Method for mutation detection in tigecycline resistance detection of klebsiella pneumoniae and application

Abstract

The invention relates to the technical field of molecular diagnosis technology, in particular to a method for detecting tigecycline resistance of klebsiella pneumoniae by mutation detection and application of the method. The method comprises the following steps: firstly, sampling a sample, adding a lysis solution containing a specific lysis enhancer, incubating at 37 DEG C, and then carrying out gradient centrifugation to take a supernatant; then extracting nucleic acid by using special bifunctional magnetic nanoparticles, washing and then eluting; preparing a PCR reaction system containing primers, enzyme and the like, and adding a novel auxiliary factor for amplification; then detecting mutation by using a compound nucleic acid dye through an HRM (High Resolution Melting) technology; and finally, comparing melting curves, and combining a machine learning algorithm to judge whether the sample is drug-resistant. The detection method has outstanding advantages; the innovative steps of sample treatment, nucleic acid extraction and detection improve the detection accuracy and efficiency, and can accurately identify low-level drug-resistant strains; meanwhile, the cost is greatly reduced, popularization in primary hospitals is facilitated, and great significance on prevention and control of drug-resistant bacteria is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular diagnosis, and specifically to a method and application of mutation detection in the detection of tigecycline resistance of Klebsiella pneumoniae. Background Art

[0002] As a common opportunistic pathogen, Klebsiella pneumoniae has caused many problems in the field of clinical infections. It has a strong environmental adaptability and often causes serious diseases such as bloodstream infections, significantly increasing the morbidity and mortality of patients. In recent years, the clinical detection rate of hypervirulent Klebsiella pneumoniae (hvKP), especially carbapenem-resistant hvKP (CR-hvKP), has been showing an increasing trend year by year, making clinical treatment face an unprecedented dilemma.

[0003] As a glycylcycline antibiotic, tigecycline is one of the important choices for the treatment of severe infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). However, tigecycline-resistant CRKP strains have emerged globally, which seriously threatens the clinical treatment effect. When hvKP infection is accompanied by dual resistance to carbapenems and tigecycline, clinical treatment options are extremely limited, and the prognosis of patients is often poor.

[0004] In the detection of tigecycline resistance of Klebsiella pneumoniae, there are many defects in the existing technologies. Traditional phenotypic drug susceptibility tests, such as the microbroth dilution method, take an extremely long time for detection, usually more than 48 hours, which is extremely likely to cause delays in clinical treatment. Moreover, the detection sensitivity of this method for low-level resistant strains is relatively low, and false-susceptible results are likely to occur, unable to provide timely and accurate basis for clinical treatment.

[0005] Existing commercial molecular detection products also have deficiencies. For example, some kits (such as the GeneXpert Carba-R kit) do not cover the ramR gene region, which makes it easy to miss strains carrying ramR gene mutations, thus affecting the accurate judgment of the drug resistance of Klebsiella pneumoniae. In addition, clinical strains often carry multiple drug resistance-related mutations, such as ramR+acrR co-mutations, and it is difficult for existing technologies to distinguish the contribution degree of single mutations to drug resistance, which brings great difficulties to the research of drug resistance mechanisms and precision treatment.

[0006] Although whole-genome sequencing can discover new mutation sites, the detection cost is extremely high, and its cost is more than 40 times that of conventional PCR. This high cost makes it difficult to popularize this technology in primary hospitals, restricting its wide application in clinical detection.

[0007] Current detection techniques for tigecycline resistance in Klebsiella pneumoniae have obvious deficiencies in terms of sensitivity, timeliness, target coverage, multi-gene mutation analysis, and cost, and cannot meet the needs of rapid and accurate clinical detection. Developing an efficient, accurate, and low-cost detection method is urgent, which is of crucial significance for guiding rational clinical drug use, controlling the spread of drug-resistant bacteria, and improving patient prognosis. Summary of the Invention

[0008] (I) Technical Problems to be Solved

[0009] In view of the deficiencies of the prior art, the present invention provides a method and application of mutation detection in the detection of tigecycline resistance in Klebsiella pneumoniae.

