CN116004792A - Nucleic acid product, kit and method for detecting human KCTD19 mutation and application of nucleic acid product - Google Patents

Abstract

The application relates to a nucleic acid product, a kit, a method and application thereof for detecting human KCTD19 mutation. The KCTD19 mutations include at least one of c.g628a and c.c893t. By detecting the mutation condition of human KCTD19, the genetic defect of the oligospermia patient can be assisted to be analyzed, and a reference is provided for selecting a proper pregnancy assisting mode for the oligospermia patient. The nucleic acid product has the advantages of low cost and high efficiency when being used for detecting the mutation of human KCTD 19.

Description

Nucleic acid product, kit and method for detecting human KCTD19 mutation and application of nucleic acid product

Technical Field

The application relates to the technical field of molecular biology, in particular to a nucleic acid product, a kit, a method and application thereof for detecting human KCTD19 mutation.

Background

Male infertility is a heterogeneous disease caused by a variety of factors including oligospermia, azoospermia, oligospermia, teratospermia, and the like. Among them, oligospermia is usually caused by genetic defects such as chromosomal abnormality, AZF microdeletion on Y chromosome, gene mutation, etc., and is one of common causes of male infertility.

Currently, there are 2300 genes specifically highly expressed in testis tissues, and it is confirmed by model animals that 800 genes are associated with male sperm disorder. With the wide application of a new generation of sequencing technology represented by whole exon sequencing in screening of pathogenic genes of male sterile patients, some pathogenic gene mutations causing oligospermia are discovered successively, such as RPL10L, M1AP, PNLDC1 and the like, and an important molecular basis is provided for diagnosis, prevention and fertility treatment of oligospermia patients. However, the identification of pathogenic genes for oligospermia patients by using a high-throughput sequencing technology has the problems of high cost, long time consumption and the like.

Intracytoplasmic sperm injection (Intracytoplasmic sperm injection, ICSI) is one of the common fertility options for severely oligospermic patients due to their low sperm count and impaired motility. However, asthenospermia patients may have genetic defects (e.g., mutations in genes), and offspring that are pregnant through ICSI will also carry the corresponding defective gene. When the carrier performs wedding after adulthood, if women also have heterozygous mutations in the gene, then their offspring of fertility are at risk of serious oligospermia vertical transmission. Therefore, it is important to clarify the genetic etiology of oligospermia patients.

In clinical practice, patients with oligospermia usually need genetic diagnosis (Gene diagnosis) to confirm the genetic etiology before selecting a suitable means of assisted pregnancy to ensure healthy offspring. For patients with a defined phenotype and known pathogenic genes, the exon sequences of the individual genes and their flanking sequences were mainly detected by PCR-Sanger sequencing technology. For patients with undefined pathogenic genes, a new generation sequencing technology including whole exome sequencing is mainly adopted, the detection flux of the technology is high, all coding genes in the genome can be detected simultaneously, but the genetic variation detected by the technology also needs to be subjected to a large amount of bioinformatics analysis and Sanger sequencing verification, and a special bioinformatics analysis engineer is needed to be matched, so that the cost is high.

Disclosure of Invention

The research of the application finds that the missense mutation c.G428A or c.C893T of KCTD19 of oligospermia patients affects the expression of KCTD19 protein (p.E210K and p.P298L), thereby affecting the normal meiosis process and finally appearing as oligospermia. The two mutation sites are not described in the database of healthy people. By detecting whether KCTD19 carried by the oligospermia patient has the mutation sites, the genetic etiology of the oligospermia patient is clarified, and a reference can be provided for selecting a proper pregnancy assisting mode for the oligospermia patient. Based on the above, the application provides a nucleic acid product, a kit, a method and application thereof for detecting human KCTD19 gene mutation with low cost and high efficiency.

Use of an agent for detecting mutations in human KCTD19, the wild type KCTD19 mRNA being numbered nm_001100915.3 in the NCBI database, with reference to the wild type KCTD19 mRNA, for the preparation of a diagnostic product for oligospermia, said mutations comprising at least one of the following mutations: g628a and c.c893t.

Use of an agent for detecting mutations in human KCTD19 protein, the mRNA of wild type KCTD19 protein in NCBI database numbered nm_001100915.3, with reference to wild type KCTD19 protein, for the preparation of a diagnostic product for oligospermia, the mutations comprising at least one of the following mutations: p.E210K and p.P298L.

