NL2030588B1 - Pretreatment method for fungal positive blood culture by using separating gel and chemical reagent - Google Patents

Pretreatment method for fungal positive blood culture by using separating gel and chemical reagent Download PDF

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NL2030588B1
NL2030588B1 NL2030588A NL2030588A NL2030588B1 NL 2030588 B1 NL2030588 B1 NL 2030588B1 NL 2030588 A NL2030588 A NL 2030588A NL 2030588 A NL2030588 A NL 2030588A NL 2030588 B1 NL2030588 B1 NL 2030588B1
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tube
aspirating
rpm
mixture
room temperature
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NL2030588A
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Zhou Menglan
Yang Qiwen
Xu Yingchun
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Peking Union Medical College Hospital
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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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi

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Abstract

A pretreatment method for fungal positive blood culture by using a separating gel and a chemical reagent is provided. The method includes: identifying a resulting bacterial suspension directly by MALDI-TOF mass spectrometry after treatment with chemical reagents, which effectively avoids the defect that the whole process takes a long time in the prior art.

Description

PRETREATMENT METHOD FOR FUNGAL POSITIVE BLOOD CULTURE BY
USING SEPARATING GEL AND CHEMICAL REAGENT
TECHNICAL FIELD
[01] The present invention relates to the technical field of fungal positive blood culture.
BACKGROUND ART
[02] According to an existing identification method for fungal positive blood culture, a positive blood culture solution needs to be transferred to a culture medium for incubation until a single pure colony is visible to naked eyes, and then the single pure colony is selected for identification by a subsequent method, so that the whole process takes a long time (about 48-72 h).
SUMMARY
[03] To overcome the defects in the prior art, the present invention provides a solution to a pretreatment method for fungal positive blood culture by using a separating gel and a chemical reagent.
[04] The method includes: extracting 8.5 ml of positive blood culture solution into a 8.5 ml gel and clot activator tube; placing the gel and clot activator tube into a centrifugal machine for centrifugation at a centrifugal force of 4000 g for 10 min; after centrifuging and discarding supernatant, adding 1 ml of Tween 20 to an EP tube for repeated suction and blowing, then allowing the EP tube to stand at room temperature for 3 min, centrifuging in the centrifugal machine at 13000 rpm for 2 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube; blowing and sucking a precipitate repeatedly with 1 ml of sterile double distilled water and 200 pl of SDS at a concentration of 10% to form a bacterial suspension; and identifying the resulting bacterial suspension directly by MALDI-TOF mass spectrometry after treatment with chemical reagents, which effectively avoids the defect that the whole process takes a long time in the prior art.
[05] The present invention has the following beneficial effects:
According to the method for fungal positive blood culture by using a separating gel in the present invention, a positive blood culture solution is identified directly by mass spectrometry after pretreatment with a separating gel rather than transferring the positive blood culture solution to a culture medium until a single colony grows for identification by a subsequent method. The whole process takes about 30-40 min, greatly shortening the identification process, and saving manpower and material resources.
BRIEFT DESCRIPTION OF THE DRAWINGS
[06] FIG. 1 is a schematic structural diagram of a scarfing device of the present invention;
[07] FIG. 2 is a schematic structural diagram of a first scarfing portion of the present invention;
[08] FIG. 3 is a partial sectional view of a second scarfing portion of the present invention; and
[09] FIG. 41s a sectional view of a scarfing device of the present invention.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[10] The present invention will be further described with reference to the drawings and embodiments.
EMBODIMENT 1
[11] A pretreatment method for fungal positive blood culture by using a separating gel and a chemical reagent, including the following specific steps: step 1: extracting a positive blood culture solution into a 8.5 ml gel and clot activator tube; step 2: placing the gel and clot activator tube into a centrifugal machine for centrifugation at a centrifugal force of 4000 g for 10 min; step 3: sucking and discarding supernatant without touching a pellicle on the surface of a separating gel in the gel and clot activator tube; step 4: blowing and sucking the pellicle on the surface of the separating gel in the gel and clot activator tube repeatedly with 1 ml of sterile double distilled water to suspend the pellicle in the sterile double distilled water to form a bacterial suspension; step S: transferring 1 ml of the bacterial suspension into a 1.5 ml EP tube, placing the EP tube into the centrifugal machine for centrifugation at 13000 rpm for 2 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube; step 6: adding another 1 ml of sterile double distilled water to the EP tube, then blowing and sucking repeatedly for washing, centrifuging in the centrifugal machine at 13000 rpm for 2 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube;
