Specific molecular target, detection primer and rapid detection method for identifying helicobacter pylori

A technology of Helicobacter pylori and molecular targets, which is applied in the field of identification of specific molecular targets of Helicobacter pylori, can solve the problems of high cost of detection, difficulty in popularization, complicated operation of high-throughput sequencing experiments, etc. Inexpensive and easy to judge results

Active Publication Date: 2021-11-26
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the problems of complicated experimental operations and high detection costs in high-throughput sequencing, the HP detection scheme proposed by Kim is currently difficult to be promoted in clinical practice.

Method used

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  • Specific molecular target, detection primer and rapid detection method for identifying helicobacter pylori
  • Specific molecular target, detection primer and rapid detection method for identifying helicobacter pylori
  • Specific molecular target, detection primer and rapid detection method for identifying helicobacter pylori

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Analysis of micro-ecosystem of gastric flora

[0030] (1) Gastric microecosystem analysis based on amplicon sequencing

[0031] ①Gastric microbial DNA amplicon sequencing: A gastric mucosal sample with a volume of about 3mm×3mm×1mm was taken, and the total microbial DNA of the mucosa was extracted using the QIAamp PowerFecal DNA Kit (Qiagen, Germany). The extracted gastric microbial DNA was amplified using 341F 5′-CCTACGGGNGGCWGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′ as amplification primers, and the UCP Multiplex PCR Kit (Qiagen) was used to amplify the V3-V4 region of the 16S rRNA gene. Afterwards, the QIAseq Ultralow Input Library Kit was used for library construction. The library was quantified and mixed with High Sensitivity DNAKit (Agilent, USA) and Qubit 1× dsDNA HS Assay Kit (Thermo-Fisher, USA). The mixed library was denatured with 1.0N NaOH, and then 14pM was used as the target concentration on the machine. HT1 for library dilution. Sequencing ...

Embodiment 2

[0039] Example 2 Mining of specific new detection targets for Helicobacter pylori

[0040] According to the gastric micro-ecological network constructed in Example 1, 190 microorganisms belonging to 32 genera were selected as the core microorganisms of the stomach. According to the GenBank database in NCBI and the Helicobacter pylori whole genome database previously established by the inventor team, comparative genomics analysis was performed to mine the unique target sequences of Helicobacter pylori in the gastric microecological environment. After annotating the complete map sequences of the analyzed 644 microbial genomes with Prokka software, the pan-genome analysis software Roary was used to cluster the functional genes with a similarity ≥ 85% into the same gene cluster, and a matrix list of all the analyzed genomes was generated. It is considered that the genes shared by all Helicobacter pylori in the matrix list but not in other species of microorganisms are the target s...

Embodiment 3

[0041] Example 3 Establishment of a rapid detection method for specific new detection targets of Helicobacter pylori

[0042] (1) This embodiment provides a method for detecting Helicobacter pylori, the specific operations are as follows:

[0043] According to the HP-specific detection target obtained in Example 2, primers SEQ ID NO.3 and SEQ ID NO.4 were designed to obtain a rapid detection method, including the following steps:

[0044] Table 1 HP-specific PCR detection primer sequences

[0045]

[0046] PCR detection system and amplification procedure:

[0047] The DNA template preparation method required for PCR detection was performed using the tissue microorganism DNA extraction scheme in Example 1. HP's PCR reaction system configuration is as follows:

[0048] 2×PCR Mix 12.5μL

[0049] SEQ.ID No.3 (10μmol / L) 1μL

[0050] SEQ.ID No.4 (10μmol / L) 1μL

[0051] Template DNA (2.5ng / μL) 1μL

[0052] wxya 2 0 9.5 μL

[0053] The following are the PCR reaction condit...

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Abstract

The invention discloses a specific molecular target, a detection primer and a rapid detection method for identifying helicobacter pylori. The molecular target for detecting helicobacter pylori has a nucleotide sequence as shown in SEQ ID NO. 1. The specificity of the molecular target and the detection scheme mentioned in the invention is obviously superior to that of a clinically universal Clayton scheme at present, the detection efficiency is similar to that of a high-throughput sequencing detection scheme proposed by Kim, but compared with the Kim scheme, the molecular target and the detection scheme have the advantages of quantitation, simplicity, convenience and rapidness in operation and low detection cost.

Description

technical field [0001] The invention belongs to the field of detection of Helicobacter pylori, and in particular relates to a specific molecular target for identifying Helicobacter pylori, a detection primer and a rapid detection method. Background technique [0002] Helicobacter pylori ( Helicobacter pylori , HP) is a microaerophilic Gram-negative bacterium that has been implicated in human gastrointestinal disease. According to statistics, most gastrointestinal ulcers and gastrointestinal bleeding are related to the infection of this bacteria, and about 90% of gastric cancers in the world are closely related to HP infection. It can be seen that HP infection seriously threatens human health and causes huge economic and social losses. Therefore, there is an urgent need for an accurate and efficient HP detection scheme for clinical detection of HP infection, which will help improve the identification of high-risk groups of infection, and is of great significance for the prev...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114
Inventor李滢吴清平庞锐商燕燕谢新强陈慧贞薛亮陈谋通杨润时王涓丁郁张菊梅陈惠元
OwnerGUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY