CN117535399A - Application of SLC30A5 gene rs164578 locus, primer probe combination and kit thereof - Google Patents
Application of SLC30A5 gene rs164578 locus, primer probe combination and kit thereof Download PDFInfo
- Publication number
- CN117535399A CN117535399A CN202311620241.1A CN202311620241A CN117535399A CN 117535399 A CN117535399 A CN 117535399A CN 202311620241 A CN202311620241 A CN 202311620241A CN 117535399 A CN117535399 A CN 117535399A
- Authority
- CN
- China
- Prior art keywords
- genotype
- locus
- gene
- slc30a5
- probe
- 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
- 239000000523 sample Substances 0.000 title claims abstract description 69
- 101150111807 Slc30a5 gene Proteins 0.000 title claims abstract description 53
- 208000007466 Male Infertility Diseases 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 239000003550 marker Substances 0.000 claims abstract description 17
- 208000000509 infertility Diseases 0.000 claims abstract description 13
- 230000036512 infertility Effects 0.000 claims abstract description 13
- 231100000535 infertility Toxicity 0.000 claims abstract description 13
- 206010021929 Infertility male Diseases 0.000 claims description 35
- 108700028369 Alleles Proteins 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 21
- 238000001917 fluorescence detection Methods 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 7
- 238000011144 upstream manufacturing Methods 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 238000010791 quenching Methods 0.000 claims description 4
- 230000000171 quenching effect Effects 0.000 claims description 4
- 239000003086 colorant Substances 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000003752 polymerase chain reaction Methods 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000003205 genotyping method Methods 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 14
- 238000011160 research Methods 0.000 abstract description 4
- 238000003759 clinical diagnosis Methods 0.000 abstract description 3
- 230000008506 pathogenesis Effects 0.000 abstract description 3
- 238000011282 treatment Methods 0.000 abstract description 3
- 238000009612 semen analysis Methods 0.000 description 12
- 210000000582 semen Anatomy 0.000 description 8
- 238000007400 DNA extraction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 description 2
- 101100483399 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) CDC34 gene Proteins 0.000 description 2
- 108091006550 Zinc transporters Proteins 0.000 description 2
- 239000003433 contraceptive agent Substances 0.000 description 2
- 230000002254 contraceptive effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 101150110276 mor gene Proteins 0.000 description 2
- 101150038440 Slc39a13 gene Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- -1 dntps Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000019100 sperm motility Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 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
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- 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)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of gene detection, and discloses application of an SLC30A5 gene rs164578 locus, a primer probe combination and a kit thereof, wherein the application is novel application of the SLC30A5 gene rs164578 locus serving as a marker in a male infertility risk detection method, and if the genotype of the SLC30A5 gene rs164578 locus is TT genotype, the risk of suffering from infertility is marked to be higher than that of CC genotype and CT genotype. The locus rs164578 of the SLC30A5 gene is used as a locus for judging male infertility risk, and the genotyping of the locus rs164578 is detected, so that the locus has very important guiding significance for male infertility pathogenesis research, clinical diagnosis and treatment.
Description
Technical Field
The invention belongs to the technical field of gene detection, and relates to application of an SLC30A5 gene, in particular to application of an rs164578 locus of the SLC30A5 gene, a primer probe combination and a kit thereof.
Background
The world health organization defines infertility as the inability to successfully gestate in regular life of 12 months without taking contraceptive measures. It was found that couples of over 5000 ten thousand bearing ages are afflicted with infertility worldwide, and that male factors account for 51% of the causes of infertility, with up to 40% diagnosed as idiopathic male infertility, i.e. unable to confirm the cause of infertility.
The current standard diagnostic procedures based on semen analysis are not sufficient to clearly distinguish between normal and sterile men, nor are they suitable for male infertility risk determination. On the one hand, in about 30% -40% of men suffering from idiopathic infertility, semen analysis and evaluation results such as sperm concentration, deformity rate and the like show a normal range; on the other hand, even if the semen analysis index is lower than the standard normal value, the fertility outcome cannot be directly indicated or predicted.
Meanwhile, in the existing semen analysis method, a testee is required to acquire semen in a masturbation mode, and psychological factors of a patient and a semen retention and sample delivery process bring uncertain factors to the link, so that sperm motility judgment can be influenced. The microscopic examination in the semen analysis and detection process not only relies on manual observation and counting, but also has the problems of low detection efficiency, complicated detection steps, long time consumption and the like when the result is error due to manual experience, and has the defects of high detection cost and the like when the special instrument is needed in other links of semen analysis and detection.
