CN114480603A - Reagent composition for detecting autosomal dominant hereditary polycystic kidney disease and detection method - Google Patents
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Abstract
A reagent composition for detecting autosomal dominant hereditary polycystic kidney disease comprises a primer group; a first reagent for extracting genomic DNA from a sample; a second reagent for performing a long-fragment PCR reaction using the primer set; a third reagent for processing the amplification product to enable the amplification product to be used in a high throughput sequencing technique; and a fourth reagent for high throughput sequencing of the treated amplification product. The method comprises the following steps: s1: amplifying the PKD1 gene by long-fragment PCR using the reagent composition of any one of claims 1 to 7; s2: performing high-throughput sequencing on the amplified sequence obtained in the step (1); s3: comparing the sequencing result obtained in the step (2) with a PKD1 gene reference sequence to determine a mutation site; s4: eliminating false gene locus interference through bioinformatics analysis, and determining a PKD1 true gene mutation locus; s5: comparing the true gene mutation site determined in step S4 with the known mutation site of PKD1 gene, and determining the new mutation site on PKD1 gene.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a reagent composition for detecting autosomal dominant hereditary polycystic kidney disease.
Background
The kidney is an organ that excretes various substances metabolized in the body and thus present in the blood, and particularly, filters the blood by glomerular filtration and tubular absorption and reabsorption, thereby discharging unnecessary waste products in the body in the form of urine. Kidney weight is only about 0.5% of body weight, but blood flow into the kidney is 20% to 25% of total cardiac output. Thus, the kidney is one of the most fragile organs due to the presence of a number of toxic xenobiotics in the blood. For example, when an excessive amount of drug is injected into the body, or various types of metabolic diseases such as heart disease, diabetes, and hypertension occur, the kidney may be very slowly damaged, and eventually the kidney function is stopped without subjective symptoms, e.g., toxic xenobiotics are not excreted outside the body, thereby causing additional complications.
Autosomal dominant polycystic kidney disease (APKD, also known as adult polycystic kidney disease) is a common congenital genetic disease, adult polycystic kidney disease is often found in middle-aged and middle-aged years, and can be developed at any age, and is a common hereditary polycystic kidney disease, the incidence rate is about 1/1,000-1/400, and accounts for about 5% of terminal kidney diseases. The disease slowly forms cysts and swells in the kidney during middle-aged years, eventually leading to renal failure. The diagnosis of autosomal dominant polycystic kidney depends mainly on imaging or molecular genetic testing. For imaging tests, sensitivity of the test was close to 100% in patients over 30 years of age or in younger patients with PKD1 mutations; while PKD2 mutant younger patients under the age of 30 were examined for sensitivity of only 67%. No obvious cysts were visible in the infant or child on the imaging examination. The patients with autosomal dominant polycystic kidney have gene mutation already in fetal stage and/or birth, but have polycystic kidney disease expression later in life, and many patients are missed before 30 years old because the B-suprajunction fruit is normal. Therefore, molecular genetic detection such as linkage analysis or direct mutation screening has clinical value, and early diagnosis of adult polycystic kidney disease through gene detection becomes one of new research hotspots. In addition, prenatal gene screening of fetal polycystic kidney disease can be used for early diagnosis, and helps to treat complications early.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a reagent composition for detecting autosomal dominant polycystic kidney disease, so as to rapidly obtain the mutation of a PKD1 true gene and ensure the specificity of detection.
The specific scheme is as follows:
a reagent composition for detecting autosomal dominant hereditary polycystic kidney disease comprises a primer group; a first reagent for extracting genomic DNA from a sample; a second reagent for performing a long-fragment PCR reaction using the primer set; a third reagent for processing the amplification product to enable the amplification product to be used in a high throughput sequencing technique; and a fourth reagent for high throughput sequencing of the treated amplification product.
