CN117517443A - Method for rapidly detecting positive blood culture direct mass spectrum - Google Patents
Method for rapidly detecting positive blood culture direct mass spectrum Download PDFInfo
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- CN117517443A CN117517443A CN202311459436.2A CN202311459436A CN117517443A CN 117517443 A CN117517443 A CN 117517443A CN 202311459436 A CN202311459436 A CN 202311459436A CN 117517443 A CN117517443 A CN 117517443A
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- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000009640 blood culture Methods 0.000 title claims abstract description 26
- 238000001819 mass spectrum Methods 0.000 title claims abstract description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000000926 separation method Methods 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 15
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000005406 washing Methods 0.000 claims abstract description 8
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 7
- 235000019253 formic acid Nutrition 0.000 claims abstract description 7
- 239000011159 matrix material Substances 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims description 15
- 238000004949 mass spectrometry Methods 0.000 claims description 12
- 239000002504 physiological saline solution Substances 0.000 claims description 9
- 239000008223 sterile water Substances 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 239000003223 protective agent Substances 0.000 claims description 3
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims description 3
- 229930182490 saponin Natural products 0.000 claims description 3
- 150000007949 saponins Chemical class 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000002344 surface layer Substances 0.000 claims description 3
- 241001052560 Thallis Species 0.000 abstract description 3
- 238000001269 time-of-flight mass spectrometry Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001085 differential centrifugation Methods 0.000 description 2
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
- G01N27/628—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas and a beam of energy, e.g. laser enhanced ionisation
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Abstract
The invention discloses a method for rapidly detecting positive blood culture direct mass spectrum, which comprises the following steps: s1: centrifugal extraction of the separation rubber tube; s2, washing with sterilized water; s3: blood culture microorganism pretreatment kit; s4, washing with ethanol; s5: formic acid and acetonitrile are cracked; s6, sample application and matrix addition; s7, MALDA-TOFMS detection. According to the invention, after the thalli are enriched by using the separation gel, the corresponding technical innovation is carried out by combining the kit, so that a better experimental result is obtained, and the positive blood culture sample is treated by combining the separation gel with the kit, so that the method has advantages and innovativeness, and is suitable for popularization and application in a clinical microorganism laboratory.
Description
Technical Field
The invention relates to the technical field of bacteria separation and identification in blood, in particular to a direct mass spectrum rapid detection method for positive blood culture.
Background
The spectrum technology is used for identifying clinical pathogenic bacteria as an emerging test technology in recent years, is more and more widely applied to clinic in recent years due to the characteristics of accuracy and rapidness, and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is an epoch-making technical innovation in clinical microbiology, and with the continuous improvement of technology and the perfection of a database, mass spectrometry can be used for directly detecting bacteria under the condition that a positive blood culture bottle is subjected to pretreatment, so that the spectrum is considered to be the most effective method for identifying bacteria. The enrichment of thalli in the pretreatment process is a key step and is also an important factor directly influencing the positive blood culture identification result.
The currently commonly adopted thallus enrichment methods comprise a common gel separation coagulation tube method, a filter membrane adsorption method and a differential centrifugation method, but all three methods have the defects that:
1. the filter membrane adsorption method requires a laboratory to be provided with a filter device, and has complex operation and high experimental environment requirements.
2. Differential centrifugation is a common method in pretreatment, but different centrifugal speeds are used, the difference of enrichment of strains is large, the standardization is difficult, and the operation method is relatively complicated.
3. The common separation gel coagulation tube method has low overall identification accuracy.
Therefore, we propose a direct mass spectrometry rapid detection method for positive blood culture.
Disclosure of Invention
1. In order to make up the defects of the prior art, the invention provides the following technical scheme: a method for rapidly detecting positive blood culture direct mass spectrum comprises the following steps:
s1: centrifugal extraction of the separation rubber tube;
s2, washing with sterilized water;
s3: blood culture microorganism pretreatment kit;
s4, washing with ethanol;
s5: formic acid and acetonitrile are cracked;
s6, sample application and matrix addition;
s7, MALDA-TOF MS detection.
Preferably, the step S1 includes the steps of:
s11: transferring 4-5ml from the positive culture flask into a plasma separation rubber tube, centrifuging at 900g level for 10min, discarding upper serum, and retaining off-white thallus enrichment on the surface layer of the separation rubber;
s13: 500ul of sterile water is added into a 1.5ml centrifuge tube, the off-white thallus enrichment obtained in the above steps is picked, added into the centrifuge tube, and centrifuged at a high speed of 16000g for 2 minutes.