step 7: adding 1 ml of Tween 20 to the EP tube, then sucking and blowing repeatedly,
allowing the EP tube to stand at room temperature for 3 min, centrifuging in the centrifugal machine at 13000 rpm for 2 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube;
step 8: adding 1 ml of sterile double distilled water and 200 ul of SDS at a concentration of
10% to the EP tube, then placing the EP tube on a vortex oscillator for vortex oscillation for 1 min, and allowing the EP tube to stand at room temperature for 5 min;
step 9: placing the EP tube into a centrifuge for centrifugation at 13000 rpm for 2 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube; step 10: adding 700 ul of ethanol at a concentration of 75% to a precipitate at the bottom of the EP tube to form a first mixture, and mixing the first mixture well by blowing and sucking; step 11: allowing the EP tube to stand at room temperature for 10 min; step 12: placing the EP tube into the centrifugal machine for centrifugation at 13000 rpm for 2 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube;
step 13: continuously centrifuging in the centrifugal machine at 13000 rpm for 1 min, and sucking and discarding supernatant without touching a precipitate at the bottom of the EP tube; then placing the EP tube in a biosafety cabinet, allowing the EP tube to stand at room temperature for 5 min, and blow-drying residual ethanol in a substance in the EP tube to completely volatilize the residual ethanol;
step 14: adding 15 ul of formic acid at a concentration of 70% to a precipitate at the bottom of each EP tube to form a second mixture, then mixing the second mixture well by blowing and sucking, and allowing the EP tube to stand at room temperature; step 15: adding 15 pl of acetonitrile to the EP tube to form a third mixture, then mixing the third mixture well by blowing and sucking, placing the EP tube into a centrifugal machine for centrifugation at 13000 rpm for 2 min; and step 16: dripping 1 ul of supernatant from the EP tube on a target plate, and drying the target plate in the air at room temperature; adding 1 pl of a-cyano-4-hydroxycinnamic acid (CHCA) matrix, and placing the target plate into a Bruker mass spectrometer for mass spectrometry.
[12] The amount of the positive blood culture solution extracted in the step 1 is 8.5 ml.
[13] In the step 4, the pellicle on the surface of the separating gel is blown and sucked repeatedly with 1 ml of sterile double distilled water for 5 times.
[14] The EP tube is allowed to stand at room temperature for 5 min in the step 14.
[15] The embodiment has the following beneficial effects:
According to the method for fungal positive blood culture by using a separating gel in the embodiment, a positive blood culture solution is identified directly by mass spectrometry after pretreatment with a separating gel rather than transferring the positive blood culture solution to a culture medium until a single colony grows for identification by a subsequent method. The whole process takes 30 min, greatly shortening the identification process, and saving manpower and material resources.
EMBODIMENT 2
[16] A pretreatment method for fungal positive blood culture by using a separating gel and a chemical reagent, characterized in that specific steps differ from Embodiment 1 in that:
[17] In the step 4, the pellicle on the surface of the separating gel is blown and sucked repeatedly with 1 ml of sterile double distilled water for 8 times.
[18] The EP tube is allowed to stand at room temperature for 8 min in the step 14.
[19] The embodiment has the following beneficial effects:
[20] The effect of the embodiment differs from that of Embodiment 1 in that the whole process takes 35 min.
EMBODIMENT 3
[21] A pretreatment method for fungal positive blood culture by using a separating gel and a chemical reagent, characterized in that specific steps differ from Embodiment 1 in that:
[22] In the step 4, the pellicle on the surface of the separating gel is blown and sucked repeatedly with 1 ml of sterile double distilled water for 10 times.
[23] The EP tube is allowed to stand at room temperature for 10 min in the step 14.
[24] The embodiment has the following beneficial effects:
The effect of the embodiment differs from that of Embodiment 1 in that the whole process takes 40 min.
EMBODIMENT 4
[25] A pretreatment method for fungal positive blood culture by using a separating gel 5 and a chemical reagent, characterized in that specific steps differ from Embodiment 1 in that:
[26] In the step 4, the pellicle on the surface of the separating gel is blown and sucked repeatedly with 1 ml of sterile double distilled water for 10 times.
[27] The EP tube is allowed to stand at room temperature for 10 min in the step 14.
[28] The embodiment has the following beneficial effects:
[29] The effect of the embodiment differs from that of Embodiment 1 in that the whole process takes 40 min.
