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Method for guiding helicobacter pylori to eradicate drug use based on PGM high throughput sequencing technology

A Helicobacter pylori, technical guidance technology, applied in the field of Helicobacter pylori drug resistance gene and host drug metabolizing enzyme CYP2C19 gene, can solve the problems of long detection cycle, easy pollution, low specificity, etc., achieve fast detection results, avoid The effect of waste and simple operation process

Inactive Publication Date: 2017-10-10
杭州致远医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although non-invasive detection technology is painless and convenient, it is prone to false positive results and has low specificity; among invasive detection technologies, the method of bacterial culture is the most accurate detection technology and is considered the "gold standard" for diagnosis, but It has problems such as long detection cycle, complicated cultivation, and easy pollution

Method used

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  • Method for guiding helicobacter pylori to eradicate drug use based on PGM high throughput sequencing technology
  • Method for guiding helicobacter pylori to eradicate drug use based on PGM high throughput sequencing technology
  • Method for guiding helicobacter pylori to eradicate drug use based on PGM high throughput sequencing technology

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Experimental program
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Effect test

Embodiment 1

[0022] Design specific primers covering VacA, 23SrRNA, gyrA, CYP2C19*2, and CYP2C19*3 gene regions to be tested. The amplified product is 180-220bp. See Table 1 for details.

[0023] Table 1 Specific primers for gene regions to be tested

[0024]

[0025]

[0026] A GCGTGTCTCCGACTCAG-bracode-GAT-specific primer was added to the 5' end of the forward primer of the primers designed above, and a CCTCTCTATGGGCAGTCGGTGAT-specific primer was added to the 5' end of each reverse primer. The sequence of barcode1 is CTAAGGTAAC. Different samples must use different barcode sequences to distinguish between samples. Other barcode sequences can be queried on the official website of Life Technologies. See Table 2 for details, where the red bold part is the barcode1 sequence.

[0027] Table 2 primers containing barcode1 sequence

[0028]

[0029] 3. The mixing ratio of these primers in the Primer Pool is: 23SrRNA:CYP2C19*2:CYP2C19*3:gyrA:VacA=2:1:1:1.2:1.5.

Embodiment 2

[0031] Genome extraction from gastric mucosa

[0032] (1) In a sterile 2ml round bottom EP tube, add 100μl tissue lysate and a sterile steel ball with a diameter of 3mm;

[0033] (2) Take out the gastric mucosal tissue from the sample preservation solution, and transfer it to a 2ml round-bottomed EP tube containing the lysate and steel beads;

[0034] (3) The tissue grinder grinds the sample, and the parameters are 50Hz and 60s at room temperature;

[0035] (4) After fully grinding, briefly centrifuge, and transfer the homogenate liquid to a new sterile 1.5ml EP tube;

[0036] (5) Incubate the 1.5ml EP tube containing the homogenized tissue in a 95°C metal bath for 10 minutes, and invert it several times in the middle;

[0037] (6) Quickly incubate EP in a refrigerator at 4°C for 30 minutes;

[0038] (7) Take out the EP tube from the refrigerator, centrifuge at 12,000 rpm for 5 minutes, collect the supernatant into a new EP tube, and then obtain the whole genome of the host a...

Embodiment 3

[0040] Amplification and purification of target fragments

[0041] (1) Prepare the following reaction system with PCR primers mixed according to the ratio: total reaction system 20 μl, including 10 μl of 2*Phusion HF Buffer PCR Master mix, 2 μl of template, 2 μl of primers, ddH 2 O 6 μl. The reaction conditions were pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds within the cycle, annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, 18 cycles, and extension at 72°C for 5 minutes at the end of the cycle.

[0042] (2) Add 24 μl Agencourt AMPure XP beads magnetic beads to 20 μL of the amplification product, mix by pipetting, and place at room temperature for 5 minutes. When the liquid becomes clear, discard the supernatant, slowly add 200 μl of 80% ethanol to wash, discard the ethanol after 30 seconds, and repeat once. After the ethanol volatilized, 40 μl of water was added for elution when the center of the magnetic beads began to cra...

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Abstract

The invention provides a method for guiding helicobacter pylori to eradicate drug use based on a PGM high throughput sequencing technology, and mainly aims to realize detection of helicobacter pylori on antibiotic drug resistance and host drug metabolic enzyme types. The method mainly comprises the steps of designing a specific primer for covering VacA, 23SrRNA, gyrA, CYP2C19*2 and CYP2C19*3 gene to-be-detected regions to form a Primer Pool; extracting host and helicobacter pylori genome to perform amplification; performing PGM library preparation, purification, quantifying and sequencing; and working out drug resistance proportion and drug metabolic enzyme types. By adoption of multiple PCR technologies, synchronous amplification of multiple sites can be realized; and by combination with PGM sequencing, guiding to clinical detection and eradiation of the helicobacter pylori can be facilitated.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a method for detecting Helicobacter pylori-specific genes, Helicobacter pylori drug-resistant genes and host drug-metabolizing enzyme CYP2C19 genes based on a PGM high-throughput sequencer. Background technique [0002] Helicobacter pylori (HP for short) is a Gram-negative helical bacterium that colonizes between the epithelium and mucus of the gastric mucosa. Since Marshall and Warren first isolated Helicobacter pylori with microaerophilic technology, it has been found that Helicobacter pylori is closely related to chronic gastritis, peptic ulcer, and gastric mucosa-associated lymphoid tissue lymphoma (MALT), and Helicobacter pylori infection is also gastric cancer. One of the important causes of occurrence. [0003] With the widespread use of antibiotics, the resistance of Helicobacter pylori to antibiotics is increasing year by year, and the eradication rate of trad...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
CPCC12Q1/6883C12Q1/689C12Q2600/106C12Q2600/156C12Q2600/158C12Q2600/16C12Q2600/178
Inventor 杨比特许佩松杨宁敏
Owner 杭州致远医学检验所有限公司
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