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Quick molecular diagnosis method and reagent kit for individualized medication guidance for eradication therapy of helicobacter pylori

A technology of Helicobacter pylori and a diagnostic method, applied in the field of diagnostic methods and kits for detecting and judging drug resistance of Helicobacter pylori, can solve the problems of low culture success rate, poor patient compliance, long culture cycle, etc., and achieves the operation process. Simple, reasonable design, fast detection results

Pending Publication Date: 2020-09-25
深圳艾普斯金基因检测合伙企业(有限合伙)
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are single or multiple antibiotic resistance gene detection methods, but the commonly used drug sensitivity test is still the isolation and culture of Helicobacter pylori in gastric biopsy tissue. Due to the uneven distribution of Helicobacter pylori in the stomach, the patient's compliance is poor, and Lead to rare Helicobacter pylori content in gastric biopsy samples and low success rate of culture
And because of the harsh culture conditions of Helicobacter pylori, specific instruments are required, the growth is slow, and the culture period is long, so it is not suitable for rapid diagnosis of clinical drug resistance and immediate medication guidance

Method used

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  • Quick molecular diagnosis method and reagent kit for individualized medication guidance for eradication therapy of helicobacter pylori
  • Quick molecular diagnosis method and reagent kit for individualized medication guidance for eradication therapy of helicobacter pylori
  • Quick molecular diagnosis method and reagent kit for individualized medication guidance for eradication therapy of helicobacter pylori

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Helicobacter pylori genome extraction process in gastric biopsy samples

[0065] 1. Sampling requirements:

[0066] 1) Confirm that the patient stopped taking antibiotics, proton pump inhibitors, and bismuth for more than four weeks before the test, and Chinese medicine for more than two weeks, and was verified positive by rapid urease test paper and urea breath test (UBT).

[0067] 2) When taking gastroscopy biopsy samples, try to take double samples of gastric antrum and gastric body lesions, put them into the transport medium test tube, and transport them under refrigeration at 2-8 degrees within 12 hours.

[0068] 3) The HP of newly treated patients is mostly distributed in the gastric antrum 2-5cm away from the pylorus, and the HP of patients undergoing salvage treatment will transfer from the gastric antrum to the gastric body and gastric fundus, so samples from the gastric fundus and gastric body should be collected at the same time for the latter .

...

Embodiment 2

[0086] Embodiment 2PCR amplification:

[0087] ddH 2 O is used as negative control, and Helicobacter pylori standard strain 26695 and 43504 are used as positive control; PCR amplification reagent can use reagent TaKaRaTaq TM Version 2.0 (TaKaRa, Code No.R004A) (Takara), is a ready-to-use PCR premix solution, which can be amplified by simply adding primers and templates to simplify the experimental steps.

[0088] 1.PCR primer amplification:

[0089] Using TaKaRaTaq TM Version 2.0 (TaKaRa, Code No.R004A), cold start method, amplified according to the components shown in Table 5 and the procedures shown in Table 6. The products were numbered P1-10, and stored at low temperature.

[0090] table 5

[0091] Reagent Usage amount DNA template (<500ng / μl)

1μl F Primer (10μM) 1μl R Primer (10μM) 1μl Premix Taq TM

25μl Nuclease-Free Water 22μl Total 50μL

[0092] Table 6

[0093]

[0094] 2. Nested primer amplifica...

Embodiment 3

[0102] Embodiment 3 sequencing part:

[0103] 1. PCR amplification

[0104] Using corresponding primers, use Tianjin Qingke 2×TsingKE Master Mix (Code No.: TSE003) system for PCR amplification, and ddH 2 O was used as a negative control, amplification was performed with the reaction components shown in Table 10, and 2 μl was taken for 1% agarose gel electrophoresis.

[0105] Table 10

[0106]

[0107] 2.PCR product purification

[0108] 1) Balance and centrifuge the checked PCR samples (4000rpm), check the volume of each sample, and replenish water to 50 μl.

[0109] 2) According to the sample: 6×Loading buffer=5:1, add 10 μl of 6×Loading buffer, if the sample volume is 50-70 μl, add 5 μl of 6×Loading buffer, and centrifuge and mix at 4000 rpm.

[0110]3) Put the sample into the pre-prepared 1.2% purified gel. The sample pointing order is: 1-8 wells in the first row of the purified gel are placed into the first column of the sample, the 9th hole is a marker, and the 10-...

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Abstract

The invention relates to a diagnosing method and reagent kit for detecting and determining reverse tolerance of helicobacter pylori in gastric biopsy samples. The method can perform a downstream PCR reaction only by simply and directly performing complete genome extraction on biopsy samples of a patient. 23 pairs of primers of which the nucleotide sequences are as shown in figures 2 and 3 are usedfor joint detection of 101 mutation types of common mutation sites of 8 reverse tolerance genes of 23SrRNA,gyrA, PBP1, 16SrRNA, porD, oorD, rpoB and rdxA and two mutation types of a host drug metabolism enzyme CYP2C19 gene so as to perform fast detection and accurate analysis on mutation situation of reverse tolerance genes and drug metabolic enzyme genes of 7 antibiotics which are clinically andfrequently used at present through combination of a nested PCR technique with a sequencing technique, and the specificity and the accuracy are high. Compared with traditional minimal inhibitory concentration culture experiments, the diagnosing method is quicker and acuter in detection, is more suitable for clinical application, and has great clinical application prospects in the respects of guiding individualized diagnosis and treatment of patients being positive in helicobacter pylori.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a diagnostic method and a kit for detecting and judging drug resistance of Helicobacter pylori by using gastric biopsy tissue as a sample. Background technique [0002] Helicobacter pylori is a Gram-negative helical microaerophilic bacterium that colonizes the gastric epithelium in about 50-70% of the world's population. In developing countries, the infection rate has reached 90%, and there are certain geographical differences. Helicobacter pylori was first discovered in gastric biopsy by Marshall and Warren in the early 1980s, and is considered to be the main cause of chronic gastritis, peptic ulcer, and gastric cancer. Eradication therapy can reduce the spread of bacteria, prevent the development of gastric cancer, and reduce the risk of other related diseases. [0003] Because H. pylori localizes to the acidic surface of the gastric mucosa, an acid inhibitor (usually ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6848
CPCC12Q1/689C12Q1/6848C12Q2600/106C12Q2600/156
Inventor 郗日沫佟悦王倍王朔张瑞肖杨坤
Owner 深圳艾普斯金基因检测合伙企业(有限合伙)
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