CN112080545A - Single-cell Raman clinical drug resistance kit and detection method - Google Patents
Single-cell Raman clinical drug resistance kit and detection method Download PDFInfo
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- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 56
- 238000001069 Raman spectroscopy Methods 0.000 title claims abstract description 38
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- 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
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Abstract
A single cell Raman clinical drug resistance fast detection kit and a detection method thereof comprise an incubation liquid, a stationary liquid I and a stationary liquid II; the incubation liquid comprises heavy water; the fixing solution I comprises physiological saline or PBS; the fixing solution II comprises absolute ethyl alcohol. The kit is used for detecting the drug resistance of the stable isotope labeled unicellular Raman, can realize proliferation-independent and rapid detection of the drug resistance phenotype of trace microorganisms in clinical samples, and can solve the problem of slow detection of clinical drug resistance pathogenic bacteria in the prior art.
Description
Technical Field
The invention relates to the technical field of microbial drug resistance detection, in particular to a unicellular Raman clinical drug resistance quick-detection kit and a detection method and application thereof.
Background
Antibiotics are indispensable clinical drugs for treating pathogenic bacteria infection, but in recent years, the treatment effect of the antibiotics on pathogenic bacteria is increasingly poor, the abuse of the antibiotics causes the wide spread of drug resistance, and the drug resistance of microorganisms is still a difficult problem in the medical field. After a human body is infected by pathogenic bacteria, if a doctor can not use antibiotics for treatment correctly and pertinently, the time and labor are consumed, the treatment cost is wasted, and probiotics in the body can be killed, so that partial conditioned pathogenic bacteria can generate pathogenicity, and super bacteria are more likely to be formed. The super bacteria is multi-drug resistant bacteria, and has drug resistance to various antibiotics. "Superpathogens" have become one of the most major threats to the human society in the twenty-first century. The reports of global review of antibiotic resistance indicate that antibiotic resistance can cause 1000 million deaths each year by 2050 worldwide; in China, where the amount of antibiotics is half of the world, without effective measures, 100 million people will die each year, and a loss of 20 trillion dollars is accumulated.
The traditional clinical detection of the drug resistance of pathogenic bacteria is mainly carried out by a culture method, and the method has the defects of long detection time, high professional requirements on experimental operators, easy occurrence of false positive and the like, and cannot well achieve the purposes of diagnosis and treatment. At present, the suppression of the spread of drug resistance not only needs to develop a novel antibiotic, but also needs to develop a drug resistance quick detection technology and a monitoring system to improve the pertinence and effectiveness of the use of the existing antibiotic, thereby postponing and suppressing the spread of the drug resistance. One of the most promising directions at present is the 'single cell' drug resistance detection technology, namely, skipping cell culture proliferation, directly aiming at the 'growth' or 'metabolism' phenotype of the original single cell in a sample, and performing the characterization of the single cell precision, thereby realizing the aims of rapidness, phenotype-based and wide application range in principle.
Disclosure of Invention
In view of the above, the present invention aims to provide a unicellular raman clinical drug resistance rapid detection kit and a detection method thereof, which can solve the problem of slow detection of clinical drug resistance pathogenic bacteria in the prior art. The kit is based on a stable isotope labeling single cell Raman technology, and can realize proliferation-independent and rapid detection of the drug-resistant phenotype of trace microorganisms in clinical samples.
In order to achieve the above object, the present invention provides, in one aspect, a kit comprising an incubation liquid, a fixative I and a fixative II; the incubation solution comprises heavy water, the fixing solution I comprises normal saline or PBS, and the fixing solution II comprises absolute ethyl alcohol.
The concentration of the physiological saline is 0.9%.
The PBS was 1 XPBS.
The concentration of the heavy water is 100%.
The kit further comprises a culture medium.
The Culture medium is one or more of Nutrient Broth Culture medium (NB), LB Culture medium (LB Culture), Brucella Broth (BB), and 7H9 liquid Culture medium.
The kit further comprises purified water.
In another preferred example, the kit further comprises a centrifuge tube and a fixing tube.
The invention also provides application of the kit in single-cell Raman clinical drug resistance quick detection.
