Candida blood culture positive sample rapid detection method and system

Abstract

This invention discloses a rapid detection method and system for Candida blood culture positive samples, belonging to the field of microbial detection technology. The rapid detection method for Candida blood culture positive samples includes the following steps: S1. Candida is enriched in the lysed Candida blood culture positive sample using magnetic beads. During enrichment, a 50%~99.5% (v / v) DMSO solution is used as a washing buffer to wash the magnetic beads containing Candida to obtain a Candida-magnetic bead complex; S2. Candida in the Candida-magnetic bead complex is eluted from the magnetic beads using an elution buffer, and Candida is lysed to obtain Candida lysate; S3. Target sites 1 and 2 are prepared from the Candida lysate and subjected to mass spectrometry detection; S4. The mass spectrometry spectrum is compared and interpreted with the microbial database of the mass spectrometry system. The detection method and system of this invention can effectively exceed 80% in both identification and detection rates of Candida, reaching over 90%.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a rapid detection method and system for Candida blood culture positive samples. Background Technology

[0002] Bloodstream infection is a serious systemic infectious disease in which pathogenic microorganisms exist transiently, intermittently, or persistently in the circulating blood, causing damage to all organs of the body. In severe cases, it can lead to shock, multiple organ failure, and even death. Currently, positive bloodstream infection samples can be rapidly and accurately detected using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The detection process is as follows: the sample is pretreated to separate and obtain the mass spectrometry target sample, which contains characteristic substances of the lysed pathogen, such as ribosomal proteins; the mass spectrometry target sample is mixed with the matrix and applied to a target plate, dried to obtain the target sample, commonly known as "targeting"; then the target sample is sent to the mass spectrometry system for detection to obtain the mass spectrum.

[0003] The mass spectrometry system compares the mass spectrum with microbial characteristic spectra stored in the mass spectrometry database (including the presence or absence of characteristic peaks, peak similarity, and peak intensity consistency) to match the species information of the microorganisms in the sample, and provides the matching result m and confidence score r. The higher the confidence score, the more accurate and reliable the corresponding matching result.

[0004] "Fungal blood culture positive samples" often have low initial bacterial counts, making it difficult to break down bacterial cells and release sufficient protein. During MALDI-TOF-MS detection, the proteins in the sample compete with fungal proteins for crystallization, generating extremely high background noise. Other components also interfere with mass spectrometry detection, severely hindering the detection of bacterial characteristic peaks and preventing the acquisition of effective microbial spectra. This leads to identification failure or unreliable results, manifesting as undetectable samples, large biases, or poor repeatability. For example, some domestic and international studies have reported:

[0005] ① Morgenthaler et al. reviewed 21 independent studies, all of which used the commercially available Sepsityper Kit (Bruker, Bremen, Germany) to pretreat positive blood culture samples from sepsis patients before detection. Eleven of these studies involved fungal identification, with an overall concordance rate of 66% for yeast species identification (the concordance rate varied slightly depending on the operational details and mass spectrometry identification scoring criteria used in different studies). [Morgenthaler NG, Kostrzewa M. Rapid identification of pathogens in positive blood culture of patients with sepsis: review and meta-analysis of the performance of the sepsityper kit[J]. Int J Microbiol, 2015, 2015: 827416. DOI: 10.1155 / 2015 / 827416.]

[0006] ② A study by Tian et al. reported that using commercially available serum separation tubing (BD Medical, USA) for pretreatment of blood culture-positive samples resulted in a fungal detection rate of 75.7%. [Tian Y, Zheng B, Wang B, et al. Rapid identification and multiple susceptibility testing of pathogens from positive-culture sterile body fluids by a combined MALDI-TOF mass spectrometry and Vitek susceptibility system[J]. Front Microbiol, 2016, 7: 523.DOI: 10.3389 / fmicb.2016.00523.]

[0007] ③ Zhang et al. reported that using the Sepsityper Kit (Brück, Bremen, Germany) to pretreat blood culture-positive samples, the species and genus identification accuracy of Candida was 44.4% (4 / 9) for blood culture samples infected with a single strain. [Zhang Feifei, Wang Qi, Li Henan, et al. Rapid identification of blood culture-positive pathogens by Sepsityper Kit or serum separation gel combined with MALDI-TOF MS [J]. Chinese Journal of Microbiology and Immunology, 2018, 38(2): 111-115. DOI: 10.3760 / cma.j.issn.0254-5101.2018.02.006.]

[0008] ④ Li et al. reported that using the Sepsityper Kit (Brück, Bremen, Germany) to pretreat positive blood culture samples before detection, the species and genus identification accuracy of Candida was 75.0% (27 / 36) for single-strain infected blood culture samples. [Li Yuanrui, Yu Jing, Liu Jingxian, et al. Direct identification of positive blood culture specimens using MSK kit-mass spectrometry [J]. Journal of Shanghai Jiaotong University (Medical Edition), 2016, 36(2): 256-263. DOI: 10.3969 / j.issn.1674-8115.2016.02.021.]

[0009] Currently, clinical pretreatment methods for rapid mass spectrometry identification of positive blood culture samples include magnetic beading and centrifugation. While magnetic beading is effective for bacterial samples, it is less effective for fungal samples, especially Candida. Centrifugation is complex, time-consuming, and lacks automation, making it inconvenient for clinical application, and its detection results for fungal samples are also unsatisfactory. Clinically, there is a lack of rapid and effective detection methods for fungal samples, especially Candida. Summary of the Invention

[0010] To address the technical problem that existing detection methods have poor accuracy and detection rates for fungi, especially Candida, and are difficult to exceed 80%, this invention provides a rapid detection method and system for Candida blood culture positive samples.

[0011] The technical solution of the present invention is as follows:

[0012] A rapid detection method for Candida blood culture-positive samples includes the following steps:

[0013] S1. Candida albicans blood culture positive samples after lysis were enriched with Candida albicans using magnetic bead method. During the enrichment process, DMSO solution with a volume ratio of 50% to 99.5% was used as a washing buffer to wash the magnetic beads bound with Candida albicans to obtain Candida albicans-magnetic bead complex.

[0014] S2. Using an elution buffer, Candida albicans in the Candida-magnetic bead complex is eluted from the magnetic beads and Candida albicans is lysed to obtain a Candida lysate;

[0015] S3. Prepare target 1 and target 2 from the Candida lysate and perform mass spectrometry detection.

[0016] S4. Compare and interpret the mass spectrometry results with the microbial database of the mass spectrometry system;

[0017] The comparison includes: the mass spectrometry system performs mass spectrometry detection on target 1 and target 2 respectively to obtain their respective mass spectra, and compares them with the bacterial species database of the mass spectrometry system to obtain the bacterial species results and genus results corresponding to target 1 and target 2, and assigns confidence scores to target 1 and target 2 respectively to obtain r1 and r2.

