CN118112250A - Method for detecting fluconazole-resistant candida otophylla and construction of protein fingerprint thereof - Google Patents

Abstract

The invention provides a detection method of candida fluoroconazole and a construction of a protein fingerprint thereof. And then, in the actual detection process, through a drug sensitivity test, carrying out MALDI TOF MS to collect a real-time mass spectrum of a sample to be detected, and carrying out full-feature peak comparison with a standard protein fingerprint, thereby rapidly realizing species identification of the fluconazole-resistant candida otophylla. The method has simple operation process, good identification capability on the fluconazole-resistant candida otophylla, and high identification efficiency, thereby greatly reducing the identification cost.

Description

Method for detecting fluconazole-resistant candida otophylla and construction of protein fingerprint thereof

Technical Field

The invention relates to the technical field of medical detection, in particular to a method for detecting fluconazole-resistant candida otophylla and construction of a protein fingerprint spectrum thereof.

Background

Candida otophylla (Candida auris, academic name candida otophylla) is the first fungus found in the secretion of the external auditory meatus of a japanese patient in 2009. Candida otophylla can enter blood of an infected person and spread to the whole body to cause serious invasive infection, and can also be planted at different parts of skin, respiratory tract, urinary tract and the like of a human body, so that blood, lung, urinary tract, surface wound infection, organ and tissue infection of the auditory canal and the like are caused, and the infection mortality rate is up to 60%. And candida otophylla also has extreme survival and reproductive capacity, long survival time in natural environment, strong adhesion capacity and difficult removal. Clinical isolates of candida otophylla are also generally multi-resistant, i.e., the fungus is resistant to a variety of antifungal agents commonly used to treat candida infections, as known from prior studies; statistical analysis of the CDC data in the united states showed that in clinical isolates of candida auriculata, 90% of the strains exhibited high resistance to fluconazole, 30% of the strains exhibited resistance to the polyene drug amphotericin B, and a minority (< 5%) of the strains exhibited resistance to the echinocandin drug caspofungin.

Candida otophylla is difficult to detect and identify by a conventional laboratory method, and before 2017, most laboratory common automatic detection system databases do not contain candida otophylla, so that candida otophylla is easy to be wrongly identified as other similar candida, for example, in a common colony morphology identification method, candida otophylla is cultivated on a komarjia color development flat plate for 48 hours to be light pink or purple, and the candida otophylla can be similar to candida parapsilosis, candida pseudopsilosis, candida glabrata and other strains in color along with the prolonged cultivation time, and delay identification and misdiagnosis are easy to cause. The most widely applied identification method for candida otophylla at present is to utilize a PCR technology, and the identity and the typing of candida otophylla strain can be determined by amplifying ITS and/or D1/D2 rDNA sequences, so that the reliable identification of candida otophylla is realized, but the PCR technology is time-consuming and is not suitable for routine diagnosis.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel identification technology which has wide application in the field of microorganism identification in recent years, can realize rapid and accurate identification of microorganisms, is mainly based on ribosomal protein fingerprint technology at present, and can identify strain level in some cases. In recent years, researchers have begun to explore the application of MALDI-TOF MS to candida otophylla identification, and by adopting a technology of combining MALDI-TOF mass spectrometry with a microbial database, a strain to be tested is measured by a MALDI-TOF mass spectrometer to obtain a unique microbial mass spectrum, and the unique microbial mass spectrum is compared with the spectral peak of candida otophylla in the database to finally obtain an identification result, and the identification method has the advantages of rapidness, high flux and low cost, but also has some disadvantages: the identification method mainly relies on mass spectrum of known strains for identification, and can have difficulty in identifying unknown new species or variant strains, and because candida otophylla belongs to a complex fungus population, the species of candida otophylla have higher similarity, and at present, a representative standard protein fingerprint is lacking, and when the standard protein fingerprint is constructed, the selection of characteristic peaks and the selection of distinguishing strains can cause confusion of mass spectrum during identification; in addition, the heterogeneity of candida otophylla may also cause differences in mass spectra of different strains of the same species, such as differences in mass spectra between candida otophylla resistant to fluconazole and other candida otophylla, thereby affecting the identification results. Meanwhile, the pretreatment process of the sample may affect the generation of the mass spectrum and the accuracy of the identification result, for example, the culture condition and the extraction method of candida otophylla may affect the metabolic products of the sample and the characteristics of the mass spectrum, so that the identification result is affected due to the difference between strains, and the identification accuracy is affected.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art, and provides a method for detecting candida otophyllum resistant to fluconazole and construction of a protein fingerprint thereof, which improves the identification accuracy of candida otophyllum resistant to fluconazole.

