WO2021239120A1 - Reagent kit and method for rapid detection of drug resistance of helicobacter pylori - Google Patents

Abstract

A reagent kit and a method for the rapid detection of the drug resistance of Helicobacter pylori: setting up different antibiotic concentration experimental groups and a negative control group, the different antibiotic concentration experimental groups referring to experimental groups in which different concentrations of antibiotics are added to a heavy water culture medium, and the negative control group referring to an experimental group without antibiotics; after incubation and centrifugation, washing and performing Raman detection on a sample; and, on the basis of the obtained Raman spectrum, calculating the C-D ratio as C-D/(C-D+C-H), the C-D of the different antibiotic concentration experimental groups minus the C-D ratio of the control group being the △C-D ratio, and comparing same with a critical value (-0.0075) to determine the sensitivity and drug resistance of Helicobacter pylori. A liquid culture method of incubating Helicobacter pylori is introduced, and a heavy water Raman technique is applied to the detection of Helicobacter pylori drug resistance, enabling more accurate and rapid determination of the sensitivity of Helicobacter pylori to antibiotics, and having great significance for guiding the medication and treatment of patients with Helicobacter pylori.

Description

A kit and method for rapidly detecting the drug resistance of Helicobacter pylori Technical field

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.

Background technique

Helicobacter pylori (Hp) is a Gram-negative Campylobacter, mainly distributed in gastric mucosal tissue. Helicobacter pylori infection is one of the common chronic bacterial infections in humans and the main cause of many chronic gastric diseases, affecting nearly half of the world's population. Currently, domestic antibiotics recommended for Hp eradication treatment include: clarithromycin, metronidazole, levofloxacin, amoxicillin, furazolidone and tetracycline. The first choice to eradicate Hp infection includes triple therapy of proton pump inhibitor (PPI), amoxicillin, clarithromycin or metronidazole, and has been agreed at international conferences. However, the increasing antibiotic resistance and unsuccessful treatment attempts in the past hindered the success of eradication of Hp and contributed to the emergence and spread of drug-resistant strains. In some geographical areas, the resistance of Helicobacter pylori has risen to worrying levels, and clarithromycin-resistant Helicobacter pylori has been included in the World Health Organization (WHO) high priority pathogen list. With the rapid spread of microbial drug resistance, patients with signs of drug resistance should be tested for drug resistance before treatment. Therefore, rapid, sensitive, and reliable rapid diagnosis methods are required, and antibiotics are used cautiously to slow down the development of antimicrobial resistance. Spread quickly.

The detection technology of Helicobacter pylori antibiotic resistance can be divided into two categories: phenotypic method and genotyping method. At present, 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. In addition, the standard susceptibility test is considered to be the "gold standard" for confirming the resistance of Helicobacter pylori. However, 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.

Summary of the invention

In view of this, 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.

In order to solve the above technical problems, 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.

The kit also contains a bacteria-enhancing agent, preferably, the bacteria-enhancing agent is BBL IsoVitaleX ^™ .

The kit also contains pure water.

In another preferred embodiment, 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:

a)(a1) Add the same initial concentration of Helicobacter pylori to the incubation system containing the medium and drug-resistant (R) concentration as the experimental group, and set up a control group without drugs, incubate, and then incubate it Add incubation solution to the system and continue incubation; (a2) Obtain the test solution after incubation; take out part of the test solution and directly perform Raman single-cell detection of drug resistance, or mix the test solution with Fixative I and Fixative II. Store in liquid nitrogen, and then take it out for Raman single cell detection for drug resistance when it is needed for testing;

b) Carry out Raman detection on the samples of the experimental group and the control group, and calculate the CD ratio (CD ratio) of the experimental group and the control group respectively according to the obtained Raman spectrum. The CD ratio of the experimental group (CD ratio) minus the CD ratio of the control group ( CD ratio) is △CD ratio (△CD ratio), and the CD ratio (CD ratio) refers to CD peak area/(CD peak area + CH peak area);

c) According to the breakpoint given in the CLSI drug susceptibility test standard, at the drug resistance (R) concentration, the ΔC-D ratio (ΔC-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.

In another preferred embodiment, the method includes the following steps:

a) Add the same initial concentration of Helicobacter pylori to an incubation system containing incubation medium, culture medium and different concentrations of drugs as the experimental group, and set up a control group without drugs, and obtain the test solution after incubation; take out a part to be tested The test solution is directly subjected to Raman single cell detection for drug resistance, or the test solution is mixed with Fixative I and Fixative II and placed in liquid nitrogen for storage. Wait for the test to be taken out for the Raman single cell test for drug resistance;

b) 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);

c) According to the breakpoint given in the CLSI drug susceptibility test standard, at the drug resistance (R) concentration, the ΔC-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.

In the present invention, 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.

In another preferred embodiment, the antibiotic drugs include but are not limited to Levofloxacin, Clarithromycin, Metronidazole, Amoxicillin, Tetracycline or Furazolidone ). Preferably, the antibiotic drug is Levofloxacin.

In another preferred embodiment, the incubation time is 0.5h-2h, and the continued incubation time is 1h-6h.

In another preferred embodiment, the incubation time is 8h.

In another preferred example, the medium is a BHI medium supplemented with 10% serum, and preferably, 1% IsoVitaleX ^{TM is} further added.

In another preferred embodiment, the ratio of the volume of the incubation solution to the total volume of the incubation system is 30-100%.

In another preferred embodiment, the ratio of the volume of the incubation solution to the total volume of the incubation system is 50%.

In another preferred embodiment, the formula of the incubation system is:

In another preferred embodiment, the formulations of the fixative liquid I and the fixative liquid II are:

In another preferred example, the parameters of the Raman detection are: objective lens 100×, pinhole diameter 125 μm, grating 300 g/mm, exposure time 6 s, laser wavelength 532 nm, spectral range 400-3500 cm ^-1 .

In another preferred example, the CD peak and the CH peak are located at 2050-2300 cm ^-1 and 2800-3050 cm ^{-1, respectively} .

In the present invention, C-D ratio (C-D ratio)=C-D/(C-D+C-H) means C-D peak area/(C-D peak area+C-H peak area).

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.

Compared with the prior art, the present invention has the following beneficial effects:

(1) 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;

(2) The detection method of the present invention has high sensitivity and high accuracy;

(3) The detection method of the present invention is simple to operate.

Description of the drawings

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 ΔC-D ratio of Helicobacter pylori ATCC26695 and clinical isolates.

Figure 4: The ΔC-D ratio of Helicobacter pylori ATCC26695 and clinical isolates after 8 hours of clarithromycin treatment.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work should fall within the protection scope of this application. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions are not specified in the following examples, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Example 1 Research on the universality of heavy water Raman technique strains

1. Prepare the experimental strain

Streak the frozen H. pylori ATCC26695 on the surface of the BHI plate (added with 7% sterile defibrinated horse blood) at 37°C under microaerobic conditions (N ₂ 85%, O ₂ 5%, CO ₂ 10%) Culture; scrape the bacteria on the plate in BHI liquid medium (add 10% FBS serum) and quantify to OD = 0.5.

2. Incubation of experimental strains under different heavy water concentration conditions

Prepare BHI liquid medium with different heavy water concentrations (0%, 30%, 50%, 100%), and inoculate the above bacteria in heavy water medium at a volume ratio of 1:10, at 0h, 2h, 4h, 8h, 24h, 48h sampling.

3. Raman detection

① Preparation: Take 1.0 mL of the test solution in the incubation system in a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 2 minutes, and remove the supernatant; _{resuspend in 500 μL ddH 2} O, continue centrifugation to remove the supernatant, repeat washing 2-3 times, and finally resuspended with ddH ₂ O to a final concentration of 10 ⁶ / mL; take 10μL dilution spotted onto a clean detecting chip (CaF ₂ slides), each sample was spotted three parallel, natural biological safety cabinet Air dry, to be tested.

②Detection: Raman measurement is performed under a 100x objective lens. The detection parameters are: pinhole diameter 125μm, 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.

4. Data processing and result analysis

After all the single-cell Raman spectra obtained were pre-processed (background reduction, baseline normalization and maximum normalization processing), the heavy water peak (CD peak) (2050-2300cm ^-1 area in the Raman spectra) and CH Peak (2800-3050cm ^{-1 in the} Raman spectrum), calculate the CD ratio (CD ratio) of different heavy water concentrations, namely CD/(C-D+CH).

The results showed that the Raman spectra of the strains were measured after being cultured in different heavy water concentration media to the stable period. Compared with the control group (without heavy water medium), the single-cell Raman spectra of the strains cultured in heavy water medium were in 2050. There ^{will be a clear heavy water peak in the area of 2300cm -1} (Figure 1). After adding heavy water medium, the CD ratio increases with the extension of the incubation time, and finally stabilizes gradually at about 8h (Figure 2).

Example 2 Detecting the drug resistance of H. pylori ATCC26695 and H. pylori clinical isolates with a rapid test kit for drug resistance of Helicobacter pylori

1. Kit information

An example of the components of a kit, 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 and BBL IsoVitaleX ^TM You can prepare by yourself.

Table 1. Composition of the kit

Table 2. The preparation of the incubation system

Table 3. Preparation of fixative

2. Rapid detection of drug resistance of H. pylori ATCC26695 and Hp clinical isolates

Using the kit of the present invention, combined with the heavy water incubation concentration and sampling time determined in Example 1, 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:

(1) Prepare the Helicobacter pylori strain

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%).

(2) Establish an incubation system

Establish an incubation system for the control group and the experimental group according to Table 4. 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.

Table 4. Co-incubation system of bacteria, heavy water and drugs

Note: The concentration of levofloxacin is 100μg/mL.

(3) Quick detection

①Sampling and processing

Instant detection: Take 1.0 mL of the test solution in the incubation system in a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 2 minutes, and remove the supernatant; _{resuspend in 500 μL ddH 2} O, continue to centrifuge to remove the supernatant, repeat washing 2-3 times, and finally use Resuspend ddH ₂ O to a final concentration of 10 ⁶ /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.

②Producing

Take 10 μL of the diluted solution and spot it on a clean detection chip (calcium fluoride glass slide), and perform three parallel spotting on each sample, and air dry in a biological safety cabinet for testing.

(4) Raman detection

Raman measurement is performed under a 100-fold objective lens. The detection parameters are: pinhole diameter 125μm, 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.

(5) Data processing and result analysis

During data processing, the collected Raman spectra are processed with the software LabSpec5 for background removal, baseline normalization and maximum normalization of the Raman spectra. According to the obtained Raman spectrum analysis 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 ΔC-D ratio. Figure 3 shows the effect of different concentrations of levofloxacin on Helicobacter pylori ATCC26695 and the ΔC-D ratio of clinical isolates.

The results showed that at a drug resistance concentration of 2μg/ml, 8h sampling and data processing to obtain H. pylori ATCC26695 and Hp isolates A004 and A105 ΔC-D ratio (ΔC-D ratio) values were -0.0283, -0.0076, 0.0002, respectively , Compared with the cutoff value of -0.0075 to determine that only A105 is a drug-resistant strain, which is consistent with the E-test result.

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

Using the kit of the present invention, combined with the drug susceptibility detection process determined in Example 2, 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:

(1) Prepare the Helicobacter pylori strain

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%). (Isolated strain reference: analysis of multi-drug resistance and mutation characteristics of Helicobacter pylori in Qingdao area)

(2) Establish an incubation system

Use the heavy water delay plan to establish the control group and the experimental group incubation system according to Table 5.

Table 5. Co-incubation system of bacteria, heavy water and drugs

Remarks: The concentration of clarithromycin is 10μg/mL.

(3) Quick detection

①Sampling and processing

Instant detection: Take 1.0 mL of the test solution in the incubation system in a 1.5 mL centrifuge tube, centrifuge at 8000 rpm for 2 minutes, and remove the supernatant; _{resuspend in 500 μL ddH 2} O, continue to centrifuge to remove the supernatant, repeat washing 2-3 times, and finally use Resuspend ddH ₂ O to a final concentration of 10 ⁶ CFU/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.

②Producing

Take 10 μL of the diluted solution and spot it on a clean detection chip (calcium fluoride glass slide), and perform three parallel spotting on each sample, and air dry in a biological safety cabinet for testing.

(4) Raman detection

Raman measurement is performed under a 100-fold objective lens. The detection parameters are: pinhole diameter 125μm, 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.

(5) Data processing and result analysis

During data processing, the collected Raman spectra are processed with the software LabSpec5 for background removal, baseline normalization and maximum standardization of the Raman spectra. According to the obtained Raman spectrum analysis 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 ΔC-D ratio. Figure 4 shows the ΔC-D ratio of Helicobacter pylori ATCC26695 and clinical isolates after 8 hours of treatment with clarithromycin.

The results showed that under the treatment of 0.5μg/ml clarithromycin, 8h sampling and data processing obtained H. pylori Hp isolates A023 and A024. The ΔC-D ratio (ΔC-D ratio) value was greater than the critical value -0.0075, while the remaining strains Both are less than the critical value, so that only A023 and A024 are drug-resistant strains, which are consistent with the E-test results.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A kit for rapidly detecting the drug resistance of Helicobacter pylori, characterized in that the kit comprises an incubation solution, a fixing solution I and a fixing solution II; the incubation solution contains heavy water, and the fixation solution I contains physiological Saline, the fixative II contains absolute ethanol.
2. The kit according to claim 1, wherein the kit further comprises a culture medium.
3. The kit according to claim 2, wherein the culture medium is Brain Heart Infusion Medium (BHI), and fetal bovine serum is further added, and more preferably, 10% fetal bovine serum is added. Bovine serum.
4. The kit according to claim 3, wherein the kit further comprises a bacteria-enhancing agent, and the bacteria-enhancing agent is BBL IsoVitaleX ^(TM) .
5. The use of the kit of claim 1 for detecting the drug resistance of Helicobacter pylori.
6. A method for detecting the drug resistance of Helicobacter pylori using the kit according to claim 1, characterized in that it comprises the following steps:

   a)(a1) Add the same initial concentration of Helicobacter pylori to the incubation system containing the medium and drug-resistant (R) concentration as the experimental group, and set up a control group without drugs, incubate, and then incubate it Add incubation solution to the system and continue incubation; (a2) Obtain the test solution after incubation; take out part of the test solution and directly perform Raman single-cell detection of drug resistance, or mix the test solution with Fixative I and Fixative II. Store in liquid nitrogen, and then take it out for Raman single cell detection for drug resistance when it is needed for testing;

   b) Carry out Raman detection on the samples of the experimental group and the control group, and calculate the CD ratios of the experimental group and the control group respectively according to the obtained Raman spectra. The CD ratio of the experimental group minus the CD ratio of the control group is the ΔC-D ratio. The ratio refers to CD peak area/(CD peak area + CH peak area);

   c) According to the breakpoint given in the CLSI drug susceptibility test standard, at the drug resistance (R) concentration, the ΔC-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 drug resistance (R) concentration is 1-2 μg/ml for levofloxacin and 0.25-0.5 μg/ml for clarithromycin.
7. The method according to claim 6, wherein the drug includes but is not limited to Levofloxacin (Levofloxacin), Clarithromycin (Clarithromycin), Metronidazole (Metronidazole), Amoxicillin, Tetracycline (Tetracycline) Or furazolidone (Furazolidone).
8. The method according to claim 6, wherein the incubation time is 0.5h-2h, and the continued incubation time is 1h-6h.
9. The method according to claim 6, wherein the volume of the incubation solution accounts for 30%-100% of the total volume of the incubation system.
10. The method according to claim 6, characterized in that a 532nm laser, a grating of 300g/mm, and a spectral range of 400-3500 cm ^-1 are used when collecting Raman spectra.

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