[0018] The interpretation includes: (1) performing an L-value calculation method according to the following steps:

[0019] when At that time, L=1;

[0020] when At that time, L=2;

[0021] Neither There is none. At that time, L=0;

[0022] in,

[0023] δ s =I( M 1= M 2) is the microbial consistency indicator function, where 1 indicates consistency and 0 indicates inconsistency;

[0024] δ g =I( g 1= g 2) is the genus consistency indicator function, where 1 represents consistency and 0 represents inconsistency;

[0025] r1 is the confidence score assigned after the mass spectrometer performs mass spectrometry detection on target 1;

[0026] r2 is the confidence score assigned after the mass spectrometer performs mass spectrometry detection on target 2;

[0027] M 1 shows the bacterial species results obtained after mass spectrometry detection of target 1;

[0028] M 2 shows the bacterial species results obtained after mass spectrometry detection of target 1;

[0029] g 1 shows the bacterial genus result obtained after mass spectrometry detection of target 1;

[0030] g 2 shows the bacterial genus results obtained after mass spectrometry detection of target 1;

[0031] (2) Obtain the test results by following these steps:

[0032] When L=1, the detection result is the mass spectrometry detection result of the bacterial genus corresponding to the target point max(r1, r2);

[0033] When L=2, the detection result is the mass spectrometry detection result of the bacterial species corresponding to the target point max(r1, r2);

[0034] When L=0, the detection result is not detected.

[0035] S1 lysis refers to: mixing the positive blood culture sample, adding lysis buffer, mixing, centrifuging to remove the supernatant to obtain the lysed Candida blood culture positive sample, and then resuspending to obtain Candida suspension.

[0036] Preferably, the volume ratio of the lysis buffer to the sample is 1:5;

[0037] Preferably, the lysis buffer solution is a saponin solution with a mass-to-volume ratio of 1% to 10%.

[0038] Preferably, the centrifugation conditions are 12000 rpm for 2 minutes;

[0039] Preferably, the resuspension refers to: obtaining a Candida suspension by mixing and centrifuging the precipitate with a binding buffer;

[0040] Preferably, the binding buffer is an aqueous solution of sodium chloride with a mass-volume ratio of 0% to 35.9%.

[0041] The magnetic beads are an amino magnetic bead solution with a mass percentage of 0.5-10% using purified water as a solvent.

[0042] The enrichment includes:

[0043] (1) Candida suspension was mixed with magnetic bead solution for adsorption;

[0044] (2) Use a magnetic rod to transfer the magnetic beads adsorbed with Candida albicans and wash them for the first time with washing buffer I;

[0045] (3) Wash a second and third time with washing buffer II and washing buffer III, respectively;

[0046] Preferably, the cleaning time is 5 to 30 seconds.

[0047] The elution and lysis of S2 refers to the addition of elution buffer to the Candida-magnetic bead complex;

[0048] Preferably, the eluent is an aqueous formic acid solution with a volume ratio of 40-100%;

[0049] Preferably, the volume ratio of the binding buffer to the elution buffer is 10:1;

[0050] Preferably, the Candida lysate refers to the supernatant obtained after eluting and lysing the Candida-magnetic bead complex with an added elution buffer;

[0051] Preferably, the elution and lysis time is 30 seconds.

[0052] Before the Candida lysis buffer is used on the instrument, target points need to be prepared. The preparation of target points refers to: applying Candida lysis buffer to the target plate, drying it, covering it with a matrix solution, and drying it again to obtain the target points.

[0053] Preferably, the matrix solution is an α-cyano-4-hydroxycinnamic acid (CHCA) solution.

[0054] A rapid detection system for Candida blood culture positive samples includes: an experimental component and a data component; the experimental component includes a microbial extraction kit; the data component includes a mass spectrometer and a result interpretation device sequentially arranged on the data main line; characterized in that at least one well in the extraction plate of the microbial extraction kit is pre-filled with DMSO at a volume ratio of 50~99.5% as a washing buffer I; the mass spectrometer outputs the confidence scores r1 and r2 of target point 1 and target point 2, as well as the corresponding genus and species results obtained by mass spectrometry comparison, to the result interpretation device via the data main line;

[0055] The result interpretation device is equipped with an L-value calculation unit and an interpretation unit connected sequentially on the data main line; the L-value calculation unit includes a computer-readable storage medium on which a computer program is stored, and the computer program, when executed by a processor, implements an L-value calculation method; the L-value calculation method calculates according to the following calculation steps:

[0056] The L-value calculation unit monitored... Output L=1 to the judgment unit;

[0057] The L-value calculation unit monitored... Output L=2 to the judgment unit;

[0058] The L-value calculation unit did not monitor... And not monitored Output L=0 to the judgment unit;

[0059] When the interpretation unit monitors L=1, it outputs the mass spectrometry detection results of the target point corresponding to max(r1, r2).

[0060] When the interpretation unit monitors L=2, it outputs the mass spectrometry detection results corresponding to the target point max(r1, r2).

[0061] When the judgment unit monitors L=0, it outputs a result indicating no detection.

[0062] The two adjacent wells downstream of the well pre-filled with 50-99.5% DMSO as washing buffer I on the extraction plate are pre-filled with purified water as washing buffer II and washing buffer III, respectively.

[0063] Preferably, the experimental components further include: a microbial extraction instrument;

[0064] Preferably, the microbial extractor is the microbial extraction system described in CN223304459U;

[0065] Preferably, the size and structure of the microbial extraction kit are adapted to the size and structure of the container placement space of the microbial extractor.

[0066] The wells adjacent to the wells on the extraction plate that are pre-filled with DMSO (50-99.5% by volume) as washing buffer I are pre-filled with magnetic bead solution.

[0067] Preferably, the magnetic bead solution is an amino magnetic bead solution with a mass percentage of 0.5-10% using purified water as a solvent;

[0068] Preferably, the adjacent well upstream of the well pre-filled with magnetic bead solution on the extraction plate is pre-filled with diluent;

[0069] Preferably, the diluent is purified water.

[0070] The wells upstream of the wells pre-filled with diluent on the extraction plate are sample wells for adding Candida suspension.

[0071] Preferably, the wells adjacent to the blank wells on the extraction plate are pre-filled with eluent;

[0072] Preferably, the eluent is a formic acid aqueous solution with a volume ratio of 40-100%.

[0073] Preferably, the microbial extractor is the microbial extraction system described in CN223304459U;

[0074] Preferably, the mass spectrometer is the EXM2600 model from the Zhongyuan Huiji brand.

[0075] The beneficial effects of this invention are as follows:

[0076] The detection method and fungal magnetic bead assay kit of this invention pretreat fungal samples using a certain concentration of DMSO solution or pure DMSO as washing buffer I, followed by two-step washing, elution and lysis, and preparation of the supernatant for mass spectrometry detection. An algorithm is used to comprehensively judge the results based on the detection matching results of two parallel target sites. Through the combination of pretreatment reagents, detection strategies, and matching interpretation algorithms, the detection rate and accuracy of fungal samples are improved. The detection method of this invention improves the accuracy and detection rate of fungal samples through improvements and optimizations in sample pretreatment reagents, mass spectrometry detection, and result interpretation algorithms. Specific advancements are reflected in the following aspects:

[0077] (1) Improvement of sample pretreatment kit to reduce interference from impurities and improve detection rate from the perspective of improving the purity of mass spectrometry test samples. In this invention, after lysing and centrifuging positive blood culture samples, the supernatant is taken out, which can remove most of the impurities in the samples. Then, the samples are resuspended with binding buffer, and microorganisms are specifically enriched with magnetic bead solution. DMSO is used as a washing agent to dissolve protein impurities and organic matter in the blood lysate in the sample. Then, two-step washing is performed to remove impurities and DMSO, and mass spectrometry is used for detection.

[0078] (2) Target 1 and Target 2 are used for each sample for parallel control, which improves the detection accuracy. On the one hand, compared with the ordinary single-target detection method, Target 1 and Target 2 increase the possibility of obtaining high-quality mass spectrometry data and avoid the probability of detection failure caused by factors such as the inhomogeneity of the test sample and the deviation of the target preparation. On the other hand, parallel control can verify each other and avoid misjudgment in some cases where the matching results of the two targets are inconsistent. The detection cost of adding a target is extremely low.

[0079] (3) The matching results of target 1 and target 2 corroborate each other. Even when the confidence scores of the matching results of target 1 and target 2 are in the upper-middle confidence interval, relatively reliable results can still be given.

[0080] This method enables rapid reporting of results within 20 minutes for positive fungal blood cultures without the need for pure culture, with an accuracy rate as high as 84-92%. It also boasts a higher mass spectrometry detection rate compared to conventional magnetic bead kit pretreatment methods. Rapid and accurate detection is fundamental to the successful treatment of bloodstream infections, immediately alerting clinicians and allowing for more effective infection control through early administration of drugs sensitive to the pathogen. This prevents the infection from spreading to other organs, directly reducing patient mortality and the cost of treating medical complications. Attached Figure Description

[0081] Figure 1 This is a flowchart illustrating a rapid detection method for Candida blood culture-positive samples, provided for some embodiments of the present invention.

[0082] Figure 2 The diagram below illustrates the structure of a rapid detection system for Candida blood culture positive samples, provided for some embodiments of the present invention. The solid black line represents the main data path, and the blue arrow indicates that the Candida lysate obtained from the enrichment and extraction of the experimental components is subjected to mass spectrometry detection.

[0083] Figure 3 This is a schematic diagram of a portion of the extraction well plate of a microbial extraction kit for a rapid detection system for Candida blood culture positive samples, provided for some embodiments of the present invention. In this kit, blank circular wells are blank wells used to hold Candida lysate. In other embodiments, there may be two or more blank circular wells.

[0084] Figure 4 The images show the results of various Candida agar plates cultured using 99.5% DMSO washing buffer, which were used as simulated samples in the experimental examples of this invention.

[0085] Figure 5 The existing method provided for experimental examples of the present invention: a typical mass spectrum of a sample containing Candida glabrata processed by magnetic bead method (using urea washing buffer) and then detected by mass spectrometry.

[0086] Figure 6 The method of the present invention provided for experimental examples of the present invention: a typical mass spectrum of a sample containing Candida glabrata after being processed by mass spectrometry using the fungal magnetic bead method (using 99.5% DMSO washing buffer).

[0087] Figure 7 The control method provided for the experimental examples of the present invention is a typical mass spectrum obtained by mass spectrometry detection after processing a sample containing Candida glabrata using the fungal magnetic bead method (using 50% DMSO washing buffer).

[0088] Figure 8 The existing method provided for experimental examples of the present invention: a typical mass spectrum of a sample containing Candida glabrata after treatment by magnetic bead method (using 36% urea washing buffer) and mass spectrometry detection.

[0089] Figure 9 The method of the present invention provided for experimental examples of the present invention: a typical mass spectrum of a sample containing Candida glabrata after being processed by mass spectrometry using the fungal magnetic bead method (using 99.5% DMSO washing buffer).

[0090] Figure 10 The method of the present invention provided for experimental examples of the present invention: a typical mass spectrum of a sample containing Candida glabrata after being processed by mass spectrometry using the fungal magnetic bead method (using 50% DMSO washing buffer). Detailed Implementation

[0091] The present invention will be further described in detail below with reference to specific embodiments and experimental examples, but this does not limit the scope of protection of the present invention.

[0092] biomaterial sources

[0093] I. The Candida glabrata strains used in the experimental examples of this invention to prepare the simulated samples were from ATCC, strain number ATCC15126; Candida parapsilosis strain number ATCC90018; Candida albicans strain number ATCC10231; Candida tropicalis strain number ATCC13803; and Candida krusei strain number ATCC14243.

[0094] II. The blood culture positive samples containing Candida albicans, Candida tropicalis, Candida glabrata, Candida glabrata, and Candida krusei used in the experimental examples of this invention were all prepared according to the Candida species corresponding to the first item above in accordance with "YY / T 0656-2008 Automated Blood Culture System".

[0095] Group 1 Examples: Rapid Detection Method for Candida Blood Culture Positive Samples of the Present Invention

[0096] This set of embodiments provides a rapid detection method for Candida blood culture-positive samples. All embodiments in this set share the following common features: Figure 1 As shown, the rapid detection method for Candida blood culture positive samples includes the following steps:

[0097] S1. Candida albicans blood culture positive samples after lysis were enriched with Candida albicans using magnetic bead method. During the enrichment process, DMSO solution with a volume ratio of 50% to 99.5% was used as a washing buffer to wash the magnetic beads bound with Candida albicans to obtain Candida albicans-magnetic bead complex.

[0098] S2. Using an elution buffer, Candida albicans in the Candida-magnetic bead complex is eluted from the magnetic beads and Candida albicans is lysed to obtain a Candida lysate;

[0099] S3. Prepare target 1 and target 2 from the Candida lysate and perform mass spectrometry detection.

[0100] S4. Compare and interpret the mass spectrometry results with the microbial database of the mass spectrometry system;

[0101] The comparison includes: the mass spectrometry system performs mass spectrometry detection on target 1 and target 2 respectively to obtain their respective mass spectra, and compares them with the bacterial species database of the mass spectrometry system to obtain the bacterial species results and genus results corresponding to target 1 and target 2, and assigns confidence scores to target 1 and target 2 respectively to obtain r1 and r2.

[0102] The interpretation includes: (1) performing an L-value calculation method according to the following steps:

[0103] when At that time, L=1;

[0104] when At that time, L=2;

[0105] Neither There is none. At that time, L=0;

[0106] in,

[0107] δ s =I( M 1= M 2) is the microbial consistency indicator function, where 1 indicates consistency and 0 indicates inconsistency;

[0108] δ g =I( g 1= g 2) is the genus consistency indicator function, where 1 represents consistency and 0 represents inconsistency;

[0109] r1 is the confidence score assigned after the mass spectrometer performs mass spectrometry detection on target 1;

[0110] r2 is the confidence score assigned after the mass spectrometer performs mass spectrometry detection on target 2;

[0111] M 1 shows the bacterial species results obtained after mass spectrometry detection of target 1;

[0112] M 2 shows the bacterial species results obtained after mass spectrometry detection of target 1;

[0113] g 1 shows the bacterial genus result obtained after mass spectrometry detection of target 1;

[0114] g 2 shows the bacterial genus results obtained after mass spectrometry detection of target 1;

[0115] (2) Obtain the test results by following these steps:

[0116] When L=1, the detection result is the mass spectrometry detection result of the bacterial genus corresponding to the target point max(r1, r2);

[0117] When L=2, the detection result is the mass spectrometry detection result of the bacterial species corresponding to the target point max(r1, r2);

[0118] When L=0, the detection result is no result detected.

