Y chromosome microdeletion detection kit

A technology for detecting kits and Y chromosomes, which is applied in the field of molecular biology, can solve problems such as unstable reaction efficiency, decreased fluorescence value of the instrument, and influence on the accuracy of results, and achieve stable reaction efficiency, objective detection results, and avoidance of polymerization Effect

Pending Publication Date: 2021-04-30
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the existing STS locus-to-Y chromosome microdeletion detection system, each sequence in the system is prone to DNA dimerization, resulting in unstable reaction efficiency
And, more importantly, the inventors have found that occasionally the amplification curve will drop suddenly (making the PCR detection curve abnormal, and under the situation of multi-sample or multi-index synchronous detection, it is easy to produce misjud...

Method used

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  • Y chromosome microdeletion detection kit
  • Y chromosome microdeletion detection kit
  • Y chromosome microdeletion detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Y chromosome microdeletion detection kit

[0028] This kit uses a fluorescent quantitative PCR instrument as a platform to design 3 sets of specific primers and probe combinations for 3 DNA regions related to infertility on the human Y chromosome, and detect 1 through two different channels in one reaction system. The two representative loci in this region, the specific detection loci are: AZFa (sY84, sY86), AZFb (sY127, sY134), AZFc (sY254, sY255). When the reaction system contains different gene templates, the PCR reaction is carried out and different fluorescent signals are released. The real-time monitoring and output of the signal intensity of the corresponding channel during the PCR process is carried out by using a fluorescent quantitative PCR instrument to realize the qualitative analysis of the detection results. The etiological analysis of patients with auxiliary diagnosis as infertility.

[0029]In this experiment, RT-PCR method was used to detect...

Embodiment 2

[0048] Embodiment 2 kit detects

[0049] 1. Sample preparation

[0050] Sample DNA extraction: refer to the purchased commercial genomic DNA extraction kit instructions for operation.

[0051] 2. Reagent preparation

[0052] 1) Take out the kit from the refrigerator and thaw at room temperature. After the components are fully thawed and mixed, centrifuge quickly for 10 seconds and place on ice.

[0053] 2) Prepare the PCR amplification reaction solution according to the ratio in the following table 2.1 (take the preparation of the amplification reaction solution for 1 person as an example):

[0054] Table 2.1

[0055]

[0056]

[0057] 3) Each of the prepared amplification reaction solutions was mixed separately, and dispensed into PCR reaction tubes according to the dispensing volume of 20 μL / well.

[0058] 4) Add the genomic DNA of the sample to be tested, the QY positive quality control product, and the blank control to the reaction tubes that have been equipped w...

Embodiment 3

[0084] Embodiment 3 kit sensitivity detection

[0085] In order to investigate the detection sensitivity of the kit in the detection of actual nucleic acid samples, male blood nucleic acid extraction samples with known AZFa(sy84) site deletion were taken and diluted to 100, 50, 25, 10, 5, 2.5, 0.5 , 0.25ng / μl, and using 10mM Tris as a negative control, operate and interpret the results according to the composition of the kit described in Example 1 and the detection method described in Example 2.

[0086] chart 3.1

[0087] test group 1 2 3 4 5 6 7 8 Sample concentration to be tested (ng / μl) 100 50 25 10 5 2.5 0.5 0.25 Sample concentration in the reaction system 20 10 5 2 1 0.5 0.1 0.05

[0088] Table 3.2

[0089] test group 1 2 3 4 5 6 7 8 sy84 channel Ct value 19.50 23.14 24.96 26.78 28.31 29.76 31.51 32.27

[0090] Test results such as figure 1 As shown in table 3.2, the detection results ...

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Abstract

The invention provides a Y chromosome microdeletion detection kit. The kit comprises a reaction solution and an enzyme solution, wherein the reaction solution comprises a PCR (Polymerase Chain Reaction) buffer solution as well as primers and a probe aiming at a Y chromosome microdeletion site, and the PCR buffer solution comprises a Tris HCl buffer solution; wherein the enzyme solution comprises a hot start DNA polymerase and a Tris-HCl buffer solution; wherein tris (2-carbonyl ethyl) phosphorus hydrochloride and/or polyoxyethylene polyoxypropanolamine ether are/is added into the Tris-HCl buffer solution. According to the Y chromosome microdeletion detection kit disclosed by the invention, the generation of bubbles in a reaction system in a reaction tube and the sudden drop of individual amplification curves caused by the generation of the bubbles can be reduced; according to the present invention, the dimer sulfhydrylation DNA terminal sulfur atom can be effectively prevented from being polymerized, and the stable reaction efficiency can be achieved even in the presence of oxygen. Therefore, the Y chromosome microdeletion detection kit provided by the invention has good sensitivity and precision.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a Y chromosome microdeletion detection kit. Background technique [0002] Studies have shown that in the worldwide population, the probability of infertility is 10%-15%, of which male factors account for about 50%. Y chromosome plays an important role in male fertility because it regulates the growth and development of male sperm cells, and abnormalities in its chromosome structure or gene quantity can cause azoospermia or oligospermia. The microdeletion of the azoosperm factor region (AZF) on the long arm of the Y chromosome is one of the most common causes of infertility associated with Y chromosome abnormalities. [0003] Deletion or mutation of AZF may lead to spermatogenesis disorders, loss of Yq11 segment or more distant segments or small deletions in the middle of this segment can lead to spermatogenic disorders at different stages of spermatogenesis...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2521/101C12Q2527/125C12Q2563/107C12Q2545/113C12Q2545/101
Inventor 许嘉森吴诗扬刘志明彭璨璨曾杰黄洁芬
Owner SUREXAM BIO TECH
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