Use of a human KCTD19 mutation in the preparation of an animal model of oligospermia, the wild type KCTD19 mRNA in the NCBI database numbered nm_001100915.3, with reference to the wild type KCTD19 mRNA, said mutation comprising at least one of the following mutations: g628a and c.c893t.

A nucleic acid product comprising a primer pair for detecting a mutation in human KCTD19, the wild type KCTD19 mRNA being numbered nm_001100915.3 in the NCBI database with reference to the wild type KCTD19 mRNA, the mutation comprising at least one of the following mutations: g628a and c.c893t.

In one embodiment, the nucleic acid product detects the mutation by one or more of the following methods:

common PCR amplification methods, sequencing methods and real-time fluorescent quantitative PCR methods.

In one embodiment, the primer pair comprises one or both of the following primer pairs:

primer pairs with nucleotide sequences shown as SEQ ID No. 1-2, and primer pairs with nucleotide sequences shown as SEQ ID No. 3-4.

A kit comprising the nucleic acid product described above.

In one embodiment, the kit further comprises at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent.

A method of detecting a human KCTD19 mutation comprising the steps of:

using the DNA of the sample to be detected as a template, and carrying out PCR amplification by using the primer pair in the nucleic acid product to obtain an amplification product; a kind of electronic device with high-pressure air-conditioning system

Analyzing the amplification product to determine the mutation status of the human KCTD 19.

In one embodiment, the method further comprises the step of sequencing the amplification product prior to analyzing the amplification product.

Drawings

FIG. 1 is a family chart and KCTD19 sequencing peak chart of the oligospermia patient 1 of example 1;

FIG. 2 is a family chart and KCTD19 sequencing peak chart of asthenozoospermia patient 2 in example 1;

FIG. 3 is a three-dimensional block diagram of KCTD19 mutein (p.E210K) of example 1;

FIG. 4 is a three-dimensional structure diagram of KCTD19 mutein (p.P298L) of example 1;

FIG. 5 is a chart of HE staining of semen from oligospermia patients in example 1;

FIG. 6 is a chart showing the FISH staining of semen from oligospermia patients in example 1;

FIG. 7 is a Western blot analysis of mutant KCTD19 of example 1.

Detailed Description

The detailed description of the embodiments of the present application will be presented in order to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, and the like used in the present application are commercially available or may be prepared by existing methods.

The term "primer" as used herein refers to an oligonucleotide, whether naturally occurring in a purified restriction digest or synthetically produced, that is capable of functioning as a point of initiation of synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand (e.g., in the presence of a nucleotide and an inducer such as a DNA polymerase and at a suitable temperature and pH). "HEK 293T cell" is a cell line derived from human embryonic kidney cells, a cell line commonly used for expression studies of foreign genes, and rarely expresses endogenous receptors required for extracellular ligands. "3×FLAG" is a tag commonly used to detect protein expression _。

"missense mutation" refers to a change in the amino acid type and sequence of a polypeptide chain by changing a codon encoding an amino acid to a codon encoding another amino acid after base substitution. The result of missense mutations is generally the loss of function of the polypeptide chain, causing protein abnormalities.

Male sterility is a disease caused by a variety of factors, has high phenotypic heterogeneity and genetic heterogeneity, and needs to be continuously supplemented with new pathogenic genes for detection.

The application finds that two mutation sites (c.G6278A, c.C893T) of KCTD19 are related to oligospermia through research. The two missense mutations are both pathogenic mutations, and the mutated amino acids are highly conserved among various species, as predicted by various bioinformatics software.

Accordingly, an embodiment of the present application provides a use of an agent for detecting a human KCTD19 mutation, wherein the wild type KCTD19 mRNA is numbered nm_001100915.3 in the NCBI database, and the human KCTD19 mutation comprises at least one of the following mutations with reference to the wild type KCTD19 mRNA: g628a and c.c893t.

The result shows that the 2 gene mutation sites are missense mutation, and the protein 3D structure shows that the gene mutation leads to abnormal hydrogen bond connection of amino acid, so that the protein space structure is unstable, and the normal function of KCTD19 protein is affected.

In addition, an embodiment of the present application further provides an application of the reagent for detecting the mutation of the human KCTD19 protein in preparing a diagnostic product for oligospermia, wherein the mRNA of the wild type KCTD19 protein is numbered nm_001100915.3 in NCBI database, and the mutation of the human KCTD19 protein includes at least one of the following mutations with reference to the wild type KCTD19 protein: p.E210K and p.P298L.

