the gene structure resulted in false Indels detection, and the accuracy of other SNP and Indels detection and haplotype construction were consistent with the WGS results.

Table 2 shows the construction results of haplotypes of PKD 1-positive samples in example 1 of the invention

TABLE 3 comparison of WGS vs. LR-PCR haplotype construction results in example 1 of the invention

Note that: the bolded sites represent the LR-PCR multiple detection sites.

The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.

Claims

1. A primer set for detecting mutation of PKD1 gene, wherein the primer set comprises 6 pairs of primers, and the sequences of the 6 pairs of primers are as follows:

2. The primer set for detecting mutation of PKD1 gene according to claim 1, wherein the primer set is used for amplifying the entire region of PKD1 gene and the upstream and downstream thereof are in the range of about 15 kb.

3. A kit for detecting a mutation in a PKD1 gene, comprising the primer set for detecting a mutation in a PKD1 gene according to claim 1.

4. A kit for PKD1 gene mutation detection according to claim 3, wherein each primer in the primer set has a concentration of 10 μm.

5. A kit for the detection of mutations in the PKD1 gene according to claim 3, wherein the kit comprises PCR reagents for amplifying the long fragment sequence of the PKD1 gene, and/or reagents for extracting genomic DNA from a sample and reagents for third generation Nanopore sequencing of the treated PCR products.

6. The kit for detecting a mutation in a PKD1 gene according to claim 5, wherein the PCR reaction reagent for amplifying a long fragment sequence of the PKD1 gene comprises 2X Phanta Flash Master Mix, which comprises a DNA polymerase, a buffer and dNTPs.

7. The kit for PKD1 gene mutation detection according to claim 6, wherein the total PCR reaction system is 50. Mu.L, comprising 2X Phanta Flash Master Mix. Mu.L of each of the upstream and downstream primers, the concentration of each primer is 10. Mu. Mol/L, DNA template 2. Mu. L, DNA, the amount of each primer is 50-60 ng, the PCR enhancement is 10. Mu.L, and the ultrapure water is made up to 50. Mu.L.

8. The kit for detecting PKD1 gene mutation according to claim 6, wherein the DNA polymerase is a DNA polymerase mixture having high fidelity, high productivity, rapid extensibility and suitability for high GC system.

9. A method for in vitro detection of mutations in the PKD1 gene for non-disease diagnostic purposes, comprising the steps of:

(1) Extracting genomic DNA from a peripheral blood sample of a subject;

(2) Setting optimized reaction time and reaction temperature by using the primer set for PKD1 gene mutation detection as claimed in claim 1 with genomic DNA as a template, wherein each primer pair individually amplifies the entire region of PKD1 gene and the total of about 15kb range;

(3) Purifying and recycling the 6 PCR products obtained in the step (2);

10. The method for in vitro detection of PKD1 gene mutations for non-disease diagnostic purposes according to claim 9, wherein in step (2), the reaction time and reaction temperature are:

PCR cycle parameter setting: 98-30 s, and cycle number 1 cycle; denaturation at 98 ℃ for 10s, tm-20s, 72-6 min, cycle number of 20-60 cycles, and extension at 72 ℃ for 5min; wherein the Tm of the amplicon primers 1, 3 and 5 is 60-70 ℃; 2. the Tm of the No. 4 and No. 6 amplicon primers is 60-70 ℃.