Therefore, there is an urgent need to find a new, simple, non-invasive and reliable method for determining the risk of male infertility more accurately, from the gene level, rather than the level of the gene expression results, which characterizes semen quality.
Disclosure of Invention
The invention aims to solve the technical problem of providing a new application of the locus rs164578 of the SLC30A5 gene, wherein the locus rs164578 of the SLC30A5 gene is used as a marker in a male sterility risk detection method, so that the aim of judging male sterility risk more accurately through detection of a gene level is fulfilled;
the invention further aims to provide a primer probe combination for detecting the rs164578 locus of the SLC30A5 gene and application of a kit thereof, so as to achieve the purposes of being simple and convenient, being applicable to various crowds, and being non-invasive and reliably detecting male infertility risks;
the inventor researches the relation between zinc and zinc transporter and male sterility for a long time, and through screening and analyzing the relation between 24 zinc transporter gene polymorphism and male sterility, finally discovers that the locus rs164578 of SLC30A5 gene on human autosomal No. 5 is highly related to the risk of male sterility, and can be used for judging the risk of male sterility. The locus has three genotypes of CC, CT and TT in the crowd, the crowd carrying the TT genotype has a risk of male infertility higher than the CC genotype and the CT genotype. By detecting the genotyping of the locus rs164578 of the SLC30A5 gene, the risk of male sterility can be judged more accurately from the gene level, and the method has very important guiding significance for the pathogenesis research of male sterility and the clinical diagnosis and treatment of patients.
In order to achieve the purpose, the technical scheme adopted by the invention is the application of the locus rs164578 of the SLC30A5 gene as a marker in a male sterility risk detection method.
As a limitation of the invention, the genotype of the locus rs164578 of the SLC30A5 gene is used for judging the risk of male infertility, and if the genotype of the locus rs164578 of the SLC30A5 gene is TT genotype, the risk of infertility is judged to be higher than CC genotype and CT genotype;
the nucleotide sequence of the TT genotype is shown as SEQ ID NO. 1;
the nucleotide sequence of the CC genotype is shown as SEQ ID NO. 2;
the nucleotide sequence of the CT genotype is shown as SEQ ID NO. 3.
The invention also provides a primer probe combination for detecting the male sterility risk detection marker, which is a primer probe combination for detecting the locus rs164578 genotype of the SLC30A5 gene and comprises a primer pair and a probe pair;
the primer pairs have the following sequences:
the upstream primer F is 5'-AAGGAAGCTGTCACTCATCTGATC-3',
a downstream primer R5'-TAATATTTTGCAAGGACTCACCCCT-3';
the sequences of the probe pairs are as follows:
c allele probe: 5'-CCATTCACACCATATGCATGG-3' the number of the individual pieces of the plastic,
t allele probe: 5'-CCATTCACATCATATGCATGG-3';
wherein, the 3 'end of the probe pair is connected with a quenching group, the 5' end is connected with a fluorescent group, and the fluorescent colors of the fluorescent groups in the C allele probe and the T allele probe are different.
The primer and probe combination provided by the invention has high specificity, and can realize rapid and accurate detection of the locus rs164578 genotype of the SLC30A5 gene.
As a definition of the invention, the quenching group is an MGB group.
As a further definition of the invention, the fluorophore comprises a CY3 group, a ROX group, or a CY5 group.
As a still further definition of the invention, the C allele probe is:
5′FAM-CCATTCACACCATATGCATGG-3′MGB；
the T allele probe is as follows:
5′VIC-CCATTCACATCATATGCATGG-3′MGB。
the invention also provides a kit for detecting male sterility risk, which detects the locus rs164578 of the male sterility risk detection marker SLC30A5 gene.
Preferably, the kit further comprises a DNA polymerase, a buffer, dntps, and deionized water.
As a limitation of the present invention, the kit for male sterility risk detection comprises any one of the primer probe combinations described above.
