CN116987761A - Biological sample treatment fluid, biological sample fungus detection kit and application thereof - Google Patents
Biological sample treatment fluid, biological sample fungus detection kit and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
- Biotechnology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Microbiology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention relates to a fungus detection technology, and discloses a biological sample treatment fluid, a biological sample fungus detection kit and application thereof. The biological sample treatment fluid comprises a reducing agent, a protein dissolving agent, a cell lysing agent, a density regulator and water, wherein the cell lysing agent comprises sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether. The biological sample fungus detection kit contains the biological sample treatment fluid. The method for detecting fungi in the biological sample comprises the following steps: mixing a biological sample to be detected with the biological sample treatment liquid for incubation to obtain a liquid to be detected; and (3) performing fluorescence staining and fluoroscopic examination on the liquid to be detected after the liquid-based thin layer tabletting. The treatment fluid has high fungus detection sensitivity, is simple and convenient to operate, and can be suitable for various body fluid samples.
Description
Technical Field
The invention relates to a fungus detection technology, in particular to a biological sample treatment fluid, a biological sample fungus detection kit and application thereof.
Background
Fungi are susceptible to fungal infections, including superficial mycoses and deep mycoses. Superficial mycosis refers to a large group of infectious diseases of human skin, mucosa, hair, nails and other accessory organs caused by pathogenic fungi, such as tinea capitis, tinea manus, tinea pedis, onychomycosis and the like. Deep mycoses are diseases in which pathogenic fungi invade organs and systems such as deep tissues, viscera, blood and the like, and also become invasive mycoses, and seriously endanger the health of patients.
Depending on the kind of pathogenic fungi, a variety of test methods are available for the diagnosis of invasive mycoses, including blood culture, smear stain (including gram stain, KOH tablet, silver hexamine stain, snow periodate stain, ink stain, fungal fluorescent stain), immunodetection (G test, GM test, cryptococcus capsular antigen test, aspergillus IgG test) and molecular detection. Among them, blood culture is the gold standard for invasive mycosis detection, and the sensitivity can reach 1CFU/mL, but it takes at least 2-3 days, and even 2-3 weeks for some fungi. Immunoassays can only detect when fungal infections are severe, releasing more fungal material into the body fluid or eliciting an immune response. The molecular detection operation is complex, and only the fungus can be detected in a targeted manner (corresponding detection primers need to be matched), and false positive or false negative results are easy to be caused for samples with low fungus concentration. The smear staining method is a method for rapidly and intuitively detecting the fungi in the sample, is not limited by the types of the fungi, and particularly the fungus fluorescent staining method is a method which is efficient, rapid, high in specificity, sensitive in the cavity and more accurate than the traditional staining method. The fungus fluorescent staining solution mainly comprises a fluorescent whitening agent 28, evan's blue, potassium hydroxide and cell penetrating components (dimethyl sulfoxide, glycerol and the like); potassium hydroxide and cell penetrating components can soften and penetrate the sample, so that the fluorescent dye can contact fungi inside the sample; fluorescent whitening agent 28 can be combined with polysaccharide and chitin on various fungus cell walls, and can emit bright blue fluorescence under the excitation of ultraviolet light (340-380 nm), so that a clear fungus structure is displayed; evan blue can inhibit background fluorescence and nonspecific fluorescence staining, and enhance contrast of fungus staining.
At present, fungus fluorescent staining is mainly applied to superficial fungal infection, and is easy to identify under the mirror because of the fact that samples of the fungus fluorescent staining are easy to obtain (nail scraps, epidermis and vaginal secretion) and the fungus quantity is large. Samples of invasive mycoses typically contain relatively low concentrations of fungi, even less than 10CFU/mL, where they cannot be detected efficiently using conventional microscopy, and where the amount of sample used is typically no more than 100 μl, and the fungi in each microscopic sample can be less than 1CFU, difficult to find under a microscope and distinguish from impurities. In addition, for the detection of samples such as blood, sputum, alveolar lavage fluid and the like, magazines such as cells, tissue fragments and the like in the samples can cause serious interference to fungus detection, so that a body fluid sample with low fungus content cannot be detected by a microscopic method.
