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Method and kit for typing detection of drug resistance of helicobacter pylori in oral cavity

A technology for Helicobacter pylori and drug resistance, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., can solve problems such as poor patient compliance, slow growth, and low success rate of culture

Pending Publication Date: 2021-05-07
深圳艾普斯金基因检测合伙企业(有限合伙)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are single or multi-antibiotic resistance detection methods, but the commonly used clinical drug sensitivity test is still to obtain gastric biopsy tissue invasively, isolate and culture Hp, because of the uneven distribution of Hp in the stomach, the patient's compliance is poor, thus Lead to less Hp content and lower success rate of culture
And because of the harsh conditions of Hp culture, slow growth, long culture period and other factors, it is not suitable for rapid diagnosis of clinical drug resistance and immediate medication guidance

Method used

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  • Method and kit for typing detection of drug resistance of helicobacter pylori in oral cavity
  • Method and kit for typing detection of drug resistance of helicobacter pylori in oral cavity
  • Method and kit for typing detection of drug resistance of helicobacter pylori in oral cavity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Extraction process of whole genome of Helicobacter pylori in saliva and dental plaque specimens in oral cavity

[0057] 1. Sampling requirements:

[0058] 1) Confirm that the subject 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).

[0059] 2) The specimens come from oral saliva and dental plaque. Specimens are collected regularly, and the simple operation is as follows: Under the basic state, the subject sits quietly wearing sterile gloves in the morning on an empty stomach (fasting except for drinking water) before brushing his teeth, and collects the saliva in the mouth by swallowing and then flowing out naturally Take 1mL saliva sample, and take a buccal swab and scrape it repeatedly 6 times on both sides of the inner wall of the cheek, all collected in a sterile EP tube, an...

Embodiment 2

[0065] Embodiment 2 PCR amplification process:

[0066] ddH 2 O as a negative control, with Helicobacter pylori standard strain ATCC700392 TM , ATCC43504 TM As a positive control; PCR amplification reagents can use reagents 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.

[0067] 1.PCR primer amplification:

[0068] Using TaKaRaTaq TM Version 2.0 (TaKaRa, Code No.R004A), cold start method, amplified according to the components and procedures shown in Table 5. Store the product at low temperature.

[0069] Table 4

[0070]

[0071]

[0072] table 5

[0073]

[0074] 2. Nested PCR primer amplification:

[0075] Using TaKaRaTaqTM Version 2.0 (TaKaRa, Code No. R004A), cold start method, according to the components shown in Table 6 and the procedures shown in Table 7, the amplification was carried out. The product is ...

Embodiment 3

[0080] Embodiment 3 sequencing part:

[0081] 1. DNA agarose gel cutting purification

[0082] According to the electrophoresis results of the PCR products, the desired DNA target bands are cut, and the purification method is shown in the SK8131 gel recovery manual.

[0083] 2. PCR sequencing reaction

[0084] (1) Take a 0.2ml PCR tube, number it and insert it into granular ice, and add the reagents in the following table:

[0085] Table 8

[0086]

[0087] Do not add light mineral oil or paraffin oil, cap the PCR tube tightly, flick the tube with your fingers to mix, and centrifuge slightly.

[0088] (2) Place the PCR tube on the BBI PCR instrument for amplification. After denaturation at 98°C for 2 minutes, PCR cycle was performed. The PCR cycle parameters were 96°C for 10s, 50°C for 5s, and 60°C for 4min, 25 cycles. After the amplification was completed, set 4°C to keep warm.

[0089] 3. Purification of PCR products by sodium acetate / ethanol method

[0090] (1) Cen...

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Abstract

Because gastric biopsy samples are difficult in endoscope sampling and high in cost, helicobacter pylori (Hp) is hard to culture under harsh growth conditions and an invasive diagnosis method of the drug resistance of the Hp is not ideal, the invention provides a molecular diagnosis method for detecting drug resistance of non-invasive helicobacter pylori. An excrement sample is quickly detected and analyzed by combining a nested PCR technology and a sequencing technology; mutation of 52 sites of seven drug-resistant genes, such as 23SrRNA, gyrA, PBP1, 16SrRNA, porD, oorD and rpoB, and random mutation of an rdxA drug-resistant gene are jointly detected by utilizing primers with nucleotide sequences shown in a table 2 and a table 3; and thus,, the drug resistance conditions of seven antibiotics commonly used in clinic at present are judged. The invention belongs to the technical field of biology, is rapid and sensitive, is suitable for clinical application, and has a great application prospect in the aspect of guiding personalized diagnosis, treatment and eradication of helicobacter pylori patients.

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 drug resistance of Helicobacter pylori in oral cavity. Background technique [0002] Helicobacter pylori (H. pylori, Hp) is a Gram-negative spiral microaerophilic bacterium whose host is mainly human beings. Human gastric pylori is a specific settlement site for H. pylori, which is in the gastric epithelium of about 50-70% of the world's population Colonization, the infection rate in developing countries has reached 90%, and there are certain geographical differences. Hp is also the only known microorganism that can survive in the human stomach, and is considered to be the main cause of chronic gastritis, peptic ulcer, and gastric cancer. [0003] In 1983, Hp was found in gastric biopsy by Marshall and Warren and successfully cultured. In 1989, Krajedn successfully isolated Hp for the first time from oral saliva and dental pla...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6858
Inventor 郗日沫佟悦王倍王朔甘奇张瑞肖
Owner 深圳艾普斯金基因检测合伙企业(有限合伙)
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