CN116804219A - A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation - Google Patents
A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation Download PDFInfo
- Publication number
- CN116804219A CN116804219A CN202310697800.2A CN202310697800A CN116804219A CN 116804219 A CN116804219 A CN 116804219A CN 202310697800 A CN202310697800 A CN 202310697800A CN 116804219 A CN116804219 A CN 116804219A
- Authority
- CN
- China
- Prior art keywords
- syndrome
- kartagner
- embryo implantation
- embryo
- sites
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000032692 embryo implantation Effects 0.000 title claims abstract description 50
- 208000011580 syndromic disease Diseases 0.000 title claims abstract description 44
- 239000000203 mixture Substances 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title claims description 31
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 57
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 239000012502 diagnostic product Substances 0.000 claims abstract description 8
- 230000035772 mutation Effects 0.000 claims description 20
- 201000009266 primary ciliary dyskinesia Diseases 0.000 claims description 18
- 208000003892 Kartagener syndrome Diseases 0.000 claims description 15
- 238000011144 upstream manufacturing Methods 0.000 claims description 15
- 210000000349 chromosome Anatomy 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 6
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 45
- 238000003745 diagnosis Methods 0.000 abstract description 12
- 208000026350 Inborn Genetic disease Diseases 0.000 abstract description 10
- 238000012360 testing method Methods 0.000 abstract description 8
- 208000016361 genetic disease Diseases 0.000 abstract description 6
- 238000009007 Diagnostic Kit Methods 0.000 abstract description 4
- 239000000090 biomarker Substances 0.000 abstract description 2
- 230000000903 blocking effect Effects 0.000 abstract description 2
- 238000012163 sequencing technique Methods 0.000 description 30
- 102000054766 genetic haplotypes Human genes 0.000 description 29
- 108090000623 proteins and genes Proteins 0.000 description 21
- 238000004458 analytical method Methods 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 239000000178 monomer Substances 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 238000005516 engineering process Methods 0.000 description 11
- 230000002068 genetic effect Effects 0.000 description 11
- 108091092878 Microsatellite Proteins 0.000 description 9
- 230000003321 amplification Effects 0.000 description 9
- 238000003199 nucleic acid amplification method Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 210000002257 embryonic structure Anatomy 0.000 description 8
- 101000866368 Homo sapiens Dynein axonemal heavy chain 5 Proteins 0.000 description 7
- 238000010276 construction Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 108700028369 Alleles Proteins 0.000 description 6
- 102100031648 Dynein axonemal heavy chain 5 Human genes 0.000 description 6
- 210000004081 cilia Anatomy 0.000 description 6
- 238000013412 genome amplification Methods 0.000 description 6
- 108091093088 Amplicon Proteins 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 4
- 210000002993 trophoblast Anatomy 0.000 description 4
- 208000012661 Dyskinesia Diseases 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 210000000625 blastula Anatomy 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000012165 high-throughput sequencing Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000007403 mPCR Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 101100439666 Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34) chrA1 gene Proteins 0.000 description 2
- 101100439668 Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34) chrB1 gene Proteins 0.000 description 2
- 102100033595 Dynein axonemal intermediate chain 1 Human genes 0.000 description 2
- 208000024556 Mendelian disease Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 206010048705 Paraneoplastic cerebellar degeneration Diseases 0.000 description 2
- 208000017787 Paraneoplastic neurologic syndrome Diseases 0.000 description 2
- 101100439667 Pseudomonas aeruginosa chrA gene Proteins 0.000 description 2
- 206010000210 abortion Diseases 0.000 description 2
- 231100000176 abortion Toxicity 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 208000032307 premature centromere division Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 201000009890 sinusitis Diseases 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 101150063022 CHRD gene Proteins 0.000 description 1
- 208000025678 Ciliary Motility disease Diseases 0.000 description 1
- 102100036615 Coiled-coil domain-containing protein 39 Human genes 0.000 description 1
- 102100036616 Coiled-coil domain-containing protein 40 Human genes 0.000 description 1
- 208000027205 Congenital disease Diseases 0.000 description 1
- 101100366182 Cupriavidus metallidurans (strain ATCC 43123 / DSM 2839 / NBRC 102507 / CH34) chrC gene Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 101150003322 DNAH5 gene Proteins 0.000 description 1
- 101100274355 Danio rerio chd gene Proteins 0.000 description 1
- 102100033596 Dynein axonemal intermediate chain 2 Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000034826 Genetic Predisposition to Disease Diseases 0.000 description 1
- 101000715279 Homo sapiens Coiled-coil domain-containing protein 39 Proteins 0.000 description 1
- 101000715283 Homo sapiens Coiled-coil domain-containing protein 40 Proteins 0.000 description 1
- 101000872267 Homo sapiens Dynein axonemal intermediate chain 1 Proteins 0.000 description 1
- 101000872272 Homo sapiens Dynein axonemal intermediate chain 2 Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 208000037340 Rare genetic disease Diseases 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001109 blastomere Anatomy 0.000 description 1
- 201000009267 bronchiectasis Diseases 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000012775 microarray technology Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 210000004508 polar body Anatomy 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000019100 sperm motility Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to the field of biotechnology, in particular to a set of loci for detecting Kartagner syndrome before embryo implantation, a primer composition, a kit and application, and the application discovers a set of SNP loci for detecting Kartagner syndrome linkage before embryo implantation and proves that genotype information of pathogenic loci related to embryo Kartagner syndrome can be detected by detecting genotype information of the set of SNP loci, so that the kit can be used as a biomarker for auxiliary diagnosis of Kartagner syndrome before clinical embryo implantation. In addition, the SNP loci related to Kartagner syndrome can be used for preparing a diagnostic kit, a diagnostic reagent or a diagnostic product for testing single-gene genetic diseases before embryo implantation of Kartagner syndrome, and the diagnostic kit, the diagnostic reagent or the diagnostic product can improve the accuracy of diagnosis of Kartagner syndrome before clinical embryo implantation, thereby avoiding embryo implantation carrying pathogenic genotypes and blocking the occurrence of Kartagner syndrome family inheritance.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a set of sites, primer compositions, kits and applications for detecting Kartagner syndrome before embryo implantation.
