WO2021239120A1 - Trousse de réactifs et procédé de détection rapide de la résistance aux médicaments d'helicobacter pylori - Google Patents

Trousse de réactifs et procédé de détection rapide de la résistance aux médicaments d'helicobacter pylori Download PDF

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Publication number
WO2021239120A1
WO2021239120A1 PCT/CN2021/096842 CN2021096842W WO2021239120A1 WO 2021239120 A1 WO2021239120 A1 WO 2021239120A1 CN 2021096842 W CN2021096842 W CN 2021096842W WO 2021239120 A1 WO2021239120 A1 WO 2021239120A1
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helicobacter pylori
ratio
drug resistance
incubation
kit
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PCT/CN2021/096842
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English (en)
Chinese (zh)
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徐健
刘敏
张丽丽
付晓婷
朱鹏飞
籍月彤
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中国科学院青岛生物能源与过程研究所
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Publication of WO2021239120A1 publication Critical patent/WO2021239120A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)

Definitions

  • the invention relates to the detection of bacterial drug resistance, in particular to a kit and method for rapidly detecting the drug resistance of Helicobacter pylori.
  • Hp Helicobacter pylori
  • PPI proton pump inhibitor
  • the detection technology of Helicobacter pylori antibiotic resistance can be divided into two categories: phenotypic method and genotyping method.
  • the drug susceptibility test of Hp can use a variety of phenotypic methods, including agar dilution method, breakpoint susceptibility test, Disk diffusion test and E-test etc.
  • the standard susceptibility test is considered to be the "gold standard" for confirming the resistance of Helicobacter pylori.
  • current diagnostic methods require sample culture to detect and identify bacteria and their susceptibility to antibiotics. This is a slow process that may take several days even in the most advanced laboratories.
  • the present invention applies heavy water single-cell Raman spectroscopy technology to the detection of Helicobacter pylori drug resistance for the first time, and provides a kit and detection method for rapid detection of Hp drug resistance.
  • the present invention first provides a kit for detecting the drug resistance of Helicobacter pylori, which is characterized in that the kit includes an incubation solution, a fixing solution I and a fixing solution II; the incubation solution Contains heavy water, the fixative I contains physiological saline, and the fixative II contains absolute ethanol.
  • the physiological saline is 0.9% sodium chloride solution.
  • the concentration of the heavy water is 100%.
  • the kit also includes a culture medium.
  • the culture medium is Brain Heart Infusion Medium (BHI), and preferably, fetal bovine serum is further added, and more preferably, 10% fetal bovine serum is added.
  • BHI Brain Heart Infusion Medium
  • the kit also contains a bacteria-enhancing agent, preferably, the bacteria-enhancing agent is BBL IsoVitaleX TM .
  • the kit also contains pure water.
  • the kit further includes a centrifuge tube and a fixed tube.
  • Another aspect of the present invention provides the use of the kit for detecting the drug resistance of Helicobacter pylori.
  • Another aspect of the present invention provides a method for detecting the drug resistance of Helicobacter pylori using the kit, the method comprising the following steps:
  • CD ratio CD ratio of the experimental group minus the CD ratio of the control group
  • CD ratio CD ratio
  • ⁇ CD ratio CD ratio
  • CD ratio refers to CD peak area/(CD peak area + CH peak area
  • the ⁇ C-D ratio ( ⁇ C-D ratio) is compared with the critical value, which is greater than the critical value for drug-resistant strains and less than the critical value.
  • the value is a sensitive strain, and the critical value is -0.0075.
  • the drugs are antibiotic drugs.
  • the method includes the following steps:
  • Raman detection is performed on the samples of the experimental group and the control group, and the CD ratios of the experimental group and the control group are calculated according to the obtained Raman maps.
  • the CD ratio of the experimental group minus the CD ratio of the control group is the ⁇ CD ratio.
  • the ratio refers to CD peak area/(CD peak area + CH peak area);
  • the ⁇ C-D ratio is compared with the cut-off value.
  • the ratio is greater than the cut-off value for resistant strains and less than the cut-off value for sensitive strains, so The critical value is -0.0075.
  • the different concentrations span the two breakpoints of Helicobacter pylori in CLSI regulations, namely the sensitive (S) and drug-resistant (R) concentrations.
  • CLSI refers to the implementation standard of antimicrobial drug susceptibility testing. At present, clinical microbiology laboratories in my country use this standard as a drug susceptibility guidance document for test operations and reports.
  • the antibiotic drugs include but are not limited to Levofloxacin, Clarithromycin, Metronidazole, Amoxicillin, Tetracycline or Furazolidone ).
  • the antibiotic drug is Levofloxacin.
  • the incubation time is 0.5h-2h, and the continued incubation time is 1h-6h.
  • the incubation time is 8h.
  • the medium is a BHI medium supplemented with 10% serum, and preferably, 1% IsoVitaleX TM is further added.
  • the ratio of the volume of the incubation solution to the total volume of the incubation system is 30-100%.