In another aspect, the invention provides a rapid detection method for single-cell clinical Raman drug resistance, which comprises the following steps:
adding the pretreated clinical sample into an incubation system containing an incubation liquid, a culture medium and a medicament to obtain a solution to be detected; and taking out part of the liquid to be detected for directly carrying out Raman single cell detection drug resistance or mixing the liquid to be detected with the stationary liquid I and the stationary liquid II, putting the mixture into liquid nitrogen for preservation, and taking out the mixture for carrying out Raman single cell detection drug resistance when detection is required.
The clinical sample comprises a urine sample, a sputum sample, an alveolar lavage fluid sample, a gastric mucosa sample, or a cerebrospinal fluid sample.
The clinical samples comprise clinical gram-positive bacteria, gram-negative bacteria and fungal infection samples.
The medicine is antibiotic medicine.
In another preferred example, the antibiotic drug includes, but is not limited to, Levofloxacin (Levofloxacin), Gentamicin (Gentamicin), Ceftazidime (Ceftazidime), Ampicillin (Ampicilin), Isoniazide (Isoniazide), rifampin (Rifamicin), Clarithromycin (Clarithromycin), or Metronidazole (Metronidazole).
In another preferred embodiment, the formulation of the incubation system is:
in another preferred embodiment, the formula of the fixing solution I and the fixing solution II is:
| components | Volume of |
| Stationary liquid I | 500μL |
| Fixative II | 500μL |
| Total amount of | 1000μL |
In another preferred embodiment, the parameters of the raman single cell detection are: 100 x of objective lens, 50 mu m of pinhole diameter, 6s of exposure time, 37mW of laser intensity and 15-20min of detection time for each group of samples.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention combines the single-cell Raman spectrum and the stable isotope labeled single-cell Raman detection technology, and can realize the rapid detection of drug resistance in a short time;
(2) the detection method of the kit can realize single cell level drug resistance detection, and has high sensitivity and high accuracy;
(3) the detection method of the kit is simple to operate.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1 kit composition information
The information of the kit in this example is shown in table 1, the incubation solution is heavy water with a concentration of 100%, the fixing solution I is physiological saline with a concentration of 0.9%, the fixing solution II is absolute ethanol, and ultrapure water and the culture medium can be prepared by themselves.
TABLE 1 kit compositional information
The formula of the incubation system prepared by using the kit for the rapid detection of the single cell Raman clinical drug resistance is shown in Table 2. Wherein the volume of the test drug is adjusted for different samples.
TABLE 2 formulation of incubation systems
| Components | Volume of |
| 2X Medium (2X Medium) | 5.0mL |
| Incubation fluid (Incubation Buffer) | 3.0mL |
| Clinical sample to be tested | 1.0mL |
| Test drugs (antibiotics) | n mL |
| Ultrapure water ddH2O | Make up to 10.0mL |
| Total amount of | 10.0mL |
The formula of the fixing solution prepared when the kit is used for the rapid detection of the single cell Raman clinical drug resistance is shown in the table 3.
TABLE 3 preparation of fixative solutions
Example 2 single cell raman clinical drug resistance fast test kit for detecting drug resistance of escherichia coli (e.coli395) in clinical urine samples
In this embodiment, a single cell raman clinical drug resistance rapid detection kit is used to detect the drug resistance of escherichia coli (e.coli395) in a clinical urine sample of a patient with urinary tract infection, and the corresponding test drug (antibiotic) is Levofloxacin (Levo), and the specific drug resistance detection steps are as follows:
1. clinical urine sample and drug pretreatment
A1.0 mL sample of clinical fresh urine was collected, centrifuged (8000rpm, 2min), and after removing the supernatant, resuspended in 1.0mL of PBS.
2. Preparation of incubation System
The establishment of incubation systems for the control and experimental groups was performed according to Table 4, with the group without levofloxacin (Levo) as the control group (with ultrapure water ddH)2O instead of Levo). After the incubation culture system is established, the culture systems are all placed in a constant-temperature shaking incubator at 37 ℃ for 1 hour.
TABLE 4 formulation of urine sample incubation System
Remarking: the levofloxacin (Levo) concentration was 1.0 mg/mL.
3. Rapid detection
(1) Sampling and processing:
if the liquid to be detected is detected immediately: the solution to be tested in 1.0mL incubation system was taken into a 1.5mL centrifuge tube, centrifuged (8000rpm, 2min) and the supernatant removed. Add 500. mu.L of ddH2Resuspend, continue to centrifuge and remove the supernatant, repeat the washing three times. Finally, 30. mu.L of ddH was added to the centrifuge tube2Resuspend and mix well to pellet the sample for the corresponding dilution.
If the liquid to be detected can not be detected immediately: the solution to be detected needs to be treated, fixed by using a fixing solution, and then frozen for later detection.
The specific method for freezing and storing the liquid to be detected comprises the following steps: the solution to be tested was centrifuged (8000rpm, 2min) and the supernatant removed, the fixative prepared according to the formulation in example 1 was added, mixed well and then stored in liquid nitrogen.
(2) Sample application and preparation: spotting 10 μ L of diluent on a detection chip (calcium fluoride CaF)2Slides), three per sampleAnd (5) carrying out parallel sample application, naturally drying in a biological safety cabinet, and detecting.
4. Analyzing the obtained result data
The sample is subjected to subsequent detection and analysis by adopting a single-cell Raman fast detector, and the detection parameters are as follows: 100 x of objective lens, 50 mu m of pinhole diameter, 6s of exposure time, 37mW of laser intensity and 15-20min of detection time for each group of samples.
5. Results of the experiment
And (3) performing data analysis according to a bacterial Raman spectrum obtained by the single-cell Raman fast detector to calculate a CD-ratio value, wherein the larger the CD-ratio value is, the higher the bacterial heavy water metabolic activity is. According to the experimental results, the CD-ratio value of the bacterial Raman spectrum in the experimental group sample is not significantly different from that of the bacterial Raman spectrum in the control group sample, which indicates that under the action of levofloxacin (Levo), the bacterial cells still survive and metabolize, so that Escherichia coli (E.coli395) in the clinical urine sample is judged to be a levofloxacin (Levo) resistant strain. The results are consistent with the types and resistance of the strains obtained by the culture method.
The experimental results show that the single-cell Raman drug resistance fast detection kit can be combined with a single-cell Raman fast detection instrument to rapidly detect the drug resistance of escherichia coli in a urine sample of a clinical urinary tract infection patient within 3 hours (the sample needs 1-2 hours for pretreatment and culture, and the pretreatment and detection need 1 hour), so that the rapidness of the method is embodied, the drug resistance of pathogenic bacteria can be rapidly detected clinically, and a reasonable medication suggestion is provided.
Example 3 Single cell Raman clinical drug resistance fast detection kit for detecting the drug resistance of Escherichia coli (E.coli865) in clinical urine samples
This embodiment adopts this unicellular raman clinical drug resistance quick test kit, detects the drug resistance of escherichia coli (e.colii 865) in the clinical urine sample of urinary tract infection patient, and corresponding test medicine (antibiotic) is Gentamicin (Gentamicin, Genta), and concrete drug resistance detection step is as follows:
1. clinical urine sample and drug pretreatment
A1.0 mL sample of clinical fresh urine was collected, centrifuged (8000rpm, 2min), and after removing the supernatant, resuspended in 1.0mL of PBS.
2. Preparation of incubation System
The incubation systems of the control and experimental groups were established as shown in Table 5, with the group without gentamicin (Genta) as the control group (using ultrapure water ddH)2O instead of Genta). After the incubation culture system is established, the culture systems are all placed in a constant-temperature shaking incubator at 37 ℃ for 1 hour.
TABLE 5 formulation of urine sample incubation System
Remarking: gentamicin (Genta) concentration was 1.0 mg/mL.
3. Rapid detection
(1) Sampling and processing:
if the liquid to be detected is detected immediately: the solution to be tested in 1.0mL incubation system was taken into a 1.5mL centrifuge tube, centrifuged (8000rpm, 2min) and the supernatant removed. Add 500. mu.L of ddH2Resuspend, continue to centrifuge and remove the supernatant, repeat the washing three times. Finally, 30. mu.L of ddH was added to the centrifuge tube2Resuspend and mix well to pellet the sample for the corresponding dilution.
