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Gastric juice multiple real-time polymerase chain reaction (PCR) detection-based helicobacter pylori (HP) individualized treatment auxiliary diagnosis method

A technology of Helicobacter pylori, detection method, applied in the field of molecular biology

Inactive Publication Date: 2014-07-09
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional culture and drug susceptibility testing, as well as other rapid detection methods, cannot meet this requirement

Method used

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  • Gastric juice multiple real-time polymerase chain reaction (PCR) detection-based helicobacter pylori (HP) individualized treatment auxiliary diagnosis method
  • Gastric juice multiple real-time polymerase chain reaction (PCR) detection-based helicobacter pylori (HP) individualized treatment auxiliary diagnosis method
  • Gastric juice multiple real-time polymerase chain reaction (PCR) detection-based helicobacter pylori (HP) individualized treatment auxiliary diagnosis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Pretreatment of gastric juice specimen and extraction of nucleic acid

[0043] 1. Pretreatment of gastric juice specimens

[0044] Take 10μl-500μl of gastric juice and add it to a 1.5ml centrifuge tube, add an equal amount of Tris buffer (0.67M, pH7.4), oscillate evenly, and place at room temperature for 2h. Centrifuge at 13000rpm for 5min, discard the supernatant and save the precipitate.

[0045] 2. The specific operation steps of nucleic acid extraction were described and improved according to QIAGEN DNA Extraction Kit 51306. The specific method is as follows:

[0046] ①. Add 180 μl ATL and 20 μl proteinase K to the pellet, shake and mix well. Place the centrifuge tube in a metal bath or air shaker and shake at 56°C for 2h.

[0047] ②. Take out the centrifuge tube from the metal bath or air shaker, and perform short centrifugation. Add 200 μl AL to the centrifuge tube and shake to mix.

[0048] ③. Place the centrifuge tube in a metal bath, shake at 70...

Embodiment 2

[0057] Embodiment 2 multiplex real-time PCR system and optimization of reaction conditions

[0058] In the present invention, the Mg in the system 2+ Concentration, annealing temperature and probe combination were optimized and verified, and the optimal reaction conditions were obtained as follows: 95°C for 10 min; 95°C for 10 s, 58°C for 45 s, 45 cycles, and plate reading. The best Mg in multiplex PCR system 2+ The concentration was 2.0 mM. The optimal multiple probe combination is as follows: ① cagH probe + RnaseP probe; ② HP23S rDNA wild-type probe + A2143G probe + A2142G probe; ③ CYP2C192*G probe + CYP2C192*A probe; ④ CYP2C193*G probe + CYP2C193 *A probe. Through the above experiments, a multiple real-time PCR method was successfully constructed, which can realize the three purposes of HP infection diagnosis, HP clarithromycin resistance evaluation, and CYP2C19 allele analysis through four reaction systems in one real-time PCR test.

Embodiment 3

[0059] The optimization of the initial amount of gastric juice of embodiment 3

[0060] Vortex and oscillate the melted gastric juice sample until uniform, take 500 μl, 100 μl, and 10 μl gastric juice samples respectively, and extract nucleic acid according to the operation in Example 1. Take 2 μl and add it to each tube of PCR system. Combination ① 106 randomly selected gastric juice samples (including 99 rapid urease positive samples and 7 negative samples) were tested, and the results are shown in Tables 2, 3, and 4. Positive predictive value (PPV) refers to the probability of a true case among those with a positive test result. Negative predictive value (NPV) refers to the probability of not having the disease if the test result is negative.

[0061] Table 2 Multiple real-time PCR detection results when the initial gastric juice volume is 500 μl

[0062]

[0063] PPV=94.29%, NPV=100%

[0064] Table 3 Multiple real-time PCR detection results when the initial gastric ...

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Abstract

The invention discloses a gastric juice multiple real-time polymerase chain reaction (PCR) detection-based helicobacter pylori (HP) individualized treatment auxiliary diagnosis method. According to the method, specific genes of HP, HP clarithromycin drug-resistant mutation sites A2143G and A2142G, and polymorphism of CYP2C19 gene sites such as CYP2C19*2(G681A) and CYP2C19*3(G636A) of corresponding patients can be simultaneously detected from 10-150mu l of gastric juice samples, and human epithelial cell ribonuclease P(Rnase P) is taken as a reference gene. The detection method is rapid, accurate and small in sample demand amount, whether HP infection exists can be detected from the 10-150mu l of gastric juice samples in a single test, the HP clarithromycin drug-resistant conditions and genotypes of CYP2C19 of the patients can be reported, and medication guide is provided for individualized eradication therapy of HP infection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a multiple real-time (Real-Time) PCR detection method for individualized treatment-assisted diagnosis of Helicobacter pylori (H. pylori or HP) infection based on gastric juice. Background technique [0002] HP can cause chronic gastritis, gastric ulcer and other digestive tract-related diseases, and is the only type I carcinogen among prokaryotes specified by the WHO Cancer Society. Triple therapy or quadruple therapy using proton pump inhibitor (PPI) combined with antibiotics such as amoxicillin and clarithromycin (or metronidazole) is a recognized first-line anti-HP infection treatment combination at home and abroad. However, the success rate of this therapy is gradually declining due to the rising rate of clarithromycin resistance. Another factor affecting the success rate of this triple drug combination is the polymorphism of the human cytochrome P450 isoenzyme C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/686C12Q2537/143C12Q2561/101
Inventor 张建中彭贤惠刘杰何利华赵飞闫笑梅张茂俊张慧芳
Owner ICDC CHINA CDC
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