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Amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and reagent kit

A technology of Helicobacter pylori and sequencing primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., which can solve the problems of multiple mutation types and incomplete detection of Helicobacter pylori, and avoid Complicated and difficult, reducing manpower and material resources, and the effect of multiple experimental information

Pending Publication Date: 2020-07-28
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the defects of many types of Helicobacter pylori mutations and incomplete detection in the existing molecular diagnostic technology, and provide a set of optimized primers for PCR amplification and sequencing, which can perform 11 purposes at one time under the same conditions Gene sequence amplification PCR, successfully amplified and sequenced Helicobacter pylori gene typing and drug resistance-related regions

Method used

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  • Amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and reagent kit
  • Amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and reagent kit

Examples

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Effect test

Embodiment 1

[0063] A set of specific primers for the amplification and sequencing of Helicobacter pylori, including the amplification and sequencing primers of UreA are SEQ ID NO.1 and SEQ ID NO.2, and the amplification and sequencing primers of 16S rRNA are SEQ ID NO. 3 and SEQ ID NO.4, rdxA amplification and sequencing primers are SEQ ID NO.5 and SEQ ID NO.6, 23S rRNA amplification and sequencing primers are SEQ ID NO.7 and SEQ ID NO.8, gyrA The amplification and sequencing primers are SEQ ID NO.9 and SEQ ID NO.10, the amplification and sequencing primers of PBP1 are SEQ ID NO.11 and SEQ ID NO.12, and the amplification and sequencing primers of cagA are SEQ ID NO.13 And SEQ ID NO.14, the amplification of vacAs and sequencing primer are SEQ ID NO.15 and SEQ ID NO.16, the amplification of vacAm and sequencing primer are SEQ ID NO.17 and SEQ ID NO.18, the amplification of vacAi The amplification and sequencing primers are SEQ ID NO.19 and SEQ ID NO.20, and the amplification and sequencing ...

Embodiment 2

[0065] A kit for amplifying and sequencing Helicobacter pylori, consisting of the following reagents:

[0066] Reagent A: Consists of 2mL buffer containing Green I dye, 0.2mM dNTPs (including dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4)2SO4, 6mM MgSO4, 100U TaqDNA polymerase;

[0067] Reagent H1: 30 μL aqueous solution of sequencing primers targeting UreA, containing 4 μM SEQ ID NO.1;

[0068] Reagent H2: 30 μL aqueous solution of sequencing primers targeting 16S rRNA, containing 4 μM SEQ ID NO.3;

[0069] Reagent H3: 30 μL aqueous solution of sequencing primers for rdxA, containing 4 μM SEQ ID NO.5;

[0070] Reagent H4: 30 μL aqueous solution of sequencing primers targeting 23S rRNA, containing 4 μM SEQ ID NO.7;

[0071] Reagent H5: 30 μL aqueous solution of sequencing primers targeting gyrA, containing 4 μM SEQ ID NO.9;

[0072] Reagent H6: 30 μL aqueous solution of sequencing primers against PBP1, containing 4 μM SEQ ID NO.11;

[0073] Reagent H7: 30 ...

Embodiment 3

[0091] Real-time fluorescent quantitative PCR (real-time FQ PCR) is used to amplify all the sequences of Helicobacter pylori with the kit of the present invention. First, calculate the required amount of each reagent according to the number of experimental specimens. The experimental steps are as follows:

[0092] 1. Amplification reaction solution configuration: Prepare the reaction solution (1 test sample) in order, add 120 μL of water, 150 μL of reagent A, and 30 μL of DNA sample, a total of 300 μL; mix well.

[0093] 2. Add 25 μL of amplification reaction solution to each tube in sequence, and cover the caps.

[0094] 3. Amplify according to the PCR program designed in the experiment: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 35 seconds. During the extension period, the fluorescence value was obtained for 35 cycles, and finally stored at 25°C. ℃; take it out in time for sequencing. ...

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Abstract

The invention discloses an amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and a reagent kit. The reagent kit comprises reaction liquid A for PCR amplification, a reaction tube for coating primer pairs, and primers H1, H2, H3, H4, H5, H6, H7, H8, H9, H10 and H11 for sequencing. The amplifying and sequencing primer for subtyping and drug resistance detection of helicobacter pylori, and the reagent kit disclosed by the invention can amplify all 11 objective sequences of the helicobacter pylori under the same PCR reaction condition at one time,and then the sequencing primers are used for performing sequencing according to a set procedure. A PCR reaction system has fluorescent dye, an amplification result can be judged directly through theelevation position of a fluorescent value, and inconvenience and pollution caused by electrophoresis can be avoided. The primers are directly coated in the reaction tube, so that tedious configurationduring primer use can be avoided. The reagent kit can be used for detecting virulence subtypes and drug resistance genes of the helicobacter pylori. The amplifying and sequencing primer and the reagent kit disclosed by the invention can be used for detecting clinical biopsy specimens and paraffin-embedded specimens and can provide bacterium subtypes and drug resistance information for helicobacter pylori treatment.

Description

technical field [0001] The invention relates to a molecular biology method based on polymerase chain reaction (PCR) and DNA sequencing technology, in particular to an amplification and sequencing primer and a kit for Helicobacter pylori genotyping and drug-resistant mutation detection. Background technique [0002] Helicobacter pylori (Hp) is the only microbial species that can survive in the human stomach. Colonized Helicobacter pylori can cause chronic inflammation, peptic ulcer and even gastric cancer. Statistics show that 50-60% of people in China have Hp infection. As one of the most important pathogenic factors of gastric cancer, Hp was classified as a class I carcinogen by the World Health Organization in 1994. Robin Warren and Barry Marshall also won the 2005 Nobel Prize in Physiology or Medicine for their discovery of the relationship between Helicobacter pylori and gastric disease. [0003] Hp includes a variety of subtypes, and bacteria carrying different genoty...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6869C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6869C12Q1/6851C12Q2600/106C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 陈必成雷颖颖
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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