The technical scheme provides a method for constructing a protein fingerprint of candida auricularia auricula-judae resistant to fluconazole, which specifically comprises the following steps:

Obtaining at least one target fluconazole-resistant candida otophylla as a representative strain, and carrying out mass spectrum detection on the representative strain to obtain an original map corresponding to the representative strain; extracting characteristic peaks from an original spectrum representing the strain according to an extraction standard to form a protein fingerprint spectrum of the candida fluconazole-resistant candida otophylla;

the representative strain is ATCC MYA-5001;

the extraction standard is as follows: the minimum signal to noise ratio is more than or equal to 500, and the minimum resolution is more than or equal to 800.

Further, the mass spectrum is a matrix-assisted laser desorption ionization time-of-flight mass spectrum.

Further, prior to mass spectrometric detection of the representative strain, a pretreatment process for the representative strain is included;

The pretreatment process adopts a formic acid wall breaking method, and specifically comprises the following steps: uniformly and respectively coating single bacterial colonies representing strains on a target plate to form a plurality of samples; and dropwise adding 0.5-2.0 mu L of formic acid aqueous solution with volume fraction of 60% -80% to the samples, naturally drying, and dropwise adding 0.5-2.0 mu L of CHCA matrix to each sample to form a plurality of targets.

Preferably, for mass spectrometry detection, 6-20 targets are prepared simultaneously for each representative strain, and an original map is acquired based on each target.

The invention also aims to provide a protein fingerprint of candida albicans resistant to fluconazole, which is obtained by constructing by the construction method provided in the technical scheme;

Characteristic peaks in the fluconazole-resistant candida otophylla protein fingerprint spectrum comprise: 17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：(3004-3007),(3016-3019),(3025-3028),(3033-3036),(3044-3047),(3062-3065),(3169-3172),(3253-3256),(3273-3276),(3325-3328),(3336-3339),(3450-3453),(3471-3474),(3505-3508),(3654-3657),(3723-3726),(3794-3797);

The molecular weight is 6000-9000, and the mass-to-charge ratio is 23 ：(6000-6003),(6026-6029),(6043-6046),(6061-6064),(6082-6085),(6103-6016),(6167-6170),(6187-6190),(6206-6209),(6266-6269),(6403-6406),(6499-6502),(6543-6546),(6582-6585),(6627-6630),(6645-6648),(6667-6670),(6684-6687),(6708-6711),(6728-6731),(6895-6898),(7304-7307),(8078-8081);

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: (9624-9627), (12339-12342).

Preferably, characteristic peaks in the candida otodeae protein fingerprint spectrum of the fluconazole-resistant candida otodeae comprise: 17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：3005.51,3017.58,3026.48,3034.85,3045.54,3063.34,3170.34,3254.06,3274.65,3326.07,3337.4,3451.67,3472.42,3506.4,3655.65,3724.37,3795.47;

The molecular weight is 6000-9000, and the mass-to-charge ratio is 23 ：6001.19,6027.62,6044.23,6062.9,6083.18,6104.75,6168.42,6188.56,6207.45,6267.2,6404.18,6500.13,6544.13,6583.53,6628.8,6646.89,6668.96,6685.45,6709.4,6729.59,6896.54,7305.38,8079.85;

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: 9625.72, 12340.65.

The invention further aims at providing a detection method of fluconazole-resistant candida otophylla, which specifically comprises the following steps:

s1, performing a fluconazole drug sensitivity test on a strain to be tested;

S2, mass spectrum detection is carried out on the strain to be detected, and a mass-charge ratio peak diagram of the strain to be detected is obtained; comparing the mass-to-charge ratio peak diagram of the strain to be detected with the protein fingerprint according to any one of claims 5-6, and identifying the strain to be detected as or containing the fluconazole-resistant candida otophylla when the mass-to-charge ratio peak diagram of the strain to be detected contains all the characteristic peaks;

The mass spectrum adopts matrix-assisted laser to analyze the time-of-flight mass spectrum;

the method is not directed to the diagnosis or treatment of a disease.

Further, before mass spectrum detection is carried out on the strain to be detected, a pretreatment process of the strain to be detected is further included; the pretreatment process adopts a formic acid wall breaking method, and specifically comprises the following steps: uniformly spreading the bacterial colony of the strain to be tested on a target plate to form a plurality of samples to be tested; and dropwise adding 0.5-2.0 mu L of formic acid aqueous solution with volume fraction of 60% -80% to the samples to be tested, naturally drying, and dropwise adding 0.5-2.0 mu L of CHCA matrix to each sample to be tested to form a plurality of targets to be tested.

Further, before mass spectrum detection is carried out on the strain to be detected, a mass calibration process for mass spectrum is further included;

The quality calibration process specifically comprises the following steps: selecting a calibration strain, and carrying out mass spectrum detection on the calibration strain to obtain a mass-to-charge ratio peak diagram of the calibration strain; selecting a plurality of calibration peaks from the mass-to-charge ratio peak diagram of the calibration strain, wherein the number of the calibration peaks is not less than 3, observing the relative deviation of each calibration peak, and considering that the calibration is successful when the relative deviation of each calibration peak is less than 300 ppm.

Further, the calibration strain used was escherichia coli, accession No. ATCC 8739;

the number of the calibration peaks is 4, and the mass-to-charge ratios of the calibration peaks are respectively as follows: 4365.40, 6255.40, 7424.50, 9538.00.

Compared with the prior art, the invention has the beneficial effects that:

1. the standard protein fingerprint of the candida albicans resistant to the fluconazole is constructed by selecting representative candida albicans resistant to the fluconazole, forming a mass spectrum based on a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDITOF MS) technology, and extracting characteristic peaks according to a specific characteristic peak extraction principle. And then, in the actual detection process, through a drug sensitivity test, carrying out MALDI TOF MS to collect a real-time mass spectrum of a sample to be detected and carrying out full-feature peak comparison on the real-time mass spectrum and a standard protein fingerprint spectrum, thereby rapidly realizing the identification of the species of the candida albicans resistant to fluconazole. The method has simple operation process, good identification capability on the fluconazole-resistant candida otophylla, and high identification efficiency, thereby greatly reducing the identification cost.

2. The formic acid wall breaking method is uniformly adopted for the pretreatment method of the candida auriculata-resistant representative strain or the sample to be tested, so that the influence of different strains on the generation of mass spectrum and the accuracy of identification results by adopting different sample pretreatment methods is avoided.

Drawings

Fig. 1 is a standard protein fingerprint of candida otophyllum resistant to fluconazole provided by the invention.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The invention will now be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention. The test specimens and test procedures used in the following examples include those (if the specific conditions of the experiment are not specified in the examples, generally according to conventional conditions or according to the recommended conditions of the reagent company; the reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified).

The MALDITOF MS technology is applied to microorganism detection, and the accuracy of the identification result is greatly dependent on the fingerprint spectrum and characteristic peak of the corresponding protein in the database. Because clinical infection of the fluconazole-resistant candida auricularis is relatively rare, a representative standard protein fingerprint is lacking, and meanwhile, the mass spectrum is often insufficient in acquired mass spectrum information and the like caused by improper pretreatment process of a sample or poor instrument performance, so that the accuracy of mass spectrum identification is influenced, and the clinical application of the fluconazole-resistant candida auricularis is greatly restricted.

In view of the above, the present technical solution provides a method for constructing a protein fingerprint of candida aureobasidium fluconazole-resistant, which specifically includes:

Obtaining at least one target fluconazole-resistant candida otophylla as a representative strain, and carrying out mass spectrum detection on the representative strain to obtain an original map corresponding to the representative strain; extracting characteristic peaks from an original spectrum representing the strain according to an extraction standard to form a protein fingerprint spectrum of the candida fluconazole-resistant candida otophylla;

Wherein, the selected representative strain in the candida albicans resistant to fluconazole can be derived from the existing strain resource library or a self-collection mode; preferably, the representative strain selected is ATCC MYA-5001.

After the original spectrum corresponding to the representative strain is obtained, characteristic peaks are extracted from the original spectrum according to an extraction standard with the minimum signal to noise ratio more than or equal to 500 and the minimum resolution more than or equal to 800.

Preferably, the mass spectrum is a matrix assisted laser desorption ionization time-of-flight mass spectrum.

Further, before mass spectrometry detection is carried out on the representative strain, a pretreatment process of the representative strain is further included, wherein the pretreatment process adopts a formic acid wall breaking method. The candida auricularia auricula is a yeast-like fungus, the cell wall of the candida auricularia auricula is thicker than that of bacteria, and the complete and good protein map is difficult to obtain by direct coating, so that the credibility of the identification result is affected, the influence of the thick and tough cell wall can be overcome by adopting a formic acid wall breaking method, so that intracellular proteins can be easily separated, the operation is simple, and meanwhile, the complete ribosomal proteins can be obtained to a large extent. The pretreatment process specifically comprises the following steps: uniformly and separately coating single bacterial colonies representing strains on a target plate to form a plurality of samples; and dropwise adding 0.5-2.0 mu L of formic acid water solution with volume fraction of 60% -80% to the samples, naturally drying, and dropwise adding 0.5-2.0 mu L of CHCA matrix to each sample to form a plurality of targets.

Preferably, the pretreatment process specifically includes: uniformly spreading single bacterial colonies representing strains cultured overnight on a target plate to form a plurality of samples, and spreading each sample as evenly as possible to form a thin layer; and 1.0 mu L of 70% formic acid aqueous solution is dripped on the dried samples as soon as possible, and after natural drying, 1.0 mu L of CHCA matrix is dripped on each sample to form a plurality of targets.

Further, in order to obtain stable, reliable and representative characteristic peaks from the obtained mass spectrogram, besides filtering peaks according to a certain extraction standard, a plurality of parallel targets are constructed based on the representative strain, so that a sufficient number of original spectrograms are obtained, wherein each representative strain can simultaneously prepare 6-20 targets, and the original spectrograms are acquired based on each target.

Preferably, not less than 6 effective original spectra are collected per strain, and the effective original spectra can be understood as the original spectra after screening.

In the screening process of the original spectrogram, the principle of the screening rule comprises the following steps: removing the patterns with fewer peaks, lower peak intensity, obviously no peaks and abnormal peaks; the judgment of the degree of "less", "lower", and "abnormal" is one skilled in the art based on practical situations and experience. Preferably, the screening rule may specifically include: removing the original spectrum with the number of effective peaks less than or equal to 20, the peak intensity less than or equal to 1000mV and/or no peak.

When extracting characteristic peaks based on the effective original spectrum, optionally, the rules of peak filtering at least comprise more than two of the following characteristics: 1. the main peak and the secondary peak are distinct, the peak distribution is staggered, the resolution (m/delta m) reaches more than 800-1200, and the peaks are in an elongated shape; 2. baseline smoothing, S/N greater than 500; 3. the main peak signal intensity is proper (1000-10000).

The construction method provided by the invention can identify and extract stable and reliable characteristic peaks with representativeness, thereby constructing the standard protein fingerprint of the fluconazole-resistant candida otophylla; further, when constructing a database, introducing as many standard protein fingerprints of approximate strains which are easy to be confused with the fluconazole-resistant candida otophylla into the database as possible; and then effectively differentiate the similar strains of the fluconazole-resistant candida otophylla, candida albicans, candida huperzia, and the like, has high sensitivity, short identification time, good identification effect and large flux, and can carry out screening identification in a large scale with low cost.

The invention also provides a protein fingerprint of the candida albicans resistant to fluconazole, which is obtained by constructing the protein fingerprint by the construction method provided in the technical scheme;

Characteristic peaks in the candida otophylla protein fingerprint spectrum of the fluconazole comprise: 17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：(3004-3007),(3016-3019),(3025-3028),(3033-3036),(3044-3047),(3062-3065),(3169-3172),(3253-3256),(3273-3276),(3325-3328),(3336-3339),(3450-3453),(3471-3474),(3505-3508),(3654-3657),(3723-3726),(3794-3797);

The molecular weight is 6000-9000, and the mass-to-charge ratio is 23 ：(6000-6003),(6026-6029),(6043-6046),(6061-6064),(6082-6085),(6103-6016),(6167-6170),(6187-6190),(6206-6209),(6266-6269),(6403-6406),(6499-6502),(6543-6546),(6582-6585),(6627-6630),(6645-6648),(6667-6670),(6684-6687),(6708-6711),(6728-6731),(6895-6898),(7304-7307),(8078-8081);

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: (9624-9627), (12339-12342).

Preferably, characteristic peaks in the fluconazole-resistant candida otoxin fingerprint spectrum comprise: 17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：3005.51,3017.58,3026.48,3034.85,3045.54,3063.34,3170.34,3254.06,3274.65,3326.07,3337.4,3451.67,3472.42,3506.4,3655.65,3724.37,3795.47;

The molecular weight is 6000-9000, and the mass-to-charge ratio is 23 ：6001.19,6027.62,6044.23,6062.9,6083.18,6104.75,6168.42,6188.56,6207.45,6267.2,6404.18,6500.13,6544.13,6583.53,6628.8,6646.89,6668.96,6685.45,6709.4,6729.59,6896.54,7305.38,8079.85;

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: 9625.72, 12340.65.

The invention further aims at providing a detection method of fluconazole-resistant candida otophylla, which specifically comprises the following steps:

s1, performing a fluconazole drug sensitivity test on a strain to be tested;

S2, mass spectrum detection is carried out on the strain to be detected, and a mass-charge ratio peak diagram of the strain to be detected is obtained; comparing the mass-to-charge ratio peak diagram of the strain to be detected with a standard protein fingerprint, and identifying the strain to be detected as or containing the fluconazole-resistant candida otophylla when the mass-to-charge ratio peak diagram of the strain to be detected contains all characteristic peaks;

wherein, the mass spectrum adopts matrix-assisted laser to analyze the time-of-flight mass spectrum.

In the step S1, the specific method for performing the fluconazole drug sensitivity test on the strain to be tested comprises the following steps:

Firstly, a micro-liquid culture medium dilution method is adopted to prepare a culture medium of fluconazole, in particular to the Fluconazole (FLC), flucytosine (5-FC), amphotericin B (AMB), itraconazole (ITC) and Ketoconazole (KTC) which are respectively dissolved by sterile distilled water and 100% DMSO (dimethyl sulfoxide) and diluted to a proper concentration of 1:9, mixing the above medicinal liquid with mould culture medium to obtain culture medium containing serial medicine concentrations, and packaging into test tubes for use.

