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Y chromosome microdeletion multiple real-time fluorescence PCR detection kit, amplimer pairs and probes

A detection kit and fluorescent probe technology, applied in the field of molecular biology, can solve the problems of false positives, time-consuming, many processes, PCR product contamination and other problems

Inactive Publication Date: 2015-11-04
SHANGHAI HENGJIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has become the industry standard for the detection of Y chromosome microdeletions, there are still many shortcomings: first, the electrophoresis method is likely to cause PCR product contamination and lead to false positives; detection
However, only a large proportion of false negatives will occur in the detection of these 6 STSs, and the coverage rate of Y chromosome microdeletions is only 95%, and the deletion type of each subregion of AZF cannot be judged, so it cannot be a good guide for clinical intervention and treat

Method used

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  • Y chromosome microdeletion multiple real-time fluorescence PCR detection kit, amplimer pairs and probes
  • Y chromosome microdeletion multiple real-time fluorescence PCR detection kit, amplimer pairs and probes
  • Y chromosome microdeletion multiple real-time fluorescence PCR detection kit, amplimer pairs and probes

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Experimental program
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Effect test

Embodiment 1

[0061] 1. Primer design

[0062] Such as figure 1 As shown, for the detection of SY14, SY82, SY84, SY86, SY88, SY105, SY121, SY127, SY134, SY143, SY145, SY152, SY153, SY160, SY254, SY255, SY157, SY242, SY1064, SY1061, SY1129 Spot-specific amplification primer pairs and fluorescent probe nucleotide sequences such as figure 1 shown.

[0063] 2. Reagent kit

[0064] Divide the above primer pairs and probes into 8 reaction tubes. The sequences of primers and probes in tubes A-H and the concentrations in the reaction system are shown in Table 1-1 to Table 1-8 respectively. F is the upstream primer, and R is the Downstream primer, P is the probe.

[0065] Table 1-1 List of Primers and Probes for Tube A

[0066]

[0067] Table 1-2 List of Primers and Probes in Tube B

[0068]

[0069] Table 1-3 C tube primer and probe list

[0070]

[0071]

[0072] Table 1-4 D tube primer and probe list

[0073]

[0074] Table 1-5 E tube primer and probe list

[0075]

[0...

Embodiment 2

[0083] (1) Reagent preparation: TaqMan2×PCR Master mix (containing 10mmol / L Tris-HCl, pH8.3, 50mmol / L KCl, Mg 2+ , hot start enzyme, UNG enzyme, dNTP), or whole blood extraction kit.

[0084] (2) Genomic DNA extraction: Human genomic DNA was extracted from anticoagulated whole blood by conventional molecular biology methods or commercially available kits.

[0085] (3) Real-time fluorescent PCR amplification and detection.

[0086] a. The real-time fluorescent PCR reaction system is shown in Table 2, including 3mmol / L Mg 2+ , 200μmol / L dATP, dCTP, dGTP, 400μmol / L dUTP, 200nmol / L each specific primer, 200nmol / L each probe, 1U hot start enzyme, 0.3U UNG enzyme, 50ng human genomic DNA, and label. Reactions in 384-well plates.

[0087] Table 2 reaction system

[0088]

[0089] b. The real-time fluorescent PCR reaction was carried out on the ABI 7900 instrument, and the amplification detection was carried out according to the following conditions.

[0090] Reaction program: ...

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Abstract

The invention relates to a biomolecule diagnostic reagent and discloses a Y chromosome microdeletion multiple real-time fluorescence PCR detection kit. The Y chromosome microdeletion multiple real-time fluorescence PCR detection kit comprises amplimer pairs and fluorescent probes for detecting SY84, SY86, SY127, SY134, SY254, SY255, SY157, SY242, SY1191 and SY1291, and further comprises amplimer pairs and fluorescent probes for detecting sites including at least one of SY82, SY88, SY1064 and SY1065, at least one of SY105, SY121, SY143 and SY153, SY160 and at least one of SY145 and SY152 for detecting AZFd area deletion. The Y chromosome microdeletion multiple real-time fluorescence PCR detection kit can detect 21 STS sites at most, the deletion coverage rate is larger than 99 percent, a multiple real-time fluorescence quantitative PCR technological platform is adopted, Y chromosome microdelection sites can be quickly detected in a high-throughput mode, whether the AZF a, b, c and d areas are deleted completely or partially is distinguished, and more comprehensive Y chromosome microdeletion information is obtained.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to biomolecular diagnostic reagents, especially a kit for detecting Y chromosome microdeletion. Background technique [0002] In recent years, the number of infertile people of childbearing age in my country has shown a large-scale increase, and the number has exceeded 20 million. Twenty years ago, the infertility rate among the population of childbearing age in my country was only 3%, but now it has climbed to 15%, of which the infertility caused by male infertility accounts for 50%, an increase of 15% compared with 10 years ago. [0003] Because the clinical manifestations, treatment methods and therapeutic effects of male infertility vary greatly depending on the cause of the disease, the key point of male infertility treatment is to find the specific cause of the disease and carry out etiological treatment. The causes of male infertility include: urogenital malformations, repro...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 鹿亚超
Owner SHANGHAI HENGJIAN BIOTECH CO LTD
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