CN110564838A - Multiplex PCR primer system for neonatal glycogen accumulation disease genotyping and application thereof - Google Patents
Multiplex PCR primer system for neonatal glycogen accumulation disease genotyping and application thereof Download PDFInfo
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Abstract
The invention discloses a primer pool for detecting the genotyping of neonatal glycogen storage disease, which is characterized by comprising a plurality of primer pairs for specifically amplifying a gene targeting sequence related to the neonatal glycogen storage disease; the neonatal glycogen accumulation disease related gene targeting sequence is at least one to-be-detected fragment selected from PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC and GAA gene exon region sequence. The kit can be used for accurately genotyping the glycogen storage disease and is used for researching and diagnosing the glycogen storage disease caused by the gene defect.
Description
Technical Field
The invention belongs to the technical field of genotyping and high-throughput screening, and particularly relates to an accurate detection method for neonatal glycogen storage disease genotyping, a kit and application.
Background
Glycogen storage diseases are a group of glycogen metabolism disorders caused by congenital enzyme defects, wherein liver glycogen storage disease is a common type of glycogen storage disease, and the glycogen storage disease is mostly autosomal recessive inheritance. Excessive accumulation of glycogen or heterotype glycogen in various tissue organs, which is caused by glycogen catabolic enzyme deficiency or synthesis disorder, occurs, and the organs mainly affected are liver, kidney, muscle, brain, small intestine, etc.
Glycogen is stored mainly in the liver and muscle. Under normal conditions, glycogen is gradually degraded into glucose under the action of glycogen phosphorylase, glycogen debranching enzyme, phosphoglucomutase and glucose-6-phosphatase in liver to provide blood sugar; in muscle, glycogen degradation produces glucose-6-phosphate which cannot be hydrolyzed to glucose, but enters the glycolytic pathway for further metabolism.
The types of glycogen accumulation diseases caused by different enzyme deficiencies, which lead to glycogen synthesis and metabolic disorders, are also different, and the corresponding enzyme gene deficiencies and the corresponding types of glycogen accumulation diseases are shown in Table 1.
Table 1: genes corresponding to different types of glycogen storage diseases
How to provide a method for accurately determining the genotype of the glycogen storage disease of the newborn is an urgent problem to be solved.
Disclosure of Invention
The invention relates to a method for carrying out PCR enrichment amplification and sequencing on exon regions of 19 disease-related genes such as PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA genes and the like through a targeted amplification technology and a high-throughput sequencing method, and bioinformatics analysis on possible gene mutation so as to judge the type of the disease.
The invention mainly aims to provide a primer pool for detecting the genotyping of the glycogen storage disease of the newborn, which is characterized by comprising a plurality of primer pairs for specifically amplifying a target sequence of a gene related to the glycogen storage disease of the newborn; the neonatal glycogen accumulation disease related gene targeting sequence is at least one to-be-detected fragment selected from PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC and GAA gene exon region sequence.
In a preferred embodiment, the primer pool comprises a continuous nucleotide sequence consisting of at least 15 continuous nucleotides in the exon region sequences of PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, and GAA genes.
In a preferred embodiment, the primer for specifically amplifying the targeting sequence of the PGM1 gene comprises a continuous nucleotide sequence shown in SEQ ID No. 1-SEQ ID No. 36.
