阅读说明：本技术 一种判断女性原发不孕的标记cdc20基因及其检测试剂盒 (Marker CDC20 gene for judging female primary infertility and detection kit thereof ) 是由 王磊 桑庆 赵琳 于 2019-12-01 设计创作，主要内容包括：本发明属于基因检测技术领域,具体为一种判断女性原发不孕的标记及其检测试剂盒。本发明涉及的标记为人类CDC20基因,它的突变是导致因卵子不成熟、受精及胚胎异常致女性不孕的原因。本发明还提供检测CDC20基因是否发生突变的的引物：SEQ ID NO.2-SEQ ID NO.17；以及检测CDC20基因发生突变筛查试剂盒。本发明可用于指导临床相应患者是否合适进行试管婴儿术。(The invention belongs to the technical field of gene detection, and particularly relates to a marker for judging female primary infertility and a detection kit thereof. The marker of the invention is human CDC20 gene, and the mutation is the cause of female sterility caused by ovum immaturity, fertilization and embryo abnormality. The invention also provides a primer for detecting whether the CDC20 gene is mutated: SEQ ID NO.2 to SEQ ID NO. 17; and a screening kit for detecting mutation of CDC20 gene. The invention can be used for guiding the clinical corresponding patient whether to carry out the test tube infant operation.)

1. A marker for judging female primary infertility is characterized in that CDC20 gene has a nucleic acid sequence shown as SEQ ID NO.1, and the female primary infertility refers to female infertility caused by ovum immaturity, fertilization and embryo abnormality.

2. A method for screening female primary infertility, which is characterized in that whether a patient is primary infertility caused by ovum immaturity, fertilization and embryo abnormality is judged by detecting whether CDC20 gene has mutation, wherein the nucleic acid sequence of CDC20 gene is shown as SEQ ID NO. 1.

3. Primers for detecting whether a mutation occurs in the CDC20 gene, wherein the PCR amplification primer pairs for detecting 10 exons of the CDC20 gene are as follows: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO. 17; the nucleic acid sequence of the CDC20 gene is shown in SEQ ID NO. 1.

4. A method for detecting whether mutation occurs in CDC20 gene is characterized by comprising the following steps:

for 10 exons in CDC20, respectively carrying out PCR amplification by using corresponding primer pairs of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, …, SEQ ID NO.16 and SEQ ID NO.17, then carrying out sequencing by using the same primers, and comparing the sequencing result with the standard sequence of CDC20 in UCSC: SEQ ID NO.1 was aligned to find a mutation.

5. A kit for detecting whether a mutation occurs in CDC20 gene, wherein the kit comprises an amplification primer for amplifying exons 1-10 of CDC20 gene and corresponding sequencing primers: SEQ ID NO.2-SEQ ID NO.17, and a reaction mixture comprising DNA polymerase, dNTP and a buffer.

6. The site of CDC20 gene mutation is applied in the research and preparation of the medicine for repairing gene mutation target site, i.e. the medicine for treating primary infertility caused by ovum immaturity, fertilization and embryo abnormality.

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a marker for detecting female primary infertility and a detection kit thereof.

Background

Normal pregnancy and reproduction are important links in maintaining and extending human populations. For female infertility, genes ZP-1, Stag3, FSHR, etc. have been found to be closely related to female infertility (Huang HL et al, Mutant ZP1 in human sterility. N Engl J Med. 2014370 (13):1220-6; de Roux N et al, A family with hypo-gonadotropic hypo and mutations in the gonadolitropin-releasehormonereceiver. N Engl J. 1997337 (22): 947 159602; calret S et al, Mutant conjugation in precursor failure. N Engl Med. 2014370 (10): 943-9). However, none have been clinically applied.

The reasons for female infertility are many, and there are some clinical reports relating to the description of female infertility characterized by egg immaturity (Rudak et al, fertility and fertility, 199054: 292-. These patients, by repeated fertilization-embryo transfer (tube transfer), fail to complete successful in vitro fertilization procedures due to the failure to obtain mature egg cells. If relevant pathogenic genes can be found, the method has important significance for clinical diagnosis and typing of diseases. Recently we have found the first causative gene TUBB8(N Engl J Med. 2016 Jan 21;374(3):223-32) that causes the disorder of maturation of human ova. However, the reason for this gene can only be explained for some patients, and the reason for a considerable number of patients is still unknown.

The search of the prior art literature shows that only a few research articles surrounding the function of CDC20 gene are found. These studies report that this gene plays an important role in meiosis in mouse eggs, and that knockout results in infertility in female mice. However, no report has been made to date on the relationship between mutations in the CDC20 gene and human diseases. No report on the female infertility is found.