The invention also provides application of the kit for detecting male sterility risk, which takes genomic DNA of a sample to be detected as a template, adopts the kit for PCR amplification, and carries out real-time fluorescence detection in the amplification process to obtain a real-time fluorescence detection result;
if the fluorescence detection result only contains the fluorescence signal of the T allele probe, the genotype of the rs164578 locus of the SLC30A5 gene of the sample to be detected is TT genotype, and the risk of suffering from infertility is judged to be higher than the CC genotype and the CT genotype.
Preferably, the PCR amplification system comprises the following reagents and amounts:
| reagent(s) | Dosage of |
| 2×morgene Master mix | 12.5μL |
| 10 mu mol/L upstream primer F | 0.5μL |
| 10 mu mol/L downstream primer R | 0.5μL |
| 10 mu mol/LC allele probe | 0.25μL |
| 10 mu mol/LT allele probe | 0.25μL |
| DNA template | 10ng-100ng |
| Deionized water | Make up to 25 μl. |
As a limitation of the present invention, the PCR amplification procedure is: the mixture was subjected to 40 cycles of 5min pre-denaturation at 95 ℃, 5s denaturation at 95℃and 40s extension at 65 ℃.
The PCR amplification system and the PCR amplification program can further improve the detection efficiency of the kit on the locus rs164578 genotype of the SLC30A5 gene.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
(1) the invention provides the application of the locus rs164578 of the SLC30A5 gene as a male sterility risk detection marker for the first time, and provides a simple, non-invasive and reliable detection method suitable for various crowds for male sterility diagnosis;
(2) the primer probe combination designed for the locus rs164578 of the SLC30A5 gene has the characteristics of high sensitivity and strong specificity, and can accurately distinguish TT genotype, CC genotype and CT genotype; through designing the fluorescent group, the primer probe combination can be used for fluorescent quantitative PCR, the genotype of the rs164578 locus can be rapidly identified, and meanwhile, the accuracy of reading the detection result is further improved.
(3) The kit for detecting male sterility risk reduces the dependence of male sterility risk detection on manual observation and manual experience, has the advantages of short time consumption, simple reaction condition and visual reaction result, can realize rapid and accurate typing of the rs164578 locus of the SLC30A5 gene, and has wide application prospect;
the invention realizes positive correlation between the quantity of the amplified product and the fluorescent signal value, only needs to carry out real-time fluorescent detection in the amplification process to carry out result judgment, is more accurate, simple, convenient, visual and practical in result judgment, accurately determines the genotype to predict the risk of male infertility, provides a tool for auxiliary diagnosis of male infertility patients, and has great clinical application value.
The present invention will be described in detail with reference to specific embodiments and drawings.
Drawings
FIG. 1 is a diagram of the detection result of genotyping rs164578 locus of SLC30A5 gene of the standard DNA1 in example 4 of the present invention;
FIG. 2 is a graph of the detection result of genotyping rs164578 locus of SLC30A5 gene of the standard DNA2 in example 4 of the present invention;
FIG. 3 is a graph of the detection result of genotyping rs164578 locus of SLC30A5 gene of the marker DNA3 in example 4 of the present invention;
FIG. 4 is a graph of the detection result of genotyping rs164578 locus of SLC30A5 gene of the standard DNA4 in example 4 of the present invention;
FIG. 5 is a graph of the detection result of genotyping rs164578 locus of SLC30A5 gene of the marker DNA5 in example 4 of the present invention;
FIG. 6 is a graph of the detection result of genotyping rs164578 locus of SLC30A5 gene of the marker DNA6 in example 4 of the present invention;
FIG. 7 is a graph of the detection result of genotyping rs164578 locus of SLC30A5 gene of the marker DNA7 in example 4 of the present invention;
FIG. 8 is a graph showing the result of genotyping the rs164578 locus of the SLC30A5 gene of the marker DNA8 in example 4 of the present invention.
Detailed Description
The invention will be described in further detail by means of specific examples. It should be understood that the described embodiments are only for explaining the present invention and do not limit the present invention.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
The experimental procedures of molecular biology in the examples include DNA extraction, PCR, etc. were mainly performed with reference to "Experimental guidelines for PCR technology" (second edition), (America) C.W. Di Fengch, G.S. Devex Foundation, scientific Press, beijing, 2004). The specific experimental conditions can be determined by simple experiments, if necessary.
Example 1
The embodiment is a male sterility risk gene detection method. The method takes the locus rs164578 of the SLC30A5 gene as a gene detection marker for male sterility risk.