Background
Primary ciliated dyskinesia (primary ciliary dyskinesia, PCD) is a rare genetic disease that causes dysfunction of ciliated tissue-containing organs due to defects in ciliated structure or function caused by genetic mutations. Kartager syndrome (Kartagener syndrome, KS) belongs to a subtype of primary ciliated dyskinesia, accounting for about 50% of primary ciliated dyskinesia. It is characterized by the triple symptoms of bronchiectasis, visceral translocation and sinusitis. It is due to abnormalities in cilia structure or function. The incidence of Kartagner syndrome is approximately 1/20000-1/30000. It can be diagnosed by clinical manifestation, imaging examination, cilia examination and genetic testing. A number of genes are currently known to be associated with Kartagner syndrome, including DNAH5, DNAI1, DNAI2, CCDC39, CCDC40, and the like. Of which DNAH5 is the highest in proportion,
since kartagner syndrome is relatively rare in clinic, and most patients often take sinusitis as a main symptom or only show single respiratory system infection symptoms when taking a doctor, misdiagnosis and missed diagnosis are often caused by lack of systematic knowledge of most doctors in clinical diagnosis and especially in outpatient screening. Currently, diagnosis of Kartagener syndrome relies mainly on clinical manifestations, imaging examinations and cilia examinations. However, these methods have certain limitations such as atypical clinical manifestations, insensitivity in imaging examinations, complicated cilia examination procedures, unstable results, etc. At present, foreign scholars basically agree to diagnose diseases by screening mutation hot spots on genes DNAH5 and DNAI 1. Genetic diagnostics plays a central role in the diagnosis of PCD.
Kartagner syndrome has a familial genetic predisposition, can develop in the same generation or in alternate generations, and has a history of close-related mating for parents. Genetic research shows that the family members derived from the common ancestor have a larger chance of carrying the same recessive pathogenic genes than family members not derived from the common ancestor, the occurrence rate of the recessive pathogenic genes is improved by the near wedding, the probability of the child suffering from congenital diseases is greatly improved compared with normal people, and the population quality is greatly influenced. In the reproductive system, the disorder of cilia causes insufficient sperm movement ability, and dysfunction of cilia in the oviduct, endometrium and the like, which causes abnormal ovulation or the failure of smooth combination of ovum with sperm, so that the disease is often combined with infertility of men and women.
Screening for single genetic disease before embryo implantation (Preimplantation genetic testing for single gene PGT-M) is a technique in which, before embryo transfer, a polar body is removed from an oocyte or fertilized egg or 1-2 blastomeres or a plurality of trophoblast cells are taken from an embryo at a stage before implantation to perform specific genetic trait detection, and then an appropriate embryo is selected for transfer. This technique aims at examining the genetic information of embryos to find abnormal genes that may lead to the infant suffering from a certain genetic disease at birth. The technology was studied and explored at the earliest of the last 80 s of the last century, and this early pre-pregnancy diagnostic approach has been widely accepted at present. Currently, single gene disease detection of pre-implantation embryos requires detection of SNP sites (single nucleotide variation sites) within 1Mb upstream and downstream of the target gene.
The second generation sequencing technology (NGS) has the advantages of rapidness, accuracy and low cost, and can detect the chromosome number, structure, single-gene diseases and other information of the embryo by carrying out second generation sequencing on the embryo blastula stage cells, thereby improving the success rate of embryo implantation and healthy birth rate and reducing the risks of abortion and genetic diseases.