Disclosure of Invention

The invention aims to provide a marker for judging female primary infertility and a detection kit thereof, which are convenient to operate and have clear effect.

In the invention, the CDC20 gene for detecting female primary infertility has a nucleic acid sequence shown in SEQ ID NO. 1; the female primary infertility refers to female infertility caused by ovum immaturity, fertilization and embryo abnormality.

The invention also relates to a method for screening primary infertility caused by ovum immaturity, fertilization and embryo abnormality, namely judging whether a patient is the primary infertility caused by the ovum immaturity, the fertilization and the embryo abnormality by detecting whether the CDC20 gene is mutated. Patients carrying CDC20 gene mutations manifest themselves as infertility, as a constant failure of the ovum to mature, fertilization and embryo abnormalities, and tube baby failure.

Thus, whether the CDC20 gene is mutated or not can be used as a marker for judging female infertility caused by ovum immaturity, fertilization and embryo abnormality.

Specifically, the method for detecting whether the CDC20 gene is mutated may be a method in which DNA is extracted from peripheral blood of a patient and then the CDC20 gene is mutated by PCR (polymerase chain reaction) in combination with DNA sequencing.

Wherein the sample to be detected is DNA, and the DNA sample is from peripheral blood of a human to be detected.

Alternatively, the sample to be tested is an RNA, protein, cell or serum sample derived from the peripheral blood of the human to be tested.

The invention relates to a primer for detecting whether CDC20 gene is mutated.

The CDC20 gene has 10 exons, and PCR amplification primer pairs for detecting the 1 st exon are as follows:

5’- CAGGCGTGTTAAAGCCGGT-3’（SEQ ID NO.2）

5’- ATGAAGGCTGCTCTCACTCAG-3’（SEQ ID NO.3）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.2 and No.3 exons is as follows:

5’-TCAGTGAGGTGCCAAAGGAA-3’（SEQ ID NO.4）

5’-AAATCAACACCTCCCTTGCCC-3’（SEQ ID NO.5）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.4 exon is as follows:

5’-AAGACCCGAAGTTCCTGGTTC-3’（SEQ ID NO.6）

5’-GCTTGCACTCCACAGGTACA-3’（SEQ ID NO.7）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.5 exon is as follows:

5’-CGGAAGACCTGCCGTTACAT-3’（SEQ ID NO.8）

5’-GGACTGGTGAGAATGACAGCA-3’（SEQ ID NO.9）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the exons 6 and 7 is as follows:

5’-GAGACGTGTCCAGTGCTGTC-3’（SEQ ID NO.10）

5’-AGCCCAACCCTACTCACCTT-3’（SEQ ID NO.11）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.8 exon is as follows:

5’-CAAGGTGAGTAGGGTTGGGC-3’（SEQ ID NO.12）

5’-GGGACAGATCAAGGTGGTGG-3’（SEQ ID NO.13）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.9 exon is as follows:

5’-GAGTCTGGAGCATGTGGCA-3’（SEQ ID NO.14）

5’-GCACTGGGCCACCCAC-3’（SEQ ID NO.15）

the sequencing primer pair is as above;

the PCR amplification primer pair for detecting the No.10 exon is as follows:

5’-TGGCTGCATCAGCATTCGTA-3’（SEQ ID NO.16）

5’-GAAGGCTGCTGGGACATAGG-3’（SEQ ID NO.17）

the sequencing primer pair is as above.

The invention also relates to a method for detecting whether the CDC20 gene is mutated, which comprises the following steps:

for exon 1, primer pairs are used: SEQ ID NO.2 and SEQ ID NO.3, PCR amplification is carried out (amplification conditions: 2 minutes at 92 ℃, 30 seconds at 92 ℃,1 minute at 57 ℃, 3 seconds at 72 ℃ (these three steps are repeated for 35 cycles), and 10 minutes at 72 ℃), then sequencing is carried out by using the same primers, and the sequencing result is compared with a standard sequence (SEQ ID NO. 1) of CDC20 in UCSC, so that mutation is found;

like the above conditions, exons 2 to 10 of CDC20 were amplified with corresponding primers (SEQ ID NO.4 to NO. 17);

sequencing and aligning with a standard sequence (SEQ ID NO. 1) of CDC20 in UCSC to find the mutation; whether the CDC20 gene is mutated or not is detected by PCR and first-generation sequencing, and alignment with a standard sequence.

The invention also provides a kit for detecting whether the CDC20 gene is mutated. The kit can guide doctors to judge the etiology of diseases, correctly classify the diseases, and inform patients whether IVF or ICSI methods are adopted for tube infant operation and whether in vitro fertilization-embryo transfer (IVF) or single sperm injection (ICSI) (tube infant) is suitable to be continuously performed.

The kit provided by the invention comprises the amplification primers SEQ ID NO.2-SEQ ID NO.17 for amplifying the exons 1-10 of CDC20 and reaction mixed liquor; the reaction mixed solution comprises DNA polymerase, dNTP and buffer solution;

the specific using method comprises the following steps: the primers were diluted with water to a working solution of 10uM concentration. The reaction system is 10ul, wherein 1ul of sample DNA, 0.5ul of forward primer, 0.5ul of reverse primer, 5ul of reaction mixture and 3ul of water. After mixing, PCR reactions were performed according to the above conditions, followed by one-generation sequencing.

The kit can be used for detecting the primary infertility caused by ovum immaturity, fertilization and embryo abnormality.

The invention also provides a gene repair target site (namely actually detected mutation sites of a patient, such as 6 mutation sites listed in table 1, in the actual detection process, new mutation sites may exist by comparing with a standard sequence) for researching the primary infertility caused by ovum immaturity, fertilization and embryo abnormality treatment by using drugs, and the CDC20 can perform normal functions by repairing the target site, so that the disease can be treated. Namely, the invention can also provide a medicine for researching and repairing the gene mutation target site, namely a medicine for treating primary infertility caused by ovum immaturity, fertilization and embryo abnormality.

The invention can also guide the clinical patients with the CDC20 gene mutation whether to be suitable for the test-tube infant operation or not by detecting the mutation of the gene. In routine clinics, infertile couples of unknown cause require test tube babies to attempt to reproduce the offspring. However, if a patient is detected as having a mutation in CDC20, the patient will fail if he attempts a tube-fed infant. Because the ovum of the patient with this mutation is immature or the embryo after fertilization of the partially mature ovum is abnormal and it is difficult to obtain a valid embryo, the subsequent manipulation of the tube baby cannot be completed. Such patients are therefore advised to donate eggs.

Drawings

FIG. 1 is a graphical representation of the distribution of patient mutations over the primary structure of CDC 20.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are only illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1: collection of samples and extraction of peripheral blood DNA

The primary infertility patients caused by ovum immaturity, fertilization and embryo abnormality come from Shanghai love heredity and sterility diagnosis and treatment center of certain subsidiary hospital of Shanghai Compound denier university in China and the reproductive center of certain subsidiary hospital of Shanghai traffic university in China. Diagnostic criteria are set forth by Rudak E et al (Rudak E., DorJ., Kimchim., Goldman B., Levran D and Mashiach S, analysts of human society from human genome understanding treatment by in vitro transfer, FertilSteril.1990 Aug; 54(2): 292-6). The sperm of a male is checked to be normal, the female reproductive organ, the ovarian function and the sex hormone of a female patient are all normal, more than 5 eggs are taken each time in more than 2 ovulation cycles, most of the eggs are in the GV stage, and normal meiosis and maturation can not be carried out, or the embryo of a partially mature egg is abnormal after fertilization. The study of the subject was performed under the premise of informed consent, blood was collected, and an informed consent was signed. All patients enrolled excluded other genital endocrine diseases by medical history. The control population of the experiment is 1000 women with normal fertility, 300ul of the blood is sampled, DNA is extracted according to the kit specification, and the concentration and the purity of the DNA are detected by an ultraviolet spectrophotometer.

Example 2: detection of mutation of CDC20 Gene

The invention searches CDC20 gene mutation by combining PCR with sequencing. The principle is that primer design (primer sequence can be specifically given) and amplification are firstly carried out on 10 exons of CDC20 gene, and CDC20 gene mutation is searched. (DNA samples and reaction working solution (DNA polymerase, dNTP, water and buffer) after mixing, according to PCR program amplification, the product, through purification and further sequencing reaction, ABI3730 sequencing, analysis of results through HLA Fusion software (One lambda, CA, USA, HLA Fusion 3.0).

Example 3: CDC20 gene mutation and primary sterility caused by ovum immaturity, fertilization and embryo abnormality

As a result: we have gathered 5 cases of patients with primary infertility caused by ovum immaturity, fertilization and embryo abnormality due to CDC20 gene mutation. The corresponding mutation site information is shown in the following table (table 1). The distribution of the mutation sites is shown in FIG. 1.

TABLE 1 mutation information of patient CDC20 gene

。

In conclusion, the invention has the following important practical significance:

(1) the CDC20 gene provided by the invention can be used as a marker gene for predicting female infertility caused by ovum immaturity, fertilization and embryo abnormality;

(2) the CDC20 gene provided by the invention can be used for evaluating or preparing a screening kit for female infertility caused by ovum immaturity, fertilization and embryo abnormality;

(3) the invention can be used for researching and preparing the medicine for repairing the gene mutation target site, namely the medicine for treating primary infertility caused by ovum immaturity, fertilization and embryo abnormality;

(4) the CDC20 gene provided by the invention can be used for guiding patients with the gene mutation clinically, judging the success possibility of test-tube infants and recommending subsequent egg donation.

Sequence listing

<110> university of Compound Dan

Zhuhai Fudan Innovation Research Institute

<120> marker CDC20 gene for judging female primary infertility and detection kit thereof

<130> 001

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1500

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcacagt tcgcgttcga gagtgacctg cactcgctgc ttcagctgga tgcacccatc 60

cccaatgcac cccctgcgcg ctggcagcgc aaagccaagg aagccgcagg cccggccccc 120

tcacccatgc gggccgccaa ccgatcccac agcgccggca ggactccggg ccgaactcct 180

ggcaaatcca gttccaaggt tcagaccact cctagcaaac ctggcggtga ccgctatatc 240

ccccatcgca gtgctgccca gatggaggtg gccagcttcc tcctgagcaa ggagaaccag 300

cctgaaaaca gccagacgcc caccaagaag gaacatcaga aagcctgggc tttgaacctg 360

aacggttttg atgtagagga agccaagatc cttcggctca gtggaaaacc acaaaatgcg 420

ccagagggtt atcagaacag actgaaagta ctctacagcc aaaaggccac tcctggctcc 480

agccggaaga cctgccgtta cattccttcc ctgccagacc gtatcctgga tgcgcctgaa 540

atccgaaatg actattacct gaaccttgtg gattggagtt ctgggaatgt actggccgtg 600

gcactggaca acagtgtgta cctgtggagt gcaagctctg gtgacatcct gcagcttttg 660

caaatggagc agcctgggga atatatatcc tctgtggcct ggatcaaaga gggcaactac 720

ttggctgtgg gcaccagcag tgctgaggtg cagctatggg atgtgcagca gcagaaacgg 780

cttcgaaata tgaccagtca ctctgcccga gtgggctccc taagctggaa cagctatatc 840

ctgtccagtg gttcacgttc tggccacatc caccaccatg atgttcgggt agcagaacac 900

catgtggcca cactgagtgg ccacagccag gaagtgtgtg ggctgcgctg ggccccagat 960

ggacgacatt tggccagtgg tggtaatgat aacttggtca atgtgtggcc tagtgctcct 1020

ggagagggtg gctgggttcc tctgcagaca ttcacccagc atcaaggggc tgtcaaggcc 1080

gtagcatggt gtccctggca gtccaatgtc ctggcaacag gagggggcac cagtgatcga 1140

cacattcgca tctggaatgt gtgctctggg gcctgtctga gtgccgtgga tgcccattcc 1200

caggtgtgct ccatcctctg gtctccccat tacaaggagc tcatctcagg ccatggcttt 1260

gcacagaacc agctagttat ttggaagtac ccaaccatgg ccaaggtggc tgaactcaaa 1320

ggtcacacat cccgggtcct gagtctgacc atgagcccag atggggccac agtggcatcc 1380

gcagcagcag atgagaccct gaggctatgg cgctgttttg agttggaccc tgcgcggcgg 1440

cgggagcggg agaaggccag tgcagccaaa agcagcctca tccaccaagg catccgctga 1500

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caggcgtgtt aaagccggt 19

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaaggctg ctctcactca g 21

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tcagtgaggt gccaaaggaa 20

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaatcaacac ctcccttgcc c 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aagacccgaa gttcctggtt c 21

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcttgcactc cacaggtaca 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cggaagacct gccgttacat 20

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggactggtga gaatgacagc a 21

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gagacgtgtc cagtgctgtc 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agcccaaccc tactcacctt 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

caaggtgagt agggttgggc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gggacagatc aaggtggtgg 20

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gagtctggag catgtggca 19

<210> 15

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcactgggcc acccac 16

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tggctgcatc agcattcgta 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gaaggctgct gggacatagg 20