Primer probe set for detecting helicobacter pylori based on LFD-RMA method

A technology of Helicobacter pylori and primer probes, which is applied in the directions of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems that are not suitable for promotion and popularization in grassroots hospitals, are not very high, and are expensive, and achieve Reduce the difficulty of sampling, save the cost of testing, and facilitate the operation

Inactive Publication Date: 2021-06-29
济南国益生物科技有限公司
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Problems solved by technology

Among them, serum antibody test is generally used for epidemiological investigation of population infection rate because it cannot distinguish between past infection and ongoing infection; the popularity of fecal Hp antigen detection is not very high; urea breath test is currently widely used in clinical judgment of infection and re-examination after treatment, but antibiotics, bismuth and acid-suppressing drugs should be stopped or prohibited within 4 weeks before the inspection, and false positives are prone to occur; fluorescent quantitative PCR method is sensitive and efficient, but it is expensive and not suitable for popularization in primary hospitals

Method used

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  • Primer probe set for detecting helicobacter pylori based on LFD-RMA method
  • Primer probe set for detecting helicobacter pylori based on LFD-RMA method
  • Primer probe set for detecting helicobacter pylori based on LFD-RMA method

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Embodiment 1

[0036] 1. Preparation of positive standard plasmid

[0037] The nucleic acid of HP was extracted as a template, and the specific gene of HP was amplified by PCR. The PCR amplification product was subjected to 1% agarose gel electrophoresis, recovered by tapping the gel, cloned and connected to the pMD18-T vector, and transformed into E. coli competent cells. Blue-white screening, picking white colonies, and colony PCR verification. Send the positive recombinant bacteria to the company for sequencing, culture the correctly sequenced recombinant bacteria overnight, extract the plasmid DNA, and obtain the positive plasmid.

[0038] 2. Design of RMA primers and probes

[0039] Specific RMA primers and probes were designed according to the 16s rRNA gene of HP, as shown in Table 1:

[0040] Table 1 Primer and Probe Sequences

[0041]

[0042] 3. Establishment of RMA reaction system

[0043] Add 42.5 μL buffer solution and 5 μL extracted nucleic acid of the sample to be tested...

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Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a primer probe set for detecting helicobacter pylori based on an LFD-RMA method, and the primer probe set comprises a primer pair for detecting helicobacter pylori and a corresponding probe. The 5' end of a downstream primer is marked with Biotin; the 5' end of the probe is marked by FAM, the 31st basse T is replaced by THF (dSpacer), and the 3' end of the probe is modified by a blocking group C3-Spacer; and the size of an amplification product does not exceed 500bp. The detection method disclosed by the invention is simple, non-invasive, rapid, high in accuracy and good in safety, and can be widely applied to rapid detection of helicobacter pylori.

Description

technical field [0001] The application belongs to the technical field of biological detection, and specifically relates to a primer probe set for detecting Helicobacter pylori based on the LFD-RMA method. Background technique [0002] Helicobacter pylori (Helicobacter pylori, HP) is a Gram-negative bacillus with extremely strong survival ability and can survive in the strong acidic environment in the stomach. It is the only bacterium found so far that can survive in the stomach. Humans are it sole host and source of infection. HP is the main cause of peptic ulcer occurrence and recurrence after treatment. Its detection rate is as high as 95%-100% in patients with duodenal ulcer, and more than 70% in patients with gastric ulcer. At the same time, HP can cause gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma, and is clearly listed as a class I carcinogen by the World Health Organization. The infection rate of HP varies around the world, mainly depending ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2600/166
Inventor 陈龙陈大为张瑶
Owner 济南国益生物科技有限公司
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