In the prior art, no site and primer combination corresponding to PGT-M diagnosis applied to Kartagner syndrome based on second generation sequencing is disclosed. Thus, the occurrence of Kartagener syndrome family inheritance is not well blocked at present.
In addition, in the prior art, when the gene generates pathogenic variation and genetic detection before embryo implantation is selected, usually fewer SNP loci can be detected at two sides of the pathogenic variation loci, so that two sides of the gene mutation cannot find a sufficient number of effective SNP loci, whether homologous recombination is possible in a pathogenic variation region of the gene, construction of a haplotype fails, whether the embryo contains the mutated pathogenic locus cannot be accurately judged, and whether the embryo is a normal embryo, an embryo carrying a pathogenic genotype or an abnormal embryo cannot be judged.
Traditional PGT-M (Preimplantation Genetic Testing-Monogenic disorders) assays typically use capillary electrophoresis-based detection of nucleic acid short tandem repeats (short tandom repeats, STR) or SNP chip technology to construct haplotypes, and determine whether an embryo carries a pathogenic mutation, and thus whether the embryo is a conforming embryo. STRs require the extraction of multiple cells from an embryo for analysis, which may affect embryo development and viability. STR may have false positive or false negative results such as heterozygosity loss, gene amplification failure, nonspecific amplification, etc., and multiple verification and confirmation are required. In the prior art, because the STR sites disclosed at present are fewer, the requirements of different clinical applications are difficult to meet, the SNP sites used by the existing SNP chip technology are fixed SNP sites, and on two sides of a pathogenic site, the effective SNP sites are limited and cannot contain the pathogenic site, so that haplotypes are difficult to construct effectively. Therefore, improvement of the haplotype construction method is needed to improve the success rate of haplotype construction and the accuracy of single gene disease detection before embryo implantation.
Disclosure of Invention
To overcome the deficiencies of the prior art, a first object of the present application is to provide a set of sites for detection of kartager syndrome before embryo implantation, which can be applied to the preparation of reagents for detection and diagnosis of kartager syndrome before embryo implantation.
A second object of the present application is to provide the use of the above-mentioned sites for the preparation of a diagnostic reagent or kit for the detection of Kartagner syndrome prior to embryo implantation.
It is a third object of the present application to provide a primer composition for site detection for detection of Kartagener syndrome before embryo implantation.
A fourth object of the present application is to provide a kit comprising the above primer composition.
It is a fifth object of the present application to provide a specific primer combination for the site detection for detection of Kartagener syndrome before embryo implantation.
A sixth object of the present application is to provide a kit comprising the above specific primer combination.
A seventh object of the present application is to provide the use of the above kit for the preparation of a diagnostic product for detecting Kartagener syndrome before embryo implantation.
In order to achieve the first object of the application, the present application adopts the following technical scheme:
the application provides a group of sites for detecting Kartagner syndrome before embryo implantation, which comprises 81 sites carried by chromosome 5, wherein the sites comprise a pathogenic site, 44 SNP sites linked with the pathogenic site are arranged at the upstream of the pathogenic site, and 36 SNP sites linked with the pathogenic site are arranged at the downstream of the pathogenic site;
the 81 sites are as follows:
。
in order to achieve the second object of the application, the present application adopts the following technical scheme:
the application provides application of the above sites in preparing a diagnostic reagent or a kit for detecting Kartagner syndrome before embryo implantation.
In order to achieve the third object of the present application, the present application adopts the following technical scheme:
the application provides a primer composition for detecting a site of Kartagner syndrome before embryo implantation, which is characterized in that the information of each primer in the primer composition is as follows:
。
in order to achieve the fourth object of the present application, the present application adopts the following technical scheme:
the application provides a kit for detecting the site of Kartagner syndrome before embryo implantation, which contains the primer composition.
In order to achieve the fifth object of the present application, the present application adopts the following technical scheme:
the application provides a specific primer combination for detecting a site of Kartagner syndrome before embryo implantation, which comprises 44 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation sites and 36 pairs of downstream primers;
the sequence information of the specific primer combination is as follows:
。
in order to achieve the sixth object of the present application, the present application adopts the following technical scheme:
the present application provides a kit for detecting the site of Kartagener syndrome prior to embryo implantation, said kit comprising a specific primer combination as described above.
In order to achieve the seventh object of the present application, the present application adopts the following technical scheme:
the application provides application of the kit in preparing a diagnostic product for detecting Kartagner syndrome before embryo implantation.