If the liquid to be detected can not be detected immediately: the solution to be detected needs to be treated, fixed by using a fixing solution, and then frozen for later detection.
The specific method for freezing and storing the liquid to be detected comprises the following steps: the solution to be tested was centrifuged (8000rpm, 2min) and the supernatant removed, the fixative prepared according to the formulation in example 1 was added, mixed well and then stored in liquid nitrogen.
(2) Sample application and preparation: spotting 10 μ L of diluent on a detection chip (calcium fluoride CaF)2Slide), each sample is sampled in three parallel points, and is naturally dried in a biological safety cabinet to be detected.
4. Analyzing the obtained result data
The sample is subjected to subsequent detection and analysis by adopting a single-cell Raman fast detector, and the detection parameters are as follows: 100 x of objective lens, 50 mu m of pinhole diameter, 6s of exposure time, 37mW of laser intensity and 15-20min of detection time for each group of samples.
5. Results of the experiment
And (3) performing data analysis according to a bacterial Raman spectrum obtained by the single-cell Raman fast detector to calculate a CD-ratio value, wherein the larger the CD-ratio value is, the higher the bacterial heavy water metabolic activity is. According to the experimental result, the CD-ratio value of the bacterial Raman spectrum in the experimental group sample is not significantly different from the CD-ratio value of the bacterial Raman spectrum in the control group sample, which indicates that under the action of gentamicin (Genta), bacterial cells still survive and metabolize, and therefore, the Escherichia coli (E.coli865) in the clinical urine sample is judged to be a gentamicin (Genta) drug-resistant strain. The results are consistent with the types and resistance of the strains obtained by the culture method.
The experimental results show that the single-cell Raman drug resistance fast detection kit can be used for rapidly detecting the drug resistance of Escherichia coli (E.coli865) in the urine sample of a clinical urinary tract infection patient within 3 hours (1-2 hours is required for sample pretreatment and culture, and 1 hour is required for pretreatment and detection), and the traditional clinical culture method is about 2 days for judging whether the drug resistance exists, so that the rapidity of the method is embodied, the drug resistance of pathogenic bacteria can be rapidly detected clinically, and a reasonable drug use suggestion is provided.
Example 4 Single cell Raman clinical drug resistance fast detection kit for detecting drug resistance of helicobacter pylori (Hp) in clinical gastric mucosa samples
In this embodiment, the single cell raman clinical drug resistance rapid detection kit is used to detect the drug resistance of helicobacter pylori (Hp) in a clinical gastric mucosa sample, and the specific drug resistance detection steps corresponding to the tested drug (antibiotic) being Clarithromycin (Clarithromycin, Clarit) are as follows:
1. clinical gastric mucosa sample and drug pretreatment
Transferring the clinical gastric mucosa sample from the frozen stock solution to a 1.5mL centrifuge tube, adding a small amount of normal saline, inserting a sterilized grinding rod into the bottom of the centrifuge tube, forcibly rotating and grinding for multiple times until the liquid in the centrifuge tube becomes turbid, and obtaining a ground bacteria solution, namely obtaining the clinical gastric mucosa sample to be detected for later use.
2. Preparation of incubation System
The incubation systems of the control and experimental groups were set up as in Table 6, with the group without clarithromycin (Clarit) as the control (ddH with ultrapure water)2O instead of Clarit). After the incubation culture system is established, the culture systems are placed in a microaerophilic environment at 37 ℃ for culture for 6 hours.
TABLE 6 formulation of gastric mucosal sample incubation systems
Remarking: clarithromycin (Clarit) concentration was 50. mu.g/mL.
3. Rapid detection
(1) Sampling and processing:
if the liquid to be detected is detected immediately: the solution to be tested in 1.0mL incubation system was taken into a 1.5mL centrifuge tube, centrifuged (8000rpm, 2min) and the supernatant removed. Add 500. mu.L of ddH2Resuspend, continue to centrifuge and remove the supernatant, repeat the washing three times. Finally, 30. mu.L of ddH was added to the centrifuge tube2Resuspend and mix well to pellet the sample for the corresponding dilution.
