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A composition of primers and probes for detecting y-chromosomal microdeletion, a detection method and a kit for non-diagnostic purposes

A Y-chromosome and primer set technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of expensive capillary electrophoresis analysis instruments, high professional requirements for operators, and low positive detection rate and other problems, to achieve the effect of easy large-scale promotion, high detection efficiency and sensitivity, and low reagent cost

Active Publication Date: 2022-03-11
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV +1
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Problems solved by technology

The method in the related art utilizes multiple PCR combined with capillary electrophoresis techniques to detect 15 sites on the Y chromosome (sY84, sY86, sY182, sY81 in the AZFa region; sY121, sY134, sY127, sY105 in the AZFb region; sY255, sY105 in the AZFc region; sY254, sY242, sY154, sY1291, sY145 and sY152 of the AZFd region) can be detected to determine the deletion of AZFa, AZFb, AZFc and AZFd regions, but the steps are complicated, the detection cycle is long, and the professional requirements for operators are high. Capillary electrophoresis analysis instruments are expensive and not suitable for general promotion in hospitals and testing institutions
[0008] In addition, there is a related technical method that uses multiplex fluorescent PCR to detect 6 sequence tag sites including sY84, sY86, sY127, sY134, sY254 and sY255. This method is simple to operate, saves time and effort, but its positive detection rate is low , this site combination is recommended by European urology, but not fully applicable to the Chinese population

Method used

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  • A composition of primers and probes for detecting y-chromosomal microdeletion, a detection method and a kit for non-diagnostic purposes
  • A composition of primers and probes for detecting y-chromosomal microdeletion, a detection method and a kit for non-diagnostic purposes
  • A composition of primers and probes for detecting y-chromosomal microdeletion, a detection method and a kit for non-diagnostic purposes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The primer probe of this Y chromosome microdeletion adopts multiplex real-time fluorescent PCR method to detect the primer probe of human Y chromosome microdeletion nucleic acid and the kit (50 reactions / box) contains components as shown in Table 1:

[0039] Table 1 Kit components

[0040]

[0041]

[0042] Nucleic acid amplification reaction solution A: containing specific primers and probe solutions for sY1324 sites, sY127 sites, sY1192 sites and RNase P gene, the nucleotide sequences of the upstream and downstream primers and the primer probe sequences are as follows: Shown in SEQ ID NO.1-SEQ ID NO.12.

[0043] Nucleic acid amplification reaction solution B: contains specific primers and probe solutions for the sY85 site, sY1233 site, sY254 site, SRY gene and RNaseP gene, and its upstream and downstream primer nucleotide sequences and primer probe sequences are specific Such as: SEQ ID NO.13-SEQ ID NO.24 and SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12 shown.

[...

Embodiment 2

[0052] The process of using the kit prepared in Example 1 to perform real-time fluorescent quantitative PCR detection of Y chromosome microdeletion nucleic acid:

[0053] (1) Nucleic acid extraction:

[0054] Obtain 200 μL whole blood isolated samples (using Tianlong automatic nucleic acid extraction instrument (NP968-3S) and matching Tianlong whole blood genomic DNA extraction kit to extract whole blood samples collected in EDTA anticoagulant tubes), and use a trace volume The purity and concentration of nucleic acid were measured by ultraviolet spectrophotometer, and its OD260 / 280 was between 1.6 and 2.0; the concentration of genomic DNA was diluted to 20ng / μL with sterile double distilled water for later use.

[0055] (2) The PCR reaction system was prepared, with a total reaction volume of 20 μL, as shown in Table 2:

[0056] Table 2 PCR reaction system

[0057]

[0058] (3) Sample testing:

[0059] Add the missing control substance, normal control substance, and sam...

Embodiment 3

[0080] SALSA MLPA Probemix P360 Y-Chromosome Microdeletions kit (MRC-Holland, which has not obtained a production license for medical devices in my country), a commercially available Y chromosome microdeletion detection kit (which has obtained a production license for medical devices in my country) and Example 1 of the present invention were used. The kit was used to detect 15 cases of male Y chromosome microdeletions. The samples came from the Department of Urology and Andrology, Beijing Chaoyang Hospital, which was in line with the characteristics of the Chinese population. The detection results of Y-stained microdeletions of the three kits are shown in Table 7:

[0081] Table 7 Detection of 15 male samples in different Y-stained microdeletion kits

[0082]

[0083]

[0084] Note: "-" indicates missing detection.

[0085] Through the detection of 15 samples, the positive detection rate of the MRC-Holland kit was 87%, that of a commercially available qPCR kit was 47%, ...

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Abstract

The invention belongs to the technical field of biological detection, and discloses a composition of primers and probes for detecting Y chromosome microdeletion, a non-diagnostic detection method and a kit. The sY1324, sY127, sY1192, sY85, sY1233, sY254 loci and the SRY gene in the three regions of AZFa, AZFb and AZFc are selected for simultaneous detection, which can identify different deletion types of the Y chromosome, adding the AZFc region b2 / b3, b1 / b3 and b2 / b4 deletion detection rates. The kit only uses nucleic acid amplification reaction A and nucleic acid amplification reaction B, and the combination of the two reactions can realize the detection, breaking through the limitations of the traditional 6-site detection method, improving the positive detection rate and accuracy, and the low cost of reagents, which promotes Domestic independent research and development products have reached the international leading level. Detection by fluorescent quantitative PCR method has high detection efficiency and sensitivity, is convenient, has low requirements for detection equipment and operators, and is convenient for large-scale promotion. Quality control can be carried out for sample extraction, PCR amplification and manual operation throughout the detection process.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a composition of primers and probes for detecting Y chromosome microdeletion, a non-diagnostic detection method and a kit. Background technique [0002] Infertility is a multifactorial global health problem affecting approximately 10% to 15% of couples worldwide. It is estimated that nearly half of all infertility cases are due to male infertility and genetic factors, especially microdeletions. Y chromosome microdeletion is the main source of more than 15% of male infertility. The primary azoospermia and oligospermia caused by it are not only difficult to cure, but also can be passed on to the next generation with assisted reproductive technology, thereby causing new infertility . [0003] Y chromosome microdeletions mainly refer to the deletion of the azoospermia factor (AZF) region on the long arm of the chromosome. The AZF region can be divided into...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 田龙于超计王倩玉李佳吴文立魏星
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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