In a preferred technical scheme, the primer for specifically amplifying the AGL gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 37-SEQ No. ID 140; the primer for specifically amplifying the GBE1 gene targeting sequence comprises a continuous nucleotide sequence shown by SEQ No. ID 141-SEQ No. ID 190; the primer for specifically amplifying the GYG1 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 191-SEQ No. ID 212; the primer for specifically amplifying the PGAM2 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 213-SEQ No. ID 224; the primer for specifically amplifying the PHKA2 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. 225-SEQ No. 302; the primer for specifically amplifying the PHKA1 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID303-SEQ No. ID 386; the primer for specifically amplifying the LDHA gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 387-SEQ No. ID 414. The primer for specifically amplifying the PYGM gene targeting sequence comprises a continuous nucleotide sequence shown by SEQ No. ID 415-SEQ No. ID 462. The primer for specifically amplifying the SLC37A4 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 463-SEQ No. ID 490. The primer for specifically amplifying the GYS2 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 491-SEQ No. ID 540. The primer for specifically amplifying the PFKM gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 541-SEQ No. ID 592. The primer for specifically amplifying the PYGL gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID593-SEQ No. ID 642. The primer for specifically amplifying the ALDOA gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. 643-SEQ No. ID 666. The primer for specifically amplifying the PHKG2 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ ID 667-SEQ ID 690. The primer for specifically amplifying the PHKB gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 691-SEQ No. ID 768. The primer for specifically amplifying the ENO3 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 769-SEQ No. ID 796. The primer for specifically amplifying the G6PC gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 797-SEQ No. ID 812. The primer for specifically amplifying the GAA gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 813-SEQ No. ID 866.
The invention also aims to provide a kit for detecting the genotype of the neonatal glycogen storage disease, which is characterized by comprising the primer pool.
In a preferred technical scheme, the kit further comprises a linker, wherein the linker contains a tag sequence, and the linker is used for directly connecting with an amplification product of the neonatal glycogen accumulation disease related gene targeting sequence.
In a preferred embodiment, the kit further comprises a sequencing primer for sequencing molecules of the PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA gene fragments.
It is still another object of the present invention to provide a detection reagent, wherein the reagent contains the primer pool.
The invention also aims to provide application of the primer pool in detecting the genotyping of the glycogen accumulation disease of the newborn.
In a preferred technical scheme, the application comprises the steps of obtaining a mutation site of a pathogenic gene of the glycogen storage disease and detecting the genotype of the neonatal glycogen storage disease according to the mutation site.
Still another object of the present invention is to provide a detection method characterized in that the method comprises determining the presence of a gene mutation by PCR-enrichment amplification and sequencing of exon regions of the specifically amplified PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA genes by a targeted amplification technique and a high throughput sequencing method, determining the neonatal glycogen accumulation disease genotyping based on the gene mutation site.
Specifically, another object of the present invention is to provide a method for detecting a genotype associated with a glycogen storage disease in a newborn, comprising the steps of:
A. Amplifying samples of selected fragments of exon regions of PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC and GAA genes respectively by using a primer group to obtain amplification products;
B. Randomly fragmenting the amplification product to obtain a plurality of double-stranded nucleic acid fragments;
C. Connecting a first adaptor with an enzyme digestion recognition sequence to one end of the double-stranded nucleic acid fragment;
D. carrying out enzyme digestion on the double-stranded nucleic acid fragment to a preset length to obtain a library fragment;
E. immobilizing one end of the first adaptor on the magnetic beads to purify the library fragments;
F. Connecting a second adaptor to the other end of the first adaptor of the library fragment to obtain library molecules;
G. And sequencing the library molecules by adopting a second generation high-throughput gene sequencing technology to determine the genotype of the site to be detected. The first joint is Ion P1 Adapter; the second joint is Ion XpressTM Barcode Adapter X。
The invention also aims to provide application of the kit in detecting the genotyping of the glycogen storage disease of the newborn.
The application discloses a multiplex PCR primer system capable of genotyping a glycogen storage disease of a newborn and application thereof. The multiple PCR primer system for genotyping of the neonatal glycogen storage disease is used for amplifying exon sites of genes of the glycogen storage disease which can cause the glycogen storage disease in a human genome, and comprises 19 genes: PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA exon regions, 433 pairs of primers were designed. And (3) sequencing the amplified fragment by combining second-generation high-throughput sequencing, and accurately genotyping the glycogen storage disease.
The invention provides a primer group capable of genotyping a glycogen storage disease of a newborn, which comprises a primer pool A and a primer pool B. The sequences contained in the two primer pools are shown in table 2. Wherein the primer sequences (SEQ No. ID 1-SEQ No. ID 36) are used for specifically amplifying the exon region sequences of the PGM gene, and the total number of the 18 fragments is. The primer sequence (SEQ No. ID 37-SEQ No. ID 140) is used for specifically amplifying the exon region sequence of the AGL gene, and the total number of the fragments is 52.