[30] In addition, the centrifugal machine includes a cuboid support. A support is often susceptible to damage by moisture if placed directly on the ground. To keep away from moisture, a bottom wall of the support is often connected to a top wall of a cuboid stainless steel base placed on the ground, and the structure in which the bottom wall of the support is connected to the top wall of the cuboid stainless steel base is connected through the bottom wall of the support and the top wall of the stainless steel base. The structure in which the bottom wall of the support is connected to the top wall of the stainless steel base is generally that a positioning hole needs to be formed in the top wall of the stainless steel base, and then a screw rod passes through the bottom wall of the base from top to bottom and is connected with a nut which is fixedly welded in the positioning hole in a screwing mode, and therefore the connection between the bottom wall of the base and the top wall of the stainless steel base is completed. Such structure not only increases the manufacturing cost, but also has low assembly accuracy and poor visual effect.
[31] The structure in which the bottom wall of the support of the centrifugal machine is connected to the top wall of the cuboid stainless steel base placed on the ground is that the bottom wall of the support is connected to the top wall of the stainless steel base through a scarfing device S00;
[32] the scarfing device SOO includes a first scarfing portion SO and a second scarfing portion TO;
[33] the first scarfing portion SO is fixedly connected to the top wall of the stainless steel base, and the second scarfing portion TO is fixedly connected to the bottom wall of the support;
[34] an upper part of the first scarfing portion SO is a cuboid plate, a lower part of the first scarfing portion SO is an arch, a top end of the arch is fixedly connected to a bottom wall of the cuboid plate, a bottom end of the arch is fixedly connected to the top wall of the stainless steel base, a scarf hole S1 extending inside the arch is formed in the top wall of the cuboid plate, and a top end interface S11 of the scarf hole S1 is formed in the top wall of the cuboid plate;
[35] an upper part of the second scarfing portion TO is a cylinder, a top end of the cylinder is fixedly connected to the bottom wall of the base, a bottom end of the cylinder is a scarf joint T1 made of glass bronze, the scarf joint T1 is scarfed into the scarf hole SI, the top end interface S11 of the scart hole S1 blocks the scarf joint T1, and an outer surface of the scarf joint T1 can be brought close when scarfed into or withdrawn from the scarf hole S1.
[36] The scarf hole S1 of the first scarfing portion SO has a spherical arc structure, and a contour of the outer surface of the scarf joint T1 also has a spherical arc structure.
[37] A contour of the scarf hole S1 of the first scarfing portion SO may be formed by smoothly connecting several segmental arc arches, or may be a truncated cone, and the outer surface of the scarf joint T1 may have the same shape as the contour of the scarf hole
S1 of the first scarfing portion SO.
[38] The scarf joint T1 has a plurality of glass bronze arch scarfers T11 connected around the bottom of the cylinder of the second scarfing portion TO, the arch scarfers T11 equidistantly surrounds the bottom of the cylinder of the second scarfing portion TO, and outer surfaces of all the arch scarfers T11 form the outer surface of the scarf joint T1.
[39] The scarf hole S1 includes a bottom surface S12 in the scarf hole S1 and an edge surface S13 in the scarf hole S1, the bottom surface S12 in the scarf hole S1 is opposite to the top end interface S11 of the scarf hole S1, and the edge surface in the scarf hole SI is connected to a bottom surface S12 in the scarf hole S1. In addition, the structure from the edge surface S13 in the scarf hole S1 to the top end interface S11 of the scarf hole SI is a tapered structure.
[40] The bottom surface S12 in the scarf hole S1 has a spherical arc shape, the edge surface S13 in the scarf hole SI has a cylindrical section S13S and a truncated cone section
S13T which are sequentially connected, a bottom of the cylindrical section S13S is connected to the bottom surface S12 in the scarf hole S1, an upper part of the truncated cone section S13T is connected to the top end interface S11 of the scarf hole S1, and the truncated cone section S13T is the tapered structure.
[41] In addition, the first scarfing portion SO and the top wall of the stainless steel base are integrally formed in one step, and the second scarfing portion TO and the base are integrally formed in one step.
[42] When the scarf joint T1 made of glass bronze is scarfed into the scarf hole S1, the outer surface of the scarf joint T1 made of glass bronze is gradually brought close to project from the top end interface S11 of the scarf hole S1, and when the scarf joint T1 made of glass bronze is withdrawn from the scarf hole S1 of the first scarfing portion SO, the outer surface of the scarf joint T1 made of glass bronze is gradually brought close to be withdrawn from the top end interface S11 of the scarf hole SI, so that the first scarfing portion SO and the second scarfing portion TO are combined more firmly, with high assembly accuracy, and efficient assembly and disassembly.