If the liquid to be detected can not be detected immediately: the solution to be detected needs to be treated, fixed by using a fixing solution, and then frozen for later detection.
The specific method for freezing and storing the liquid to be detected comprises the following steps: the solution to be tested was centrifuged (8000rpm, 2min) and the supernatant removed, the fixative prepared according to the formulation in example 1 was added, mixed well and then stored in liquid nitrogen.
(2) Sample application and preparation: spotting 10 μ L of diluent on a detection chip (calcium fluoride CaF)2Slide), each sample is sampled in three parallel points, and is naturally dried in a biological safety cabinet to be detected.
4. Analyzing the obtained result data
The sample is subjected to subsequent detection and analysis by adopting a single-cell Raman fast detector, and the detection parameters are as follows: 100 x of objective lens, 50 mu m of pinhole diameter, 6s of exposure time, 37mW of laser intensity and 15-20min of detection time for each group of samples.
5. Results of the experiment
And (3) performing data analysis according to a bacterial Raman spectrum obtained by the single-cell Raman fast detector to calculate a CD-ratio value, wherein the larger the CD-ratio value is, the higher the bacterial heavy water metabolic activity is. According to the experimental results, the CD-ratio value of the bacterial Raman spectrum in the experimental group sample is not significantly different from that of the bacterial Raman spectrum in the control group sample, which shows that under the action of clarithromycin (Clarit), bacterial cells still survive and metabolize, so that helicobacter pylori (Hp) in the clinical gastric mucosa sample is judged to be a clarithromycin (Clarit) resistant strain. The results are consistent with the types and resistance of the strains obtained by the culture method.
The experimental results show that the single-cell Raman drug resistance fast detection kit can be combined with a single-cell Raman fast detection instrument to rapidly detect the drug resistance of helicobacter pylori (Hp) in a clinical gastric mucosa sample within 8 hours (the sample pretreatment and culture needs 6-7 hours, and the pretreatment and detection needs 1 hour) (while the conventional clinical culture method needs about 2 weeks for judging whether the drug resistance exists), so that the rapidity of the method is embodied, the drug resistance of pathogenic bacteria can be rapidly detected clinically, and a reasonable drug use suggestion is provided.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. A kit, characterized in that: comprises an incubation solution, a fixing solution I and a fixing solution II; the incubation solution comprises heavy water, the fixing solution I comprises normal saline or PBS, and the fixing solution II comprises absolute ethyl alcohol.
2. The kit of claim 1, wherein: the concentration of the physiological saline is 0.9%.
3. The kit of claim 1, wherein: the PBS was 1 XPBS.
4. The kit of claim 1, wherein: the concentration of the heavy water is 100%.
5. The kit of claim 1, wherein: also comprises a culture medium.
6. The kit of claim 5, wherein: the Culture medium is one or more of Nutrient Broth Culture medium (NB), LB Culture medium (LB Culture), Brucella Broth (BB), and 7H9 liquid Culture medium.
7. The use of the kit of claims 1-6 in the rapid detection of single cell raman clinical drug resistance.
8. A single cell clinical Raman drug resistance rapid detection method is characterized in that:
adding the pretreated clinical sample into an incubation system containing an incubation liquid, a culture medium and a medicament to obtain a solution to be detected; and taking out part of the liquid to be detected for directly carrying out Raman single cell detection drug resistance or mixing the liquid to be detected with the stationary liquid I and the stationary liquid II, putting the mixture into liquid nitrogen for preservation, and taking out the mixture for carrying out Raman single cell detection drug resistance when detection is required.
9. The single-cell clinical raman resistance rapid detection method according to claim 8, characterized in that: the clinical sample comprises a urine sample, a sputum sample, an alveolar lavage fluid sample, a gastric mucosa sample, or a cerebrospinal fluid sample.
10. The single-cell clinical raman resistance rapid detection method according to claim 8, characterized in that: the medicine is antibiotic medicine.
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