Although various sample enrichment and tabletting methods have been developed for fluorescent staining detection of samples of invasive mycoses, the method still has the defects of limited enrichment effect on fungi in the samples, few applicable sample types, complicated treatment and detection processes and the like, so that detection of low-concentration invasive fungal samples is difficult to realize.
Disclosure of Invention
The invention aims to solve the problem that the detection of low-concentration invasive fungus samples is difficult to realize in the prior art, and provides a biological sample treatment fluid, a biological sample fungus detection kit and application thereof.
In order to achieve the above object, a first aspect of the present invention provides a biological sample processing liquid containing a reducing agent, a proteolytic agent, a cell lysis agent, a density regulator and water, wherein the cell lysis agent contains sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether.
Preferably, the weight ratio of sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether in the cell lysis agent is 0.2-15:0.2-2:1, more preferably 0.5-10:0.5-1:1.
Preferably, the content of the reducing agent in the biological sample treatment liquid is 5-25mM, preferably 8-20mM; the protein dissolving agent is contained in an amount of 1 to 8 wt%, preferably 2 to 5 wt%; the cell lysis agent is contained in an amount of 0.3 to 3 wt%, preferably 1 to 3 wt%; the density regulator is contained in an amount of 20 to 50% by weight, preferably 30 to 40% by weight.
Preferably, the reducing agent is selected from at least one of beta-mercaptoethanol, dithiothreitol, N-acetylcysteine, and tris (2-carboxyethyl) phosphine.
Preferably, the protein dissolving agent is sodium hydroxide and/or potassium hydroxide.
Preferably, the density regulator is selected from at least one of methanol, ethanol, propanol and butanol.
In a second aspect, the present invention provides a biological sample fungus detection kit, which contains the biological sample treatment fluid.
Preferably, the kit further comprises an adsorption slide and a fungal fluorescent staining solution.
Preferably, the adsorption glass slide is a cation adsorption glass slide, and the fungus fluorescence staining solution is a chemical staining solution and/or an immunostaining solution.
The third aspect of the present invention provides an application of the biological sample processing liquid and/or the biological sample fungus detection kit in preparing a fungus detection product.
In a fourth aspect, the present invention provides a method for detecting fungi in a biological sample, the method comprising the steps of:
s1, mixing a biological sample to be detected with the biological sample treatment liquid for incubation to obtain a liquid to be detected;
s2, performing fluorescent staining and fluoroscopic examination on the liquid to be detected after the liquid-based thin layer flaking.
Preferably, in step S1, the volume ratio of the biological sample to be tested to the biological sample treatment fluid is 1:0.5-3.
Preferably, the incubation conditions include at least: the temperature is 5-40deg.C, and the time is 20-40min.
Preferably, in step S2, the process of the liquid-based thin layer tablet comprises: and settling the liquid to be detected on a glass slide to obtain a glass slide sample, wherein the settling mode adopts centrifugal settling, natural settling or membrane filtration.
Preferably, the slide glass adopts a cation adsorption slide glass, the centrifugal sedimentation is carried out by adopting a centrifugal pelleter, and the membrane filtration adopts a filter membrane with the pore size of 4-6 mu M.
Preferably, the centrifugal sedimentation or the natural sedimentation is performed for 20 to 60 minutes.
Preferably, the fluorescent staining is performed by using a chemical staining solution and/or an immunostaining solution, and the fluorescent microscopy is performed by using a fluorescent microscope and fluorescent photographing software.
