Method and detection kit for detecting helicobacter pylori drug resistance gene

A technology of Helicobacter pylori and drug resistance gene, applied in the field of molecular biology detection of Helicobacter pylori in digestive tract diseases, can solve problems such as failure of eradication treatment, achieve high specificity, high sensitivity, and reduce the effect of antibiotic abuse

Inactive Publication Date: 2014-11-26
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
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  • Claims
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Problems solved by technology

In recent years, the drug resistance of Hp has been distributed globally and has

Method used

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  • Method and detection kit for detecting helicobacter pylori drug resistance gene
  • Method and detection kit for detecting helicobacter pylori drug resistance gene
  • Method and detection kit for detecting helicobacter pylori drug resistance gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1. Collection of saliva

[0040] After obtaining the informed consent of the patients, the oral saliva of the patients was collected before gastroscopy in the morning before upper gastrointestinal symptoms were prepared.

[0041] 2. Genomic DNA extraction from saliva

[0042] Genomic DNA was extracted from saliva using a rapid genomic DNA preparation kit (Shanghai Bocai Biotechnology Co., Ltd.) and used as a template for the following polymerase chain reaction (PCR) amplification. The specific experimental operation process is as follows:

[0043] (1) Processing of specimens

[0044] 1) 25-100 mg tissue samples were cut into small pieces with a scalpel, then crushed with liquid nitrogen, or homogenized with a tissue homogenizer. Collect the powder into a 1.5mL centrifuge tube and suspend with 400μL Disgestion Buffer.

[0045] 2) Take 1 mL of fresh saliva, add 2 mL of PBS (or saline), and soak at room temperature for 2-5 hours.

[0046] (2) Add 3 μL of Proteinase K ...

Embodiment 2

[0092] Example 2 The saliva and gastric mucosal tissues of 60 patients with upper gastrointestinal symptoms and positive rapid urease test were collected. Genomic DNA was extracted from saliva and gastric mucosal tissue using a rapid genomic DNA preparation kit (Shanghai Bocai Biotechnology Co., Ltd.), and detected by two PCR methods. The first is the common PCR method, using genomic DNA in saliva (or gastric mucosal tissue) as a template, and using allele-specific primers for direct PCR amplification. The second method is the nested PCR method, that is, the first round of PCR uses genomic DNA in saliva (or gastric mucosal tissue) as a template, and the outer primers of nested PCR are used for amplification; the second round of PCR uses the first round of PCR products as templates , amplified using allele-specific primers. After the PCR, the common PCR products and nested PCR products were electrophoresed in 1.5% agarose gel, and the presence of specific amplification bands w...

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Abstract

The invention relates to a method and detection kit for detecting a helicobacter pylori drug resistance gene. The method comprises the following steps: carrying out PCR reaction on to-be-detected sample genome DNA serving as a template by using nested PCR outer primers; carrying out PCR by using the outer product of nested PCR as a template and using allele specific primers as inner primers; carrying out electrophoresis on the product (obtained after inner PCR amplification) in 1.5% agarose gel containing ethidium bromide; detecting whether a specific amplification strip exists or not under an ultraviolet ray transilluminator, wherein the nested PCR outer primer sequences are as shown in SEQ ID NO.1 and 2; the inner primer sequences are as shown in SEQ ID NO.3-9; and the kit comprises the nested PCR outer primers and inner primers. According to the method, the target of distinguishing whether drug resistance mutation sites exist in different specimens can be achieved, and the method is high in detection rate, high in sensitivity, simple and convenient to operate, cannot cause pain, is of a noninvasive type and has good patients' compliance.

Description

technical field [0001] The invention relates to the application of nested PCR combined with allele-specific primer amplification technology to the detection of Helicobacter pylori drug-resistant genes in human oral saliva, and belongs to the technical field of molecular biology detection of Helicobacter pylori in digestive tract diseases. Background technique [0002] The pathogenesis of chronic gastritis, gastroduodenal ulcer, gastric cancer, gastric mucosa-associated lymphoid tissue malignancy, etc. is closely related to Helicobacter pylori (Hp) infection, so eradicating Hp can reduce the occurrence and development of Hp-related diseases. In recent years, the drug resistance of Hp has been distributed globally and is becoming more and more serious, often leading to the failure of eradication therapy. More and more drug-resistant gene mutation sites have been found (for example, the resistance of Hp to clarithromycin is related to point mutations such as 23SrRNA gene A2142G...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
CPCC12Q1/6848C12Q1/6858C12Q2531/113C12Q2549/119
Inventor 季旻珺焦健华普汉明骆晓凤屈保进钱明芳杨丙雅侯敏
Owner NANJING MEDICAL UNIV
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