Furthermore, humans and mice are highly conserved at these two KCTD19 mutation sites. Since the phenotype of the full knockout (i.e., whole gene knock-out) of KCTD19 is azoospermia, it cannot be used for studies in oligospermia. Thus, based on the above, an embodiment of the present application further provides an application of the human KCTD19 mutation in preparing an animal model of oligospermia, the mRNA of wild type KCTD19 in NCBI database is numbered nm_001100915.3, the mRNA of wild type KCTD19 is used as a reference, and the human KCTD19 mutation includes at least one of the following mutations: g628a and c.c893t.

The animal model of oligospermia can be used for clinically assisting in understanding disease origin, screening target drugs and verifying safety and effectiveness of the drugs. Animal models of oligospermia can also be used as research models for the molecular mechanisms of meiosis and oligospermia.

In addition, an embodiment of the present application further provides a method for preparing an animal model for oligospermia, the method comprising the steps of:

a mouse model of oligospermia was constructed from the mouse mutation sites (NM-177791.3: G627A and c.C 962T) corresponding to the mutation sites (c.G 6276A and c.C 893T) of human KCTD 19.

In addition, an embodiment of the present application further provides a nucleic acid product, which includes a primer pair for detecting a mutation of human KCTD19, wherein the wild type KCTD19 mRNA is numbered nm_001100915.3 in the NCBI database, and the wild type KCTD19 mRNA is used as a reference, and the mutation of human KCTD19 includes at least one of the following mutations: g628a and c.c893t.

In one embodiment, the above nucleic acid product detects the mutation by one or more of the following methods: common PCR amplification methods, sequencing methods and real-time fluorescent quantitative PCR methods.

In one embodiment, the primer pair comprises one or both of the following primer pairs:

primer pairs with nucleotide sequences shown as SEQ ID No. 1-2, and primer pairs with nucleotide sequences shown as SEQ ID No. 3-4.

Specifically, the nucleotide sequence shown in SEQ ID No.1 is 5'-CATGGACACACCCCTGTTAGAC-3'; the nucleotide sequence shown in SEQ ID No.2 is 5'-GTCATGCTTCCGCTACCTC-3'; the nucleotide sequence shown as SEQ ID No.3 is 5'-AAGCCTCCAATCCAGCTATGACC-3'; the nucleotide sequence shown as SEQ ID No.4 is 5'-GCTGAAAGAGGACGCCATTCC-3'.

The primer pair for detecting the KCTD19 mutation of the human has the characteristics of simplicity and rapidness in operation, low cost and the like.

In addition, an embodiment of the present application also provides a kit for detecting human KCTD19 mutation, including the nucleic acid product of any of the above examples.

In one embodiment, the kit further comprises at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent.

In one embodiment, the positive control is a DNA sample of human KCTD19 in which either or both of the mutations are present.

In one embodiment, the negative control is deionized water. It will be appreciated that in other specific examples, the negative control may be a sample of other human KCTD19 that has not been mutated.

In one embodiment, the PCR amplification reagent comprises a PCR premix. In an alternative specific example, the PCR premix includes Mg ²⁺ Reaction buffer, DNA polymerase and dNTPs.

In one embodiment, the reaction buffer may be 1×,2×,5×, 10×; the concentration of dNTPs is 8 mM-12 mM; mgCl ₂ 20 mM-30 mM; taqDNA polymerase is 4U/. Mu.L to 6U/. Mu.L.

In a specific example, the concentration of the nucleic acid product is 10 μm; PCR buffer was 5×; the concentration of dNTPs was 10mM; mgCl ₂ 25mM; taqDNA polymerase was 5U/. Mu.L.

It will be appreciated that in other specific examples, the above kit may not include any of the positive control, the negative control, the PCR amplification reagents, the sequencing reagents, and the electrophoresis detection reagents, and the reagents not included may be reasonably obtained from the outside.

The kit for detecting the KCTD19 mutation of the human can be used for assisting in judging male oligospermia, and has the characteristics of simplicity and rapidness in operation and the like.

An embodiment of the present application also provides a method for detecting a mutation of human KCTD19, including steps S10, S20, S30, S40 and S50. Specifically:

step S10: and obtaining DNA of the sample to be tested.

In one embodiment, in step S10, the sample to be tested is one or more of peripheral blood, semen, blastula culture solution, and prenatal diagnosis-related samples.