Through the technical scheme, the invention has the beneficial effects that:
the biological sample treatment fluid provided by the invention takes the sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether as cell lysis agents, can efficiently lyse cells and tissue fragments contained in a sample, releases fungi existing in the cells, and removes nonfungal tangible substances interfering with detection flaking; the treatment fluid formed by the cell lysis agent, the reducing agent, the protein dissolving agent and the density regulator not only realizes that biological samples can be flaked for fungus fluorescence detection only by one-step treatment, is simple and convenient to operate, has good enrichment effect on fungi, can detect low-concentration fungus samples, and has the minimum detection limit of 10CFU/mL; the treatment fluid can treat various body fluid samples such as blood, sputum, bronchial lavage, hydrothorax and ascites, pus and wound infected tissues, cerebrospinal fluid, joint fluid, vaginal secretion or urine and the like, and is widely applicable to sample types.
Drawings
FIG. 1 is a partial microscopic image of Aspergillus staining of blood sample 1 in example 1.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The first aspect of the present invention provides a biological sample treatment liquid comprising a reducing agent, a protein-dissolving agent, a cell-lysing agent, a density-adjusting agent and water, wherein the cell-lysing agent comprises Sodium Dodecyl Sulfate (SDS), sodium deoxycholate and polyethylene glycol octyl phenyl ether (Triton X-100).
In the research process of fungus detection in body fluid, the inventor of the invention surprisingly finds that when sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether are matched to be used as cell lysing agents, cells and tissue fragments contained in a biological sample can be efficiently lysed to release fungi existing in the cells, and non-fungus tangible substances such as all human sources except the fungi in the sample are removed, so that interference caused by sample impurities in the process of detecting the fungi with low concentration is avoided; the cell lysing agent is further mixed with a reducing agent, a protein dissolving agent and a density regulator to form a treatment fluid, disulfide bonds among proteins in a biological sample are opened by the reducing agent and the protein dissolving agent, the proteins are hydrolyzed, so that viscous samples such as sputum, pus and the like are liquefied, the density of the treated samples can be reduced by the density regulator, and fungus cells and spores can be fully enriched and precipitated for tabletting; the biological sample can be tabletted for fungus fluorescence detection only by one-step treatment, the operation is simple and convenient, the enrichment effect on fungi is good, the fungus sample with low concentration can be detected, and the sensitivity of fungus detection is improved; the treatment fluid can treat various body fluid samples such as blood, sputum, bronchial lavage, hydrothorax and ascites, pus and wound infected tissues, cerebrospinal fluid, joint fluid, vaginal secretion or urine and the like, and is widely applicable to sample types.
In the present invention, the preparation method of the biological sample processing liquid adopts a conventional reagent solution preparation method, for example, a reducing agent, a protein dissolving agent, a cell lysis agent, a density regulator and water can be directly mixed; alternatively, the reducing agent, the proteolytic agent, the cell lysing agent, the density adjusting agent and one part of the water may be mixed with the other part.
According to the invention, preferably, the weight ratio of sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether in the cell lysis agent is 0.2-15:0.2-2:1, more preferably 0.5-10:0.5-1:1. The inventors have found that in this preferred embodiment, the lysis effect on cells is better and non-fungal tangible substances interfering with the tableting can be better removed.
According to the present invention, the reducing agent is preferably contained in the biological sample processing liquid in an amount of 5 to 25mM, more preferably 8 to 20mM; the protein dissolving agent is contained in an amount of 1 to 8 wt%, preferably 2 to 5 wt%; the cell lysis agent is contained in an amount of 0.3 to 3 wt%, more preferably 1 to 3 wt%; the density regulator is contained in an amount of 20 to 50% by weight, more preferably 30 to 40% by weight. The inventors have found that in this preferred embodiment the biological sample treatment fluid is a low density fluid which provides a beneficial environment for fungal cell sedimentation when tableting, thereby enhancing the enrichment effect on fungi.
According to the present invention, the reducing agent may employ a substance having a reducing effect, which is conventional in the biological sample detection field, preferably, the reducing agent is selected from at least one of β -mercaptoethanol, dithiothreitol, N-acetylcysteine, and tris (2-carboxyethyl) phosphine (TCEP). The inventors have found that in this preferred embodiment it is advantageous to further increase the sensitivity of detecting low concentrations of fungal sample.