Preferably, the step S2 includes the steps of:
s21, adding 500ul of sterile water into a 1.5ml centrifuge tube, picking the gray-white thallus enrichment obtained in the steps, adding the gray-white thallus enrichment into the centrifuge tube, and centrifuging at a high speed of 16000g for 2 minutes;
s22: the supernatant was removed by pipetting with a sterile lance, 500ul of sterile water was added, mixed well by shaking, and centrifuged at 16000g for 2 minutes.
Preferably, the step S3 includes the steps of:
s31, adding 1.0mL of sterile physiological saline into a centrifuge tube of the thallus enrichment;
s32, absorbing 200uL of reagent containing saponin and protective agent dimethyl sulfoxide, adding into the centrifuge tube in the step 5, fully oscillating and uniformly mixing, standing for 3-5 minutes at room temperature, centrifuging for 10 minutes, and rotating at 2600rpm;
s33, sucking the supernatant by using a micropipette, adding 1mL of physiological saline, and re-suspending and precipitating;
s34, separating for 2 minutes at 13000m, removing the upper part, re-suspending by using 1mL of physiological saline, and centrifuging for 2 minutes at 13000rpm.
Preferably, the step S4 includes the steps of:
s41, removing the supernatant fully, adding 500ul of 75% alcohol, shaking and mixing uniformly, and centrifuging for 2 minutes at 16000 g;
s42, discarding the supernatant, then 16000g, centrifuging for 2 minutes, uncovering at room temperature, standing and airing to remove the ethanol.
Preferably, the method of S5 is as follows: 20ul of 70% formic acid was added, mixed well with shaking, 20ul of acetonitrile was added, 16000g was added, and centrifuged for 2 minutes.
Preferably, the method of S6 is as follows: and (3) adding matrix liquid into 1ul of supernatant after centrifugation, spotting a target plate, and performing mass spectrometry.
Advantageous effects
Compared with the prior art, the invention provides a direct mass spectrum rapid detection method for positive blood culture, which has the following beneficial effects:
1. according to the method for rapidly detecting the positive blood culture direct mass spectrum, after a positive blood culture specimen is processed through a bacteria enrichment technology, MALDA-TOF MS can be directly used for identification, so that the identification speed is greatly improved, the reporting time is shortened, the requirement of clinical rapid diagnosis is met, the MALDA-TOF MS system is utilized, pathogenic bacteria in body fluid culture are directly identified by adopting the method recorded by the invention, the identification accuracy is improved, the separation gel can be used for carrying out corresponding technical innovation by combining a kit after enriching the thalli, a better experimental result is obtained, and the positive blood culture specimen is treated by combining the separation gel with the kit in view of advantages and innovativeness, so that the method is suitable for popularization and application in a clinical microorganism laboratory.
Drawings
FIG. 1 is a flow chart of a method for rapid detection of a direct mass spectrum of positive blood culture in the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1, the invention provides a method for rapid detection of positive blood culture direct mass spectrometry, which comprises the following steps:
s1: centrifugal extraction of the separation rubber tube;
s2, washing with sterilized water;
s3: blood culture microorganism pretreatment kit;
s4, washing with ethanol;
s5: formic acid and acetonitrile are cracked;
s6, sample application and matrix addition;
s7, MALDA-TOF MS detection.
In this embodiment, the step S1 includes the following steps:
s11: transferring 4-5ml from the positive culture flask into a plasma separation rubber tube, centrifuging at 900g level for 10min, discarding upper serum, and retaining off-white thallus enrichment on the surface layer of the separation rubber;
s13: 500ul of sterile water is added into a 1.5ml centrifuge tube, the off-white thallus enrichment obtained in the above steps is picked, added into the centrifuge tube, and centrifuged at a high speed of 16000g for 2 minutes.
In this embodiment, the step S2 includes the following steps:
s21, adding 500ul of sterile water into a 1.5ml centrifuge tube, picking the gray-white thallus enrichment obtained in the steps, adding the gray-white thallus enrichment into the centrifuge tube, and centrifuging at a high speed of 16000g for 2 minutes;
s22: the supernatant was removed by pipetting with a sterile lance, 500ul of sterile water was added, mixed well by shaking, and centrifuged at 16000g for 2 minutes.
In this embodiment, the step S3 includes the following steps:
s31, adding 1.0mL of sterile physiological saline into a centrifuge tube of the thallus enrichment;
s32, absorbing 200uL of reagent containing saponin and protective agent dimethyl sulfoxide, adding into the centrifuge tube in the step 5, fully oscillating and uniformly mixing, standing for 3-5 minutes at room temperature, centrifuging for 10 minutes, and rotating at 2600rpm;
s33, sucking the supernatant by using a micropipette, adding 1mL of physiological saline, and re-suspending and precipitating;
s34, separating for 2 minutes at 13000m, removing the upper part, re-suspending by using 1mL of physiological saline, and centrifuging for 2 minutes at 13000rpm.