  • the ratio of the volume of the incubation solution to the total volume of the incubation system is 50%.
  • the formula of the incubation system is:
  • the formulations of the fixative liquid I and the fixative liquid II are:
  • the parameters of the Raman detection are: objective lens 100 ⁇ , pinhole diameter 125 ⁇ m, grating 300 g/mm, exposure time 6 s, laser wavelength 532 nm, spectral range 400-3500 cm -1 .
  • the CD peak and the CH peak are located at 2050-2300 cm -1 and 2800-3050 cm -1, respectively .
  • the present invention has the following beneficial effects:
  • the present invention combines single-cell Raman spectroscopy and stable isotope-labeled single-cell Raman detection technology, which can realize rapid detection of Helicobacter pylori resistance in a short time;
  • the detection method of the present invention has high sensitivity and high accuracy
  • Figure 1 Changes in the Raman profile of Helicobacter pylori ATCC26695 single cells in heavy water media with different concentrations.
  • Figure 2 The relationship between the proportion of heavy water peaks (C-D ratio) and incubation time in the Raman spectrum of Helicobacter pylori ATCC26695 single cells in heavy water media with different concentrations.
  • Figure 3 The effect of different concentrations of levofloxacin on the ⁇ C-D ratio of Helicobacter pylori ATCC26695 and clinical isolates.
  • Figure 4 The ⁇ C-D ratio of Helicobacter pylori ATCC26695 and clinical isolates after 8 hours of clarithromycin treatment.
  • Raman measurement is performed under a 100x objective lens.
  • the detection parameters are: pinhole diameter 125 ⁇ m, grating 300g/mm, exposure time 6s, laser wavelength 532nm, spectral range 400 ⁇ 3500cm -1 .
  • the Raman spectra of about 20 bacterial single cells were randomly measured and collected in different fields of view.
  • CD ratio CD/(C-D+CH).
  • the incubation solution is 100% heavy water
  • the fixative I is 0.9% sodium chloride solution
  • the fixation II is absolute ethanol
  • ultrapure water medium
  • BBL IsoVitaleX TM You can prepare by yourself.
  • the drug resistance of H. pylori ATCC26695 and H. pylori clinical isolates are detected.
  • the corresponding test drug (antibiotic) is levofloxacin (Levofloxacin, Levo) ,
  • the specific drug resistance detection steps are as follows:
  • the frozen H. pylori ATCC26695 and Hp clinical isolates were streaked on the surface of the BHI plate and cultured at 37°C under microaerobic conditions (N 2 85%, O 2 5%, CO 2 10%).
  • the experimental group with different antibiotic concentrations refers to the experimental group with different concentrations of antibiotics added to the heavy water medium, and the negative control group refers to the experimental group without antibiotics.
  • the concentration of levofloxacin is 100 ⁇ g/mL.
  • Freeze to be tested If the test solution cannot be detected immediately, take 1.0 mL of the test solution, centrifuge at 8000 rpm for 2 min, remove the supernatant, resuspend in the prepared fixative solution and store in liquid nitrogen.
  • Raman measurement is performed under a 100-fold objective lens.
  • the detection parameters are: pinhole diameter 125 ⁇ m, grating 300g/mm, exposure time 6s, laser wavelength 532nm, spectral range 400 ⁇ 3500cm -1 .
  • the Raman spectra of about 20 bacterial single cells were randomly measured and collected in different fields of view.
  • the collected Raman spectra are processed with the software LabSpec5 for background removal, baseline normalization and maximum normalization of the Raman spectra.
  • CD peak and CH peak located at 2050-2300cm -1 and 2800-3050cm -1 respectively , calculate the CD ratio (CD ratio) of the experimental group and the control group, namely CD/(C-D+CH) , So as to calculate the ⁇ C-D ratio.
  • Figure 3 shows the effect of different concentrations of levofloxacin on Helicobacter pylori ATCC26695 and the ⁇ C-D ratio of clinical isolates.
  • Example 3 The rapid detection kit for Helicobacter pylori resistance to detect the resistance of H. pylori ATCC26695 and H. pylori clinical isolates to clarithromycin
  • the heavy water delay protocol is more optimally applied to the detection of H. pylori ATCC26695 and H. pylori clinical isolate clarithromycin (Clarithromycin, Clr) resistance ,
  • the specific detection steps are as follows:
  • the concentration of clarithromycin is 10 ⁇ g/mL.
  • Freeze to be tested If the test solution cannot be detected immediately, take 1.0 mL of the test solution, centrifuge at 8000 rpm for 2 min, remove the supernatant, resuspend in the prepared fixative solution and store in liquid nitrogen.
  • Raman measurement is performed under a 100-fold objective lens.
  • the detection parameters are: pinhole diameter 125 ⁇ m, grating 300g/mm, exposure time 6s, laser wavelength 532nm, spectral range 400 ⁇ 3500cm -1 .
  • the Raman spectra of about 20 bacterial single cells were randomly measured and collected in different fields of view.
  • the collected Raman spectra are processed with the software LabSpec5 for background removal, baseline normalization and maximum standardization of the Raman spectra.
  • CD peak and CH peak located at 2050-2300cm-1 and 2800-3050cm-1, respectively, calculate the CD ratio (CD ratio) of the experimental group and the control group, namely CD/(C-D+CH) , So as to calculate the ⁇ C-D ratio.
  • Figure 4 shows the ⁇ C-D ratio of Helicobacter pylori ATCC26695 and clinical isolates after 8 hours of treatment with clarithromycin.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