Further, in step S10, the sample to be measured is peripheral blood of the patient.

Step S20: and (3) taking the DNA of the sample to be detected as a template, and carrying out PCR amplification by using the primer pair in the nucleic acid product of any embodiment to obtain an amplification product.

In some embodiments, in step S20, the amplification procedure of PCR is set to: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 30-45 s, annealing at 56-60 ℃ for 30-60 s, extension at 72 ℃ for 45-60 s, and co-circulation for 30-35 times; finally, the PCR amplification product is extended for 3 to 7 minutes at 72 ℃, and is stored at 4 ℃. It will be appreciated that in some other specific examples, the PCR procedure may be rationally adjusted.

In one specific example, the amplification procedure for PCR is set to: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 56.5℃for 30s and extension at 72℃for 60s, followed by 34 cycles; finally, the PCR amplification product was stored at 4℃after 5min extension at 72 ℃.

Step S30: agarose gel electrophoresis was performed on the PCR amplified products.

In one embodiment, in step S30, 3. Mu.L to 5. Mu.L of PCR amplification product and DL2000 DNA Marker (TSJ 011-100, optimago) are respectively dispensed into the gel well, and electrophoresis is carried out for 9min to 10min at 220V, and imaging and photographing are carried out.

In one embodiment, in step S30, the band size of the PCR amplification product is determined by agarose gel electrophoresis.

Step S40: sequencing the amplified product.

In one embodiment, in step S40, the sequencing method is Sanger sequencing.

In one embodiment, in step S40, a sequencing analysis is performed using the same primer pair as the PCR amplification primer pair.

Step S50: the mutation status of KCTD19 was analyzed.

In one embodiment, in step S50, the sequence sequenced in step S40 is aligned with the mRNA of wild type KCTD19 (NCBI database No. nm_ 001100915.3) to analyze whether KCTD19 is mutated.

The method for detecting the human KCTD19 mutation has at least the following advantages: (1) high detection efficiency: the KCTD19 mutation can be detected by only designing a pair of primers, amplifying by PCR and carrying out sequencing analysis, and the mutation detection can be completed within 1-2 days. (2) simple operation and low cost: compared with DNA library construction and total exon sequencing combined bioinformatics analysis, the PCR amplification combined sequencing technology is simpler and has lower cost.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the present application.

EXAMPLE 1 screening of pathogenic genes related to oligospermia

1. Obtaining DNA of a sample to be tested

Clinically, peripheral blood samples of 2 patients suffering from idiopathic male infertility are obtained, and the conventional detection results of semen of the patients show that the sperm concentration is 6.7X10 respectively ⁶ /mL and 0.5X10 ⁶ Per mL, and sperm without forward movement, chromosome karyotyping and Y chromosome AZF microdeletion detection were not abnormal, and other physical examinations (including height, weight, intelligence and development, etc.) were not abnormal (table 1). Wherein semen analysis is assessed according to the world health organization fifth edition (2010) guidelines.

TABLE 1 sterility-related detection of two oligospermia patients

A DNA extraction kit (DNA QIAamp DNA Blood Mini Kit; 51106) from QIAGEN, germany, was used to extract DNA from a peripheral blood sample of a patient and their family. The specific extraction steps are as follows:

1) 200. Mu.L of whole blood and 20. Mu.L of proteinase K were added to a 1.5mL EP tube and mixed well;

2) Adding 200 μL AL, mixing, shaking for 30s, centrifuging for 3s instantaneously, and water-bathing at 56 deg.C for 15min;

3) Adding 200 mu L of absolute ethyl alcohol, reversing, uniformly mixing, oscillating for 30s, and performing instantaneous centrifugation for 3s;

4) Pouring the liquid into a filter column with a centrifuge tube, centrifuging at 13000 Xg for 1min, and discarding the filtrate and the centrifuge tube;

5) Placing the filter column into a new centrifuge tube, adding 500 mu L of AW1, centrifuging for 1min at 13000 Xg, and discarding the filtrate and the centrifuge tube;

6) Placing the filter column into a new centrifuge tube, adding 500 mu L of AW2, centrifuging for 1min at 13000 Xg, and discarding the filtrate; centrifuging for 3min, and discarding filtrate and centrifuging tube;

7) The filter cartridge was placed in a new EP tube, 80. Mu.L of AE was added, left standing at room temperature for 5min, centrifuged at 13000 Xg for 1min, and stored at 4℃for a short period or at-70℃for a long period.