[0119] In some embodiments, the symbols in the above calculation methods, calculation steps, and interpretation steps are all common logical operators in the computer field, and have conventional technical meanings that are generally understood by those skilled in the art in the computer or mathematics fields. For example, ∧ means "and", ∨ means "or", and ¬ means "not".

[0120] In other embodiments, the microbial consistency indicator function δ s Genera-identity indicator function δ g Both are simple types of indicator functions in mathematics, which determine whether two values ​​are the same.

[0121] In some specific embodiments, the L-value calculation method and interpretation steps can be integrated into existing mass spectrometry systems, or stored on additional computer-readable storage media for execution by additional computer programs or software. Conventional mass spectrometry detection methods prepare one target for each sample and perform detection, directly matching the mass spectrum with a microbial fingerprint protein spectrum database to obtain the identification result. However, this conventional method is not effective for identifying Candida. This invention simultaneously improves the reagent kit and detection strategy: pretreatment is performed using a fungal-specific reagent kit, and target 1 and target 2 are prepared for each sample. The mass spectra of the two targets are matched separately to obtain the bacterial species result (this part is the same as conventional mass spectrometry detection), and then combined with interpretation to give the final identification result.

[0122] In other embodiments, the bacterial strain database of S4 refers to the protein database of the bacterial strain, and the comparison is performed by comparing the Candida protein released by lysis with the protein database of the bacterial strain.

[0123] In some more specific embodiments, when the interpretation unit monitors L=1, the output of max(r1, r2) corresponds to the genus result of mass spectrometry detection of the target point, or the genus result to which both belong. This should be understood as follows: when the interpretation unit monitors L=1, the genus result of mass spectrometry detection of the target point corresponding to the maximum value of confidence scores r1 and r2 for each of target point 1 and target point 2 is the genus result finally output by the interpretation unit, or the genus result to which both target point 1 and target point 2 belong. It cannot be identified at the species level. For example, when the interpretation unit monitors L=1, the confidence scores r1 and r2 corresponding to target point 1 and target point 2 are 2.02 and 2.14, respectively. The mass spectrometry detection results corresponding to target point 1 and target point 2 are Candida glabrata and Candida parapsilosis, respectively. Both have high confidence scores. Therefore, the final output result of the interpretation unit is not a reliable species but a reliable genus, namely Candida genus.

[0124] In some other specific embodiments, when the interpretation unit monitors L=2, the mass spectrometry detection result of the target corresponding to the target point max(r1, r2) should be understood as follows: when the interpretation unit monitors L=2, the target point whose mass spectrometry detection result corresponds to the maximum value of the confidence scores r1 and r2 corresponding to target point 1 and target point 2 is the final output result of the interpretation unit; for example, when the interpretation unit monitors L=2, the confidence scores r1 and r2 corresponding to target point 1 and target point 2 are 2.10 and 1.47 respectively, and the mass spectrometry detection results corresponding to target point 1 and target point 2 are Candida spicata and Candida glabrata respectively, then the final output result of the interpretation unit is Candida spicata.

[0125] In some embodiments, the term "on-machine mass spectrometry detection" has a conventional technical meaning well known to those skilled in the art. For example, "on-machine" can mean the term "on-machine" as recorded in the article "Research on the Method for Direct Determination of Carbaryl and Aldicarb in Water by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry," and "mass spectrometry detection" can also mean the term "mass spectrometry detection" as recorded in the article "Research on the Method for Direct Determination of Carbaryl and Aldicarb in Water by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry," or it can mean detection using a mass spectrometer. In some preferred embodiments of the present invention, the mass spectrometer is a Zhongyuan Huiji EXM2600 model mass spectrometer.

[0126] In specific embodiments, such as Figure 1 As shown, the lysis of S1 refers to: mixing the positive blood culture sample, adding lysis buffer, mixing, centrifuging to remove the supernatant to obtain the lysed Candida blood culture positive sample, and then resuspending to obtain Candida suspension.

[0127] Preferably, the volume ratio of the lysis buffer to the sample is 1:5;

[0128] Preferably, the lysis buffer solution is a saponin solution with a mass-to-volume ratio of 1% to 10%.

[0129] Preferably, the centrifugation conditions are 12000 rpm for 2 minutes;

[0130] Preferably, the resuspension refers to: obtaining a Candida suspension by mixing and centrifuging the precipitate with a binding buffer;

[0131] Preferably, the binding buffer is a sodium chloride aqueous solution with a mass-volume ratio of 0% to 35.9%;

[0132] In some embodiments, a 0% (w / v) sodium chloride aqueous solution, i.e., pure water or double-distilled water, can also be used as a binding buffer.

[0133] In a further embodiment, the magnetic beads are an amino magnetic bead solution with a mass percentage of 0.5-10% using purified water as a solvent;

[0134] Preferably, the amino magnetic beads are the amino magnetic beads described in patent document CN119437858A;

[0135] In a specific embodiment, the enrichment includes:

[0136] (1) Candida suspension was mixed with magnetic bead solution for adsorption;

[0137] (2) Use a magnetic rod to transfer the magnetic beads adsorbed with Candida albicans and wash them for the first time with washing buffer I;

[0138] (3) Wash a second and third time with washing buffer II and washing buffer III, respectively;

[0139] Preferably, the cleaning time is 5 to 30 seconds.

[0140] In some specific embodiments, the enrichment can be performed manually using a pipette and a magnetic rod, or it can be performed using the microbial extraction system described in CN223304459U. This microbial extraction system is commercially available, specifically the EXM3000Duo fully automated nucleic acid and microbial extractor manufactured by the second applicant, Zhongyuan Huiji Biotechnology Co., Ltd. The instruction manual for the EXM3000Duo fully automated nucleic acid and microbial extractor also describes the specific enrichment steps, but unlike the present invention, the washing buffer I does not use DMSO.

[0141] In other embodiments, the elution and lysis of S2 refers to the addition of an elution buffer to the Candida-magnetic bead complex.

[0142] In some embodiments, the elution and lysis function of S2 is mainly to break the proteins released by Candida cells, and at the same time, to separate the cell fragments from the magnetic beads.

[0143] Preferably, the eluent is an aqueous formic acid solution with a volume ratio of 40-100%;

[0144] Preferably, the volume ratio of the binding buffer to the elution buffer is 10:1;

[0145] Preferably, the Candida lysate refers to the supernatant obtained after eluting and lysing the Candida-magnetic bead complex with an added elution buffer;

[0146] Preferably, the elution and lysis time is 30 seconds.

[0147] In a further embodiment, such as Figure 1 As shown, the Candida lysate needs to be prepared as a target before it is used in the extraction plate; the preparation of the target refers to: applying the Candida lysate to the blank wells of the extraction plate, drying it, covering it with a matrix solution, and drying it again to obtain the target.