[0010] (II) Technical Solutions

[0011] A method for detecting the resistance of Klebsiella pneumoniae to tigecycline, comprising the following steps:

[0012] S1: Sample pretreatment

[0013] Take a Klebsiella pneumoniae sample, add a lysis solution containing lysis enhancer X, where X is a complex of 2-(2-aminoethoxy)ethanol, dithiothreitol, and compound Z, and the structure of 2-(2-aminoethoxy)ethanol is:

[0014]

[0015] The structural formula of compound Z is:

[0016]

[0017] After lysis is completed, use gradient centrifugation to remove large impurity precipitates, then centrifuge the supernatant, and take the supernatant for standby;

[0018] S2: Nucleic acid extraction

[0019] Add nucleic acid adsorbent Y to the supernatant, where nucleic acid adsorbent Y is a bifunctional magnetic nanoparticle surface-modified with carboxyl and amino groups; mix by shaking at room temperature; use an external magnetic field to separate the nanoparticles adsorbed with nucleic acid, and wash them successively with an ethanol solution containing NaCl and deionized water; finally, elute the nucleic acid with an appropriate amount of Tris-HCl buffer solution to obtain a Klebsiella pneumoniae nucleic acid extract;

[0020] S3: PCR amplification

[0021] Configure a PCR reaction system, including a specific primer pair, a hot-start Taq DNA polymerase, dNTPs, a PCR buffer, and the above-extracted nucleic acid extract; among them, the specific primer pair is designed for the 517-518 site deletion mutation of the ramR gene, the forward primer sequence is 5'-GCCGCCAGGTAAAAG-3', and the reverse primer sequence is 5'-GTAAACGGGTAGGTCAGG-3'; the PCR amplification program is: pre-denaturation at 95°C for 3-5 minutes; denaturation at 95°C for 30-45 seconds, annealing at 58-62°C for 30-45 seconds, extension at 72°C for 30-45 seconds, for a total of 30-35 cycles; finally, extension at 72°C for 5-10 minutes;

[0022] S4: Mutation detection

[0023] Add nucleic acid dye Z to the amplification product, and on the HRM instrument, heat from 60°C to 95°C, and monitor the change of fluorescence signal in real time to draw a melting curve;

[0024] S5: Result determination

[0025] Compare and analyze the melting curve of the test sample with the melting curves of the known positive and negative control samples of the 517-518 site deletion mutation of the ramR gene; at the same time, combine a machine learning algorithm to analyze the characteristic parameters of the melting curve and establish a prediction model.

[0026] Preferably, it also includes enriching and culturing the sample before the S1 sample pretreatment step; inoculating the sample into an LB medium containing a nutritional additive, and the nutritional additive is yeast extract, casein amino acids, and a synthetic oligopeptide mixture, and the oligopeptide mixture is a tripeptide composed of alanine, glycine, and serine, with a concentration of 1-2 g / L, and culturing with shaking at 37°C and 180-220 rpm for 6-8 hours.

[0027] Preferably, it also includes evaluating the quality of the nucleic acid extract in the S2 nucleic acid extraction step; using an ultraviolet spectrophotometer to measure the absorbance of the nucleic acid extract at wavelengths of 260 nm, 280 nm, and 230 nm, and calculate the A 260 / A 280 and A 260 / A 230 ratios, and it is required that the A 260 / A 280 ratio is between 1.8 and 2.0, and the A 260 / A 230 ratio is between 2.0 and 2.2.

[0028] Preferably, in the S3 PCR amplification step, the specific primer pair is modified with phosphorothioate and 2'-O-methyl; meanwhile, a locked nucleic acid LNA-modified probe with a concentration of 0.1-0.5 μmol / L is added to the PCR reaction system.

