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ALDH2 (aldehyde dehydrogenase 2) Gene polymorphism detection kit

A site polymorphism and reagent technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of complicated operation steps and long time-consuming, and achieve high detection specificity and easy operation. , the effect of high conversion rate

Inactive Publication Date: 2018-07-10
滨江华康(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main methods for detecting ALDH2*2 include Sanger sequencing, PCR-taqman fluorescent probe Ct value method, PCR capillary electrophoresis fragment analysis method, gene chip hybridization method, etc., but these methods require nucleic acid extraction, and the operation steps are complicated and time-consuming. long, and these methods need to be performed in specialized gene amplification laboratories

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  • ALDH2 (aldehyde dehydrogenase 2) Gene polymorphism detection kit
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  • ALDH2 (aldehyde dehydrogenase 2) Gene polymorphism detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1, ALDH2 gene polymorphism detection kit and detection method

[0087] 1. ALDH2 Gene Polymorphism Detection Primers

[0088] 1. Design of primers

[0089] According to the rs671 site of the target gene ALDH2 gene, and in order to shorten the amplification time of PCR, the length of the PCR amplification product is controlled at about 150bp, and the following three pairs of primer sequences are designed (the three pairs of primer sequences are all synthesized by Bioserch Company ):

[0090] ALDH2-F1: TTTGGAGCCCAGTCACCCCTTTG;

[0091] ALDH2-R1:AGCCACCAGCAGACCCTCAA;

[0092] ALDH2-F2: TGTTTGGAGCCCAGTCACCCTT;

[0093] ALDH2-R2:AGCCACCAGCAGACCCTCAA;

[0094] ALDH2-F3: TGATGTGTTTGGAGCCCAGTCA;

[0095] ALDH2-R3:AGCCACCAGCAGACCCTCAA.

[0096] Table 2, rs671 site information

[0097]

[0098] 2. PCR amplification

[0099] (1) Phire Hotstart II DNA polymerase for PCR amplification

[0100] Using throat swab liquid samples (ALDH2*1*1 carriers) and the DNA s...

Embodiment 2

[0149] Example 2, Sensitivity Detection of ALDH2 Gene Polymorphism Detection Kit

[0150] Extract DNA from throat swab samples of ALDH2*1*2 carriers, quantify the obtained DNA extract by Nanodrop1000 (Thermo Scientific, ND-1000) and then serially dilute the concentration to 250pg / μL, 125pg / μL, 62.5pg / μL μL, 31.25pg / μL, 15.625pg / μL, 7.8pg / μL, 3.9pg / μL, 1.95pg / μL DNA solution. Then use a pipette gun to draw 2 μL of the above DNA solution and detect the ALDH2 gene polymorphism in DNA solutions with different concentrations according to the method in step 3 of Example 1.

[0151] The result is as Figure 5 shown. The minimum detection DNA content of the ALDH2 gene polymorphism detection kit of the present invention reaches 7.8pg / μL.

Embodiment 3

[0152] Example 3, Application of ALDH2 Gene Polymorphism Detection Kit

[0153] The oral swab samples of 6 patients with angina pectoris (from Beijing Fuwai Hospital, all patients with angina pectoris were confirmed by clinical diagnosis and informed consent) were taken, and the oral swab samples of each patient were divided into two equal parts, and one was used The ALDH2 gene polymorphism detection kit of the present invention detects the genotype of the patient sample to be tested according to the method in Step 3 of Example 1, and another copy is sent to Sanger Sequencing Company for sequencing.

[0154] The test results are shown in Table 7. The melting curve results are as Figure 7 As shown, the sequencing results are as follows Figure 8 shown. As can be seen from the figure and the table: the detection result of the kit of the present invention is consistent with the detection result of the sequencing method, indicating that the kit of the present invention can eff...

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Abstract

The invention discloses an ALDH2 (aldehyde dehydrogenase 2) Gene polymorphism detection kit, having the advantages that detection sensitivity is high, the lowest detection limit is 7.8 pg DNA; the kitis simple to operate and allows point-of-care test without professional knowledge; the detection time is short, down to 45 min; the detection specificity is high, and the detection accuracy is very high since a specific fluorescent probe is used; nucleic acid extraction is not required, low-melting-point agarose is added, and PCR (polymerase chain reaction) is greatly avoided. When used with ParaDNA gene amplification detectors to carry out POCT (point-of-care test), the kit can be used to clinically provide guidance on the administration of nitroglycerin and the notification on the risk of alcohol drinking.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a detection kit for ALDH2 gene polymorphism. Background technique [0002] The full name of ALDH2 is aldehyde dehydrogenase 2 family (mitochondrial), that is, mitochondrial acetaldehyde dehydrogenase 2. ALDH2 has both acetaldehyde dehydrogenase and esterase activities, and participates in the metabolism of drugs such as ethanol and nitroglycerin. ALDH2*2(rs671, NM_000690.3:c.1510G>A) is the base mutation of the rs671 site of the wild-type allele ALDH2*1 from G to A, resulting in the 504th glutamic acid of the encoded protein being mutated Lysine is substituted. The ALDH2 enzyme activity of individuals carrying the mutant allele (ALDH2*2) decreased, the enzyme activity of mutant heterozygous individuals was only 10% of that of wild-type individuals, and the enzyme activity of mutant homozygous individuals was absent. Therefore, individuals carrying mutant ALDH2*2 alleles have de...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/106C12Q2600/156C12Q2563/107C12Q2527/107C12Q2531/113
Inventor 戴维·约翰·弗伦奇莫妮卡·帕纳苏杨松江
Owner 滨江华康(北京)生物科技有限公司
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