Compared with the prior art, the application has the beneficial effects that:
(1) The application detects SNP locus linked with Kartagner syndrome before embryo implantation and proves that the SNP locus can detect genotype information of pathogenic locus related to embryo Kartagner syndrome by detecting genotype information of the SNP locus, so that the SNP locus can be used as a biomarker for detecting Kartagner syndrome before clinical embryo implantation. In addition, the SNP locus related to Kartagner syndrome and the related primer combination can be used for preparing a diagnostic kit, a diagnostic reagent or a diagnostic product for testing single-gene genetic disease before Kartagner syndrome embryo implantation, and the diagnostic kit, the diagnostic reagent or the diagnostic product can improve the accuracy of detecting Kartagner syndrome diagnosis before clinical embryo implantation, thereby avoiding embryo implantation carrying pathogenic genotype and blocking the occurrence of Kartagner syndrome family inheritance. In addition, the application can be used for analyzing 3-5 cells, thus being suitable for detecting embryo before transplanting in the technique of test tube infants.
(2) The primer composition for detecting the locus for detecting Kartagner syndrome before embryo implantation has better universality because 81 SNP are adopted for analysis, and can be used for embryo transplantation diagnosis of different families.
(3) The specific primer combination for detecting the locus of Kartagner syndrome before embryo implantation comprises 44 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation loci and 36 pairs of downstream primers, wherein the specific primer combination has unique sequence on a target chromosome, the Tm values of the primers are similar, the primers have no complementary or partially complementary sequence with the primers, a dimer or hairpin structure can be avoided, and in addition, a targeted amplification interval at least comprises one selected SNP locus or pathogenic locus, so that the specific primer combination has high specificity.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
To aid in a better understanding of the application, the following concepts are explained:
PGT-M, a genetic test of monogenic disease before embryo implantation, is an Assisted Reproduction (ART) technique for embryo genetic testing. IVF/ICSI operation was performed first, and then gene detection was performed on the embryos obtained. Finally, embryos without genetic pathogenic mutations are screened and transplanted to the mother's uterus.
Reads refers to short sequences generated by a high throughput sequencing platform, namely base sequences obtained by single sequencing by a sequencer; single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to DNA sequence diversity at the genomic level caused by variation of a single nucleotide. SNPs are the most common genetic variations.
The Short Tandem Repeat (STR) is a DNA repeat 12 comprising 1-6 base units. STRs have a high degree of variability among individuals and can be used as DNA fingerprints of cells.
SNP chip (SNP chip) technology refers to technology in which high-density DNA fragments are attached to a solid phase surface such as a film, a glass plate or the like in a certain sequence or arrangement manner by a high-speed robot or an in-situ synthesis manner through a Microarray technology, and a large number of SNP locus genotypes are detected by using DNA probes labeled with isotopes or fluorescence and by means of a base complementary hybridization principle.
Minimum Allele Frequency (MAF), SNP or STR has multiple alleles, the sum of which in the population is 1, the minimum allele frequency is between 0 and 0.5, the higher the frequency, the higher the position gene polymorphism.
The amplicon is a pair of PCR primers that can amplify a DNA fragment in the genome by PCR. Amplicon panel is a group of amplicons that can simultaneously amplify multiple regions in the genome in one PCR experiment.
Haplotypes (Haplotype) are genotypes of a set of closely related alleles on a chromosome, usually inherited as a unit. Haplotypes may reflect the combination and distribution of Single Nucleotide Polymorphisms (SNPs) on a chromosome.
The sequencing depth refers to the ratio of the total number of bases sequenced to the size of the genome to be tested, and can be understood as the average number of times each base in the genome is sequenced. The higher the sequencing depth, the higher the quality and reliability of the sequencing data, but also increases the cost and data volume of sequencing. For example, in one embodiment, a sequencing depth of 1000x represents that the strip of specific PCR amplification product is sequenced 1000 times.
The effective site refers to a SNP site or an STR site of both man and woman, wherein one of the SNP site and the STR site is homozygous and the other is heterozygous, and the site is called as the effective site.
In addition, when mutations occur at certain sites in a gene, single gene defects can result in the potential risk of disease in the body or its progeny. The incidence of genetic diseases caused by single gene defects is high, so that the method has great significance for researching single gene defects. Through single gene defect detection, DNA fragments to be detected of the foreigner, the parent and the offspring embryo can be analyzed to determine haplotypes of the foreigner, the parent and the offspring embryo, so that qualified offspring embryo is selected for implantation, and the risk of diseases of offspring and offspring is effectively reduced.
The following description is made with reference to specific embodiments.
Example 1
A set of sites for detecting Kartagener syndrome prior to embryo implantation, comprising 81 sites carried by chromosome 5, comprising one pathogenic site, 44 SNP sites linked to said pathogenic site upstream and 36 SNP sites linked to said pathogenic site downstream;
the 81 sites are as follows:
example 2
The use of a set of sites for detection of Kartagener syndrome prior to embryo implantation in the preparation of a diagnostic reagent or kit for Kartagener syndrome prior to embryo implantation, example 1.
The primer composition for detection is designed for the set of sites for detecting Kartagner syndrome before embryo implantation, and the information of each primer in the designed primer composition is as follows:
wherein a kit for detecting the site of Kartagner syndrome before embryo implantation is designed, which contains the above primer composition.
In addition, a specific primer combination for detection is designed for the group of sites for detecting Kartagner syndrome before embryo implantation, and comprises 44 pairs of upstream primers, 1 pair of specific primers containing pathogenic mutation sites and 36 pairs of downstream primers; the sequence information of this specific primer combination is as follows:
wherein a kit for detecting the site of Kartagner syndrome before embryo implantation is designed, which contains the specific primer combination described above.
In addition, the kit is applied to preparing a diagnostic product for detecting Kartagner syndrome before embryo implantation.
Experiment:
1. amplification and sequencing of a Single nucleotide sequence on DNAH5
The specific method comprises the following steps:
(1) 1Mb of SNP sites with MAF value larger than 0.3 are selected at the upstream and downstream of DNAH5 gene pathogenic sites respectively; (2) Designing an amplicon panel around these sites and pathogenic sites; (3) Extracting DNA from a family blood sample (parent, precursor), multiplex amplifying, establishing a library, and sequencing on a machine; (4) Single-cell whole genome amplification, multiplex amplification, library establishment and on-machine sequencing are carried out on the embryo sample; (5) performing mutation analysis on 81 sites according to the sequencing data; (6) SNP linkage analysis is carried out on the family sample and the embryo sample according to the 81 locus genotypes determined by mutation analysis to determine haplotypes; (7) And selecting the embryo with the target site pathogenic genotype detected as a precursor, and determining the pathogenic haplotype according to the detected genotype and haplotype analysis results of the pathogenic locus in the family sample and the precursor embryo sample, thereby determining whether the embryo carries the pathogenic haplotype and the pathogenic gene.
The method can quickly and accurately determine candidate SNP with high mutation rate by combining polymorphism information of known individuals or groups with existing data. Adopts multi-SNP linkage haplotype analysis, thereby more accurately establishing haplotype and realizing embryo implantation pre-diagnosis, fetal diagnosis and abortion tissue diagnosis of different partners.
Wherein, single-cell whole genome amplification technology (SCWGA), multiplex PCR capture sequencing, high-throughput sequencing method and SNP-based monomer linkage analysis method are adopted for the sample. Therefore, compared with other methods, the method has the advantages of universality, high flux, low cost, high sensitivity and strong specificity, and can rapidly and accurately detect Kartagner syndrome before embryo transfer by sequencing multi-site SNP and preventing detection misdiagnosis caused by amplified allele release (ADO).
The multi-site SNP sequencing in the application is based on a second generation sequencing technology, and a plurality of SNPs near the DNAH51 gene are analyzed without depending on known probes. The specific steps of multi-site SNP sequencing include: 1) According to the pathogenic sites and genotypes determined by the gene detection reports of both parents, respectively selecting SNP sites with high polymorphism (MAF > 0.3) in Chinese population in the 1Mb region upstream and downstream of the sites; 2) Designing a multi-targeting PCR amplification panel aiming at the sites; 3) Performing multiplex PCR amplification and sequencing on genome targeted intervals of the family samples and the embryo samples; 4) SNP analysis was performed based on the data obtained by sequencing.
In addition, the application enables high throughput haploid construction of DNAH5 mutations based on high throughput sequencing technology, enabling analysis of a large number of samples at once by adding different tag sequences to each sample.
Along with the continuous development of sequencing technology and continuous reduction of sequencing cost and analysis of a large number of samples at a time, the cost of haploid analysis of DNAH5 mutation is also continuously reduced.
The present application uses embryos as a precursor to determine haplotypes carrying pathogenic genotypes. Some target heterozygous sites of a partial embryo may be detected as homozygous sites due to allele tripping (ADO) caused by WGA amplification. Resulting in detection of false negatives. The application innovatively selects the embryo which is definitely detected to carry the pathogenic genotype as the precursor. The pathogenic haplotypes are then determined based on the gene flow from parent to embryo. Judging whether other embryos carry pathogenic genes or not according to pathogenic haplotypes.
2. The criteria for screening the effective sites are:
(1) Considering that the MAF values of SNP loci among different human species have obvious difference, the self-built 15 ten thousand Chinese crowd SNP database is combined on the basis of a thousand genome database so as to better adapt to the requirements of Chinese crowd.
(2) And selecting SNP with MAF larger than 0.3 for research, and improving the probability of the searched SNP locus as an effective locus.
(3) In the human genome, sequences around SNP sites do not show homology.
(4) Removing SNP sites containing polynucleotides can increase the success rate of amplification of SNP detection sites.
(5) SNP loci with GC content more than 70% in the upstream and downstream 100bp range are removed, and the success rate of SNP detection locus amplification can be increased.