2. Screening candidate pathogenic genes

Taking 30 mug of peripheral blood DNA of the 2 oligospermia patients, sending the peripheral blood DNA to Shenzhen Dada gene technology Co., ltd for whole exon sequencing and analysis (including DNA library construction, whole exon sequencing machine and data analysis), and using a genome analysis kit for mutation identification; mutations found by sequencing were annotated using ANNOVAR software and pathogenic genes were screened based on the annotation results.

The screening strategy for candidate pathogenic genes is approximately as follows: (1) Filtering mutations with a frequency greater than 1% in a public genetic mutation database (1000 genome, dbSNP, exAC database); (2) Selecting bioinformatics software prediction (Mutation master, SIFT, ploypen-2) as a Mutation which is likely to cause diseases; (3) prioritizing homozygous mutations or complex heterozygous mutations; (4) The selection of the gene whose mutation is associated with spermatogenesis, or is specifically highly expressed in testis tissue, or is indicative of a male sterile phenotype in model animals.

Screening to obtain a pathogenic gene KCTD19 related to male oligospermia based on the steps; mutation sites associated with oligospermia include c.g628a and c.c893t.

PCR amplification

According to the pathogenic genes and pathogenic mutation sites thereof obtained by screening, specific amplification primers are designed, and the primer information is shown in Table 2. Preparing a PCR reaction system of a sample to be tested according to the table 3, wherein the PCR premix is

Green PCR Master Mix (REF M7123, promega, USA); the DNA template is peripheral blood gDNA of oligospermia patients or families thereof.

TABLE 2 primer information

TABLE 3 PCR reaction System

Composition of the components	Volume of
		Green Master Mix，2×	15μL
RNase-free Water	13μL
		DNA template	1μL
Primer F (10. Mu. Mol/L)	0.5μL
		Primer R (10. Mu. Mol/L)	0.5μL
Totals to	30μL

The prepared PCR reaction system is amplified according to the following procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 56.5℃for 30s, extension at 72℃for 60s, 34 cycles; finally, the PCR amplification product was stored at 4℃after 5min extension at 72 ℃.

4. Agarose gel electrophoresis

(1) Preparing 2% agarose gel: weighing 2g agarose (TSJ 001, optimus Praeparatus) in conical flask, adding 100mL 1 xTAE buffer (TSG 001, optimus Praeparatus) and heating with strong fire for 3min for dissolving, cooling to about 35deg.C, adding 4 μl of nucleic acid dye (TSJ 003, optimus of Optimus) and mixing, pouring the mixture into a mounted glue making mold, and cooling and forming at room temperature.

(2) Respectively taking 3 μl of PCR amplification product and DL2000 DNA Marker (TSJ 011-100, optimago) and dispensing into gel well, electrophoresis at 220V for 9min, imaging and photographing.

5. Sequencing analysis

And (3) sending the PCR amplification product qualified by agarose gel electrophoresis and the corresponding PCR amplification primer pair to the Soaceae biotechnology Co., ltd (Beijing, china) for Sanger sequencing analysis. As shown in FIG. 1, the oligospermia patient 1 (F1 II-5) and two sterile brogers (F1 II-1 and F1 II-4) were homozygous missense mutations of KCTD19 (c.G628A: p.E210K); as shown in FIG. 2, oligospermia patient 2 (F2 II-1) carries a homozygous missense mutation at another site of KCTD19 (c.C893T: p.P298L). The black squares in fig. 1 and 2 represent oligospermia patients.

6. Protein structural analysis

In order to determine the pathogenicity of two homozygous missense mutations, a three-dimensional structural model of the wild type KCTD19 protein was downloaded from the AlphaFold Protein Structure database and the three-dimensional structure of the KCTD19 mutant protein was predicted using PyMOL visualization software. As shown in fig. 3, the c.g628a mutation mutates the corresponding amino acid (p.e210k) and causes abnormal hydrogen bond connection, so that the protein space structure is unstable; as shown in FIG. 4, the c.C893T mutation mutated the corresponding amino acid (p.P298L), and the hydrogen bond connection between adjacent amino acids was abnormal after the mutation.

7. Staining analysis

HE staining (hematoxylin-eosin staining) was performed on semen from oligospermia patients, as shown in fig. 5, in which sperm with various morphologies, including head abnormality and tail abnormality, were seen in the semen.