Secondly, the strain to be tested is strictly prepared into bacterial liquid according to the requirement of CLSIM-27-A broth dilution method, and is inoculated into each tube after double ratio dilution, and is respectively incubated for 48 hours at 35 ℃. And a positive control tube of menstruum, a blank tube of culture medium and candida is arranged. MIC (minimum inhibitory concentration) determination criterion for candida in vitro drug sensitive assay prescribed by CLSIM-a protocol: the MIC of FLC is less than or equal to 8.0mg/L and is sensitive, 16.0-32.0mg/L is medium, 64.0mg/L is drug resistant; MIC 1.0mg/L of AMB is sensitive, 2.0-4.0mg/L is medium; 8.0mg/L is drug resistance; MIC of ITC is less than or equal to 0.125mg/L and is sensitive, 0.25-0.50mg/L is medium, 1.0mg/L is drug resistant; the MIC of KTC is less than or equal to 0.5mg/L and is sensitive, 1.0-2.0mg/L is medium, and 4.0mg/L is drug resistant. MIC of 5-FC is less than or equal to 4.0mg/L and is sensitive, 8.0-16.0mg/L is medium, and 32.0mg/L is drug resistant. The result judges that the sterile growing medicine-containing tube is the MIC of the bacterium, and simultaneously judges the MIC50 (the medicine concentration with the bacteriostasis rate of 50 percent) and the MIC90; wherein, pyrrole and fluorocytosine adopt the loose standard of MIC50, is used for reducing the influence of tailing or partial inhibition on result interpretation, improve the repeatability among rooms; amphotericin B adopts the loose standard of MIC 90.

The detection method does not take diagnosis or treatment of diseases as a direct aim, is applied to the situation that a sample to be detected is an artificial sample (such as a positive sample or a negative sample) or an environmental sample, and can directly and effectively acquire and compare the performance (such as the effectiveness, the accuracy, the stability and other parameters) of the detection method, or detect and acquire whether the sample contains the fluconazole-resistant candida otophyllum, so as to effectively distinguish the fluconazole-resistant candida otophyllum, candida albicans, candida similaris and other similar strains.

Further, before mass spectrum detection is carried out on the strain to be detected, a pretreatment process of the strain to be detected is further included; the pretreatment process adopts a formic acid wall breaking method, and specifically comprises the following steps: uniformly spreading bacterial colonies of the strain to be tested on a target plate to form a plurality of samples to be tested; and dropwise adding 0.5-2.0 mu L of formic acid aqueous solution with volume fraction of 60% -80% to the samples to be tested, naturally drying, and dropwise adding 0.5-2.0 mu L of CHCA matrix to each sample to be tested to form a plurality of targets to be tested. Preferably, the pretreatment process specifically includes: uniformly spreading colonies of the strains to be tested, which are cultured overnight, on a target plate to form a plurality of samples to be tested, and spreading each sample to be tested as evenly as possible to form a thin layer; and 1.0 mu L of formic acid aqueous solution with the volume fraction of 70% is dripped onto the dried samples to be detected as soon as possible, after natural drying, 1.0 mu L of CHCA matrix is dripped into each sample to be detected, a plurality of target spots to be detected are formed, and then the samples are detected by a machine.

Further, before mass spectrum detection is carried out on the strain to be detected, a mass calibration process for mass spectrum is further included; the method specifically comprises the following steps: selecting a calibration strain, and carrying out mass spectrum detection on the calibration strain to obtain a mass-to-charge ratio peak diagram of the calibration strain; selecting a plurality of calibration peaks from a mass-to-charge ratio peak diagram of the calibration strain, wherein the number of the calibration peaks is not less than 3, observing the relative deviation of each calibration peak, and considering that the calibration is successful when the relative deviation of all the calibration peaks is less than 300 ppm. Before each batch of sample detection, the calibration strain should be analyzed first to ensure the reliability of the mass spectrometer, so the mass spectrometer should be calibrated before the representative strain or the strain to be detected is detected on-line.

The selection and determination principle of the calibration strain and the calibration characteristic peak which can be used as the candida albicans with fluconazole resistance is as follows: the molecular weight range of the calibration strain should cover more than 60% of the identification detection range (2000-20000 Da) of the strain to be detected, and at least more than 8 standard peaks detected by the calibration strain should be identified. Wherein, the selected calibration characteristic peak is positioned in the concentrated range of the characteristic peak of the standard protein fingerprint of the target fluconazole-resistant candida otodea.