(6) The removal of SNP sites with homology in the human genome to the upstream and downstream 100bp sequences can eliminate the interference of homologous sequences that may exist.
(7) The SNP sites are uniformly distributed on the upper and lower stream of the gene pathogenic mutation site in the 1Mb region on the upper and lower stream of the DNAH5 pathogenic mutation site, and one SNP site is uniformly distributed every 4-5Kb, so that the haplotype obtained by SNP linkage analysis is tightly related with the pathogenic site.
Experimental cases:
experimental example 1: determination of haplotype by upstream and downstream SNP loci
1. Method of operation
(1) Sample acquisition: the selected parent parties adopt whole blood samples; extracting blood genome DNA, measuring DNA concentration by using Qubit 4.0, and taking a certain amount as a DNA sample to be measured. 4 cases of embryo to be detected are selected, the embryo develops to the blastula stage, 3-5 external trophoblast cells are taken out by adopting a conventional embryo biopsy technology, and the trophoblast cells of the blastula cultured in vitro enrich genomic DNA in the cells through whole genome amplification, and the whole genome amplification adopts Fapon Single Cell genome-Ampli Kit (NK 023).
(2) According to Ion AmpliSeq TM Library kit 2.0 standard Library construction procedure. That is, the DNA molecules of the multiplex PCR amplified products are subjected to cluster growth by adding linkers for sequencing at both ends thereof under a certain condition, and then sequenced on Ion Torrent PGM to obtain a DNA fragment sequence with fragment length distribution in 130-150 target regions.
(3) Haplotype linkage analysis: capturing and sequencing a whole genome amplification product (DNA) of an embryo biopsy cell and a family sample, carrying out SNP (single nucleotide polymorphism) cloning on a target site by using bwa-mem and bcftools, thereby determining the genotype of the SNP site in the target region, wherein the genotype of the pathogenic site detected by the embryo is from a chromosome monomer of a parent, and the embryo can only inherit one of two chromosome monomers of the parent respectively, so that linkage haplotype information of couples and embryos and the genotype information of the pathogenic site are obtained through SNP haplotype linkage analysis, thereby determining the pathogenic haplotype, and determining whether to carry the pathogenic gene related to Kartagner syndrome or not according to the haplotype information of the embryo sample, and classifying and diagnosing. The specific method comprises the following steps: the original data are filtered by using fastp-0.21.0 software, the low alignment quality sequence is removed by using BWA-mem software to align to human GRCh38 reference genome, the sequencing depth of the target site is counted, the site with the depth less than 100 is removed, and the genotype of the SNP site in the target region is determined. And screening effective loci to construct haplotypes by utilizing genotypes of SNP loci of parent and embryo target regions. And determining the pathogenic haplotype by using the established haplotype and the genotypes of pathogenic sites carried by parents and embryos, and judging whether the embryos carry mutation or not.
2. Judgment result
The haplotypes were constructed by selecting 48 effective sites of the target region, and the results are shown in Table 1.
TABLE 1 upstream and downstream SNP locus determination monomer type result data sheet
Upstream and downstream SNP locus judgment monomer type result data sheet (upper sheet)
Wherein, the column of the chrysD of the male genotype and the column of the chrysD of the embryo 1 are the staining monomers carrying the pathogenic deletion gene. Wherein chrA and chrB are normal chromosome monomers, chrC is normal chromosome monomer, and chrD is a risk-carrying parent stain monomer.
The judgment result shows that: the male dyeing monomers are classified into chrysC and chrysD, wherein chrysD is a dyeing monomer carrying pathogenic mutation, and chrysC is an normal chromosome monomer; female dyeing monomers are classified into chrA and chrB, and are all normal chromosome monomers. Embryo 1 is heterozygous mutation, carries pathogenic locus genotype, and embryo 2, embryo 3 and embryo 4 are all monomer without father source risk staining, and have no pathogenic mutation.
Experimental example 2: panel capture performance index detection
The experimental example uses the same 4 cases of genome amplification products of trophoblast cells in embryo blastocyst stage and 4 cases of DNA samples of peripheral blood cells of parents, uses the primer composition (81 pairs of SNP primers) designed by the application, carries out library construction according to the Fapon Single Cell genome-Ampli Kit (NK 023) standard library construction flow, and carries out sequencing on Ion Torrent PGM to obtain the DNA fragment sequence of the target region with the fragment length distributed between 130 and 150 bp. And performing quality control and analysis of conventional parameters on the obtained sequencing off-machine data.
Detection result:
the samples in the experimental example 1 are tested, so that the full coverage of the target interval can be obtained, the target full coverage is realized, the lowest sequencing depth is 100x,mapping rate-99%, the ontarget rate is 95% or more, and the uniformity of 100x is 95% or more. Specific information is shown in table 2 below.