[0148] Preferably, the matrix solution is an α-cyano-4-hydroxycinnamic acid (CHCA) solution.

[0149] In some embodiments, 1 μL of Candida lysate is applied to the target plate, dried, and then 1 μL of matrix solution is added. After drying, the sample is analyzed by mass spectrometry. "Target application" means that 1 μL of Candida lysate is applied to the target plate.

[0150] In other embodiments, the target plate, also known as the sample target, can be a commercially available mass spectrometry target plate, such as a "disposable sample target plate for bioMérieux 410893VITEKMS mass spectrometer".

[0151] Group 2 Examples: Rapid Detection System for Candida Blood Culture Positive Samples of the Present Invention

[0152] This set of embodiments provides a rapid detection system for Candida blood culture positive samples. All embodiments in this set share the following common features: the rapid detection system for Candida blood culture positive samples includes: an experimental component and a data component; the experimental component includes a microbial extraction kit; the data component includes a mass spectrometer and a result interpretation device sequentially arranged on the data main line; characterized in that at least one well in the extraction plate of the microbial extraction kit is pre-filled with DMSO at a volume ratio of 50-99.5% as washing buffer I; at least two wells in the extraction plate are blank wells reserved for adding Candida lysis buffer to prepare target point 1 and target point 2; the mass spectrometer outputs the confidence scores r1 and r2 of target point 1 and target point 2, along with their corresponding genus and species results obtained from mass spectrometry alignment, to the result interpretation device via the data main line;

[0153] The result interpretation device is equipped with an L-value calculation unit and an interpretation unit connected sequentially on the data main line; the L-value calculation unit includes a computer-readable storage medium on which a computer program is stored, and the computer program, when executed by a processor, implements an L-value calculation method; the L-value calculation method calculates according to the following calculation steps:

[0154] The L-value calculation unit monitored... Output L=1 to the judgment unit;

[0155] The L-value calculation unit monitored... Output L=2 to the judgment unit;

[0156] The L-value calculation unit did not monitor... And not monitored Output L=0 to the judgment unit;

[0157] When the interpretation unit monitors L=1, it outputs the mass spectrometry detection results of the target point corresponding to max(r1, r2).

[0158] When the interpretation unit monitors L=2, it outputs the mass spectrometry detection results corresponding to the target point max(r1, r2).

[0159] When the judgment unit monitors L=0, it outputs a result indicating no detection.

[0160] In a specific embodiment, the two adjacent wells downstream of the well pre-filled with DMSO at a volume ratio of 50~99.5% as washing buffer I on the extraction plate are respectively pre-filled with purified water as washing buffer II and washing buffer III.

[0161] In a preferred embodiment, both washing buffer II and washing buffer III are purified water.

[0162] In a further embodiment, magnetic bead solution is pre-filled in the wells adjacent to the wells pre-filled with 50-99.5% DMSO as washing buffer I on the extraction plate.

[0163] Preferably, the magnetic bead solution is an amino magnetic bead solution with a mass percentage of 0.5-10% using purified water as a solvent;

[0164] Preferably, the amino magnetic beads are the amino magnetic beads described in patent document CN119437858A;

[0165] Preferably, the adjacent well upstream of the well pre-filled with magnetic bead solution on the extraction plate is pre-filled with diluent;

[0166] Preferably, the diluent is purified water.

[0167] In a further embodiment, the well adjacent to the well pre-filled with diluent on the extraction plate is used to add Candida suspension (also referred to as cell suspension in some embodiments, e.g.) Figure 3 The sample feeding well (as shown);

[0168] Preferably, the wells adjacent to the blank wells on the extraction plate are pre-filled with eluent;

[0169] Preferably, the eluent is an aqueous formic acid solution with a volume ratio of 40-100%;

[0170] In a more preferred embodiment, such as Figure 3 As shown, on the extraction plate, the sample loading wells, the wells pre-filled with diluent, the wells pre-filled with magnetic bead solution, the wells pre-filled with DMSO (50-99.5% by volume) as washing buffer I, the two wells pre-filled with purified water as washing buffer II and washing buffer III respectively, one or two or more blank wells for holding Candida lysate (which can correspond to the well positions of target point 1 and target point 2 on the target plate), and the wells pre-filled with elution buffer are arranged in a straight line.

[0171] Preferably, the microbial extractor is the microbial extraction system described in CN223304459U;

[0172] Preferably, the mass spectrometer is a Zhongyuan Huiji EXM2600 mass spectrometer.

[0173] In a further preferred embodiment, the experimental components further include: a microbial extractor;

[0174] Preferably, the microbial extractor can be the microbial extraction system described in CN223304459U, or the nucleic acid and microbial extractor EXM3000Duo produced by the second applicant, Zhongyuan Huiji Biotechnology Co., Ltd.

[0175] In a further embodiment, the size and structure of the microbial extraction kit are adapted to the size and structure of the container placement space of the microbial extractor.

[0176] In a more specific embodiment, the size and structure of the microbial extraction kit are consistent with the size and structure of the microbial extraction container of the microbial extraction system described in CN223304459U.

[0177] In some embodiments, the structure and material of the extraction plate can be similar to those of commercially available 6-well or 96-well plates, or it can be the microbial extraction container of the microbial extraction system described in CN223304459U. This microbial extraction system is commercially available as the EXM3000Duo nucleic acid and microbial extractor manufactured by the second applicant, Zhongyuan Huiji Biotechnology Co., Ltd. The instruction manual for the EXM3000Duo also describes specific microbial extraction (enrichment) procedures and steps, but unlike this invention, the washing buffer I does not use DMSO.

[0178] Experimental examples, rapid detection method of the present invention, and performance testing

[0179] This experimental example provides a rapid detection kit for positive Candida blood cultures, as detailed below:

[0180] 1.1 The components and concentration ranges of the kit are shown in Table 1 below:

[0181] Table 1

[0182]

[0183] 2. Preparation method of the reagent kit

[0184] 2.1 Prepare lysis buffer and binding buffer according to the above embodiments.

[0185] 2.2 The diluent, magnetic bead solution, washing buffer I, washing buffer II, and washing buffer III were prepared according to the above embodiments and dispensed into 6 / 96-well plates. The magnetic beads were amino magnetic beads, manufactured in-house by the second applicant company (the preparation method can be found in patent document CN119437858A).

[0186] 2.3 Assemble the components into boxes and label them.

[0187] 3. Microbial identification

[0188] The detection method in this embodiment is as follows: Figure 1 As shown.

[0189] The testing was performed using simulated blood culture positive samples, and the specific process is as follows.

[0190] 1) Preparation of simulated positive blood culture samples

[0191] Simulated positive blood culture samples are prepared according to the following procedure:

[0192] ① Add 10 mL of sterile defibrinated sheep blood to a blood culture bottle.

[0193] ② Prepare a 0.5 mcf physiological saline suspension from the freshly cultured bacterial strain using a turbidimeter, and dilute it 1*10. 7 After doubling, aspirate 1 mL and inoculate it into a blood culture bottle containing sheep blood (inoculation concentration is approximately 5-30 CFU / mL).

[0194] ③ Place the blood bottle into a fully automated microbial culture system and incubate until a positive result is obtained.

[0195] ④ Remove it as soon as possible after a positive result is reported.

[0196] 2) Sample pretreatment

[0197] (1) Take 1 mL of the simulated blood culture positive sample, add 200 μL of lysis buffer, mix and lyse for 30 s.

[0198] (2) Centrifuge at 12000 rpm for 2 min and remove the supernatant.

[0199] (3) Add 500 μL of binding buffer and mix the binding buffer by pipetting.

[0200] (4) Take 250 μL of sample and add it to column 1 / 7 of the pre-amplified bacterial enrichment reagent (diluent, magnetic bead solution, washing buffer I and washing buffer II are aliquoted according to the set amount).

[0201] (5) Add 50 μL of eluent to column 6 / 12 of the pre-amplified bacterial enrichment reagent and run the enrichment program (Zhongyuan Huiji Fully Automated Nucleic Acid and Nucleic Acid Microbial Extractor EXM3000Duo). The enrichment program includes the following steps:

[0202] ① Mix the magnetic bead solution with the sample resuspension and allow it to adsorb for 0.1-2 min. Use a magnetic rod and a magnetic rod sleeve to adsorb the magnetic beads and the pathogens they bind, and then transfer them to washing buffer I.

[0203] ②Wash the mixture for 5-30 seconds, then use a magnetic rod and magnetic rod sleeve to adsorb the magnetic beads and the pathogens they bind, and transfer them to washing buffer II;

[0204] ③ Wash for 5-30 seconds, then use a magnetic rod and magnetic rod sleeve to adsorb the magnetic beads and the pathogens they bind, and transfer them to washing buffer III;

[0205] ④ During the mixed washing for 5-30 seconds, the magnetic beads and the pathogens they bind are adsorbed by the magnetic rod and magnetic rod sleeve and transferred to the eluent.

[0206] ⑤ Lysis of pathogens.

[0207] (6) After the program finishes running, immediately prepare the sample solution.

[0208] It is worth noting that during the washing step, with a mixing time of only 5 seconds, the DMSO eluent, while removing impurities, did not significantly affect the activity of pathogens bound to the magnetic beads. This experimental example, using simulated samples, showed that after washing, the pathogens on the magnetic beads could still form colonies through plate culture. The specific procedure for the simulated sample experiment was as follows: after lysing blood cells and centrifuging and resuspending the simulated sample, magnetic beads were added for binding, followed by washing with 99.5% DMSO. The magnetic beads, along with the bound bacteria, were then mixed in physiological saline and diluted 1*10. 6 Double the amount of bacterial suspension, take 100 μL, spread it onto a plate for incubation, and the results are as follows. Figure 4 As shown. The simulated samples refer to blood culture samples containing Candida glabrata, Candida parapsilosis, Candida albicans, Candida tropicalis, and Candida krusei, respectively. The preparation of blood culture samples containing these Candida species can be referred to "YY / T 0656-2008 Automated Blood Culture System".

[0209] Therefore, in step (5), after washing, the magnetic beads and the pathogens they bind can be frozen without transferring them to the elution buffer for elution and lysis. If the subsequent test results are abnormal, they can be thawed and cultured directly, and other methods can be used for testing.

[0210] 3) Mass spectrometry detection and result interpretation

[0211] (1) Take 1 μL of sample and spot it onto the target plate (i.e., take 1 μL of sample and drop it onto the target plate; this term has the conventional technical meaning known to those skilled in the art, for example, it can be the meaning of the term "spotting" recorded in patent texts such as "CN219917066U", "CN114539362B", and "CN120468270B", also known as "spotting"), dry it, cover it with 1 μL of matrix solution, dry it again, and then perform mass spectrometry detection. Spot two samples of each sample onto the target plate as parallel samples.

[0212] (2) Detection. A MALDI-TOF MS mass spectrometry system (fully automated microbial mass spectrometry detection system, EXM2600, Zhongyuan Huiji) was used for detection. The mass spectra were compared with the bacterial database of the mass spectrometry system to obtain the bacterial matching results m and confidence scores r for target 1 and target 2, respectively. M 1, r 1), ( M 2, r 2); where M 1, M 2∈M, where M is the database set of bacterial species for the mass spectrometry system. r 1, r2∈[0,3.0], the confidence score of the mass spectrometry detection system is 0~3.0; further, we can obtain M 1、 M2 Corresponding genus name g 1、 g 2。

[0213] (3) Interpretation. Based on the fungal matching results and confidence scores of target 1 and target 2, the following function is used to make a judgment and output the interpretation result (L, M∗):

[0214] ,

[0215] ,

[0216] in,

[0217] L represents the species confidence level, where L∈{0,1,2} (0=not detected, 1=genus, 2=species).

[0218] M* represents the species identification result:

[0219] δ s =I( M 1= M 2) is the microbial consistency indicator function, where 1 indicates consistency and 0 indicates inconsistency;

[0220] δ g =I( g 1= g 2) is the genus consistency indicator function, where 1 represents consistency and 0 represents inconsistency;

[0221] This is a simple type of indicator function in mathematics, which determines whether two values ​​are the same.

[0222] max( r 1, r 2) is the maximum score of the target point;

[0223] Min( r 1, r 2) is the minimum target score;

[0224] i The index of the two parallel target points, with a value of 1 or 2.

[0225] Interpretation results ( L , M The corresponding detection result for *) is:

[0226] like L =0, detection result M * It is null, that is Not detected;

[0227] like L =1, Detection result M * indicates the genus of bacteria corresponding to species with higher confidence scores. g i ;

[0228] like L =2, Detection result M * indicates strains with higher confidence scores. M i .

[0229] 4. Detection Principle

[0230] Positive samples from simulated fungal blood cultures were lysed using lysis buffer. After centrifugation, the supernatant was removed to remove most impurities. The precipitate was then resuspended in binding buffer, and a magnetic bead solution (the magnetic beads were self-made by the company, and the preparation method was based on patent document CN119437858A) was added to enrich the fungi on the solid-phase magnetic beads. The samples were then washed with a highly polar organic solvent (DMSO), followed by a two-step water wash to further remove residual impurities. Finally, the microorganisms bound to the magnetic beads were eluted and lysed using elution buffer, and then detected by mass spectrometry. For the mass spectrometry system used in this invention, it is generally considered that a matching result of r ≥ 2.0 is reliable at the species level, and a matching result of 1.7 ≤ m ≤ 2.0 is reliable at the genus level. However, under different experimental conditions such as sample type, microbial type, and equipment operating parameters, the confidence score threshold can be appropriately adjusted. This method uses two parallel targets for detection, and the matching results of target 1 and target 2 are combined for comprehensive judgment to provide the interpretation result.