Further staining of patient chromosome 18, chromosome X, and chromosome Y by FISH (fluorescence in situ hybridization, fluorescent in situ hybridization) showed significant increase of aneuploidy sperm in the patient semen, including abnormal 18, 18+18+XX, 18+18+X, 18+XXY, 18+XX sperm, as shown in FIG. 6. In FIG. 6, chromosome 18 is shown in the circle, chromosome X is shown in the square box, and chromosome Y is shown in the arrow.

8. Western blot analysis

Expression levels of wild type KCTD19 protein and mutant KCTD19 protein in HEK 293T cells were analyzed by Western Blot (WB). As shown in FIG. 7, EV, WT, M1 and M2 represent blank plasmids, plasmids carrying KCTD19-3 xFLAG, p.E210K_KCTD19-3 xFLAG and p.P298L_KCTD19-3 xFLAG, respectively, and GADPH is an internal reference protein. Statistical analysis of the relative expression level of KCTD19 protein, the expression level of KCTD19 (p.e210k) protein was significantly lower than that of wild type; the expression level of KCTD19 (p.p298l) protein was very significantly lower than that of the wild type; two missense mutations in KCTD19 are shown to reduce the expression of KCTD19 protein, so that the function of KCTD19 protein is abnormal, thereby disrupting the normal meiosis process and finally appearing as oligospermia. In fig. 7, significant differences at p <0.05 level are indicated, and significant differences at p <0.01 level are indicated.

According to the application, research shows that missense mutation is generated in KCTD19 of oligospermia patients, and the missense mutation is mainly expressed as c.G428A mutation and c.C893T mutation, so that hydrogen bond connection between adjacent amino acids is abnormal, the expression level of KCTD19 protein is obviously reduced, normal meiosis process is influenced, various aneuploidy sperms are formed, and the misshapen sperms are finally expressed as sperm malformation and oligospermia.

The method for detecting the KCTD19 mutation of the human body, disclosed by the application, has the advantages of low cost, short time consumption and the like, and can assist in diagnosis or prediction of the curative effect of the oligospermia patient.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims (10)

1. Use of an agent for detecting mutations in human KCTD19 for the preparation of a diagnostic product for oligospermia, characterized in that the mRNA of wild-type KCTD19 is numbered nm_001100915.3 in the NCBI database, with reference to the mRNA of wild-type KCTD19, said mutations comprising at least one of the following mutations: g628a and c.c893t.

2. Use of an agent for detecting mutations in human KCTD19 protein, characterized in that the mRNA of wild type KCTD19 protein in the NCBI database is numbered nm_001100915.3, with reference to wild type KCTD19 protein, for the preparation of a diagnostic product for oligospermia, said mutations comprising at least one of the following mutations: p.E210K and p.P298L.

3. Use of a human KCTD19 mutation in the preparation of an animal model of oligospermia, characterized in that the mRNA of wild type KCTD19 in the NCBI database is numbered nm_001100915.3, with reference to the mRNA of wild type KCTD19, said mutation comprising at least one of the following mutations: g628a and c.c893t.

4. A nucleic acid product comprising a primer pair for detecting a mutation in human KCTD19, the wild type KCTD19 mRNA being numbered nm_001100915.3 in the NCBI database, with reference to the wild type KCTD19 mRNA, said mutation comprising at least one of the following mutations: g628a and c.c893t.

5. The nucleic acid product of claim 4, wherein the mutation is detected by one or more of the following methods:

common PCR amplification methods, sequencing methods and real-time fluorescent quantitative PCR methods.

6. The nucleic acid product of claim 4, wherein the primer pair comprises one or both of the following primer pairs:

primer pairs with nucleotide sequences shown as SEQ ID No. 1-2, and primer pairs with nucleotide sequences shown as SEQ ID No. 3-4.

7. A kit comprising the nucleic acid product of any one of claims 4 to 6.

8. The kit of claim 7, further comprising at least one of a negative control, a positive control, a nucleic acid extraction reagent, a PCR amplification reagent, a sequencing reagent, and an electrophoresis detection reagent.

9. A method for detecting a human KCTD19 mutation, comprising the steps of:

performing PCR amplification by using the primer pair in the nucleic acid product according to any one of claims 4 to 6 with DNA of a sample to be detected as a template to obtain an amplified product; a kind of electronic device with high-pressure air-conditioning system

Analyzing the amplification product to determine the mutation status of the human KCTD 19.

10. The method of claim 9, further comprising the step of sequencing the amplification product prior to analyzing the amplification product.

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