The information of locus rs164578 of the SLC30A5 gene is shown in table 1, and the locus has three genotypes of CC, CT and TT in the crowd. Wherein:
the nucleotide sequence of the TT genotype is shown as SEQ ID NO. 1;
the nucleotide sequence of the CC genotype is shown as SEQ ID NO. 2;
the nucleotide sequence of CT genotype is shown in SEQ ID NO. 3.
TABLE 1 SLC30A5 Gene rs164578 site information
| SNPs | Position | Allele | Location | MAF(east-asian) |
| rs164578 | chr5:68417643 | C/T | Intron | 0.319 |
In Table 1, MAF (east-asan) represents the minimum allele frequency in the east Asian population.
In this example, whole genome DNA was extracted from a peripheral blood sample using a small amount of genomic DNA extraction kit (Tian-mo organism) according to the instructions, and the concentration of the extracted DNA was measured to be 34 ng/. Mu.L, A 260 /A 280 ＝1.889。
The primer and the probe aiming at the locus rs164578 of the SLC30A5 gene are designed, the gene sequence is amplified by a PCR method, and the risk of male infertility is judged according to the genotype of locus rs164578 of the SLC30A5 gene.
The judgment principle is as follows: if the genotype of the locus rs164578 of the SLC30A5 gene is TT genotype, judging that the risk of suffering from infertility is higher than that of CC genotype and CT genotype.
Wherein, the sequences of the primer pairs are as follows:
the upstream primer F is 5'-AAGGAAGCTGTCACTCATCTGATC-3',
a downstream primer R5'-TAATATTTTGCAAGGACTCACCCCT-3';
the sequences of the probe pairs are as follows:
c allele probe: 5'FAM-CCATTCACACCATATGCATGG-3' MGB,
t allele probe: 5'VIC-CCATTCACATCATATGCATGG-3' MGB.
The gene sequence is amplified to obtain the male peripheral blood specimen, and the genotype of the locus rs164578 of the SLC30A5 gene is TT genotype. It is considered to be at higher risk for infertility than individuals of the CC and CT genotypes in the population.
The result shows that the invention provides a new application of using the locus rs164578 of the SLC30A5 gene as a gene detection marker for male sterility risk.
Example 2
The embodiment is a primer probe combination for detecting male sterility risk detection markers, which is a primer probe combination for detecting SLC30A5 gene rs164578 locus genotype and comprises a primer pair and a probe pair;
wherein, the sequences of the primer pairs are as follows:
the upstream primer F is 5'-AAGGAAGCTGTCACTCATCTGATC-3',
a downstream primer R5'-TAATATTTTGCAAGGACTCACCCCT-3';
the sequences of the probe pairs are as follows:
the C allele probes were: 5'FAM-CCATTCACACCATATGCATGG-3' MGB;
the T allele probes were: 5'CY5-CCATTCACATCATATGCATGG-3' MGB.
In addition to the above embodiments, the fluorescent groups in the probe pair sequences can be replaced by genetic engineering techniques, as required, by a person skilled in the art, under the premise of ensuring that the fluorescent colors of the fluorescent groups in the C-allele probe and the T-allele probe are different. Wherein the fluorescent group can also be selected from VIC group, CY3 group, ROX group, etc.
Example 3
The embodiment is a kit for male sterility risk detection and application thereof. The reagent composition in the kit comprises a primer probe combination for detecting the locus rs164578 genotype of the SLC30A5 gene, DNA polymerase, a buffer solution, dNTPs and deionized water, wherein the amounts of the components are shown in Table 2.
In the application process of the embodiment, the genome DNA of a sample to be detected is used as a template, the kit of the embodiment is adopted for PCR amplification, and real-time fluorescence detection is carried out in the amplification process, so that a real-time fluorescence detection result is obtained, and the relationship between genotypes of different experimental objects and male infertility is analyzed. The method specifically comprises the following steps:
(1) The experimental object:
the subjects were in two groups, 142 in sterile group and 71 in normal group. Wherein, the sterile male patient in the outpatient service is selected as a sterile group, and the sterile group inclusion standard is as follows: the man can live for more than one year after wedding, and the man can not be inoculated with any contraceptive measure, and the man is sterile due to man factors. The normal group is the donor of human sperm pool in Hebei province.
