Identification of specific molecular targets, detection primers and rapid assays for Helicobacter pylori
A Helicobacter pylori and detection method technology, applied in the field of Helicobacter pylori detection, can solve the problems of high-throughput sequencing experiments such as complicated operation, difficulty in popularization, and high detection cost, and achieve low detection cost, short detection time, and result judgment simple effect
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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 using 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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