[0231] Comparison of existing methodologies with this invention

[0232] (1) Comparison of the detection effects of magnetic bead pretreatment using different types of cleaning solutions

[0233] The magnetic bead (urea) kit is similar in composition to the fungal magnetic bead (DMSO) kit of this invention, except that the washing buffer I is a 36% urea aqueous solution (commonly used for bacterial detection). The fungal magnetic bead (5% Tween 20) kit is similar in composition to the fungal magnetic bead (DMSO) kit of this invention, except that the washing buffer I is a 5% Tween 20 aqueous solution (commonly used for molecular detection). The enrichment steps in both the magnetic bead and fungal magnetic bead methods are performed using a fully automated nucleic acid extractor (Zhongyuan Huiji, EXM3000 / 3200). After sample processing, target preparation is performed. In the magnetic bead (36% urea) method, one target spot is prepared for each sample, and the identification result is directly obtained after mass spectrometry detection. In the fungal magnetic bead (5% Tween 20) and fungal magnetic bead (99.5% / 50% DMSO) methods, target spot 1 and target spot 2 are prepared for each sample, and the identification result is obtained after mass spectrometry detection and interpretation of the detection results.

[0234] The specific operating methods and testing results are compared in Tables 2 and 3 below. Figures 5-10 As shown:

[0235] Table 2

[0236]

[0237] Table 3

[0238]

[0239] Comparison of microbial species identification results shows that the fungal magnetic bead kit of this invention achieves a fungal detection rate no lower than that of general-purpose magnetic bead kits, with even better results achieved using 99.5% DMSO as the washing buffer. Currently, clinically used blood culture pretreatment kits combined with MALDI-TOF mass spectrometry have an accuracy rate of approximately 40-75% for fungal species identification in positive blood culture samples. This invention offers a higher and more reliable detection rate. Although washing with DMSO solution improves mass spectrometry detection, the specific mechanism is not yet clear. It is speculated that DMSO solution can dissolve small amounts of impurities adsorbed on the magnetic beads, thereby reducing interference from impurities in mass spectrometry detection and improving the detection rate.

[0240] Mass spectra detected by mass spectrometry ( Figures 5-10 As can be seen, compared with the mass spectra obtained by existing methods, the mass spectra obtained by the detection method of this invention have fewer impurity peaks, higher characteristic peak intensities, and higher resolution.

[0241] (2) Comparison of the detection effects of magnetic bead pretreatment using different organic reagents as cleaning solutions

[0242] Simultaneously, two other organic solvents, CCl4 (purity ≥99.5%) and acetonitrile (purity ≥99.5%), were used as eluents to prepare detection kits, named the fungal magnetic bead method (carbon tetrachloride) and the fungal magnetic bead method (acetonitrile), respectively. The same methods were used for testing, with a total of 25 cases tested. Compared with the fungal magnetic bead method (99.5% DMSO) group, both had lower detection rates, as shown in Table 4 below.

[0243] Table 4

[0244]

[0245] (3) The comparison of the pretreatment effects of fungal magnetic bead detection using different concentrations of DMSO as washing buffer is shown in Table 5 below:

[0246] Table 5

[0247]

[0248] Increasing the DMSO concentration in washing buffer I from 50% to 90% resulted in a higher fungal detection rate in the samples. While the overall detection rate did not significantly improve with DMSO concentrations above 90%, the identification score did increase. This is presumably because higher concentrations of DMSO solution effectively cleaned the magnetic bead-bacterial complex, resulting in better impurity removal, less interference, and higher reliability of the mass spectrometry identification results.

[0249] (4) Impact of the interpretation algorithm

[0250] The interpretation algorithm of this invention plays a crucial role in improving detection accuracy and detection rate. Experimental verification shows that the identification results obtained using the interpretation algorithm are reliable. Several typical samples are shown in Table 6 below:

[0251] Table 6

[0252]

[0253] Note: In each sample, -1 and -2 represent the mass spectrometry detection matching results of target 1 and target 2, respectively.

[0254] In Sample 1, the detection results for target 1 and target 2 were consistent, both being Candida spicata, but the confidence score was less than 1.7, and the interpretation result was determined to be at the level of Candida spp.

[0255] In Sample Example 2, the species detected by target 1 and target 2 are inconsistent, but the genus is consistent, both being Candida. However, the confidence scores for both targets are less than 2.0. According to conventional methods, the match result for 2-1 would be Candida glabrata, with a species level of confidence; the match result for 2-2 would be Candida subglabrata, also with a species level of confidence. That is, Sample Example 2 might be misclassified using conventional single-target detection methods. However, the comprehensive interpretation result of this method is reliable at the genus level.

[0256] In Sample Example 3, the detection results for target 1 and target 2 were inconsistent. The 3-1 match result was (Candida glabrata, 2.10), and the 3-2 match result was (Candida glabrata, 1.47). This reflects the characteristic of large deviations in fungal detection results. According to conventional single-target detection judgment methods, Sample Example 3 would be considered undetectable with a low confidence score, meaning identification failure is possible. However, the comprehensive interpretation result of this method indicates that the Candida glabrata species level is reliable.

[0257] In Sample Example 4, the bacterial species detected by target 1 and target 2 were inconsistent. Specifically, the match result for 4-1 was (Candida glabrata, 1.81), and the match result for 4-2 was (Candida parapsilosis, 1.77), showing a significant deviation. According to conventional single-target detection methods, Sample Example 3 might have been classified as *Candida parapsilosis* or *Candida parapsilosis* at the genus level. However, the overall interpretation result of this method was "not detected," thus avoiding misclassification.

[0258] Therefore, addressing the issues of large biases and poor repeatability in conventional mass spectrometry detection methods for fungal samples, this invention employs a dual-target matching result interpretation algorithm to improve detection rate and accuracy. It is worth noting that even when detecting only Candida species (i.e., L=1) at the genus level, the rapid detection method of this invention still has clinical application value. This is because different antifungal drugs are preferred clinically for different species. If accurate identification at the species level is possible, the preferred drug for that species should be selected first; if only the genus level can be identified, a broader-spectrum (covering multiple Candida species) antifungal drug should be considered to avoid inappropriate medication.

Claims

1. A rapid detection method for Candida blood culture-positive samples, characterized in that, Includes the following steps: S1. Candida albicans blood culture positive samples after lysis were enriched with Candida albicans using magnetic bead method. During the enrichment process, DMSO solution with a volume ratio of 50% to 99.5% was used as a washing buffer to wash the magnetic beads bound with Candida albicans to obtain Candida albicans-magnetic bead complex. S2. Using an elution buffer, Candida albicans in the Candida-magnetic bead complex is eluted from the magnetic beads and Candida albicans is lysed to obtain a Candida lysate; S3. Prepare target 1 and target 2 from the Candida lysate and perform mass spectrometry detection. S4. Compare and interpret the mass spectrometry results with the microbial database of the mass spectrometry system; The comparison includes: the mass spectrometry system performs mass spectrometry detection on target 1 and target 2 respectively to obtain their respective mass spectra, and compares them with the bacterial species database of the mass spectrometry system to obtain the bacterial species results and genus results corresponding to target 1 and target 2, and assigns confidence scores to target 1 and target 2 respectively to obtain r1 and r2. The interpretation includes: (1) performing an L-value calculation method according to the following steps: when At that time, L=1; when At that time, L=2; Neither There is none. At that time, L=0; in, δ s =I( M 1= M 2) is the microbial consistency indicator function, where 1 indicates consistency and 0 indicates inconsistency; δ g =I( g 1= g 2) is the genus consistency indicator function, where 1 represents consistency and 0 represents inconsistency; r1 is the confidence score assigned after the mass spectrometer performs mass spectrometry detection on target 1; r2 is the confidence score assigned after the mass spectrometer performs mass spectrometry detection on target 2; M 1 shows the bacterial species results obtained after mass spectrometry detection of target 1; M 2 shows the bacterial species results obtained after mass spectrometry detection of target 1; g 1 shows the bacterial genus result obtained after mass spectrometry detection of target 1; g 2 shows the bacterial genus results obtained after mass spectrometry detection of target 1; (2) Obtain the test results by following these steps: When L=1, the detection result is the mass spectrometry detection result of the bacterial genus corresponding to the target point max(r1, r2); When L=2, the detection result is the mass spectrometry detection result of the bacterial species corresponding to the target point max(r1, r2); When L=0, the detection result is not detected.

2. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, S1 lysis refers to: mixing the positive blood culture sample, adding lysis buffer, mixing, centrifuging to remove the supernatant to obtain the lysed Candida blood culture positive sample, and then resuspending to obtain Candida suspension.

3. The rapid detection method for Candida blood culture positive samples according to claim 2, characterized in that, The volume ratio of the lysis buffer to the sample is 1:

5.

4. A rapid detection method for Candida blood culture positive samples according to claim 2 or 3, characterized in that, The lysis buffer solution is a saponin solution with a mass-volume ratio of 1% to 10%.

5. The rapid detection method for Candida blood culture positive samples according to claim 2, characterized in that, The centrifugation conditions were 12,000 rpm for 2 minutes.

6. The rapid detection method for Candida blood culture positive samples according to claim 2, characterized in that, The resuspension refers to the Candida suspension obtained by mixing the precipitate with binding buffer and centrifuging.

7. The rapid detection method for Candida blood culture positive samples according to claim 6, characterized in that, The binding buffer solution is an aqueous solution of sodium chloride with a mass-volume ratio of 0% to 35.9%.

8. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, The magnetic beads are an amino magnetic bead solution with a mass percentage of 0.5-10% using purified water as a solvent.

9. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, The enrichment includes: (1) Candida suspension was mixed with magnetic bead solution for adsorption; (2) Use a magnetic rod to transfer the magnetic beads adsorbed with Candida albicans and wash them for the first time with washing buffer I; (3) Wash a second and third time with washing buffer II and washing buffer III, respectively; Washing buffer I is a 50%~99.5% DMSO solution; washing buffer II and washing buffer III are purified water.

10. A rapid detection method for Candida blood culture positive samples according to claim 9, characterized in that, The cleaning time is 5-30 seconds.

11. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, The elution and lysis of S2 refers to the addition of elution buffer to the Candida-magnetic bead complex.

12. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, The eluent is an aqueous solution of formic acid with a volume ratio of 40-100%.

13. A rapid detection method for Candida blood culture-positive samples according to claim 6 or 7, characterized in that, The volume ratio of the binding buffer to the elution buffer is 10:

1.

14. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, The Candida lysate refers to the supernatant obtained after eluting and lysing the Candida-magnetic bead complex with an added elution buffer.

15. A rapid detection method for Candida blood culture positive samples according to any one of claims 1, 11, and 14, characterized in that, The elution and lysis time is 30 seconds.

16. The rapid detection method for Candida blood culture positive samples according to claim 1, characterized in that, Before the Candida lysate is used on the instrument, target points need to be prepared. The preparation of target points refers to: applying Candida lysate to a target plate, drying it, covering it with a matrix solution, and drying it again to obtain the target points.

17. A rapid detection method for Candida blood culture positive samples according to claim 16, characterized in that, The matrix solution is an α-cyano-4-hydroxycinnamic acid (CHCA) solution.

18. A rapid detection system for Candida blood culture-positive samples, comprising: Experimental components and data components; the experimental components include a microbial extraction kit; the data components include a mass spectrometer and a result interpretation device sequentially arranged on the data main line; characterized in that at least one well in the extraction plate of the microbial extraction kit is pre-filled with DMSO at a volume ratio of 50~99.5% as a washing buffer I; the mass spectrometer outputs the confidence scores r1 and r2 of target point 1 and target point 2, as well as the corresponding genus and species results obtained by mass spectrometry comparison, to the result interpretation device via the data main line; The result interpretation device is equipped with an L-value calculation unit and an interpretation unit connected sequentially on the data main line; the L-value calculation unit includes a computer-readable storage medium on which a computer program is stored, and the computer program, when executed by a processor, implements an L-value calculation method; the L-value calculation method calculates according to the following calculation steps: The L-value calculation unit monitored... Output L=1 to the judgment unit; The L-value calculation unit monitored... Output L=2 to the judgment unit; The L-value calculation unit did not monitor... And not monitored Output L=0 to the judgment unit; When the interpretation unit monitors L=1, it outputs the mass spectrometry detection results of the target point corresponding to max(r1, r2). When the interpretation unit monitors L=2, it outputs the mass spectrometry detection results corresponding to the target point max(r1, r2). When the judgment unit monitors L=0, it outputs a result indicating no detection.

19. A rapid detection system for Candida blood culture positive samples according to claim 18, characterized in that, The two adjacent wells downstream of the well pre-filled with 50-99.5% DMSO as washing buffer I on the extraction plate are pre-filled with purified water as washing buffer II and washing buffer III, respectively.

20. A rapid detection system for Candida blood culture positive samples according to claim 18, characterized in that, The experimental components also include: a microbial extraction instrument.

21. A rapid detection system for Candida blood culture positive samples according to claim 20, characterized in that, The size and structure of the microbial extraction kit are adapted to the size and structure of the container placement space of the microbial extractor.

22. The rapid detection system for Candida blood culture positive samples according to claim 18, characterized in that, The wells adjacent to the wells on the extraction plate that are pre-filled with DMSO (50-99.5% by volume) as washing buffer I are pre-filled with magnetic bead solution.

23. The rapid detection system for Candida blood culture positive samples according to claim 22, characterized in that, The magnetic bead solution is an amino magnetic bead solution with a mass percentage of 0.5-10% using purified water as a solvent.

24. A rapid detection system for Candida blood culture positive samples according to claim 22, characterized in that, The wells upstream of the wells pre-filled with magnetic bead solution on the extraction plate are pre-filled with diluent.

25. A rapid detection system for Candida blood culture positive samples according to claim 24, characterized in that, The diluent is purified water.

26. A rapid detection system for Candida blood culture positive samples according to claim 24, characterized in that, The wells upstream of the wells pre-filled with diluent on the extraction plate are sample wells for adding Candida suspension.

27. A rapid detection system for Candida blood culture positive samples according to any one of claims 18, 22, 24, and 26, characterized in that, The wells adjacent to the blank wells on the extraction plate are pre-filled with eluent.

28. A rapid detection system for Candida blood culture positive samples according to claim 27, characterized in that, The eluent is an aqueous solution of formic acid with a volume ratio of 40-100%.

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