Claims (1)

ConclusiesConclusions 1. Voorbehandelingswerkwijze voor schimmelpositieve bloedkweek met behulp van een scheidingsgel en een chemisch reagens, die de volgende specifieke stappen omvat: 1: het plaatsen van een 8,5 ml gel en bloedpropactivatorbuis, die een positieve bloedkweekoplossing bevat, in een centrifugemachine voor centrifugatie met een centrifugale kracht van 4000 g gedurende 10 min; 2: het zuigen en weggooien van supernatant zonder het aanraken van een vlies op het oppervlak van een scheidingsgel in de gel en bloedpropactivatorbuis; 3: het herhaaldelijk blazen en zuigen met 1 ml steriel dubbel gedestilleerd water om een bacteriële suspensie te vormen; 4: het overbrengen van 1 ml van de bacteriële suspensie in een 1,5 ml EP-buis voor centrifugatie bij 13000 rpm gedurende 2 min en het zuigen en weggooien van supernatant; 5: het toevoegen van 1 ml steriel dubbel gedestilleerd water aan de EP-buis, daarna het herhaaldelijk blazen en zuigen voor het wassen, het centrifugeren bij 13000 rpm gedurende 2 min en het zuigen en weggooien van supernatant, 6: het toevoegen van 1 ml Tween 20 aan de EP-buis, daarna het herhaaldelijk zuigen en blazen, het toestaan aan de EP-buis om te staan bij kamertemperatuur gedurende 3 min, daarna het centrifugeren bij 13000 rpm gedurende 2 min en het zuigen en weggooien van supernatant; 7: het toevoegen van 1 ml steriel dubbel gedestilleerd water en 200 ul SDS in een concentratie van 10% aan de EP-buis, daarna het plaatsen van de EP-buis op een vortexoscillator voor vortexoscillatie gedurende | min en het toestaan aan de EP-buis om te staan bij kamertemperatuur gedurende 5 min; 8: het plaatsen van de EP-buis in de centrifugemachine voor centrifugatie bij 13000 rpm gedurende 2 min en het zuigen en weggooien van supernatant; 9: het toevoegen van 700 ul ethanol in een concentratie van 75% aan een neerslag op de bodem van de EP-buis om een eerste mengsel te vormen, en het goed mengen van het eerste mengsel door blazen en zuigen; 10: het toestaan aan de EP-buis om te staan bij kamertemperatuur gedurende 10 min;1. Pretreatment method for fungal-positive blood culture using a separation gel and a chemical reagent, which includes the following specific steps: centrifugal force of 4000 g for 10 min; 2: Aspirating and discarding supernatant without touching a pellicle on the surface of a separating gel in the gel and clot activator tube; 3: blowing and sucking repeatedly with 1 ml of sterile double-distilled water to form a bacterial suspension; 4: Transfer 1 ml of the bacterial suspension to a 1.5 ml EP tube for centrifugation at 13000 rpm for 2 min and aspirate and discard supernatant; 5: adding 1 ml of sterile double-distilled water to the EP tube, then blowing and aspirating repeatedly for washing, centrifuging at 13000 rpm for 2 min and aspirating and discarding supernatant, 6: adding 1 ml Tween 20 to the EP tube, then repeatedly aspirate and blow, allow the EP tube to stand at room temperature for 3 min, then centrifuge at 13000 rpm for 2 min and aspirate and discard supernatant; 7: Adding 1 ml of sterile