(2) The specific information of the primer probe combination is as follows:
the upstream primer F is 5'-AAGGAAGCTGTCACTCATCTGATC-3',
a downstream primer R5'-CCATTCACATCATATGCATGG-3';
the probe pair sequences are as follows:
c allele probe 5'-FAM-CCATTCACACCATATGCATGG-MGB-3',
t allele probe 5'-VIC-CCATTCACATCATATGCATGG-MGB-3'.
(3) Fluorescent quantitative PCR reaction:
(301) DNA extraction
The whole genome DNA extraction was performed on peripheral blood specimens using a universal genome DNA miniprep kit (desert organism) according to the instructions for use. The concentration of the extracted DNA was required to be not less than 10 ng/. Mu.L, A260/A280=1.9.+ -. 0.1.
(302) PCR reaction conditions
The extracted 10ng-100ng DNA sample was added to the following amplification system, and amplified by fluorescent quantitative PCR, and the reaction system was as shown in Table 2.
TABLE 2 PCR reaction System
| Component name | Volume/. Mu.L is added |
| 2×morgene Master mix | 12.5 |
| Upstream primer F (10. Mu.M) | 0.5 |
| Downstream primer R (10. Mu.M) | 0.5 |
| C allele probe (10. Mu.M) | 0.25 |
| T allele probe (10. Mu.M) | 0.25 |
| H 2 O | Adding the system to 25 |
| DNA template | 100ng |
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 5s and extension at 65℃for 40s were performed for a total of 40 cycles.
(303) Reaction principle and judgment criterion
Two different color fluorescent signals were designed based on both the C and T alleles of rs 164578. If only the C allele exists in the rs164578 locus, the real-time fluorescence detection only shows the fluorescence signal of the C allele; if only T allele exists in the rs164578 locus, the real-time fluorescence detection only shows the fluorescence signal of the T allele; if the locus rs164578 is a C/T heterozygous genotype, then real-time fluorescence detection reveals both fluorescent signals for the C allele and the T allele.
(304) Results of the implementation
The difference of different gene frequencies of Hardy-Weinberg equilibrium and normal group and sterile group is detected by using SPSS21.0 software and using chi-square test, and p <0.05 is used as the difference to have statistical significance.
The frequencies of genes carried by the normal group and the sterile group were statistically analyzed, and the results are shown in Table 3.
TABLE 3 comparison of Gene frequencies in Normal and sterile groups
As can be seen from the above table, the frequency of TT genotypes was significantly increased in the sterile group compared to the normal group, and the above results indicate that the TT genotypes increased the risk of male infertility (p < 0.05).
Verification experiment
To fully verify the relationship between TT genotype and male sterility, semen quality in normal and sterile groups was confirmed by conventional semen analysis methods.
Conventional semen analysis method: after the normal group and the sterile group have no appetite for 2-7 days, semen is reserved by a masturbation method, and semen analysis is carried out by using a CASA system, so that the semen volume, the semen concentration and the forward movement semen percentage (PR%) are detected.
The results of routine semen analysis for normal and sterile group subjects are shown in table 4.
TABLE 4 results of routine semen analysis
As can be seen from the above table, there was no significant difference in sperm amount and age (P > 0.05) and significant difference in sperm concentration and PR% (P < 0.05) in the normal group compared with the sterile group. The experimental object selection of the embodiment is effective, and the reliability of the embodiment results is high.
Among these, there is a significant difference between the two indices of sperm concentration and PR%, but conventional semen analysis results are not straightforward for individuals to judge their risk of infertility. The male sterility risk detection kit based on gene detection has a certain clinical value.
Comparative example
The rs164578 locus genotyping detection is carried out on the two groups of patient specimens by using a Sanger sequencing method, and the experimental results prove that the detection results of the Sanger sequencing method and the detection results of the kit method of the embodiment 3 are completely consistent.
The result shows that the kit of the embodiment can be used for detecting the locus rs164578 of the SLC30A5 gene, has high accuracy of detection results, reduces the dependence of male sterility risk detection on manual observation and manual experience, has the advantages of short time consumption, simple reaction condition and visual reaction results, and can realize rapid and accurate typing of locus rs164578 of the SLC30A5 gene.
Example 4
In the embodiment, the primer probe combination and the PCR reaction conditions in the embodiment 3 are adopted to carry out rs164578 locus genotyping detection on 8 sterile patient specimens. Wherein:
peripheral blood DNA, DNA concentration and A are extracted by using a general genome DNA small-scale extraction kit (desert organism) 260 /A 280 See table 5.