Table 2 sample test information table
In table 2, "Mapping rate" represents the comparison rate at the time of resequencing; "Ontarget rate" represents the mid-target rate, representing the ratio of sequencing data aligned to the targeted amplicon; ">0.2x Average Depth Rate" represents a proportion of sites greater than 0.2x average sequencing depth, the higher the value, the better the amplification uniformity; ">30 xLepth Rate" represents coverage of target sites, the higher this value, the higher the target site coverage.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the application.
Claims (7)
1. A set of sites for detection of Kartagener syndrome prior to embryo implantation comprising 81 sites carried by chromosome 5 comprising a single pathogenic site, 44 SNP sites linked to said pathogenic site upstream and 36 SNP sites linked to said pathogenic site downstream;
the 81 sites are as follows:
2. use of the locus according to claim 1 for the preparation of a diagnostic reagent or kit for the detection of Kartagener syndrome prior to embryo implantation.
3. The set of primer compositions for detecting the site of Kartagener syndrome before embryo implantation according to claim 1, wherein the information of each primer in the primer composition is as follows:
4. a kit for detecting the site of kartager syndrome prior to embryo implantation, comprising the primer composition of claim 3.
5. The set of specific primer combinations for site detection for detection of Kartagener syndrome prior to embryo implantation according to claim 1, wherein the specific primer combinations comprise 44 pairs of upstream primers, 1 pair of specific primers containing a pathogenic mutation site, 36 pairs of downstream primers;
the sequence information of the specific primer combination is as follows:
6. a kit for detecting the site of kartager syndrome prior to embryo implantation comprising a specific primer combination according to claim 5.
7. Use of a kit according to any one of claims 4 or 6 for the preparation of a diagnostic product for detecting Kartagener syndrome prior to embryo implantation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310697800.2A CN116804219A (en) | 2023-06-13 | 2023-06-13 | A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310697800.2A CN116804219A (en) | 2023-06-13 | 2023-06-13 | A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116804219A true CN116804219A (en) | 2023-09-26 |
Family
ID=88079291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310697800.2A Pending CN116804219A (en) | 2023-06-13 | 2023-06-13 | A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116804219A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010111360A2 (en) * | 2009-03-24 | 2010-09-30 | The Research Foundation Of State University Of New York | Diagnosis of ciliopathies |
| US20100292088A1 (en) * | 2009-05-15 | 2010-11-18 | The University Of North Carolina At Chapel Hill | Methods and compositions for detecting genetic markers associated with primary ciliary dyskinesia |
| CN104120133A (en) * | 2013-04-28 | 2014-10-29 | 中国人民解放军总医院 | Gene mutant and application thereof |
| CN108220403A (en) * | 2017-12-26 | 2018-06-29 | 北京科迅生物技术有限公司 | Detection method, detection device, storage medium and the processor in specific mutation site |
| CN108841950A (en) * | 2018-07-13 | 2018-11-20 | 上海市第人民医院 | A kind of autosomal recessive inheritance Kartagener syndrome mutated gene and its application |
| CN109055532A (en) * | 2018-09-12 | 2018-12-21 | 苏州贝康医疗器械有限公司 | Hereditary hearing impairment genetic test Primer composition, kit and application before embryo implantation |
| CN110438220A (en) * | 2019-08-16 | 2019-11-12 | 深圳市人民医院 | The motionless syndrome gene panel kit of cilium and its application |
| CN111500697A (en) * | 2020-03-20 | 2020-08-07 | 四川省人民医院 | Immobile cilium syndrome screening kit |
| CN113005195A (en) * | 2021-04-27 | 2021-06-22 | 北京嘉宝仁和医疗科技有限公司 | Primer composition, product and method for detecting embryo carrying risk chromosome |
| CN113151284A (en) * | 2020-12-23 | 2021-07-23 | 首都医科大学附属北京妇产医院 | DNAH5 gene mutant and application thereof |
| CN113436680A (en) * | 2020-05-22 | 2021-09-24 | 复旦大学附属妇产科医院 | Method for simultaneously identifying chromosome structural abnormality and carrier state of pathogenic gene of embryo |
-
2023
- 2023-06-13 CN CN202310697800.2A patent/CN116804219A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010111360A2 (en) * | 2009-03-24 | 2010-09-30 | The Research Foundation Of State University Of New York | Diagnosis of ciliopathies |
| US20100292088A1 (en) * | 2009-05-15 | 2010-11-18 | The University Of North Carolina At Chapel Hill | Methods and compositions for detecting genetic markers associated with primary ciliary dyskinesia |