[0029] Preferably, in the PCR reaction system, the concentration of hot-start Taq DNA polymerase is 0.5-1.5 U / μL, the concentration of each nucleotide in dNTPs is 0.2-0.4 mmol / L, and the PCR buffer contains KCl, Tris-HCl, and MgCl2; and a PCR cofactor W is also added, which is a complex of tris(hydroxymethyl)methylglycine and betaine mixed at a molar ratio of 1:2, and its concentration is 5-10 mmol / L; the nucleic acid dye Z is a complex of EvaGreen and a fluorescence enhancer Q mixed at a molar ratio of 1:0.1, and the Q is rhodamine B modified with 4-(dimethylamino)benzaldehyde.

[0030] Preferably, in S5, if the model predicts positive, and the melting temperature of the test sample is offset by ≥1.5 °C compared with the negative control and the ΔCt value is ≥5 compared with the internal reference gene, it is determined to be positive for the deletion mutation at positions 517-518 of the ramR gene, that is, Klebsiella pneumoniae is resistant to tigecycline; otherwise, it is negative.

[0031] Preferably, in the S1 sample pretreatment step, if the sample is a blood sample, first add erythrocyte lysate, incubate at room temperature for 5-10 minutes to lyse erythrocytes, then centrifuge at 3000-5000 g for 5-10 minutes, discard the supernatant, and perform subsequent lysis treatment on the precipitate.

[0032] Preferably, in the S3 PCR amplification step, nested PCR technology is used, and an inner primer pair and an outer primer pair are designed; the outer primer pair is first used for the first-round amplification, and the amplification product is diluted 10-100 times and used as the template for the second-round amplification, and the inner primer pair is used for amplification.

[0033] Preferably, in the S2 nucleic acid extraction step, when using microfluidic chip technology for nucleic acid extraction, the surface of the chip is modified with a hydrophilic polymer.

[0034] Preferably, the application of the method according to any one of the above in the preparation of a kit for detecting the resistance of Klebsiella pneumoniae to tigecycline, the kit is used for the detection of the resistance of Klebsiella pneumoniae to tigecycline in clinical diagnosis, epidemiological investigation, and antimicrobial susceptibility monitoring, and the kit is equipped with special data analysis software, which can automatically import melting curve data, use machine learning algorithms for result determination, and generate a test report.

[0035] (III) Beneficial technical effects

[0036] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0037] 1. Through innovative sample pretreatment, nucleic acid extraction, PCR amplification, and mutation detection steps, the method of the present invention greatly improves the detection efficiency and accuracy. For example, the use of the novel lysis enhancer X can more effectively lyse Klebsiella pneumoniae samples and improve the nucleic acid release efficiency; the bifunctional magnetic nanoparticles as the nucleic acid adsorbent Y have higher nucleic acid adsorption specificity and efficiency, and through optimizing the washing steps, the nucleic acid purity is further improved.

[0038] 2. In the PCR amplification step, the addition of the novel PCR cofactor W, primer modification, and the use of locked nucleic acid probes enhance the amplification stability and specificity. Combining with the HRM analysis of multi-channel fluorescence detection technology, the 517-518 site deletion mutation of the ramR gene can be detected more accurately, effectively avoiding missed detection and misjudgment, breaking through the sensitivity limitation of traditional detection methods, and realizing the accurate identification of low-level drug-resistant strains.

