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21 hydroxylase gene mutation detection method and 21 hydroxylase gene mutation detection kit

A detection method and hydroxylase technology, which are applied in biochemical equipment and methods, and the determination/inspection of microorganisms, can solve the problems of difficult and effective amplification of long fragments, and achieve high detection throughput, simple and rapid detection process, and improved detection. The effect of reliability

Inactive Publication Date: 2018-10-23
GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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Problems solved by technology

[0007] Standard PCR can usually only amplify short fragments of 2-3kb, and it is difficult to effectively amplify long fragments above 5kb

Method used

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  • 21 hydroxylase gene mutation detection method and 21 hydroxylase gene mutation detection kit
  • 21 hydroxylase gene mutation detection method and 21 hydroxylase gene mutation detection kit
  • 21 hydroxylase gene mutation detection method and 21 hydroxylase gene mutation detection kit

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Experimental program
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Embodiment 1

[0041] 1. According to the characteristics of 21-hydroxylase gene, design specific primers, the primer sequence is as follows:

[0042] Upstream primer: 5'-AGGTGGGCTGTTTTCCCTTTCA-3' (SEQ ID NO: 1);

[0043] Downstream primer: 5'-CTGTGCCTGGCTATAGCAAGC-3' (SEQ ID NO: 2).

[0044] The RCCX module where the 21-hydroxylase gene is located and the primers used are as follows: figure 1 shown. figure 1 The middle dotted box is the pseudogene CYP21A1P, TNXA and RP2, the upstream primer SEQ ID NO: 1 and the downstream primer SEQ ID NO: 2 can theoretically amplify the pseudogene and the true gene, but if the pseudogene is amplified together, this The length of the amplified fragment is 41kb. The fragment is too long and requires special amplification conditions. It cannot be amplified according to the long-fragment PCR amplification conditions described later in this embodiment. Therefore, using the primers and amplifiers of this embodiment The amplification conditions can only amplif...

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Abstract

The invention discloses a 21 hydroxylase gene mutation detection method and a 21 hydroxylase gene mutation detection kit. According to the method and the kit, based on the human 21 hydroxylase gene mutation characteristics, the hot-spot mutation long-segment deletion is used as a detection object, a RCCX module is subjected to long-fragment PCR amplification by selecting a specific long-fragment PCR amplification primer, the long-fragment amplification product is subjected to enzymatic cleavage reaction detection, whether the detected gene has long-fragment deletion can be intuitively detectedthrough an electrophoresis map, and by combining a high-throughput sequencing technology, the point mutation, the short-fragment insertion of deletion, and other mutation information can be detected.According to the present invention, the whole detection process is simple and rapid, the detection flux is high, the specificity is strong, and the detection result is accurate and reliable.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a detection method and a detection kit for 21 hydroxylase gene mutation. Background technique [0002] Traditional methods for detecting 21 hydroxylase gene mutations include blot hybridization, multiple ligation probe amplification technology, restriction fragment length polymorphism analysis and sequencing technology. [0003] Southern blot (SB) is a traditional method for detecting the recombination of long-fragment 21 hydroxylase genes, that is, the process of transferring the electrophoresis-separated DNA fragments to a certain solid-phase support for detection. After the DNA molecule is digested with a restriction endonuclease, the obtained DNA fragments are separated by agarose gel electrophoresis according to the molecular weight, and then the agarose gel containing the DNA fragments is denatured, and the denatured single-stranded DNA is transferred by siphoning and i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6869C12Q2521/301C12Q2535/122C12Q2525/191
Inventor 张巍
Owner GUANGZHOU JIAJIAN MEDICAL TESTING CO LTD
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