Further, the first reagent comprises:
a first buffer solution: 0.2M EDTA2Na, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH2PO4 and 0.1M Na2HPO4, adjusting the pH to 7.5, carrying out volume fixing with deionized water to 1L, and sterilizing at high temperature and high pressure;
30mg/ml lysozyme: 20mM Tris-HCl, 2mM EDTA, 1.2% volume fraction Triton x-100;
10wt％CTAB；
a second buffer solution: weighing 0.5g CTAB, and fixing the volume to 50ml by using a first buffer solution;
20% anionic surfactant: weighing 5g of sodium stearate and 5g of sodium dodecyl sulfate, metering the volume to 50ml with sterile water, heating in a water bath at 68 ℃ to dissolve, and adjusting the pH to 7.5;
phenol: chloroform: isoamyl alcohol (25: 24: 1);
chloroform: isoamyl alcohol (24: 1);
pre-cooling with isopropanol (4 ℃), and adding 70% ethanol;
RNase-free water；
OMEGA Bacterial DNA Kit；
proteinase K20 mg/ml.
Further, the primer group comprises primers for amplifying exons 2-7 of the PKD1 gene, and a forward primer and a reverse primer of the primer are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2;
the primers for amplifying exons 8-12 of the PKD1 gene comprise a forward primer and a reverse primer which are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4;
the primers for amplifying exons 13-21 of the PKD1 gene comprise a forward primer and a reverse primer which are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6;
the primers for amplifying exons 22-34 of the PKD1 gene comprise a forward primer and a reverse primer which are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8;
the forward primer and the reverse primer of the primer for amplifying exons 35-46 of the PKD1 gene are respectively shown as SEQ ID NO. 9 and SEQ ID NO. 10.
Further, the second reagent comprises template DNA, DNA polymerase, buffer solution and a dNTP mixture, wherein the DNA polymerase is DNA polymerase II.
Furthermore, the content of the DNA template is more than or equal to 100ng, 2 mul of each of the upstream primer and the downstream primer, 0.5 mul of DNA polymerase II, 5 mul of 10 Xbuffer II and 8 mul of dNTP mixture.
The invention provides a method for detecting an autosomal dominant hereditary polycystic kidney disease gene, which comprises the following steps:
s1: amplifying the PKD1 gene by long-fragment PCR using the reagent composition of any one of claims 1 to 7;
s2: performing high-throughput sequencing on the amplified sequence obtained in the step (1);
s3: comparing the sequencing result obtained in the step (2) with a PKD1 gene reference sequence to determine a mutation site;
s4: eliminating false gene locus interference through bioinformatics analysis, and determining a PKD1 true gene mutation locus;
s5: comparing the true gene mutation site determined in step S4 with the known mutation site of PKD1 gene, and determining the new mutation site on PKD1 gene.
Further, the long fragment amplification reaction condition in the step S1 is preferably 98 ℃ for 1min, then 10 cycles each of 98 ℃ for 10S and 68 ℃ for 10min, then 20 cycles each of 98 ℃ for 10S and 68 ℃ for 10min for 20S, then 72 ℃ for 10min, and then 4 ℃ until use; and/or wherein the high throughput sequencing is preferably Ion Torrent sequencing.
Compared with the prior art, the invention has the beneficial effects that:
the invention can detect all the mutations of the No. 2-46 exon region on the PKD1 gene, the experimental method is simple, and the operator can be familiar with the process quickly and realize high-efficiency detection; solves the clinical problems and can be applied in large-scale industrialization. The detection result not only can assist in diagnosing adult polycystic kidney disease, but also can obtain a plurality of previously unknown new mutations on the PKD1 true gene and provide the new mutations for doctors or researchers to study the relevance between the mutations and the adult polycystic kidney disease.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the scope of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
Long-fragment PCR and high-throughput sequencing detection of PKD1 gene mutations using the true gene-specific primers (E2, T3, K3, R5, K5) from exon regions 2-46 of the PKD1 gene long-fragment PCR was used in combination with high-throughput sequencing to detect PKD1 gene mutations in 70 subjects with informed consent of the subjects.
