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
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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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