According to the present invention, the reducing agent may be a substance having a protein-solubilizing effect, which is conventional in the biological sample detection field, preferably, sodium hydroxide and/or potassium hydroxide. The inventors have found that in this preferred embodiment, it is advantageous to increase the enrichment effect on fungi and to increase the sensitivity of detecting low concentration fungal samples.
According to the present invention, the reducing agent may employ a substance capable of adjusting density, which is conventional in the biological sample detection field, preferably, the density adjusting agent is selected from at least one of methanol, ethanol, propanol and butanol. The inventors have found that in this preferred embodiment, it is advantageous to adjust the biological sample treatment fluid to a low density fluid, providing a favorable environment for fungal cell sedimentation when tableting, thereby improving the enrichment effect and detection sensitivity for fungi.
The above materials are commercially available.
In a second aspect, the present invention provides a biological sample fungus detection kit, which contains the biological sample treatment fluid.
In the invention, the biological sample fungus detection kit can only contain the biological sample treatment liquid, and can also be prepared into biological sample treatment liquid, other corresponding detection reagents, consumables and the like according to a fungus detection method applicable to the kit.
According to the invention, when the biological sample fungus detection kit is used for liquid-based thin-layer smear detection, the kit also contains an adsorption glass slide and fungus fluorescent staining solution; furthermore, the kit also contains consumables such as centrifugal precipitation flaking combination and the like.
In the invention, the adsorption glass slide can be selected from conventional test glass slides and is provided with a corresponding cover glass; preferably, the adsorption slide is a cation adsorption slide.
In the invention, the fungus fluorescent staining solution can adopt a conventional fungus staining method so as to be capable of carrying out staining identification on fungi on a glass slide. Preferably, the fungal fluorescent staining solution is a chemical staining solution and/or an immunostaining solution.
The third aspect of the present invention provides an application of the biological sample processing liquid and/or the biological sample fungus detection kit in preparing a fungus detection product.
In the invention, the prepared fungus detection product can be an independent kit or a detection product matched with equipment or instruments adopted in fungus detection such as a centrifugal pelleter, a fluorescence microscope and the like.
In a fourth aspect, the present invention provides a method for detecting fungi in a biological sample, the method comprising the steps of:
s1, mixing a biological sample to be detected with the biological sample treatment liquid for incubation to obtain a liquid to be detected;
s2, performing fluorescent staining and fluoroscopic examination on the liquid to be detected after the liquid-based thin layer flaking.
Based on the biological sample treatment liquid provided by the invention, the biological sample can be flaked for fungus fluorescence detection only by one-step treatment, the operation is simple and convenient, the enrichment effect on fungi is good, and the fungus sample with low concentration can be detected. The method can be applied to fungus detection in various body fluid samples such as blood, sputum, bronchus lavage, hydrothorax and ascites, pus and wound infected tissues, cerebrospinal fluid, joint fluid, vaginal secretion or urine and the like, and has wide application range.
Fungus detection on biological samples is generally standard, and 3 or more are generally positive in view of unclean production of consumables and reagents used.
According to the invention, in step S1, if the biological sample to be tested is a non-liquid sample such as wound infected tissue or vaginal secretion, the swab dipped with the non-liquid sample can be directly immersed into 2-5mL of the biological sample treatment liquid, and stirring is performed for 10-60S to ensure that the biological sample on the swab is fully released into the biological sample treatment liquid. If the biological sample to be measured is a liquid sample, the liquid sample can be directly mixed with the biological sample treatment liquid, preferably, the volume ratio of the biological sample to be measured to the biological sample treatment liquid is 1:0.5-3.
According to the present invention, preferably, the conditions of the incubation include at least: the temperature is 5-40deg.C, specifically 5 deg.C, 10 deg.C, 15 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, or any value between the two values; the time is 20-40min, and can be specifically 20min, 25min, 30min, 35min, 40min, or any value between the two values. The inventors have found that in this preferred embodiment, it is advantageous to enhance the processing of biological samples to remove interference of non-fungal tangible substances with fungal detection.
According to the present invention, the process of preparing the liquid-based thin film may be performed in a conventional manner, and preferably, in step S2, the process of preparing the liquid-based thin film includes: and settling the liquid to be detected on a glass slide to obtain a glass slide sample, wherein the settling mode adopts centrifugal settling, natural settling or membrane filtration.
According to the present invention, preferably, the slide is a cation-adsorbing slide, and the centrifugal sedimentation is performed by a centrifugal pelleter. Illustratively, centrifugal sedimentation can be performed using a KRD-12 centrifugal slide machine and a mating centrifugal sedimentation slide silo, and a cation-adsorbing slide.
According to the present invention, it is preferable that the membrane filtration employs a filter membrane having a pore size of 4 to 6. Mu.M, preventing spherical fungi having a small diameter from flowing through the filter membrane, resulting in deterioration of detection sensitivity, and failure to detect a low concentration of the fungus sample.
According to the present invention, the centrifugal sedimentation or the natural sedimentation is preferably performed for 20 to 60 minutes, and may specifically be performed for 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, or any value between the two values.
According to the invention, the fluorescent staining may be performed by conventional means of fungal detection fluorescent staining, preferably by chemical staining and/or immunostaining. The fluorescent staining solution is prepared by mixing the following raw materials in percentage by weight: 0.001-0.005% of fluorescent whitening agent, 5-15% of potassium hydroxide, 2-6% of dimethyl sulfoxide, 2-5% of sodium chloride, 5-15% of glycerol, 0.01-0.05% of Evan's blue and the balance of purified water; for another example, the fluorescent staining solution is prepared by mixing the following raw materials in parts by weight: phenol 5-12 parts, glycol 20-40 parts, PBS buffer solution 100-200 parts, phycocyanin or phycoerythrin 30-50 parts and deionized water 100-200 parts.
In the invention, the fluorescence microscopy is performed by using a fluorescence microscope and fluorescence imaging software.
According to a particularly preferred embodiment of the invention, the method for the detection of fungi in biological samples comprises the following steps:
s1, mixing a reducing agent, a protein dissolving agent, a cell lysis agent, a density regulator and water to form a biological sample treatment solution, wherein the cell lysis agent contains sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether, the weight ratio of the sodium dodecyl sulfate to the sodium deoxycholate to the polyethylene glycol octyl phenyl ether is 0.2-15:0.2-2:1, the content of the reducing agent in the biological sample treatment solution is 5-25mM, the content of the protein dissolving agent is 1-8 wt%, the content of the cell lysis agent is 0.3-3 wt%, and the content of the density regulator is 20-50 wt%;
mixing a biological sample to be detected and a biological sample treatment fluid in a volume ratio of 1:0.5-3, and incubating for 20-40min at a temperature of 5-40 ℃ to obtain a liquid to be detected;
s2, centrifugally settling the liquid to be detected for 20-60min to obtain a slide sample, and performing fluorescence staining on the slide sample, and performing microscopic examination by using a conventional fluorescence microscope and fluorescence imaging software.
The present invention will be described in detail by examples.
In the following examples, sodium Dodecyl Sulfate (SDS) was purchased from Shanghai Ala Biotechnology Co., ltd, product number S108349, sodium deoxycholate was purchased from Shanghai Ala Biotechnology Co., ltd, product number S104198, polyethylene glycol octyl phenyl ether (Triton X-100) was purchased from Shanghai Ala Biotechnology Co., ltd, product number T109026, beta-mercaptoethanol was purchased from Shanghai Ala Biotechnology Co., ltd, product number M301573, dithiothreitol was purchased from Shanghai Ala Biotechnology Co., ltd, product number D265376, N-acetylcysteine was purchased from Shanghai Ala Biotechnology Co., product number A105420, tris (2-carboxyethyl) phosphine (TCEP) was purchased from Shanghai Ala Biotechnology Co., ltd, product number T107252, NP-40 was purchased from Shanghai Ala Biotechnology Co., ltd, product number N274337; without specific description, the remaining reagents or raw materials are all conventional biochemical reagent grade products.
In the following examples, the centrifugal pelleter was purchased from Shenzhen Kangruide medical science, inc., model KRD-12, the fluorescence microscope was purchased from Guangzhou Ming Mei photoelectric technology, inc., model MF-31, the fluorescence imaging software was purchased from Guangzhou Ming Mei photoelectric technology, inc., software name dermatophyte mirror detection system (version number v 1.0), and the cation adsorption slide was purchased from Jiangsu shitai laboratory equipment, inc., model liquid-based slide (LBC series).
In the following examples, the fungus fluorescent staining solution is prepared by mixing the following raw materials in percentage by weight: fluorescent whitening agent 0.005%, potassium hydroxide 5%, dimethyl sulfoxide 5%, sodium chloride 2%, glycerin 5%, evan's blue 0.05%, and the balance being purified water.
Without specific explanation, the room temperature was 25.+ -. 5 ℃.
Example 1
10 blood samples positive by blood culture were taken, and the fungus concentration obtained by blood culture was 10-50CFU/mL, as shown in Table 1.
The fungus detection process by the method provided by the invention comprises the following steps:
s1, preparing biological sample treatment fluid, wherein the formula of the biological sample treatment fluid comprises the following components: 10mM dithiothreitol, 4 weight percent of sodium hydroxide, 1 weight percent of SDS, 1 weight percent of sodium deoxycholate, 100 weight percent of Triton X-100, 40 weight percent of ethanol and the balance of water; mixing 1mL of biological sample treatment liquid with 1mL of blood sample, and performing shaking incubation at room temperature for 30min to obtain liquid to be detected;
s2, carrying out liquid-based thin-layer slide preparation on the liquid to be detected by using a KRD-12 centrifugal slide preparation machine and a matched centrifugal sedimentation method slide preparation bin, and obtaining a slide sample, wherein the centrifugal slide preparation time is 30min; the slide sample is dyed by using a fungus fluorescence staining solution, and after the dyeing, the microscopic examination is carried out by using a fluorescence microscope and fluorescence imaging software, 10 blood samples are positive, and the fungus number in the blood sample is determined, wherein the result is shown in a table 1, and an aspergillus staining local microscopic examination picture of the blood sample 1 is shown in fig. 1.
TABLE 1
| Numbering device | CFU/mL for detecting fungus quantity by blood culture | The number of fungi CFU/mL detected in example 1 |
| 1 | 18 | 10 |
| 2 | 22 | 12 |
| 3 | 15 | 11 |
| 4 | 39 | 29 |
| 5 | 41 | 31 |
| 6 | 33 | 21 |
| 7 | 42 | 36 |
| 8 | 25 | 22 |
| 9 | 21 | 17 |
| 10 | 30 | 20 |
Example 2
Taking 12 sputum samples positive in sputum culture, and concentrating fungus obtained by sputum cultureThe degree is 10-10 3 CFU/mL, table 2.
The fungus detection process by the method provided by the invention comprises the following steps:
s1, preparing biological sample treatment fluid, wherein the formula of the biological sample treatment fluid comprises the following components: 10mM dithiothreitol, 10mM TCEP, 2% sodium hydroxide, 0.5% SDS, 0.5% sodium deoxycholate, 100% Triton X-100%, 30% methanol, and the balance water; mixing 3mL of biological sample treatment liquid with 1mL of sputum sample, and vibrating and incubating for 20min at room temperature to obtain liquid to be detected;
s2, carrying out liquid-based thin-layer slide preparation on the liquid to be detected by using a KRD-12 centrifugal slide preparation machine and a matched centrifugal sedimentation method slide preparation bin, and obtaining a slide sample, wherein the centrifugal slide preparation time is 20min; the slide samples were stained with a fungal fluorescent staining solution, and after staining, were subjected to microscopic examination using a fluorescence microscope and fluorescence imaging software, to obtain 12 sputum samples, each of which was positive, and the number of fungi in the sputum samples was determined, and the results are shown in table 2.