[0039] 3. The method of the present invention combines conventional PCR technology with innovative optimization, greatly reducing the detection cost. Compared with whole-genome sequencing, the cost is reduced by more than 95%, and the operation is simple, without the need for expensive special equipment, and ordinary PCR instruments can be used. This enables the detection method to be widely promoted and applied in primary hospitals, improving the popularity of tigecycline resistance detection for Klebsiella pneumoniae, and is of great significance for improving the overall medical level and public health status. Description of the Drawings

[0040] Figure 1 is the method flow chart of the mutation detection in the tigecycline resistance detection of Klebsiella pneumoniae proposed by the present invention;

[0041] Figure 2 is the columnar comparison chart of the detection time of the example and the comparative example;

[0042] Figure 3 is the columnar comparison chart of the missed detection rate and misjudgment rate of the example and the comparative example;

[0043] Figure 4 is the nuclear magnetic resonance hydrogen spectrum diagram of the compound Z proposed by the present invention. Detailed Embodiments

[0044] The Klebsiella pneumoniae tigecycline-resistant mutant strain ATCC43816-16 involved in the present invention has submitted its whole-genome sequencing data to the International Nucleotide Sequence Database Collaboration (DDBJ / ENA / GenBank), and the project accession number is JAVKOT000000000. The gene mutation sequence of ramR of the strain has been separately submitted to the GenBank database, and the accession number is OR509661 (the ramR gene sequence containing a double-base deletion mutation at positions 517-518).

[0045] According to Figures 1 to 4 , the specific implementation manner of the present invention is as follows:

[0046] Example 1

[0047] Sample collection: Sputum samples of patients suspected of Klebsiella pneumoniae infection were collected from the respiratory medicine ward of a certain hospital. The sputum samples were collected in sterile sputum cups and immediately sent to the laboratory for processing.

[0048] Sample pretreatment: Take 2 mL of sputum sample, add an equal volume of 5% NaOH solution, vortex for 1 minute, and let it stand at room temperature for 15 minutes for liquefaction treatment. Then, centrifuge the liquefied sample at 3000 g for 5 minutes and discard the supernatant. Add 1 mL of lysis solution containing a novel lysis enhancer X (a complex of 2-(2-aminoethoxy)ethanol, dithiothreitol, and N-(3-mercaptopropyl)-4-methoxybenzamide mixed at a molar ratio of 1:1:0.5) to the precipitate, and incubate at 37 °C for 20 minutes to fully lyse the sample. First, centrifuge at 8000 g for 3 minutes to remove large impurity precipitates, and then centrifuge the supernatant at 12000 g for 5 minutes, and take the supernatant for use.

[0049] Nucleic acid extraction: Add 100 μL of nucleic acid adsorbent Y (bifunctional magnetic nanoparticles with carboxyl and amino groups modified on the surface and a molar ratio of 2:1, with a particle size between 20-50 nm) to the supernatant, mix by shaking at room temperature for 10 minutes to adsorb the nucleic acid on the surface of the nanoparticles. Use an external magnetic field to separate the nanoparticles adsorbed with nucleic acid, and wash twice with 500 μL of 70% ethanol solution containing 0.1 mol / L NaCl and 500 μL of deionized water to remove impurities. Finally, elute the nucleic acid with 50 μL of Tris-HCl buffer (pH 8.0) to obtain the Klebsiella pneumoniae nucleic acid extract.

[0050] PCR Amplification: Prepare a 25 μL PCR reaction system, including 2.5 μL of 10× PCR buffer (containing 50 mmol / L KCl, 10 mmol / L Tris-HCl (pH 8.3), 1.5 mmol / L MgCl2), 2 μL of dNTPs (each nucleotide concentration is 0.2 mmol / L), 0.5 μL each of forward primer (5'-GCCGCCAGGTAAAAG-3') and reverse primer (5'-GTAAACGGGTAGGTCAGG-3') (both at a concentration of 10 μmol / L), 0.5 U of hot-start Taq DNA polymerase, 1.25 μL of a novel PCR cofactor W (a complex of tris(hydroxymethyl)methylglycine and betaine mixed at a molar ratio of 1:2, with a concentration of 5 mmol / L), and 5 μL of nucleic acid extract, and make up to 25 μL with deionized water. The PCR amplification program is as follows: pre-denaturation at 95 °C for 3 minutes; denaturation at 95 °C for 30 seconds, annealing at 58 °C for 30 seconds, extension at 72 °C for 30 seconds, for a total of 30 cycles; finally, extension at 72 °C for 5 minutes.