The primer sequences (SEQ No. ID 141-SEQ No. ID 190) were used to specifically amplify the exon region sequences of GBE1 gene, for a total of 25 fragments. The primer sequence (SEQ No. ID 191-SEQ No. ID 212) is used for specifically amplifying the exon region sequence of the GYG1 gene, and 11 fragments are obtained in total. The primer sequences (SEQ No. ID 213-SEQ No. ID 224) were used to specifically amplify the exon region sequences of the PGAM2 gene, for a total of 6 fragments. The primer sequence (SEQ No. ID 225-SEQ No. ID 302) is used for specifically amplifying the exon region sequence of the PHKA2 gene, and the total number of the primers is 39. The primer sequence (SEQ No. ID303-SEQ No. ID 386) is used for specifically amplifying the exon region sequence of the PHKA1 gene, and 42 fragments are used. The primer sequence (SEQ No. ID 387-SEQ No. ID 414) is used for specifically amplifying the exon region sequence of the LDHA gene, and the total number of the fragments is 14. The primer sequence (SEQ No. ID 415-SEQ No. ID 462) is used for specifically amplifying the sequence of the exon region of the PYGM gene, and the total number of the sequences is 24. The primer sequence (SEQ No. ID 463-SEQ No. ID 490) is used for specifically amplifying the sequence of the exon region of the SLC37A4 gene, and the total number of the 14 fragments is. The primer sequence (SEQ No. ID 491-SEQ No. ID 540) is used for specifically amplifying the exon region sequence of the GYS2 gene, and the total number of the 25 fragments is total. The primer sequences (SEQ No. ID 541-SEQ No. ID 592) were used to specifically amplify the exon region sequences of the PFKM gene, for a total of 26 fragments. The primer sequence (SEQ No. ID593-SEQ No. ID642) is used for specifically amplifying the sequence of the exon region of the PYGL gene, and the sequences comprise 25 segments. The primer sequence (SEQ No. ID 643-SEQ No. ID 666) is used for specifically amplifying the exon region sequence of the ALDOA gene, and 12 fragments are obtained in total. The primer sequence (SEQ No. ID 667-SEQ No. ID 690) is used for specifically amplifying the exon region sequence of the PHKG2 gene, and the total number of the fragments is 12. The primer sequence (SEQ No. ID 691-SEQ No. ID 768) is used for specifically amplifying the exon region sequence of the PHKB gene, and the total number of the fragments is 39. The primer sequence (SEQ No. ID 769-SEQ No. ID 796) is used for specifically amplifying exon region sequences of the ENO3 gene, and the sequences comprise 14 segments. The primer sequence (SEQ No. ID 797-SEQ No. ID 812) was used to specifically amplify the exon region sequence of the G6PC gene for 8 fragments. The primer sequence (SEQ No. ID 813-SEQ No. ID 866) was used to specifically amplify the exon region sequence of the GAA gene for 27 fragments in total.
The invention also provides application of the primer group in detecting the pathogenic gene mutation site of the glycogen storage disease and application of the primer group in preparing a kit for detecting the glycogen storage disease genotyping.
The invention also provides a kit for simultaneously detecting the glycogen accumulation disease related gene mutation, which comprises the primer group.
The invention relates to a glycogen storage disease gene mutation detection kit.
Compared with the prior art, the invention has the following remarkable characteristics:
The invention adopts a high-throughput sequencing technology, which is also called a next generation sequencing technology, and can sequence tens of millions of DNA molecules at a time. The invention provides a method for carrying out PCR enrichment amplification and sequencing on exon regions of glycogen accumulation disease related genes in a table 1 through a targeted amplification technology and a high-throughput sequencing method, and carrying out bioinformatics analysis on possible gene mutation so as to judge the types of diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the results of deep sequencing analysis according to the present invention.
FIG. 2 shows the results of C/G content analysis of sequencing;
FIG. 3 shows the results of the length analysis of the sequenced read fragments.