Extracting the genomic DNA of the peripheral blood of a detected person by adopting an OMEGA genomic DNA extraction kit (purchased from OMEGA, USA), and extracting the genomic DNA by adopting a first reagent; the extracted DNA is detected by a spectrophotometer or other detection instruments to obtain the DNA concentration and purity, wherein the DNA concentration is more than 50 ng/mu l, the volume is more than 30 mu l, and A260/A280 is between 1.6 and 2.0 and is used as a DNA template.
A DNA template from each subject was aliquoted into five separate reaction tubes, one of the long-fragment PCR primers E2, T3, K3, R5 and K5 (the sequence is shown in Table 1) was added into each of the five reaction tubes, the reagents were added according to the reaction system shown in Table 2, and the reaction was carried out in a PTC-200PCR apparatus (Bio-Rad Co.) under the following conditions: 1min at 98 ℃, then 10 cycles of 10s at 98 ℃ and 10min at 68 ℃, then 20 cycles of 10s at 98 ℃ and 10min at 68 ℃, then 10min at 72 ℃ and then 4 ℃ until use; wherein the high throughput sequencing is preferably Ion Torrent sequencing; long-fragment PCR reactions for different exon regions for multiple subject samples can be performed in the same PCR instrument under the same reaction conditions.
TABLE 1 primer sequences
TABLE 2 reaction System
After the amplification is finished, carrying out agarose gel electrophoresis on the amplification product, wherein the gel concentration is 0.8%, spotting 5 mu 1PCR products, detecting whether the amplification reaches the target length, and the result shows that the gel cutting recovery and purification are carried out after a single strip is detected, wherein the electrophoresis images of the amplification products of the No. 2-46 exon regions of the PKD l genes of 4 samples are shown, wherein lanes 2, 8, 14 and 20 are the results of amplifying the No. 2-7 exon regions by using E2 primers and the size is 4041 bp; 3. lanes 9, 15 and 21 show the results of amplification of the 8-12 exon region with T3 primer, which is 4200bp in size; 4. lanes 10, 16 and 22 show the results of amplification of the 12-21 exon regions with the primer K3, and the size is 7800 bp; 5. lanes 11, 17 and 23 show the results of amplification of the exon regions 22-34 with the primer R5, with a size of 7503 bp; 6. lanes 13, 18 and 24 show the results of amplification of the 35-46 exon region with K5 primer, with size 5200 bp; the l lane M is 15Kb Marker, and the fragment length is 500bp, l000bp, 1500bp, 3000bp, 5000bp, 7500bp, l0000bp, and 15000bp sequentially from bottom to top.
Length of epiblast region fragments from Table 2-46
| Segment of interest | Length (kb) |
| 2-7 number episcope region | 4 |
| Number 8-12 episcope region | 4.2 |
| Number 13-21 episcope region | 7.8 |
| Number 22-34 episcope region | 7.5 |
| Number 35-46 episcope region | 5.2 |
The building blocks of the library and high throughput sequencing and analysis were then performed according to prior art kits.
Comparing the PKD1 gene mutation sites analyzed above with the dbSNP138 database, and determining which mutation sites are reported mutation sites and which mutation sites are new mutation sites.
Through multiple sample verification, the method can determine the new mutation site, and has high accuracy.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
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Claims (7)
1. A reagent composition for detecting autosomal dominant hereditary polycystic kidney disease, which is characterized by comprising a primer set; a first reagent for extracting genomic DNA from a sample; a second reagent for performing a long-fragment PCR reaction using the primer set; a third reagent for processing the amplification product to enable the amplification product to be used in a high throughput sequencing technique; and a fourth reagent for high throughput sequencing of the treated amplification product.