[0051] Mutation Detection: Add 2.5 μL of nucleic acid dye Z (a complex of EvaGreen and rhodamine B modified with 4-(dimethylamino)benzaldehyde mixed at a molar ratio of 1:0.1, with a final concentration of 1:1000) to the PCR amplification product. On an HRM instrument, increase the temperature from 60 °C to 95 °C at a rate of 0.1 °C / second, and simultaneously monitor the change in fluorescence signal in real-time to plot the melting curve.

[0052] Result Judgment: Compare and analyze the melting curve of the test sample with the melting curves of the known positive and negative control samples of the deletion mutation at positions 517 - 518 of the ramR gene. At the same time, combined with the support vector machine (SVM) model, analyze the characteristic parameters of the melting curve (such as melting temperature, peak height, peak width, etc.). The melting temperature of this sample is offset by 2 °C compared to the negative control, and the ΔCt value (compared to the internal reference gene) is 6. The model predicts it as positive, and it is determined to be positive for the deletion mutation at positions 517 - 518 of the ramR gene, that is, Klebsiella pneumoniae is resistant to tigecycline.

[0053] Example 2

[0054] Sample Collection: Collect blood samples from patients suspected of Klebsiella pneumoniae infection in the intensive care unit of a certain hospital. Use a sterile syringe to draw 5 mL of the patient's venous blood and inject it into a sterile test tube containing anticoagulant.

[0055] Sample Pretreatment: Take 1 mL of blood sample, add 2 mL of red blood cell lysate (ammonium chloride-potassium bicarbonate buffer), incubate at room temperature for 5 minutes to lyse red blood cells. Then centrifuge at 3000g for 5 minutes and discard the supernatant. Add 1 mL of lysate containing novel lysis enhancer X to the precipitate, incubate at 37 °C for 25 minutes to fully lyse the sample. First, centrifuge at 9000g for 4 minutes to remove large impurity precipitates, and then centrifuge the supernatant at 13000g for 6 minutes. Take the supernatant for standby.

[0056] Nucleic Acid Extraction: Add 120 μL of nucleic acid adsorbent Y to the supernatant, mix by oscillation at room temperature for 12 minutes to adsorb nucleic acids on the surface of nanoparticles. Use an external magnetic field to separate the nanoparticles adsorbed with nucleic acids, and wash them 3 times successively with 500 μL of 75% ethanol solution containing 0.2 mol / L NaCl and 500 μL of deionized water to remove impurities. Finally, elute the nucleic acids with 60 μL of Tris-HCl buffer (pH 8.2) to obtain Klebsiella pneumoniae nucleic acid extract.

[0057] PCR Amplification: Prepare a 25 μL PCR reaction system, including 2.5 μL of 10×PCR buffer (containing 60 mmol / L KCl, 15 mmol / L Tris-HCl (pH 8.5), 2.0 mmol / L MgCl2), 2 μL of dNTPs (each nucleotide concentration is 0.3 mmol / L), 0.5 μL each of forward primer and reverse primer (both concentrations are 10 μmol / L), 1 U of hot-start Taq DNA polymerase, 1.75 μL of novel PCR cofactor W (concentration is 7 mmol / L), 5 μL of nucleic acid extract, and make up to 25 μL with deionized water. The PCR amplification program is: pre-denaturation at 95 °C for 4 minutes; denaturation at 95 °C for 35 seconds, annealing at 60 °C for 35 seconds, extension at 72 °C for 35 seconds, for a total of 32 cycles; finally, extension at 72 °C for 7 minutes.

[0058] Mutation Detection: Add 2.5 μL of nucleic acid dye Z (final concentration is 1:1500) to the PCR amplification product. On the HRM instrument, increase the temperature from 60 °C to 95 °C at a heating rate of 0.2 °C / second, and simultaneously monitor the change of fluorescence signal in real time to draw a melting curve.