Preferably, the calibration strain is Escherichia coli, accession number ATCC 8739; the number of the adopted calibration peaks is 4, and the mass-to-charge ratios are respectively as follows: 4365.40, 6255.40, 7424.50, 9538.00.

Preferably, the method for identifying whether the strain to be detected is or contains the candida albicans resistant to fluconazole can be carried out through comparison software, for example, CMI TOF software can be adopted for data identification, and the comparison score is more than or equal to a set threshold value, and the judgment is positive; otherwise, the result is negative. The software automatically calculates the selectable threshold and divides the result confidence level according to the standard, and the technician can choose to set the threshold based on the result confidence level. Optionally, the set threshold includes, but is not limited to, 2.0.

Example 1

The method for constructing the fluconazole-resistant candida otophylla protein fingerprint specifically comprises the following steps:

The selected representative strain of the candida albicans resistant to fluconazole is ATCC MYA-5001;

Pretreatment process: uniformly and separately coating single bacterial colonies representing strains cultured overnight on a target plate by adopting a formic acid wall breaking method to form a plurality of samples, and uniformly spreading each sample as much as possible to form a thin layer; and 1.0 mu L of 70% formic acid aqueous solution is dripped on the dried samples as soon as possible, after natural drying, 1.0 mu L of CHCA matrix is dripped on each sample to form a plurality of targets, and the samples are completely dried and then are detected by a machine.

The adopted mass spectrometer is CMI 1600 of He Xin kang Yuan medical science and technology Co., ltd, and the mass spectrum detection conditions are as follows: acceleration voltage is 22100V, focusing voltage is 5000V, and detection voltage is 1600V; pulse 1 is 1100V, pulse 2 is 700V, ion screening is 477Da, ion delay is 500ns, sampling rate is 2000MH, and mass spectrum range is 2000 Da-20000 Da; laser energy 20%70%, laser frequency 50200Hz, accumulation times 100500, peak smoothness 50%95%, background noise 20%60%, and minimum signal to noise ratio 3.0.

The calibration process comprises the following steps: before the original spectrum of the representative strain is collected, the mass spectrometer is calibrated through a calibration strain, wherein the calibration strain adopts escherichia coli with the number of ATCC 8739; mass spectrum detection is carried out on the calibration strain to obtain a mass-to-charge ratio peak diagram of the calibration strain, the adopted calibration peaks are 4 known peaks, and the mass-to-charge ratios are respectively as follows: 4365.40, 6255.40, 7424.50, 9538.00, and observing the relative deviation magnitudes of the calibration peaks, a calibration is considered successful when the relative deviations of the calibration peaks are less than 300 ppm.

The detection process comprises the following steps: each representative strain prepares 8 targets, and after the instrument is calibrated, the original map of each target is collected in a manual or automatic mode; removing spectrograms with fewer effective peaks (less than 20), lower peak intensity (less than 1000 mv) or no peak, selecting an original spectrogram with optimal quality from the spectrograms, collecting at least 6 effective spectrograms for each representative strain, and carrying out data supplementary acquisition on low-quality sample points by adopting a manual mode.

The original map meeting the requirements of library construction is imported by using characteristic peak extraction software (HXKY. MicroCreate. Exe), and the peak filtering standard is as follows: the main peak and the secondary peak are distinct, the peak distribution is staggered, the resolution (m/delta m) reaches more than 800-1200, and the peaks are in an elongated shape; baseline smoothing, S/N greater than 500; the main peak signal intensity is proper (1000-10000), and the extracted standard fluconazole-resistant candida otophylla protein fingerprint is shown in figure 1.

The standard fluconazole-resistant candida otophylla protein fingerprint comprises polypeptide or protein characteristic peaks with molecular weights of 3000-20000, and the standard protein fingerprint is drawn by different characteristic peaks and relative intensities:

17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：3005.51,3017.58,3026.48,3034.85,3045.54,3063.34,3170.34,3254.06,3274.65,3326.07,3337.4,3451.67,3472.42,3506.4,3655.65,3724.37,3795.47;

The molecular weight is 6000-9000, and the mass-to-charge ratio is 23 ：6001.19,6027.62,6044.23,6062.9,6083.18,6104.75,6168.42,6188.56,6207.45,6267.2,6404.18,6500.13,6544.13,6583.53,6628.8,6646.89,6668.96,6685.45,6709.4,6729.59,6896.54,7305.38,8079.85;

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: 9625.72, 12340.65.

Example 2

The detection method of the fluconazole-resistant candida otophylla specifically comprises the following steps:

collecting 23 candida otophylla from a clinical laboratory of a hospital as a strain to be tested;

s1, performing a fluconazole drug sensitivity test on a strain to be tested;

The specific method comprises the following steps: firstly, a micro-liquid culture medium dilution method is adopted to prepare a culture medium of fluconazole, in particular to the Fluconazole (FLC), flucytosine (5-FC), amphotericin B (AMB), itraconazole (ITC) and Ketoconazole (KTC) which are respectively dissolved by sterile distilled water and 100% DMSO (dimethyl sulfoxide) and diluted to a proper concentration of 1:9, mixing the above medicinal liquid with mould culture medium to obtain culture medium containing serial medicine concentrations, and packaging into test tubes for use.