2. The reagent composition for detecting autosomal dominant polycystic kidney disease according to claim 1, wherein said first reagent comprises:
a first buffer solution: 0.2M EDTA2Na, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH2PO4 and 0.1M Na2HPO4, adjusting pH to 7.5, diluting with deionized water to 1L, and sterilizing at high temperature and high pressure;
30mg/ml lysozyme: 20mM Tris-HCl, 2mM EDTA, 1.2% volume fraction Triton x-100; 10 wt% CTAB;
a second buffer solution: weighing 0.5g CTAB, and fixing the volume to 50ml by using a first buffer solution;
20% anionic surfactant: weighing 5g of sodium stearate and 5g of sodium dodecyl sulfate, metering the volume to 50ml with sterile water, heating in a water bath at 68 ℃ to dissolve, and adjusting the pH to 7.5;
phenol: chloroform: isoamyl alcohol (25: 24: 1);
chloroform: isoamyl alcohol (24: 1);
pre-cooling with isopropanol (4 ℃), and adding 70% ethanol;
RNase-free water；
OMEGA Bacterial DNA Kit；
proteinase K20 mg/ml.
3. The reagent composition for detecting the autosomal dominant polycystic kidney disease according to claim 1, wherein the primer set comprises primers for amplifying exons 2-7 of PKD1 gene, and the forward primer and the reverse primer are shown as SEQ ID NO. 1 and SEQ ID NO. 2, respectively;
the primers for amplifying exons 8-12 of the PKD1 gene comprise a forward primer and a reverse primer which are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4;
the primers for amplifying exons 13-21 of the PKD1 gene comprise a forward primer and a reverse primer which are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6;
the primers for amplifying exons 22-34 of the PKD1 gene comprise a forward primer and a reverse primer which are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8;
the forward primer and the reverse primer of the primer for amplifying exons 35-46 of the PKD1 gene are respectively shown as SEQ ID NO. 9 and SEQ ID NO. 10.
4. The reagent composition for detecting autosomal dominant polycystic kidney disease according to claim 1, wherein said second reagent comprises a template DNA, a DNA polymerase, a buffer and a dNTP mixture, wherein the DNA polymerase is DNA polymerase ii.
5. The reagent composition for detecting autosomal dominant polycystic kidney disease according to claim 4, wherein the amount of said DNA template is not less than 100ng, 2. mu.l each of the upstream and downstream primers, 0.5. mu.l of DNA polymerase II, 5. mu.l of 10 XBuffer II, and 8. mu.l of dNTP mixture.
6. The method for detecting the autosomal dominant hereditary polycystic kidney disease gene is characterized by comprising the following steps of:
s1: amplifying the PKD1 gene by long-fragment PCR using the reagent composition of any one of claims 1 to 7;
s2: performing high-throughput sequencing on the amplified sequence obtained in the step (1);
s3: comparing the sequencing result obtained in the step (2) with a PKD1 gene reference sequence to determine a mutation site;
s4: eliminating false gene locus interference through bioinformatics analysis, and determining a PKD1 true gene mutation locus;
s5: comparing the true gene mutation site determined in step S4 with the known mutation site of PKD1 gene, and determining the new mutation site on PKD1 gene.
7. The method for detecting an autosomal dominant hereditary polycystic kidney disease gene according to claim 6, wherein the long fragment amplification reaction condition in step S1 is preferably 98 ℃ for 1min, followed by 10 cycles of 98 ℃ for 10S and 68 ℃ for 10min, followed by 20 cycles of 98 ℃ for 10S and 68 ℃ for 10min for 20S, followed by 72 ℃ for 10min, and then left at 4 ℃ until use; and/or wherein the high throughput sequencing is preferably Ion Torrent sequencing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116536325A (en) * | 2023-03-29 | 2023-08-04 | 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） | Mutant of PKD1 gene and application thereof |
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2020
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116536325A (en) * | 2023-03-29 | 2023-08-04 | 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） | Mutant of PKD1 gene and application thereof |
| CN116536325B (en) * | 2023-03-29 | 2023-10-24 | 青岛市妇女儿童医院（青岛市妇幼保健院、青岛市残疾儿童医疗康复中心、青岛市新生儿疾病筛查中心） | Mutant of PKD1 gene and application thereof |
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