Detailed Description
The present invention will be more fully understood from the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed embodiment.
the aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as open-ended and not limiting.
The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. Furthermore, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In addition, where the term "about" is used before a quantity, the present teachings also include the particular quantity itself unless specifically stated otherwise.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
The invention aims to provide a primer kit for detecting glycogen storage disease gene mutation, which combines multiple PCR enrichment technology and high-throughput sequencing technology, comprehensively and systematically detects genes which can cause different types of glycogen storage diseases, such as PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA and other 19 gene mutations. The kit can accurately genotype the glycogen storage disease and is used for researching and diagnosing the glycogen storage disease caused by the gene defect.
The design scheme of the mutation site detection system aiming at the glycogenosis genotyping is as follows:
1, designing PCR primers for exon regions of 19 genes causing glycogen storage disease, PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA; 433 pairs of primers were obtained, and each pair of primers was mixed in equimolar amounts into two primer pools, primer pool a and primer pool B, and the specific information is shown in table 2.
table 2: primer sequence information Table
2, performing multiplex PCR using the primer set according to claim 1 using whole genomic DNA of peripheral blood as a template to obtain a plurality of specific target sequences of PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, and the exon region of GAA gene;
3, constructing a library by using the amplified specific target sequence, and sequencing by using a high-throughput sequencing method based on the constructed library;
Comparing the sequenced sequence with a reference sequence to obtain PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC and GAA mutation conditions of the gene causing the glycogen storage disease, finding out mutation sites and carrying out functional annotation on the mutation sites; thereby judging whether the patient suffers from the glycogen storage disease and the corresponding type thereof.
Wherein, the library creating reagent used in the step 3 is preferably Ion AmpliSeq from Thermo Fisher ScientificTMDNA libraries kit 2.0 or Ion AmpliSeqTMDNA libraries plus。
The sequencing Chamber used in step 3 was derived from 520 at Thermo Fisher Scientific IonTM&Ion 530TMKit or Ion 540TMKit, the sequencing platform used was ion S5TMSystem or Ion S5TMXL System。
The primer group designed by the invention establishes a method for simultaneously detecting common pathogenic mutation sites of the glycogen storage disease and genotyping the disease by applying a multiplex PCR amplification technology and a high-throughput sequencing technology.
The technical scheme of the invention is further explained by combining the attached drawings and a plurality of embodiments.
example 1 design of primer set
1, aiming at 19 genes causing glycogen storage disease by taking human genome Hg19 as a reference sequence: (PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA) exon region design primers, avoiding the formation of dimers and high GC content primers between the primers. And (2) allocating primers adjacent to the reference sequence position to different primer pools to avoid overlap, allocating primers with far positions and similar GC content to a uniform primer pool, and optimizing to obtain the primer group of the invention, wherein the primer group comprises two primer pools: a primer pool A and a primer pool B. The two primer pools were used for PCR amplification of the genome.
The target region amplified by the designed primer group covers the exon regions of 19 genes, and the total length is 87 kb.
Example 2 detection method of genotyping of glycogen accumulation disease
The detection method comprises the steps of carrying out multiplex PCR by using the primer group designed in the embodiment 1 and peripheral whole blood genome DNA as a template, constructing a library of PCR products, sequencing the library by using a high-throughput sequencing method, and analyzing sequencing data so as to detect and analyze the gene mutation condition.
1, extracting DNA of a sample to be detected: the genomic DNA of the subject is derived from a peripheral blood sample (including a dry blood spot sample), and the genomic DNA is extracted by using a commercially available blood genomic DNA (dry blood spot genomic DNA) extraction Kit, the concentration of the extracted DNA is measured by using a Qubit dsDNA HS Assay Kit, and the purity of the extracted DNA is detected by using a Nanodrop 2000 c.
2, multiplex PCR amplification of target sequence: each sample was prepared with two reactions in primer pool A and primer pool B, respectively, and each reaction was prepared in a PCR tube according to Table 3. The prepared system was placed on a PCR instrument to perform a reaction under the reaction conditions shown in Table 4.