Secondly, the strain to be tested is strictly prepared into bacterial liquid according to the requirement of CLSIM-27-A broth dilution method, and is inoculated into each tube after double ratio dilution, and is respectively incubated for 48 hours at 35 ℃. And a positive control tube of menstruum, a blank tube of culture medium and candida is arranged. MIC (minimum inhibitory concentration) determination criterion for candida in vitro drug sensitive assay prescribed by CLSIM-a protocol: the MIC of FLC is less than or equal to 8.0mg/L and is sensitive, 16.0-32.0mg/L is medium, 64.0mg/L is drug resistant; MIC 1.0mg/L of AMB is sensitive, 2.0-4.0mg/L is medium; 8.0mg/L is drug resistance; MIC of ITC is less than or equal to 0.125mg/L and is sensitive, 0.25-0.50mg/L is medium, 1.0mg/L is drug resistant; the MIC of KTC is less than or equal to 0.5mg/L and is sensitive, 1.0-2.0mg/L is medium, and 4.0mg/L is drug resistant. MIC of 5-FC is less than or equal to 4.0mg/L and is sensitive, 8.0-16.0mg/L is medium, and 32.0mg/L is drug resistant. The result judges that the sterile growing medicine-containing tube is the MIC of the bacterium, and simultaneously judges the MIC50 (the medicine concentration with the bacteriostasis rate of 50 percent) and the MIC90; wherein, pyrrole and fluorocytosine adopt the loose standard of MIC50, is used for reducing the influence of tailing or partial inhibition on result interpretation, improve the repeatability among rooms; amphotericin B adopts the loose standard of MIC 90.

The results show that the MIC of the 23 candida otophylla FLC tested is 256mg/L, the MIC of the AMB is 2mg/L, the MIC of the ITC is 0.5mg/L, the MIC of the KTC is 1mg/L, and the MIC of the 5-FC is 8mg/L; thus indicating that all 23 candida otobacteria tested were fluconazole-resistant candida otobacteria.

S2, mass spectrum detection is carried out on the strain to be detected, and a mass-charge ratio peak diagram of the strain to be detected is obtained according to the consistent pretreatment process, calibration process and detection process as in the embodiment 1; comparing the mass-to-charge ratio peak diagram of the strain to be detected with the standard protein fingerprint constructed in the embodiment 1, and identifying the strain to be detected as or containing the fluconazole-resistant candida otophylla when the mass-to-charge ratio peak diagram of the strain to be detected contains all characteristic peaks; the CMI TOF software is adopted to carry out data identification on the acquired mass-to-charge ratio peak diagram of the strain to be detected, the threshold value is set to be 2.0, the data identification result shows that all characteristic peaks corresponding to the comparison standard protein fingerprint are successfully identified in all effective mass-to-charge ratio peak diagrams of the candida 23 strains, the comparison score is more than 2.7 and is far greater than the set threshold value of 2.0, the positive result of the candida 23 strains to be detected when the candida strains to be detected are identified as the candida bifonazole is prompted, and the identification result is highly reliable. In order to further verify the identity of the obtained 23 candida auriculata and candida auriculata-type candida fluconazole-resistant strains, the color development identification is carried out by carrying out the color development flat-plate culture for 48 hours, meanwhile, the sequencing identification is carried out on the obtained 23 candida auriculata, and the results show that the 23 candida auriculata to be detected is candida auriculata-type candida fluconazole-resistant.

Claims (10)

1. The method for constructing the protein fingerprint of the candida auricularia fluconazole-resistant candida is characterized by comprising the following steps of:

Obtaining at least one target fluconazole-resistant candida otophylla as a representative strain, and carrying out mass spectrum detection on the representative strain to obtain an original map corresponding to the representative strain; extracting characteristic peaks from an original spectrum representing the strain according to an extraction standard to form a protein fingerprint spectrum of the candida fluconazole-resistant candida otophylla;

the extraction standard is as follows: the minimum signal to noise ratio is more than or equal to 500, and the minimum resolution is more than or equal to 800.

2. The method of claim 1, wherein the mass spectrum is a matrix-assisted laser desorption ionization time-of-flight mass spectrum.

3. The method of any one of claims 1-2, further comprising a pretreatment of the representative strain prior to mass spectrometric detection of the representative strain;

The pretreatment process adopts a formic acid wall breaking method, and specifically comprises the following steps: uniformly and respectively coating single bacterial colonies representing strains on a target plate to form a plurality of samples; and dropwise adding 0.5-2.0 mu L of formic acid aqueous solution with volume fraction of 60% -80% to the samples, naturally drying, and dropwise adding 0.5-2.0 mu L of CHCA matrix to each sample to form a plurality of targets.

