Kit used for detecting helicobacter pylori drug resistance gene mutation via multiple fluorescent PCR

A Helicobacter pylori, multiple fluorescence technology, applied in the field of fluorescence quantitative PCR, can solve the problems of complicated operation of sequencing method, long detection period, complicated steps, etc., and achieve the effects of improving the success rate of treatment, reducing the cost of testing, and reducing the economic burden.

Inactive Publication Date: 2017-08-29
嘉兴雅康博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(1) PCR-sequencing method: This method is to sequence the PCR products after the PCR is completed, and judge the mutation status from the sequencing results. The sequencing method is complex in operation, long in the...

Method used

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  • Kit used for detecting helicobacter pylori drug resistance gene mutation via multiple fluorescent PCR
  • Kit used for detecting helicobacter pylori drug resistance gene mutation via multiple fluorescent PCR
  • Kit used for detecting helicobacter pylori drug resistance gene mutation via multiple fluorescent PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Reagent preparation

[0044] (1) Preparation of clarithromycin resistance reaction buffer

[0045] Take a 10mL volumetric flask and add 0.5mol / L Trizma ® HCl 80μL, 0.5mol / L Trizma ® Base 720μL, 100mmol / L MgCl 2 200 μL, 5mol / L KCl 160 μL, formamide 300 μL, 1mol / L ammonium sulfate 400 μL, 100 mmol / L dNTPs 90 μL, 100 μmol / L CLW upstream primer 33.3 μL, 100 μmol / L 2142, 2143, 2182 downstream primers each 33.3 µL, 10 µL of 100 µmol / L CLW probe, 20 µL of 100 µmol / L internal control upstream primer, 20 µL of 100 µmol / L internal control downstream primer, 5 µL of 100 µmol / L internal control probe. Make up the volume to 10mL with sterilized ultrapure water, mix well, dispense 1.5mL / tube into centrifuge tubes, and store at -20°C for later use.

[0046] (2) Preparation of Tetracycline Resistance Reaction Buffer

[0047] Take a 10mL volumetric flask and add 0.5mol / L Trizma ® HCl 80μL, 0.5mol / L Trizma ® Base 720μL, 100mmol / L MgCl 2 200 μL, 5mol / L KCl 160 μL, formamide...

Embodiment 2

[0075] 1. Reagent specificity verification

[0076] (1) Experimental samples

[0077] Six specific samples were taken to verify the specificity of the reagents, namely Escherichia coli, Campylobacter jejuni, Salmonella, Enterovirus 71, Coxsackievirus 16, and Rotavirus.

[0078] (2) Experimental process

[0079]The reagents described in this method were used to detect the above six specific samples, and the test results were analyzed to verify the specificity of the reagents.

[0080] (3) Experimental results

[0081] The test results of 6 specific samples were all negative, indicating that the reagent has good specificity and no cross-reaction. The specific results are shown in the table below:

[0082] Specific test results

[0083]

[0084] 2. Reagent precision verification

[0085] (1) Experimental samples

[0086] The precision of the reagents was verified by the HP drug-resistant positive control.

[0087] (2) Experimental process

[0088] Repeat the detection...

Embodiment 3

[0101] Embodiment 3: Detect the result of 60 routine clinical samples

[0102] 1. According to the preparation method shown in Example 1, the relevant components of the kit were prepared and stored at -20°C for later use.

[0103] 2. Obtained 60 cases of clinical gastric mucosal paraffin section samples from the Fuzhou General Hospital of Nanjing Military Region, and used the paraffin tissue nucleic acid extraction reagent (magnetic bead method) of Jiaxing Yakangbo Beinan Biotechnology Co., Ltd. to extract the genomic DNA of 60 cases of clinical samples. The purity of the DNA samples was tested by a photometer, and the OD260 / OD280 of 60 samples were all between 1.6 and 2.0.

[0104] 3. According to the steps shown in Example 1, add DNA samples and perform detection on a fluorescent quantitative PCR instrument. The instrument used this time is Bio-rad CFX96.

[0105] 4. According to the judgment standard shown in Example 1, the results are interpreted and counted, and the resu...

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Abstract

The invention provides a kit used for detecting helicobacter pylori drug resistance gene mutation via multiple fluorescent PCR. The kit can be used for detecting helicobacter pylori drug resistance gene mutation of gastric mucosa tissue samples, and determining whether drug resistance is generated. The kit comprises following drugs and drug resistance gene mutation sites: clarithromycin (2142A>G, 2143A>G, 2182C>T), tetracycline (926-928AGA>TTC), quinolones (Asn87-Lys, Ala88-Val, Asp91-Gly), and amoxicillin (S414R, Y484C, and P600T). The kit can be used for obtaining accurate results in a short time, and is suitable for guidance of personalized medicine of helicobacter pylori in clinical treatment.

Description

technical field [0001] The invention belongs to the field of fluorescence quantitative PCR, and in particular relates to a kit for detecting mutations of drug-resistant genes of Helicobacter pylori by multiple fluorescence PCR. Background technique [0002] Helicobacter pylori (HP) infection is a global problem, affecting 50% of the world's population. It is closely related to the occurrence and development of digestive tract diseases such as chronic gastritis, peptic ulcer and gastric adenocarcinoma, and the eradication of HP is an important link in the prevention and treatment of the above diseases. In many consensus opinions on HP treatment in China, Europe, etc., triple therapy containing PPI and two antibiotics (two of amoxicillin, clarithromycin, and metronidazole) is recommended as the first-line regimen for the eradication of HP . With the popularization and promotion of HP eradication therapy, more and more patients cannot eradicate HP after receiving the first tr...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/01
CPCC12Q1/6858C12Q1/689C12Q2600/106C12Q2600/156C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2545/101
Inventor 陈智林童超魏莎莎高幼冷
Owner 嘉兴雅康博生物技术有限公司
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