阅读说明：本技术 Y染色体微缺失检测引物组及试剂盒 (Y chromosome microdeletion detection primer group and kit ) 是由 张惠丹 于 2019-12-03 设计创作，主要内容包括：本发明公开了Y染色体微缺失检测引物组及试剂盒,针对19个Y染色体微缺失突变位点,选择X/Y连锁锌指蛋白基因(ZFX/Y)为内对照位点。本发明可以在可操作性、灵敏度、节省时间方面解决现有技术的不足,技术实验设计灵活,覆盖突变位点多,更适合中国人,结果准确,检测速度快,适合批量筛查。(The invention discloses a Y chromosome microdeletion detection primer group and a kit, aiming at 19Y chromosome microdeletion mutation sites, an X/Y linkage zinc finger protein gene (ZFX/Y) is selected as an internal control site. The invention can solve the defects of the prior art in the aspects of operability, sensitivity and time saving, has flexible technical experiment design, more mutation sites, accurate result and high detection speed, is more suitable for Chinese people and is suitable for batch screening.)

A Y chromosome microdeletion detection primer set for each of 19Y chromosome microdeletion mutation sites, wherein:

when the mutation sites are sY82, sY84, sY86 and sY88 sites of the AZFa region, the primer group comprises:

sY82-F CAGTGTCCACTGATGGATGA，

sY82-R ATCCTGCCCTTCTGAATCTC；

sY84-F AGAAGGGTCTGAAAGCAGGT，

sY84-R GCCTACTACCTGGAGGCTTC；

sY86-F GTGACACACAGACTATGCTTC，

sY86-R ACACACAGAGGGACAACCCT；

sY88-F CACCCAGCCATTTGTTTTAC，

sY88-R TTGTAATCCAAATACATGGGC；

when the mutation sites are sY121, sY123, sY124, sY127, sY133 and sY134 sites of the AZFb region, the primer group comprises:

sY121-F CCTGTGACTCCAGTTTGGTC，

sY121-R AGTTCACAGAATGGAGCCTG；

sY123-F GGGGTTTATACTGACCTGCC，

sY123-R CCGTGTGTTGCTGGGCTGTC；

sY124-F ACTGTGGCAAAGTTGCTTTC，

sY124-R CAGGCAGGACAGCTTAAAAG；

sY127-F GGCTCACAAACGAAAAGAAA，

sY127-R CTGCAGGCAGTAATAAGGGA；

sY133-F TGATGATTGCCTAAAGGGAA，

sY133-R ATTTCTCTGCCCTTCACCAG；

sY134-F GTCTGCCTCACCATAAAACG，

sY134-R ACCACTGCCAAAACTTTCAA；

when the mutation sites are sY145, sY152, sY153, sY157, sY239, sY242, sY254, sY255 and sY277 sites of the AZFc region, the primer group comprises:

sY145-F ACAGGAGGGTACTTAGCAGT，

sY145-R AAGACAGTCTGCCATGTTTCA；

sY152-F ACAGGAGGGTACTTAGCAGT，

sY152-R AAGACAGTCTGCCATGTTTCA；

sY153-F TGCAGGGTACTTAGCAGTA，

sY153-R ATTAGACAGTCTGCCATGA；

sY157-F CCTGCTGTCAGCAAGATACA，

sY157-R CTTAGGAAAAAGTGAAGCCG；

sY239-F CATGTTTCACAGCAAGATACA，

sY239-R CTTAATAAAACGAAGCCG；

sY242-F TCTGCCACTAAACTGTAAGCTCC，

sY242-R ACACAGTAGCAGCGGGAGTT；

sY254-F GAACCGTATCTACCAAAGCAGC，

sY254-R GGGTGTTACCAGAAGGCAAA；

sY255-F GTTACAGGATTCGGCGTGAT，

sY255-R CTCGTCATGTGCAGCCAC；

sY277-F GTTACAGTCTGCCATGTTT，

sY277-R ACAGAATGGAGCCACATT。

2. the Y chromosome microdeletion detection primer set according to claim 1, wherein the primer set further comprises a primer set for a control site in an X/Y-linked zinc finger protein gene ZFX/Y:

ZFX/Y-F ACCRCTGTACTGACTGTGATTACAC，

ZFX/Y-R GCACYTCTTTGGTATCYGAGAAAGT。

a Y chromosome microdeletion detection kit comprising the Y chromosome microdeletion detection primer set according to claim 1 or 2.

Technical Field

The invention belongs to the technical field of biological gene detection, and relates to a Y chromosome microdeletion detection primer group and a kit.

Background

About 15% of couples are infertile all over the world, and the male infertility factors account for about 50%. The microdeletion of the Y chromosome is the second leading factor in idiopathic male infertility, except for the kreb's syndrome. In 1976, scientists discovered azoospermia factor (AZF), which they believed was the gene site of the long arm of the Y chromosome that controls spermatogenesis. Subsequent studies have demonstrated the presence of spermatogenesis controlling genes on Y chromosome yq11.23 and the division of AZF into 3 regions, AZFa, AZFb and AZFc, each of which contains several AZF candidate genes, such as USP9Y and DBY in the AZFa region, RBMY1 and HSFY in the AZFb region, DAZ in AZFc, etc., which are all likely to cause abnormal spermatogenesis leading to male infertility. Even though some AZF-deficient patients may be gestated by normal or assisted reproductive techniques, AZF-deficiency is passed on to their male offspring. Therefore, the application of the Y chromosome microdeletion detection in clinic is very important.

At present, 6 sites recommended by the European Male Association/European molecular genetics quality network are adopted clinically. More than 6 sites are suitable for European and American people, and the condition of missing detection may exist for Chinese people.

Disclosure of Invention

The invention aims to provide a Y chromosome microdeletion detection primer group and a kit, which can solve the problems of the prior art in the aspects of operability, sensitivity and time saving.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a Y chromosome microdeletion detection primer group which aims at 19Y chromosome microdeletion mutation sites, wherein:

when the mutation sites are sY82, sY84, sY86 and sY88 sites of the AZFa region, the primer group comprises:

sY82-F CAGTGTCCACTGATGGATGA，

sY82-R ATCCTGCCCTTCTGAATCTC；

sY84-F AGAAGGGTCTGAAAGCAGGT，

sY84-R GCCTACTACCTGGAGGCTTC；

sY86-F GTGACACACAGACTATGCTTC，

sY86-R ACACACAGAGGGACAACCCT；

sY88-F CACCCAGCCATTTGTTTTAC，

sY88-R TTGTAATCCAAATACATGGGC；

when the mutation sites are sY121, sY123, sY124, sY127, sY133 and sY134 sites of the AZFb region, the primer group comprises:

sY121-F CCTGTGACTCCAGTTTGGTC，

sY121-R AGTTCACAGAATGGAGCCTG；

sY123-F GGGGTTTATACTGACCTGCC，

sY123-R CCGTGTGTTGCTGGGCTGTC；

sY124-F ACTGTGGCAAAGTTGCTTTC，

sY124-R CAGGCAGGACAGCTTAAAAG；

sY127-F GGCTCACAAACGAAAAGAAA，

sY127-R CTGCAGGCAGTAATAAGGGA；

sY133-F TGATGATTGCCTAAAGGGAA，

sY133-R ATTTCTCTGCCCTTCACCAG；

sY134-F GTCTGCCTCACCATAAAACG，

sY134-R ACCACTGCCAAAACTTTCAA；

when the mutation sites are sY145, sY152, sY153, sY157, sY239, sY242, sY254, sY255 and sY277 sites of the AZFc region, the primer group comprises:

sY145-F ACAGGAGGGTACTTAGCAGT，

sY145-R AAGACAGTCTGCCATGTTTCA；

sY152-F ACAGGAGGGTACTTAGCAGT，

sY152-R AAGACAGTCTGCCATGTTTCA；

sY153-F TGCAGGGTACTTAGCAGTA，

sY153-R ATTAGACAGTCTGCCATGA；

sY157-F CCTGCTGTCAGCAAGATACA，

sY157-R CTTAGGAAAAAGTGAAGCCG；

sY239-F CATGTTTCACAGCAAGATACA，

sY239-R CTTAATAAAACGAAGCCG；

sY242-F TCTGCCACTAAACTGTAAGCTCC，

sY242-R ACACAGTAGCAGCGGGAGTT；

sY254-F GAACCGTATCTACCAAAGCAGC，

sY254-R GGGTGTTACCAGAAGGCAAA；

sY255-F GTTACAGGATTCGGCGTGAT，

sY255-R CTCGTCATGTGCAGCCAC；

sY277-F GTTACAGTCTGCCATGTTT，

sY277-R ACAGAATGGAGCCACATT。

preferably, the primer set further comprises a primer set for a control site within an X/Y-linked zinc finger protein gene (ZFX/Y):

ZFX/Y-F ACCRCTGTACTGACTGTGATTACAC，

ZFX/Y-R GCACYTCTTTGGTATCYGAGAAAGT。

the invention also provides a Y chromosome microdeletion detection kit comprising the primer group.

The invention has the following beneficial effects:

the method has the advantages of flexible technical experiment design, more mutation sites covered, more suitability for Chinese people, accurate result, high detection speed and suitability for batch screening.

Detailed Description

The present invention will be further illustrated by the following specific examples, which should be understood as not limiting the scope of the invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The inventor collects the common mutations of Chinese according to professional genetic databases such as Clinvar, HGMD, OMIM, DisGeNET, PheGenI, dbNSFP, UniProtKB and the like and consults Chinese databases such as the Hopkinson and the like, and finally determines 19Y chromosome microdeletion mutation sites:

sY82, sY84, sY86, sY88 of AZFa region;

sY121, sY123, sY124, sY127, sY133, sY134 of AZFb region;

sY145, sY152, sY153, sY157, sY239, sY242, sY254, sY255, sY277 of AZFc region.

In addition, X/Y-linked zinc finger protein gene (ZFX/Y) was selected as an internal control site.

According to the position information, the inventor adopts different primer design software, such as Vector NTI Suit, Dnasis, Primr premier, Oligo, Omiga, Dnastar and the like to design primers, and the details are shown in Table 1.

TABLE 1 primer sequences for different sites and their PCR groupings