Table 5 8 sample DNA concentrations and A 260 /A 280
| Specimen numbering | DNA concentration (ng/. Mu.L) | A 260 /A 280 |
| Specimen DNA1 | 40.0 | 1.905 |
| Specimen DNA2 | 49.0 | 1.885 |
| Specimen DNA3 | 23.0 | 1.917 |
| Specimen DNA4 | 39.0 | 1.857 |
| Specimen DNA5 | 27.0 | 1.800 |
| Specimen DNA6 | 34.0 | 1.889 |
| Specimen DNA7 | 46.0 | 1.917 |
| Specimen DNA8 | 91.0 | 1.896 |
The test results are shown in Table 6:
table 6 rs164578 locus genotyping results
Wherein, the genotyping fluorescence detection result graphs of the specimens of each patient are sequentially shown in fig. 1-8.
In addition, rs164578 locus genotyping detection is carried out on the 8 patient specimens by using a Sanger sequencing method, and experimental results prove that the detection results of the two methods are completely consistent.
The result shows that the genetic type of the SLC39A13 gene SNP locus rs164578 of the population in the abnormal group and the normal group is detected, and compared with the CC/CT genetic type group, the genetic type TT of the sterile group is higher in frequency, and the risk of male sterility is obviously increased. Therefore, the rs164578 locus can be used as a marker for male sterility risk judgment.
The invention designs the specific primer and the probe of the locus rs164578 of the SLC30A5 gene, carries out fluorescent quantitative PCR detection on the locus rs164578 of the SLC30A5 gene, carries out genetic analysis and judgment on the locus rs164578 of the SLC30A5 gene of a tester according to fluorescent signals, thereby judging the risk of male infertility, judging the risk of male infertility more accurately from the genetic level, and having important guiding significance for pathogenesis research, clinical diagnosis and treatment of male infertility and higher clinical application value.
It should be noted that the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but the present invention is described in detail with reference to the foregoing embodiment, and those skilled in the art may modify the technical solutions described in the foregoing embodiments or substitute some of the technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
- Application of the locus rs164578 of the SLC30A5 gene as a marker in a male sterility risk detection method.
- 2. The use according to claim 1, wherein the genotype at locus rs164578 of the SLC30A5 gene is used for risk determination of male infertility, and if the genotype at locus rs164578 of the SLC30A5 gene is the TT genotype, the risk of infertility is determined to be higher than the CC genotype and the CT genotype;the nucleotide sequence of the TT genotype is shown as SEQ ID NO. 1;the nucleotide sequence of the CC genotype is shown as SEQ ID NO. 2;the nucleotide sequence of the CT genotype is shown as SEQ ID NO. 3.
- 3. A primer probe combination for detecting the male sterility risk detection marker according to claim 1 or 2, which is characterized by comprising a primer pair and a probe pair for detecting the locus rs164578 genotype of the SLC30A5 gene;the primer pairs have the following sequences:the upstream primer F is 5'-AAGGAAGCTGTCACTCATCTGATC-3',a downstream primer R5'-TAATATTTTGCAAGGACTCACCCCT-3';the sequences of the probe pairs are as follows:c allele probe: 5'-CCATTCACACCATATGCATGG-3' the number of the individual pieces of the plastic,t allele probe: 5'-CCATTCACATCATATGCATGG-3';wherein, the 3 'end of the probe pair is connected with a quenching group, the 5' end is connected with a fluorescent group, and the fluorescent colors of the fluorescent groups in the C allele probe and the T allele probe are different.
- 4. The primer probe combination of claim 3, wherein the quenching group is an MGB group.
- 5. The primer probe combination of claim 4, wherein the fluorescent group comprises a CY3 group, a ROX group, or a CY5 group.
- 6. The primer probe combination of claim 3, wherein the C allele probe is: 5'FAM-CCATTCACACCATATGCATGG-3' MGB;the T allele probe is as follows: 5'VIC-CCATTCACATCATATGCATGG-3' MGB.
- 7. A kit for male sterility risk detection, characterized in that the marker for male sterility risk detection detected by the kit is the rs164578 locus of the SLC30A5 gene as set forth in any one of claims 1 to 6.
- 8. The kit for male sterility risk detection according to claim 7, characterized by comprising a primer probe combination according to any one of claims 4-6.