In this embodiment, the step S4 includes the following steps:
s41, removing the supernatant fully, adding 500ul of 75% alcohol, shaking and mixing uniformly, and centrifuging for 2 minutes at 16000 g;
s42, discarding the supernatant, then 16000g, centrifuging for 2 minutes, uncovering at room temperature, standing and airing to remove the ethanol.
In this embodiment, the method of S5 is: 20ul of 70% formic acid was added, mixed well with shaking, 20ul of acetonitrile was added, 16000g was added, and centrifuged for 2 minutes.
In this embodiment, the method of S6 is: and (3) adding matrix liquid into 1ul of supernatant after centrifugation, spotting a target plate, and performing mass spectrometry.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. A method for rapidly detecting positive blood culture direct mass spectrum is characterized by comprising the following steps:
s1: centrifugal extraction of the separation rubber tube;
s2, washing with sterilized water;
s3: blood culture microorganism pretreatment kit;
s4, washing with ethanol;
s5: formic acid and acetonitrile are cracked;
s6, sample application and matrix addition;
s7, MALDA-TOF MS detection.
2. The method for rapid detection of positive blood culture direct mass spectrometry according to claim 1, wherein the method comprises the following steps: the step S1 comprises the following steps:
s11: transferring 4-5ml from the positive culture flask into a plasma separation rubber tube, centrifuging at 900g level for 10min, discarding upper serum, and retaining off-white thallus enrichment on the surface layer of the separation rubber;
s13: 500ul of sterile water is added into a 1.5ml centrifuge tube, the off-white thallus enrichment obtained in the above steps is picked, added into the centrifuge tube, and centrifuged at a high speed of 16000g for 2 minutes.
3. The method for rapid detection of positive blood culture direct mass spectrometry according to claim 1, wherein the method comprises the following steps: the step S2 comprises the following steps:
s21, adding 500ul of sterile water into a 1.5ml centrifuge tube, picking the gray-white thallus enrichment obtained in the steps, adding the gray-white thallus enrichment into the centrifuge tube, and centrifuging at a high speed of 16000g for 2 minutes;
s22: the supernatant was removed by pipetting with a sterile lance, 500ul of sterile water was added, mixed well by shaking, and centrifuged at 16000g for 2 minutes.
4. The method for rapid detection of positive blood culture direct mass spectrometry according to claim 1, wherein the method comprises the following steps: the step S3 comprises the following steps:
s31, adding 1.0mL of sterile physiological saline into a centrifuge tube of the thallus enrichment;
s32, absorbing 200uL of reagent containing saponin and protective agent dimethyl sulfoxide, adding into the centrifuge tube in the step 5, fully oscillating and uniformly mixing, standing for 3-5 minutes at room temperature, centrifuging for 10 minutes, and rotating at 2600rpm;
s33, sucking the supernatant by using a micropipette, adding 1mL of physiological saline, and re-suspending and precipitating;
s34, separating for 2 minutes at 13000m, removing the upper part, re-suspending by using 1mL of physiological saline, and centrifuging for 2 minutes at 13000rpm.
5. The method for rapid detection of positive blood culture direct mass spectrometry according to claim 1, wherein the method comprises the following steps: the step S4 comprises the following steps:
s41, removing the supernatant fully, adding 500ul of 75% alcohol, shaking and mixing uniformly, and centrifuging for 2 minutes at 16000 g;
s42, discarding the supernatant, then 16000g, centrifuging for 2 minutes, uncovering at room temperature, standing and airing to remove the ethanol.
6. The method for rapid detection of positive blood culture direct mass spectrometry according to claim 1, wherein the method comprises the following steps: the method of S5 is as follows: 20ul of 70% formic acid was added, mixed well with shaking, 20ul of acetonitrile was added, 16000g was added, and centrifuged for 2 minutes.
7. The method for rapid detection of positive blood culture direct mass spectrometry according to claim 1, wherein the method comprises the following steps: the method of S6 is as follows: and (3) adding matrix liquid into 1ul of supernatant after centrifugation, spotting a target plate, and performing mass spectrometry.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311459436.2A CN117517443A (en) | 2023-11-03 | 2023-11-03 | Method for rapidly detecting positive blood culture direct mass spectrum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311459436.2A CN117517443A (en) | 2023-11-03 | 2023-11-03 | Method for rapidly detecting positive blood culture direct mass spectrum |
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| Publication Number | Publication Date |
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| CN117517443A true CN117517443A (en) | 2024-02-06 |
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| CN202311459436.2A Pending CN117517443A (en) | 2023-11-03 | 2023-11-03 | Method for rapidly detecting positive blood culture direct mass spectrum |
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| Country | Link |
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| CN (1) | CN117517443A (en) |
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- 2023-11-03 CN CN202311459436.2A patent/CN117517443A/en active Pending
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