4. A method of constructing according to claim 3, wherein in mass spectrometry detection, 6 to 20 targets are prepared simultaneously for each representative strain, and an original profile is acquired based on each target.

5. A protein fingerprint of fluconazole-resistant candida otophylla, characterized in that the protein fingerprint is constructed by the construction method according to any one of claims 1-4;

characteristic peaks in the fluconazole-resistant candida otophylla protein fingerprint spectrum comprise: 17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：(3004-3007),(3016-3019),(3025-3028),(3033-3036),(3044-3047),(3062-3065),(3169-3172),(3253-3256),(3273-3276),(3325-3328),(3336-3339),

(3450-3453)，(3471-3474)，(3505-3508)，(3654-3657)，(3723-3726)，(3794-3797)；

The molecular weight is 6000-9000, 23 characteristic peaks are provided, and the mass-to-charge ratios are respectively as follows: (6000-6003), (6026-6029),

(6043-6046)，(6061-6064)，(6082-6085)，(6103-6016)，(6167-6170)，(6187-6190)，

(6206-6209)，(6266-6269)，(6403-6406)，(6499-6502)，(6543-6546)，(6582-6585)，

(6627-6630)，(6645-6648)，(6667-6670)，(6684-6687)，(6708-6711)，(6728-6731)，

(6895-6898)，(7304-7307)，(8078-8081)；

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: (9624-9627), (12339-12342).

6. The protein fingerprint according to claim 5, wherein the characteristic peaks in the fluconazole-resistant candida otoxin fingerprint comprise: 17 characteristic peaks with molecular weight of 3000-6000 and mass-to-charge ratio of respectively ：3005.51,3017.58,3026.48,3034.85,3045.54,3063.34,3170.34,3254.06,3274.65,3326.07,3337.4,3451.67,3472.42,3506.4,3655.65,3724.37,3795.47;

The molecular weight is 6000-9000, and the mass-to-charge ratio is 23 ：6001.19,6027.62,6044.23,6062.9,6083.18,6104.75,6168.42,6188.56,6207.45,6267.2,6404.18,6500.13,6544.13,6583.53,6628.8,6646.89,6668.96,6685.45,6709.4,6729.59,6896.54,7305.38,8079.85;

The molecular weight has 2 characteristic peaks in 9000-13000, and the mass-to-charge ratios are respectively as follows: 9625.72, 12340.65.

7. The method for detecting the candida otophylla resistant to fluconazole is characterized by comprising the following steps of:

s1, performing a fluconazole drug sensitivity test on a strain to be tested;

S2, mass spectrum detection is carried out on the strain to be detected, and a mass-charge ratio peak diagram of the strain to be detected is obtained; comparing the mass-to-charge ratio peak diagram of the strain to be detected with the protein fingerprint according to any one of claims 5-6, and identifying the strain to be detected as or containing the fluconazole-resistant candida otophylla when the mass-to-charge ratio peak diagram of the strain to be detected contains all the characteristic peaks;

The mass spectrum adopts matrix-assisted laser to analyze the time-of-flight mass spectrum;

the method is not directed to the diagnosis or treatment of a disease.

8. The method according to claim 7, further comprising a pretreatment process for the strain to be tested before mass spectrometry; the pretreatment process adopts a formic acid wall breaking method, and specifically comprises the following steps: uniformly spreading the bacterial colony of the strain to be tested on a target plate to form a plurality of samples to be tested; and dropwise adding 0.5-2.0 mu L of formic acid aqueous solution with volume fraction of 60% -80% to the samples to be tested, naturally drying, and dropwise adding 0.5-2.0 mu L of CHCA matrix to each sample to be tested to form a plurality of targets to be tested.

9. The method according to claim 7, further comprising a mass calibration process for mass spectrometry prior to mass spectrometry of the strain to be tested;

The quality calibration process specifically comprises the following steps: selecting a calibration strain, and carrying out mass spectrum detection on the calibration strain to obtain a mass-to-charge ratio peak diagram of the calibration strain; selecting a plurality of calibration peaks from the mass-to-charge ratio peak diagram of the calibration strain, wherein the number of the calibration peaks is not less than 3, observing the relative deviation of each calibration peak, and considering that the calibration is successful when the relative deviation of each calibration peak is less than 300 ppm.

10. The method according to claim 9, wherein the calibration strain is escherichia coli, ATCC 8739;

the number of the calibration peaks is 4, and the mass-to-charge ratios of the calibration peaks are respectively as follows: 4365.40, 6255.40, 7424.50, 9538.00.