| CN104120133A (en) * | 2013-04-28 | 2014-10-29 | 中国人民解放军总医院 | Gene mutant and application thereof |
| CN106282195A (en) * | 2013-04-28 | 2017-01-04 | 中国人民解放军总医院 | Gene mutant and application thereof |
| CN108220403A (en) * | 2017-12-26 | 2018-06-29 | 北京科迅生物技术有限公司 | Detection method, detection device, storage medium and the processor in specific mutation site |
| CN108841950A (en) * | 2018-07-13 | 2018-11-20 | 上海市第人民医院 | A kind of autosomal recessive inheritance Kartagener syndrome mutated gene and its application |
| CN109055532A (en) * | 2018-09-12 | 2018-12-21 | 苏州贝康医疗器械有限公司 | Hereditary hearing impairment genetic test Primer composition, kit and application before embryo implantation |
| CN110438220A (en) * | 2019-08-16 | 2019-11-12 | 深圳市人民医院 | The motionless syndrome gene panel kit of cilium and its application |
| CN111500697A (en) * | 2020-03-20 | 2020-08-07 | 四川省人民医院 | Immobile cilium syndrome screening kit |
| CN113436680A (en) * | 2020-05-22 | 2021-09-24 | 复旦大学附属妇产科医院 | Method for simultaneously identifying chromosome structural abnormality and carrier state of pathogenic gene of embryo |
| CN113151284A (en) * | 2020-12-23 | 2021-07-23 | 首都医科大学附属北京妇产医院 | DNAH5 gene mutant and application thereof |
| CN113005195A (en) * | 2021-04-27 | 2021-06-22 | 北京嘉宝仁和医疗科技有限公司 | Primer composition, product and method for detecting embryo carrying risk chromosome |
Non-Patent Citations (3)
| Title |
|---|
| LILI ZHANG等: "Co-occurrence of Moyamoya syndrome and Kartagener syndrome caused by the mutation of DNAH5 and DNAH11: a case report", 《BMC NEUROLOGY》, vol. 20, pages 1 - 5 * |
| XIAN-DONG CHENG等: "Novel homozygous mutations of DNAH5 in Kartagener syndrome", 《QJM: AN INTERNATIONAL JOURNAL OF MEDICINE》, vol. 115, no. 5, pages 319 - 320 * |
| 徐保平等: "Kartagener综合征儿童DNAll、DNAH5基因的研究", 《中华医学会第五次全国儿科中青年学术交流大会论文集》, pages 932 - 939 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6760917B2 (en) | Analysis of genomic fractions using polymorphic counts | |
| Pös et al. | Recent trends in prenatal genetic screening and testing | |
| Vialard et al. | Prenatal BACs‐on‐Beads™: a new technology for rapid detection of aneuploidies and microdeletions in prenatal diagnosis | |
| Vialard et al. | Prenatal BACs‐on‐BeadsTM: the prospective experience of five prenatal diagnosis laboratories | |
| WO2024027569A1 (en) | Haplotype construction method independent of proband | |
| CN102171565A (en) | Methods for allele calling and ploidy calling | |
| CN106661609B (en) | Method for predicting congenital heart defects | |
| JP2014531208A (en) | Gene copy number polymorphism testing method | |
| CN114480667A (en) | Method for detecting fetal chromosome balance structural variation through pregnant woman peripheral blood free DNA | |
| WO2021237105A1 (en) | Methods for determining a genetic variation | |
| WO2024114253A1 (en) | Kit for determining whether individual to which sample to be tested belongs has genetic disease | |
| CN106939334A (en) | A kind of detection method of fetal DNA in maternal plasma DNA content | |
| CN117025752A (en) | Single-gene defect detection method for eliminating risk chromosomes of embryo before implantation and application | |
| CN104087672A (en) | Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique | |
| CN117248030A (en) | PKD1 variant molecule detection method based on single-cell whole genome amplification and application thereof | |
| CN116804219A (en) | A set of sites, primer compositions, and kits and applications for detection of Kartagner syndrome prior to embryo implantation | |
| CN101407843B (en) | Reagent kit for detecting chromosome numerical abnormalities resulting in natural abortion | |
| CN111172248B (en) | General kit for verifying copy number variation based on fragment analysis technology | |
| Tian et al. | Robust evidence reveals the reliable rate of normal/balanced embryos for identifying reciprocal translocation and Robertsonian translocation carriers | |
| Halder et al. | Mosaicism in 22q11. 2 Microdeletion Syndrome. | |
| Li et al. | Diagnosis of prenatal 22q11. 2 duplication syndrome: a two-case study | |
| CN116555417A (en) | Site for detecting congenital deafness before embryo implantation, primer combination and application thereof | |
| CN109112211A (en) | A kind of the primer combination and method of human embryos Chediak-Higashi syndrome LYST detection in Gene Mutation | |
| CN113621695B (en) | Marker of endometrial receptivity of RIF patient, application of marker and detection kit | |
| CN117925820B (en) | Method for detecting variation before embryo implantation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |