Azoospermia chromosome variation detection kit

A mutation detection and chromosome technology, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long cycle, many detection sites, complex process, etc., to reduce detection cost and detection sensitivity. The effect of improving and high detection sensitivity

Pending Publication Date: 2022-01-28
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV +1
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Problems solved by technology

[0004] In order to solve the current situation that peripheral blood karyotype analysis, capillary electrophoresis or multiplex fluorescent quantitative PCR combination is mostly used in the detection of Chinese male azoospermia in the prior art, the above-mentioned technologies adopt different method systems; the capillary electrophoresis method requires the detection site more, the process is complicated and the cycle is longer; the traditional European detection 6-site multiple fluorescent quantitative PCR detection site is not completely suitable for the characteristics of the Chinese population. These are the problems faced by the reagents in clinical testing. Oligospermia Chromosomal Variation Detection Kit

Method used

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  • Azoospermia chromosome variation detection kit
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  • Azoospermia chromosome variation detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0028] The multiple real-time fluorescent PCR method of the present embodiment, the primer probe and the kit (48 reactions / box) for detecting the chromosomal variation of azoospermia and oligospermia include components, as shown in Table 1:

[0029] (1) Y detection tube: dry powder containing primer probes for detecting Y chromosome microdeletions AZFa, AZFb, AZFc, Taq enzyme, and PCR reaction buffer substances. The sequences of the primer probes included are:

[0030] SEQ ID NO.1: AZFa1-F: 5'-AGTGAAGGAATAACATGCCGAG-3'

[0031]SEQ ID NO.2: AZFa1-R: 5'-AAACTGTATATACCATAATCCCT-3'

[0032] SEQ ID NO.3: AZFa1-P:5'-FAM-AAGTGGTGATGGATGAGGAGT-3'-QSY

[0033] SEQ ID NO.4: AZFa2-F: 5'-CTGGGCCCAAGACACATTGT-3'

[0034] SEQ ID NO.5: AZFa2-R: 5'-ACAACTCTGGGAAGCCATTACC-3'

[0035] SEQ ID NO.6: AZFa2-P:5'-FAM-CCATGGATCTCACTTTGCAGGACAGAGAC-3'-QSY

[0036] SEQ ID NO.7: AZFb1-F:5'-TATAGCCCAAAACTAATCAGCATC-3'

[0037] SEQ ID NO.8: AZFb1-R: 5'-TGGTAGATTCCAGTGGGTGCTATC-3'

[0038] SEQ ID NO....

Embodiment 2

[0066] The method for real-time fluorescent quantitative PCR detection of human azoospermia and oligospermia chromosomal variation using the kit described in Example 1:

[0067] (1) Nucleic acid extraction:

[0068] Take 200 μL whole blood in vitro sample, use Tianlong automatic nucleic acid extractor (NP968-3S) and matching Tianlong whole blood genomic DNA extraction kit to extract the whole blood in vitro sample collected by EDTA anticoagulant tube, and use Micro-volume ultraviolet spectrophotometer to determine the purity and concentration of nucleic acid, its OD 260 / 280 Between 1.6-2.0; dilute the genomic DNA concentration to 5ng / μL with sterile double distilled water for later use.

[0069] (2) In vitro sample detection:

[0070] Add the absence control substance, normal control substance, and the isolated sample DNA to be tested into the reaction wells of the Y detection tube and the KS detection tube, respectively, with a sample volume of 20 μL.

[0071] (3) Amplific...

Embodiment 3

[0096]Two negative samples were tested using the kit described in Example 1 and a commercially available Y chromosome microdeletion detection kit (fluorescent PCR method). The DNA concentration used was serially diluted to 20ng / μL, 10ng / μL, 5ng / μL, 2ng / μL, 1ng / μL respectively, and the Ct value of the internal reference signal was detected. The internal reference Ct values ​​of the two kits are as follows:

[0097] Table 6 Comparison of the detection limit of the internal reference signal between this kit and a commercially available Y staining kit

[0098]

[0099] It can be seen from the above results that after the drying process is adopted in this kit, the total sample consumption is greatly reduced, and the sensitivity is 5 times higher than that of the traditional fluorescence quantitative kit.

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Abstract

The invention belongs to the technical field of biological detection, and discloses an azoospermia chromosome variation detection kit. According to the invention, on the basis of retrospective analysis of detection results of chromosome karyotype and Y chromosome microdeletion of patients suffering from azoospermia and establishment of an azoospermia genetic database in China, a multiplex fluorescent quantitative PCR method is adopted to establish a primer probe and a kit for realizing azoospermia chromosome variation detection at one time, the clinical male infertility reason detection efficiency is improved, the cost is reduced, and the period is shortened; meanwhile, the research specially aims at Chinese populations, the clinical actual requirements of China are better met, and the positive detection rate is increased. The cause of male infertility is determined from the molecular level, so that unnecessary treatment is avoided, and the problem that the clinical mutation deletion rate of male offspring is increased due to the application of ICSC is reduced. In addition, the most advanced drying technology is adopted, the detection sensitivity can be improved, the clinical detection process is simplified, and actual use and popularization are more convenient.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a detection kit for chromosome variation of azoospermia and oligospermia. Background technique [0002] With the rapid development of society, the incidence of infertility is on the rise. Among them, about 50% of infertility is caused by male factors, and about 20% are caused by genetic factors. Abnormalities in male reproductive genetics include chromosomes, genes, sperm DNA integrity and epigenetics, which are important causes of male infertility. With the advancement of gene sequencing technology and the wide application of assisted reproductive technology, male reproductive genetics has become a research hotspot in recent years. Carrying out research on male reproductive genetics can not only give a genetic explanation for the cause of infertility, but also have important guiding significance for the selection of treatment methods. [0003] Klinefe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q1/686C12Q2600/156C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 田龙于超计王倩玉雷洪恩吴文立肖江山魏星
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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