Kit de réactifs et procédé pour la détection rapide de la résistance aux médicaments de Helicobacter pylori comportant les étapes suivantes : mise en place de groupes expérimentaux à différentes concentrations d'antibiotiques et d'un groupe témoin négatif, les groupes expérimentaux à différentes concentrations d'antibiotiques se référant à des groupes expérimentaux dans lesquels différentes concentrations d'antibiotiques sont ajoutées à un milieu de culture d'eau lourde, et le groupe témoin négatif se référant à un groupe expérimental sans antibiotiques ; après incubation et centrifugation, lavage et réalisation d'une détection Raman sur un échantillon ; et, sur la base du spectre Raman obtenu, calcul du rapport C-D tel que C-D/(C-D+C-H), le C-D des groupes expérimentaux à différentes concentrations d'antibiotiques moins le rapport C-D du groupe témoin étant le rapport △C-D, et comparaison de celui-ci avec une valeur critique (-0. 0075) pour déterminer la sensibilité et la résistance aux médicaments de Helicobacter pylori. Un procédé de culture liquide pour l'incubation de Helicobacter pylori est présenté, et une technique Raman à eau lourde est appliquée à la détection de la résistance aux médicaments de Helicobacter pylori, permettant une détermination plus précise et rapide de la sensibilité de Helicobacter pylori aux antibiotiques, et ayant une grande importance pour guider la médication et le traitement des patients atteints de Helicobacter pylori.
PCT/CN2021/096842 2020-05-28 2021-05-28 Trousse de réactifs et procédé de détection rapide de la résistance aux médicaments d'helicobacter pylori WO2021239120A1 (fr)

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CN116377096A (zh) * 2023-03-08 2023-07-04 上海粒盛生物科技有限公司 幽门螺旋杆菌克拉霉素耐药性分析试剂盒及检测方法
CN116656574A (zh) * 2023-07-17 2023-08-29 南京松天盛科生物科技有限公司 一种幽门螺杆菌液体培养方法及其作为抗原的制备

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Publication number Priority date Publication date Assignee Title
CN115807051A (zh) * 2022-11-30 2023-03-17 右江民族医学院 检测幽门螺杆菌耐药性的培养基及其制备方法和应用
CN115807051B (zh) * 2022-11-30 2024-01-16 右江民族医学院 检测幽门螺杆菌耐药性的培养基及其制备方法和应用
CN116377096A (zh) * 2023-03-08 2023-07-04 上海粒盛生物科技有限公司 幽门螺旋杆菌克拉霉素耐药性分析试剂盒及检测方法
CN116377096B (zh) * 2023-03-08 2024-04-26 上海粒盛生物科技有限公司 幽门螺旋杆菌克拉霉素耐药性分析试剂盒及检测方法
CN116656574A (zh) * 2023-07-17 2023-08-29 南京松天盛科生物科技有限公司 一种幽门螺杆菌液体培养方法及其作为抗原的制备
CN116656574B (zh) * 2023-07-17 2024-02-06 南京松天盛科生物科技有限公司 一种幽门螺杆菌液体培养方法及其作为抗原的制备

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