doubly distilled water and 200 µl of SDS at a concentration of 10% to the EP tube, then placing the EP tube on a vortex oscillator for vortex oscillation for | min and allowing the EP tube to stand at room temperature for 5 min; 8: Placing the EP tube in the centrifuge machine for centrifugation at 13000 rpm for 2 min and aspirating and discarding supernatant; 9: adding 700 µl of ethanol at a concentration of 75% to a precipitate at the bottom of the EP tube to form a first mixture, and thoroughly mixing the first mixture by blowing and aspirating; 10: Allowing the EP tube to stand at room temperature for 10 min; 11: het centrifugeren van de EP-buis bij 13000 rpm gedurende 2 min en het zuigen en weggooien van supernatant;11: Centrifugation of the EP tube at 13000 rpm for 2 min and aspirating and discarding supernatant; 12: het centrifugeren bij 13000 rpm gedurende 1 min, en het zuigen en weggooien van supernatant; daarna het plaatsen van de EP-buis in een bioveiligheidskast,12: Centrifugation at 13000 rpm for 1 min, aspirating and discarding supernatant; then placing the EP tube in a biosafety cabinet, het toestaan aan de EP-buis om te staan bij kamertemperatuur gedurende 5 min en het blaasdrogen van resterend ethanol in een stof in de EP-buis;allowing the EP tube to stand at room temperature for 5 minutes and blow drying residual ethanol into a substance in the EP tube; 13: het toevoegen van 15 ul mierenzuur in een concentratie van 70% aan een neerslag op de bodem van de EP-buis om een tweede mengsel te vormen, daarna het goed mengen van het tweede mengsel door het blazen en zuigen en het toestaan aan het mengsel om te staan bij kamertemperatuur;13: adding 15 µl of formic acid at a concentration of 70% to a precipitate at the bottom of the EP tube to form a second mixture, then thoroughly mixing the second mixture by blowing and aspirating and allowing it to mixture to stand at room temperature; 14: het toevoegen van 15 ul acetonitril aan de EP-buis om een derde mengsel te vormen, daarna het goed mengen van het derde mengsel door het blazen en zuigen en het centrifugeren bij 13000 rpm gedurende 2 min; en14: adding 15 µl of acetonitrile to the EP tube to form a third mixture, then thoroughly mixing the third mixture by blowing and aspirating and centrifuging at 13000 rpm for 2 min; and 15: het druppelen van 1 ul supernatant van de EP-buis op een doelplaat en het drogen van de doelplaat in de lucht bij kamertemperatuur; het toevoegen van 1 pl van een 0-cyaan-4-hydroxykaneelzuur- (CHCA-) matrix en het plaatsen van de doelplaat in een Bruker massaspectrometer voor massaspectrometrie.15: Dropping 1 µl of supernatant from the EP tube onto a target plate and drying the target plate in air at room temperature; adding 1 µl of an O-cyano-4-hydroxycinnamic acid (CHCA) matrix and placing the target plate in a Bruker mass spectrometer for mass spectrometry.
NL2030588A 2022-01-18 2022-01-18 Pretreatment method for fungal positive blood culture by using separating gel and chemical reagent NL2030588B1 (en)

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