Table 3: reaction system
table 4: reaction conditions
After amplification, the amplification tube was removed from the PCR instrument, the two amplification tubes, primer pool A and primer pool B, were centrifuged slightly, and then the two tubes were combined into one tube with a total volume of 20ul and immediately proceeded to the next step.
3, primer digestion: to the pooled PCR product tubes, FuPa Reagent 2ul was added, at which time the total volume in the reaction tube was 22ul, the tube caps were closed, vortexed slightly, centrifuged, placed on a PCR instrument, and the following temperature program was performed: 50 ℃ for 10 min; 55 deg.C, 10min, 60 deg.C, 20 min; 10 ℃ and Hold. After completion, the next step was immediately carried out.
4, connecting joint: after the primer digestion is completed, Ion P1Adapter and Ion Xpress are connectedTMBarcode Adapter X. Wherein Barcode X is a specific linker constructed by the library and used for distinguishing different samples, and a proper linker can be properly selected according to the conditions of the samples.
According to table 5, a linker solution was prepared:
Table 5: joint solution preparation system
According to Table 6, a ligation reaction system was prepared, and the total volume was 30 ul. The prepared reaction system was placed on a PCR instrument and the reaction conditions shown in Table 7 were followed.
Table 6: ligation reaction System
table 7: reaction conditions
| Temperature (. degree.C.) | Time of day |
| 22 | 30min |
| 68 | 5min |
| 72 | 5min |
| 10 | Hold |
after the reaction was completed, the PCR tube was taken out, slightly centrifuged, and immediately subjected to the next purification.
5, purifying a connection product: adding 45uL of AMPure XP magnetic beads (the volume of the connecting product is 1.5 times that of the connecting product) into a PCR tube of the connecting product, blowing, stirring uniformly, transferring into a new centrifugal tube of 1.5mL, and standing for 5min at room temperature; placing the centrifuge tube on a magnetic frame, discarding the supernatant after the solution is clarified, keeping the centrifuge tube on the magnetic frame, washing the magnetic beads with a freshly prepared 70% ethanol solution for 2 times, air-drying the magnetic beads at room temperature for 3min, adding 50ul low TE buffer after the excess alcohol is volatilized, taking the centrifuge tube off the magnetic frame, blowing and uniformly mixing, incubating at room temperature for 2min, and placing the centrifuge tube back to the magnetic frame to clarify the solution. At this time, the supernatant solution was the constructed library, and 48ul of the supernatant was aspirated into a new centrifuge tube and stored at-20 ℃.
6, library quality inspection and sequencing: after library construction was complete, library concentrations were quantified using a qPCR instrument and sequenced using the Ion S5 system/Ion S5 XL system according to sequencing kit instructions.
7, data analysis: after sequencing is completed, the sequencer automatically starts an analysis program, compares data to a human genome reference sequence hg19, performs sequencing depth, coverage analysis and mutation analysis on a target region, and gives an analysis chart, such as sequencing data statistical analysis results shown in table 8 and sequencing results shown in fig. 1-3. Wherein FIG. 1 is the results of a deep sequencing analysis; FIG. 2 shows the results of C/G content analysis of sequencing; FIG. 3 shows the results of the length analysis of the sequenced read fragments.
Table 8: statistical results of sequencing data
| Size of target region (bp) | 104,263 |
| total data volume (Mb) | 162.41 |
| Total read number | 800，027 |
| Average read Length (bp) | 203 |
| Target area data volume (Mb) | 149.72 |
| Ratio of target area data amount to total data amount | 92.19％ |
| Depth of sequencing of target region | 1,436 |
| Target region sequencing 1 × coverage | 99.74％ |
| Target region sequencing 2x coverage | 99.27％ |
| Target area sequencing 100x coverage | 98.92％ |
| Target region sequencing 500x coverage | 90.14％ |
8, performing functional annotation on the screened mutation sites to obtain a final analysis result, and finding out a pathogenic mutation in the GBE1 gene, wherein the result indicates that the patient suffers from glycogen storage disease caused by the GBE1 gene defect.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.