[0059] Result Judgment: Compare and analyze the melting curve of the test sample with the melting curves of the known positive and negative control samples of the 517-518 site deletion mutation of the ramR gene. Analyze the characteristic parameters of the melting curve in combination with the SVM model. The melting temperature of this sample is shifted by 1.8 °C compared with the negative control, and the ΔCt value (compared with the internal reference gene) is 5.5. The model predicts it as positive, and it is determined to be positive for the 517-518 site deletion mutation of the ramR gene, that is, Klebsiella pneumoniae is resistant to tigecycline.

[0060] Example 3

[0061] Sample collection: Urine samples were collected from patients suspected of Klebsiella pneumoniae infection in a community hospital. The patients collected 10 mL of midstream urine in a sterile urine cup.

[0062] Sample pretreatment: Take 2 mL of urine sample, centrifuge at 5000 g for 10 minutes, and discard the supernatant. Add 1 mL of lysis buffer containing the novel lysis enhancer X to the precipitate, and incubate at 37 °C for 30 minutes to fully lyse the sample. First, centrifuge at 10000 g for 5 minutes to remove large impurity precipitates, and then centrifuge the supernatant at 15000 g for 10 minutes. Take the supernatant for use.

[0063] Nucleic acid extraction: Add 150 μL of nucleic acid adsorbent Y to the supernatant, mix by shaking at room temperature for 15 minutes to adsorb the nucleic acid on the surface of the nanoparticles. Use an external magnetic field to separate the nanoparticles adsorbed with nucleic acid, and wash 3 times with 500 μL of 80% ethanol solution containing 0.3 mol / L NaCl and 500 μL of deionized water in turn to remove impurities. Finally, elute the nucleic acid with 80 μL of Tris-HCl buffer (pH 8.5) to obtain the Klebsiella pneumoniae nucleic acid extract.

[0064] PCR amplification: Prepare a 25 μL PCR reaction system, including 2.5 μL of 10×PCR buffer (containing 70 mmol / L KCl, 20 mmol / L Tris-HCl (pH 8.8), 3.0 mmol / L MgCl2), 2 μL of dNTPs (each nucleotide concentration is 0.4 mmol / L), 0.5 μL of forward primer and reverse primer each (concentrations are both 10 μmol / L), 1.5 U of hot start Taq DNA polymerase, 2.5 μL of the novel PCR cofactor W (concentration is 10 mmol / L), and 5 μL of nucleic acid extract. Add deionized water to make up to 25 μL. The PCR amplification program is: pre-denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 45 seconds, annealing at 62 °C for 45 seconds, extension at 72 °C for 45 seconds, for a total of 35 cycles; finally, extension at 72 °C for 10 minutes.

[0065] Mutation detection: Add 2.5 μL of nucleic acid dye Z (final concentration is 1:2000) to the PCR amplification product. On the HRM instrument, raise the temperature from 60 °C to 95 °C at a heating rate of 0.3 °C / second, and simultaneously monitor the change of fluorescence signal in real time to draw a melting curve.

[0066] Result determination: Compare and analyze the melting curve of the test sample with the melting curves of the known positive and negative control samples of the ramR gene deletion mutation at positions 517 - 518. Analyze the characteristic parameters of the melting curve in combination with the SVM model. The melting temperature of this sample is offset by 2.2 °C compared with the negative control, and the ΔCt value (compared with the internal reference gene) is 7. The model predicts it as positive, and it is determined to be positive for the ramR gene deletion mutation at positions 517 - 518, that is, Klebsiella pneumoniae is resistant to tigecycline.

[0067] Comparative example

[0068] The traditional microbroth dilution method was used to detect the tigecycline resistance of the samples from the same source in the above three examples. The total duration was about 48 hours.