- 9. The use of the kit for male sterility risk detection as claimed in claim 7 or 8, characterized in that the kit is used for PCR amplification by taking genomic DNA of a sample to be detected as a template, and real-time fluorescence detection is performed in the amplification process to obtain a real-time fluorescence detection result;if the fluorescence detection result only contains the fluorescence signal of the T allele probe, the genotype of the rs164578 locus of the SLC30A5 gene of the sample to be detected is TT genotype, and the risk of suffering from infertility is judged to be higher than the CC genotype and the CT genotype.
- 10. Use of the kit for male sterility risk detection according to claim 9, characterized in that the PCR amplification procedure is: the mixture was subjected to 40 cycles of 5min pre-denaturation at 95 ℃, 5s denaturation at 95℃and 40s extension at 65 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311620241.1A CN117535399A (en) | 2023-11-30 | 2023-11-30 | Application of SLC30A5 gene rs164578 locus, primer probe combination and kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311620241.1A CN117535399A (en) | 2023-11-30 | 2023-11-30 | Application of SLC30A5 gene rs164578 locus, primer probe combination and kit thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117535399A true CN117535399A (en) | 2024-02-09 |
Family
ID=89787908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311620241.1A Pending CN117535399A (en) | 2023-11-30 | 2023-11-30 | Application of SLC30A5 gene rs164578 locus, primer probe combination and kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117535399A (en) |
-
2023
- 2023-11-30 CN CN202311620241.1A patent/CN117535399A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6523375B2 (en) | How to assess liver involvement | |
| CN103205488B (en) | Method and reagent for prediction of ankylosing spondylitis susceptibility | |
| KR102637032B1 (en) | Composition for diagnosing bladder cancer using CpG methylation status of specific gene and uses thereof | |
| CN115216539A (en) | Maternal cell pollution detection kit and application thereof | |
| CN107513577A (en) | A kind of method of efficient detection EGFRT790M mutant and probe and kit for detection | |
| CN110564861A (en) | Fluorescence labeling composite amplification kit for human Y chromosome STR locus and InDel locus and application thereof | |
| CN114410810A (en) | Kit for detecting non-tuberculous mycobacteria, detection method and application thereof | |
| CN116987791B (en) | Application of plasma markers in identification of benign and malignant thyroid nodule | |
| CN113151434A (en) | SNP marker, application, primer, probe and lung transplant rejection risk kit | |
| KR102086204B1 (en) | Methods and kits for diagnosing or assessing the risk of cervical cancer | |
| CN110863040A (en) | Method for detecting CYP3A5 gene polymorphism by fluorescent quantitative PCR | |
| CN116064842A (en) | Composite amplification box for degradation material deducing biological geographical ancestor DIPs and sex identification | |
| CN117535399A (en) | Application of SLC30A5 gene rs164578 locus, primer probe combination and kit thereof | |
| CN111621557B (en) | Kit for detecting osteoporosis susceptibility gene by cocktail and using method thereof | |
| CN114606311B (en) | Application of SLC39A13 gene rs755555 locus, detection primer and probe combination thereof and kit | |
| CN106811545A (en) | The method and reagent of a kind of neurological susceptibility for predicting hypertriglyceridemia | |
| CN113166810A (en) | SNP marker for diagnosing cerebral aneurysm including single base polymorphism of GBA gene | |
| KR102254341B1 (en) | methods for diagnosing the high risk group of Diabetes based on Genetic Risk Score | |
| CN113817818B (en) | Tool for diagnosing allergic airway inflammation | |
| CN114231620B (en) | Application of UQCC1 gene polymorphism in diagnosis of moderate and severe MAFLD (myeloproliferative disorder) | |
| US11939630B2 (en) | Fluorescent PCR method for detecting HLA-B*15:02 allele and specific primer probe combination thereof | |
| KR102543976B1 (en) | HLA-B genotype analysis method using Korean-specific SNPs and optimized pipeline | |
| CN112553318B (en) | Taqman probe-based deletion type alpha-thalassemia detection kit and detection method thereof | |
| CN113817819B (en) | Application of LINC01996 in diagnosis of allergic airway inflammation | |
| KR20230101467A (en) | Single-nucleotide polymorphic biomarkers for predicting obesity risk according to eating habits and use thereof |
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 |