PCR amplification primer and probe for detecting AMY2B gene copy number and application of PCR amplification primer and probe

A technology of gene copy number and amplification primers, applied in the field of biomedical clinical molecular detection, can solve the problems of affecting accuracy and prone to false positives.

Pending Publication Date: 2020-10-27
中国人民解放军联勤保障部队第九〇〇医院
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  • Application Information

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Problems solved by technology

However, considering that the homology between the amylase genes is very high, the homology of AMY1 gene sequence and AMY2B is 93.2%, the homology with AMY2B is 93.6%, and the homology of AMY2B and AMY2B genes is 94%. , the result is prone to false positives, which affects its accuracy

Method used

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  • PCR amplification primer and probe for detecting AMY2B gene copy number and application of PCR amplification primer and probe
  • PCR amplification primer and probe for detecting AMY2B gene copy number and application of PCR amplification primer and probe
  • PCR amplification primer and probe for detecting AMY2B gene copy number and application of PCR amplification primer and probe

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Experimental program
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Embodiment 1

[0055] Embodiment 1: The establishment of the amylase AMY2B gene copy number TaqMan real-time fluorescent quantitative PCR detection system (the present invention takes the establishment of TaqMan real-time fluorescent quantitative PCR method as an example)

[0056] 1. Materials

[0057] 1.1 Experimental reagents and consumables: Real-time fluorescent quantitative eight-row tubes were purchased from BIO-RAD; Probe PCR Mix was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; T vector was purchased from Bao Bioengineering (Dalian) Co., Ltd.; whole blood DNA The extraction kit was purchased from QIAGEN; the gel recovery kit was purchased from Omega.

[0058] 1.2 Source of samples: collected whole blood samples.

[0059] 2. Establishment of TaqMan real-time fluorescent quantitative PCR method

[0060] 2.1 DNA sample preparation

[0061] Use the QIAGEN DNA extraction kit to extract the corresponding DNA samples from the collected whole blood samples, wherein th...

Embodiment 2

[0108] Embodiment two: specificity analysis:

[0109] The present invention takes the DNA sample extracted in the whole blood as template, utilizes PCR amplification primer and probe to carry out real-time fluorescent quantitative PCR reaction by 2.2.2 optimized AMY2B real-time fluorescent quantitative PCR reaction system and reaction conditions and obtains corresponding amplification curve ( Such as figure 1 shown), from figure 1 It can be seen that the amplification curve is a single S-type amplification curve, and no non-specific amplification band appears. It shows that the PCR amplification primers and probes of the present invention have good specificity, and they do not amplify other genes in whole blood DNA samples.

[0110] The present invention also uses the whole blood DNA sample as a template, utilizes the AMY2B real-time fluorescent quantitative PCR reaction system and reaction conditions optimized by PCR amplification primers and probes according to 2.2.2 to pe...

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Abstract

The invention relates to a PCR amplification primer and a probe for detecting AMY2B gene copy number and an application of the PCR amplification primer and the probe. The PCR amplification primer comprises an upstream primer and a downstream primer, wherein the sequences of the upstream primer and the downstream primer are as follows respectively: the upstream primer is 5'-GAACCATGATCACCCACCTGCATTCT-3', and the downstream primer is 5'-TGTGCTACACCTCCCAACCTTAATCAATCA-3' the sequence of the probe is 5'-CTGGGTGACAGAGTGAGACCCTCCTCCCTCC-3'. The PCR amplification primer designed by the invention hashigh specificity, can specifically amplify the AMY2B gene, and does not amplify other genes in a whole blood DNA sample, and the PCR amplification primer is combined with a probe, so that the copy number of the AMY2B gene can be rapidly detected.

Description

technical field [0001] The invention belongs to the field of biomedical clinical molecular detection, in particular to a PCR amplification primer and probe for detecting the copy number of AMY2B gene and application thereof. Background technique [0002] The full name of amylase (AMY) is 1,4-α-D-glucan hydrolase, which is the most important enzyme for hydrolyzing carbohydrates, catalyzing the hydrolysis of starch and glycogen to generate glucose, maltose and α1,6-glucosidic bonds Branched chain dextrins. Blood amylase is mainly secreted by pancreas and salivary glands, so amylase isozymes are mainly divided into two types: salivary type S-AMY and pancreatic type P-AMY. The amylase gene clusters on human autosome 1 mainly include: salivary type (AMY1A / 1B / 1C), pancreatic type (AMY2B / 2B) and AMYP. There are extensive copy number variations (CNV) in different races and races, and there are large differences between individuals, such as AMY1 copy number ranges from 1-27, AMY2B ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851C12N15/11
CPCC12Q1/6883C12Q1/6851C12Q2600/156
Inventor 詹芳洁兰小鹏徐冬闽
Owner 中国人民解放军联勤保障部队第九〇〇医院
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