Claims (10)
1. A primer pool for detecting the genotyping of the glycogen accumulation disease of the newborn, which is characterized by comprising a plurality of primer pairs for specifically amplifying a target sequence of a gene related to the glycogen accumulation disease of the newborn; the neonatal glycogen accumulation disease related gene targeting sequence is at least one to-be-detected fragment selected from PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37A4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC and GAA gene exon region sequence.
2. the primer pool of claim 1, wherein the primer pool comprises a contiguous nucleotide sequence consisting of at least 15 contiguous nucleotides of the sequence of the exon of the PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA gene, respectively.
3. The primer pool of claim 1, wherein the primer that specifically amplifies the PGM1 gene targeting sequence comprises a contiguous nucleotide sequence shown in SEQ ID No. 1-SEQ ID No. 36.
4. The primer pool of claim 1, wherein the primer for specific amplification of the AGL gene targeting sequence comprises a contiguous nucleotide sequence shown in SEQ No. ID 37-SEQ No. ID 140; the primer for specifically amplifying the GBE1 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 141-SEQ No. ID 190; the primer for specifically amplifying the GYG1 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 191-SEQ No. ID 212; the primer for specifically amplifying the PGAM2 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 213-SEQ No. ID 224; the primer for specifically amplifying the PHKA2 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 225-SEQ No. ID302; the primer for specifically amplifying the PHKA1 gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID303-SEQ No. ID 386; the primer for specifically amplifying the LDHA gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 387-SEQ No. ID 414. The primer for specifically amplifying the PYGM gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 415-SEQ No. ID 462. The primer for specifically amplifying the SLC37A4 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 463-SEQ No. ID 490. The primer for specifically amplifying the GYS2 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 491-SEQ No. ID 540. The primer for specifically amplifying the PFKM gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 541-SEQ No. ID592. The primer for specifically amplifying the PYGL gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID593-SEQ No. ID 642. The primer for specifically amplifying the ALDOA gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. 643-SEQ No. ID 666. The primer for specifically amplifying the PHKG2 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 667-SEQ No. ID 690. The primer for specifically amplifying the PHKB gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 691-SEQ No. ID 768. The primer for specifically amplifying the ENO3 gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 769-SEQ No. ID 796. The primer for specifically amplifying the G6PC gene targeting sequence comprises a continuous nucleotide sequence shown in SEQ No. ID 797-SEQ No. ID 812. The primer for specifically amplifying the GAA gene targeting sequence comprises a continuous nucleotide sequence shown as SEQ No. ID 813-SEQ No. ID 866.
5. A kit for detecting neonatal glycogen accumulation disease genotyping, wherein the kit comprises the primer pool of any one of claims 1 to 4.
6. A detection reagent comprising the primer pool according to any one of claims 1 to 4.
7. Use of the primer pool of claim 1 for detecting neonatal glycogen accumulation disease genotyping.
8. the use according to claim 7, wherein the use comprises the steps of obtaining a mutation site of a gene causing glycogen storage disease, and detecting a genotype of the glycogen storage disease in the newborn based on the mutation site.
9. An assay method comprising determining the presence of a genetic mutation by PCR-rich amplification and sequencing of the exon regions of the specifically amplified PGM1, AGL, GBE1, GYG1, PGAM2, PHKA2, PHKA1, LDHA, PYGM, SLC37a4, GYS2, PFKM, PYGL, ALDOA, PHKG2, PHKB, ENO3, G6PC, GAA genes by targeted amplification techniques and high throughput sequencing, determining the genotype of the neonatal glycogen accumulation disease based on the locus of the genetic mutation.
10. use of the kit of claim 5 for detecting neonatal glycogen accumulation disease genotyping.
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Effective date of registration: 20230926 Address after: No. 1018 Huahua Road, Maogang Town, Songjiang District, Shanghai, May 2016 Applicant after: Shanghai Jia more medical equipment Co.,Ltd. Address before: 201612 building 52, Lane 255, siziannan Road, Xinqiao Town, Songjiang District, Shanghai Applicant before: Shanghai Qianbei Medical Technology Co.,Ltd. |
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