TABLE 2
| Numbering device | Sputum culture for detecting fungus quantity CFU/mL | Example 2 number of fungi CFU/mL detected |
| 1 | 5×10 | 3×10 |
| 2 | 1×10 3 | 5×10 2 |
| 3 | 2×10 2 | 1×10 2 |
| 4 | 5×10 2 | 2×10 2 |
| 5 | 5×10 2 | 3×10 2 |
| 6 | 1×10 3 | 6×10 2 |
| 7 | 4×10 2 | 2×10 2 |
| 8 | 8×10 2 | 5×10 2 |
| 9 | 5×10 2 | 4×10 2 |
| 10 | 1×10 2 | 6×10 |
| 11 | 1×10 3 | 6×10 2 |
| 12 | 5×10 2 | 4×10 2 |
Example 3
10 samples of cerebrospinal fluid positive by culture were taken, and the fungus concentration obtained by culture was 10-30CFU/mL, as specified in Table 3.
The fungus detection process by the method provided by the invention comprises the following steps:
s1, preparing biological sample treatment fluid, wherein the formula of the biological sample treatment fluid comprises the following components: beta-mercaptoethanol 4mM, TCEP 4mM, potassium hydroxide 5 wt%, SDS1 wt%, sodium deoxycholate 0.1 wt%, triton X-100.1 wt%, ethanol 35 wt%, and water for the rest; mixing 1mL of biological sample treatment liquid with 1mL of cerebrospinal fluid sample, and performing shake incubation at room temperature for 40min to obtain liquid to be detected;
s2, carrying out liquid-based thin-layer slide preparation on the liquid to be detected by using a KRD-12 centrifugal slide preparation machine and a matched centrifugal sedimentation method slide preparation bin, and obtaining a slide sample, wherein the centrifugal slide preparation time is 60min; the slide samples were stained with a fungus fluorescent staining solution, and after staining, were subjected to microscopic examination using a fluorescence microscope and fluorescence imaging software, 10 samples of cerebrospinal fluid were obtained, and the number of fungi in the cerebrospinal fluid samples was determined, and the results are shown in table 3.
TABLE 3 Table 3
| Numbering device | Culture detection of fungus quantity CFU/mL | Example 3 number of fungi detected CFU/mL |
| 1 | 26 | 18 |
| 2 | 12 | 8 |
| 3 | 30 | 20 |
| 4 | 28 | 22 |
| 5 | 14 | 9 |
| 6 | 11 | 6 |
| 7 | 21 | 15 |
| 8 | 10 | 5 |
| 9 | 17 | 12 |
| 10 | 24 | 15 |
Example 4-example 16
Fungus detection was performed on the blood sample (sample No. 1 in Table 1) in the same manner as in example 1, except that the contents of the respective components in the biological sample treatment liquid were as shown in tables 4 to 6, and the results of the fungus detection were as shown in Table 7.
TABLE 4 Table 4
TABLE 5
TABLE 6
Comparative example 1-comparative example 5
Fungus detection was performed on the blood sample (No. 1 in table 1) according to the method of example 1, except that the contents of the respective components in the biological sample treatment liquid are shown in table 6, and the results of the fungus detection are shown in table 7.
TABLE 7
| Numbering device | Quantity of fungi CFU/mL | Numbering device | Quantity of fungi CFU/mL | Numbering device | Quantity of fungi CFU/mL |
| Example 1 | 10 | Example 10 | 2 | Comparative example 1 | 0 |
| Example 4 | 5 | Example 11 | 4 | Comparative example 2 | 0 |
| Example 5 | 5 | Example 12 | 3 | Comparative example 3 | 0 |
| Example 6 | 3 | Example 13 | 4 | Comparative example 4 | 0 |
| Example 7 | 3 | Example 14 | 2 | Comparative example 5 | 0 |
| Example 8 | 4 | Example 15 | 4 | ||
| Example 9 | 5 | Example 16 | 4 |
As can be seen from the results in Table 7, the biological sample treatment solution provided by the invention is adopted in the embodiment, the sodium dodecyl sulfate, the sodium deoxycholate and the polyethylene glycol octyl phenyl ether are matched to be used as a cell lysis agent, and are matched with a reducing agent, a protein dissolving agent and a density regulator, so that the biological sample can be tabletted for fungus fluorescence detection only by one-step treatment, the operation is simple and convenient, the enrichment effect on fungi is good, the fungus sample with low concentration can be detected, the sensitivity of fungus detection is improved, and the application range is wide.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (10)
1. The biological sample treatment liquid is characterized by comprising a reducing agent, a protein dissolving agent, a cell lysis agent, a density regulator and water, wherein the cell lysis agent comprises sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether.
2. The biological sample processing fluid according to claim 1, wherein the weight ratio of sodium dodecyl sulfate, sodium deoxycholate and polyethylene glycol octyl phenyl ether in the cell lysis agent is 0.2-15:0.2-2:1, preferably 0.5-10:0.5-1:1.
3. Biological sample processing liquid according to claim 1 or 2, characterized in that the content of the reducing agent in the biological sample processing liquid is 5-25mM, preferably 8-20mM; the protein dissolving agent is contained in an amount of 1 to 8 wt%, preferably 2 to 5 wt%; the cell lysis agent is contained in an amount of 0.3 to 3 wt%, preferably 1 to 3 wt%; the density regulator is contained in an amount of 20 to 50% by weight, preferably 30 to 40% by weight.
4. The biological sample processing liquid according to claim 1 or 2, wherein the reducing agent is at least one selected from the group consisting of β -mercaptoethanol, dithiothreitol, N-acetylcysteine, and tris (2-carboxyethyl) phosphine;
preferably, the protein dissolving agent is sodium hydroxide and/or potassium hydroxide;
preferably, the density regulator is selected from at least one of methanol, ethanol, propanol and butanol.
5. A biological sample fungus detection kit, characterized in that the kit contains the biological sample treatment liquid according to any one of claims 1 to 4.
6. The biological sample fungus detection kit according to claim 5, further comprising an adsorption slide and a fungus fluorescent staining solution;
preferably, the adsorption glass slide is a cation adsorption glass slide, and the fungus fluorescence staining solution is a chemical staining solution and/or an immunostaining solution.
7. Use of a biological sample treatment fluid according to any one of claims 1 to 4 and/or a biological sample fungus detection kit according to claim 5 or 6 for the preparation of a fungus detection product.
8. A method for detecting fungi in a biological sample, the method comprising the steps of:
s1, mixing a biological sample to be detected with the biological sample treatment liquid according to any one of claims 1 to 4 for incubation to obtain a liquid to be detected;
s2, performing fluorescent staining and fluoroscopic examination on the liquid to be detected after the liquid-based thin layer flaking.
9. The method according to claim 8, wherein in step S1, the volume ratio of the biological sample to be tested to the biological sample treatment fluid is 1:0.5-3;
preferably, the incubation conditions include at least: the temperature is 5-40deg.C, and the time is 20-40min.
10. The method according to claim 8 or 9, wherein in step S2, the process of producing the liquid-based thin layer tablet comprises: settling the liquid to be detected on a glass slide to obtain a glass slide sample, wherein the settling mode adopts centrifugal settling, natural settling or membrane filtration;
preferably, the glass slide adopts a cation adsorption glass slide, the centrifugal sedimentation is carried out by adopting a centrifugal pelleter, and the membrane filtration adopts a filter membrane with the pore diameter of 4-6 mu M;
preferably, the centrifugal sedimentation or the natural sedimentation is carried out for 20-60min;
preferably, the fluorescent staining is performed by using a chemical staining solution and/or an immunostaining solution, and the fluorescent microscopy is performed by using a fluorescent microscope and fluorescent photographing software.
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