[0069] Sample treatment: Inoculate the collected sputum, blood, and urine samples onto blood agar plates respectively, and culture them at 37 °C for 24 hours. Pick the colonies suspected to be Klebsiella pneumoniae, perform Gram staining and biochemical identification. After determining it as Klebsiella pneumoniae, adjust the bacterial liquid concentration to 0.5 McFarland turbidity with sterile normal saline.

[0070] Drug susceptibility test: Inoculate the adjusted-concentration bacterial liquid into broth media containing different concentrations of tigecycline, and set three replicates for each concentration. At the same time, set the quality control strain Escherichia coli ATCC 25922, positive control (broth medium without drug), and negative control (broth medium without bacteria). After incubating the inoculated media at 37 °C for 16 - 20 hours, observe the bacterial growth. The lowest drug concentration at which no bacterial growth is observed with the naked eye is the minimum inhibitory concentration (MIC).

[0071] Comparison of the detection results and time between the examples and the comparative example is as follows in the table:

[0072] Table 1

[0073]

[0074] Conclusion: This table shows the detection results and the required duration of the examples and the comparative example for different sample types. The results show that both the examples and the comparative example detected the resistance of the samples to tigecycline, but the detection duration of the examples was significantly shorter than that of the comparative example, reflecting the advantage of the detection method of the present invention in terms of timeliness. Comparison of the detection accuracy-related indicators between the examples and the comparative example is as follows in the table:

[0075] Table 2

[0076]

[0077]

[0078] Conclusion: This table compares the key indicators of the examples and comparative examples in terms of detection accuracy. The mean melting temperature shift, mean ΔCt, etc. of the examples reflect that they can accurately detect mutations. The SVM model has a high prediction accuracy, low missed detection rate and misjudgment rate, and has more advantages in detection accuracy compared with the comparative examples.

[0079] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the resistance of Klebsiella pneumoniae to tigecycline, characterized in that: The following steps are involved: S1: Sample pretreatment A Klebsiella pneumoniae sample was taken and a lysis solution containing a lysis enhancer X was added, wherein X was a complex of 2-(2-aminoethoxy)ethanol, dithiothreitol and compound Z, and the structure of 2-(2-aminoethoxy)ethanol was: The structural formula of the compound Z is: After the lysis is completed, the larger impurities are removed by gradient centrifugation, and the supernatant is centrifuged and taken for later use; S2: Nucleic acid extraction Adding a nucleic acid adsorbent Y to the supernatant, wherein the nucleic acid adsorbent Y is a bifunctional magnetic nanoparticle with a carboxyl group and an amino group modified on the surface; oscillating and mixing at room temperature; separating the nanoparticles adsorbed with nucleic acid using an external magnetic field, and washing with an ethanol solution containing NaCl and deionized water in sequence; and finally eluting the nucleic acid with an appropriate amount of Tris-HCl buffer to obtain a Klebsiella pneumoniae nucleic acid extract; S3: PCR amplification A PCR reaction system is configured, including a specific primer pair, a hot-start Taq DNA polymerase, dNTPs, a PCR buffer, and the nucleic acid extract extracted above; wherein the specific primer pair is designed for the deletion mutation at position 517-518 of the ramR gene; based on the whole genome data of Klebsiella pneumoniae, the mutation hotspot of the ramR gene is accurately located, and through multiple sequence alignment, it is found that the deletion causes an open reading frame shift, which is highly correlated with tigecycline resistance, the forward primer sequence is 5'-GCCGCCAGGTAAAAG-3', and the reverse primer sequence is 5'-GTAAACGGGTAGGTCAGG-3'; The PCR amplification program was as follows: pre-denaturation at 95°C for 3-5 minutes; denaturation at 95°C for 30-45 seconds, annealing at 58-62°C for 30-45 seconds, extension at 72°C for 30-45 seconds, for a total of 30-35 cycles; and finally extension at 72°C for 5-10 minutes. S4: Mutation Detection Add nucleic acid dye Z to the amplified product, raise the temperature from 60°C to 95°C on the HRM instrument, monitor the change of fluorescence signal in real time, and draw a melting curve; S5: Result determination The melting curve of the test sample was compared with the melting curves of the known ramR gene 517-518 deletion mutation positive and negative control samples; at the same time, the characteristic parameters of the melting curve were analyzed in combination with the machine learning algorithm to establish a prediction model.

2. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: The method also includes enriching and culturing the sample before the S1 sample pretreatment step; inoculating the sample into an LB medium containing nutrient additives, wherein the nutrient additives are yeast extract, casamino acids and a synthetic oligopeptide mixture, wherein the oligopeptide mixture is a tripeptide composed of alanine, glycine and serine, and the concentration is 1-2 g / L, and culturing with shaking at 37° C. and 180-220 rpm for 6-8 hours.

3. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: The method further includes, in the S2 nucleic acid extraction step, evaluating the quality of the nucleic acid extract; using an ultraviolet spectrophotometer to measure the absorbance of the nucleic acid extract at wavelengths of 260 nm, 280 nm and 230 nm, and calculating A 260 / A 280 and A 260 / A 230 Ratio, requirement A 260 / A 280 The ratio is between 1.8 and 2.

0. 260 / A 230 The ratio is between 2.0-2.

2.

4. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: In the S3 PCR amplification step, the specific primer pair is modified with phosphorothioate and 2'-O-methyl; at the same time, a locked nucleic acid LNA-modified probe is added to the PCR reaction system at a concentration of 0.1-0.5 μmol / L.

5. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: The concentration of hot-start Taq DNA polymerase in the PCR reaction system is 0.5-1.5U / μL, the concentration of each nucleotide in dNTPs is 0.2-0.4mmol / L, and the PCR buffer contains KCl, Tris-HCl, and MgCl2; and a PCR auxiliary factor W is also added, which is a complex of tris(hydroxymethyl)methylglycine and betaine mixed in a molar ratio of 1:2, and its concentration is 5-10mmol / L; the nucleic acid dye Z is a complex of EvaGreen and a fluorescence enhancer Q mixed in a molar ratio of 1:0.1, and Q is rhodamine B modified with 4-(dimethylamino)benzaldehyde.

6. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: In S5, if the model predicts a positive result, and the melting temperature of the test sample is shifted by ≥1.5°C compared with the negative control, and the ΔCt value is ≥5 compared with the internal reference gene, then the deletion mutation at position 517-518 of the ramR gene is judged to be positive, that is, Klebsiella pneumoniae is resistant to tigecycline; otherwise, it is negative.

7. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: In the S1 sample pretreatment step, if the sample is a blood sample, first add red blood cell lysis solution, incubate at room temperature for 5-10 minutes to lyse the red blood cells, then centrifuge at 3000-5000g for 5-10 minutes, discard the supernatant, and precipitate for subsequent lysis treatment.

8. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: In the S3 PCR amplification step, nested PCR technology is used to design inner primer pairs and outer primer pairs; the outer primer pair is first used for the first round of amplification, and the amplified product is diluted 10-100 times and used as a template for the second round of amplification, and amplification is performed using the inner primer pair.

9. The method for mutation detection in tigecycline resistance detection of Klebsiella pneumoniae according to claim 1, characterized in that: In the S2 nucleic acid extraction step, when nucleic acid extraction is performed using microfluidic chip technology, the chip surface is modified with a hydrophilic polymer.

10. Use of the method according to any one of claims 1 to 9 in preparing a kit for detecting the resistance of Klebsiella pneumoniae to tigecycline, characterized in that: The kit is used for the detection of tigecycline resistance of Klebsiella pneumoniae in clinical diagnosis, epidemiological surveys and antimicrobial sensitivity monitoring, and the kit is equipped with special data analysis software that can automatically import melting curve data, use machine learning algorithms to determine results, and generate test reports.

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