CN108102966A - MALDI-TOFMS instant Quality Control viable bacteria standard items - Google Patents

MALDI-TOFMS instant Quality Control viable bacteria standard items Download PDF

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Publication number
CN108102966A
CN108102966A CN201810018888.XA CN201810018888A CN108102966A CN 108102966 A CN108102966 A CN 108102966A CN 201810018888 A CN201810018888 A CN 201810018888A CN 108102966 A CN108102966 A CN 108102966A
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CN
China
Prior art keywords
standard items
viable bacteria
bacteria standard
maldi
quality control
Prior art date
Application number
CN201810018888.XA
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Chinese (zh)
Inventor
崔生辉
刘娜
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中国食品药品检定研究院
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Priority to CN201810018888.XA priority Critical patent/CN108102966A/en
Publication of CN108102966A publication Critical patent/CN108102966A/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04Preserving or maintaining viable microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14Streptococcus; Staphylococcus

Abstract

The invention discloses a kind of MALDI TOF MS instants Quality Control viable bacteria standard items, solve existing positive control strain obtain the time it is long the technical issues of, belong to the technical fields of mass spectrum standard items.The viable bacteria standard items, are prepared by following steps:(1) the second-generation bacterial strain that purpose obtains single bacterium colony source is cultivated, identification confirms that kind is correct;(2) fresh second-generation bacterial strain is collected, is suspended in freeze drying protectant, viable bacteria standard items mother liquor is made;(3) viable bacteria standard items mother liquor is diluted, calculates bacterial strain concentration in viable bacteria standard items mother liquor;Meanwhile bacterium ball is made in the viable bacteria standard items mother liquor after dilution, it calculates and obtains lyophilized survival rate;(4) according to lyophilized survival rate, the Quality Control viable bacteria standard items containing target bacterium number is prepared, are stored in 20 DEG C.The characteristics of present invention can make full use of MALDI TOF MS identification bacterial strain kinds quick, high-throughput, shortens the time of Micro biological Tests, improves checkability.

Description

MALDI-TOFMS instant Quality Control viable bacteria standard items

Technical field

Technical field more particularly to a kind of MALDI-TOF MS instants Quality Control viable bacteria the present invention relates to mass spectrum standard items Standard items.

Background technology

Matrix-assisted laser desorption ionization (Matrix-Assisted Laser Desorption/ Ionization Time ofFlight Mass Spectrometry, MALDI-TOF-MS) it is developed in recent years one The new mass-spectrometric technique identified by ionization bacterial strain kind of kind, principle is to be formed with laser irradiating sample with matrix Cocrystallization film, matrix absorbs energy from laser and passes to biomolecule, and life is transferred an electron in ionization process The sub or slave biomolecule of object point obtains electronics, and charged biomolecule accelerates to fly over dirft tube under electric field action, and to be measured The mass-to-charge ratio (M/Z) of biomolecule is directly proportional to the flight time, so as to different according to the flight time for reaching detector to life Object molecule is detected.

MALDI-TOF MS technologies are due to having the technologies such as quick, high-throughput a little, in terms of daily bacterial strain identifies kind Receive favor, from environment bacterial strain screening to common pathogen Rapid identification, MALDI-TOF MS all play it is increasingly heavier The effect wanted.But MALDI-TOF MS are generally divided into several grades to the qualification result of sample, i.e., for some bacterial strain kinds Identification being only possible to property judgement, for it needs to be determined that kind bacterial strain identify, especially need to provide the bacterium of reading report Strain, it is necessary to which precise Identification is carried out to strain kind.It, can be by strain to be tested and positive control strain if setting positive control Peak figure is compared, to determine strain kind.And positive control strain, it generally requires and is cultivated to obtain, generation bacterium Incubation time at least needs 18h.And experiment bacterial strain, especially control strain are typically chosen the second-generation bacterial strain of monoclonal, so Available positive control strain acquisition at least needs 36h, this is for routine check, and especially emergent to examine, there are larger drawbacks.

Efficiently utilize the technical characterstics such as the quick, high throughput of MALDI-TOF MS identification bacterial strain kinds, it is ensured that bacterial strain kind is reflected Determine reliable results, be one of effective way for improving checkability.By setting positive control to suspicious bacterial strain, bacterium can be met Strain Species estimation accurately requires, if the incubation of positive control strain is avoided to meet instantaneity requirement, is undoubtedly efficient Utilize the important method of MALDI-TOF MS.

The content of the invention

The present invention for existing MALDI-TOF MS with positive control strain obtain the time it is long the technical issues of, provide one Kind of MALDI-TOF MS instant Quality Control viable bacteria standard items, can make full use of MALDI-TOF MS identification bacterial strain kinds it is quick, The characteristics of high-throughput, shortens the time of Micro biological Tests, improves checkability.

To achieve these goals, the present invention provides following technical solution:

MALDI-TOF MS instant Quality Control viable bacteria standard items, are prepared by following steps:

(1) the second-generation bacterial strain that purpose obtains single bacterium colony source is cultivated, by biochemical identification, confirms that kind is correct;

(2) fresh second-generation bacterial strain to be collected, is suspended in freeze drying protectant, vortex makes second-generation bacterial strain be dispersed into single bacterium colony, And be filtered through 5 μm of filter membranes, removing fails to be dispersed into single bacterium colony cell mass, and viable bacteria standard items mother liquor is made;

(3) viable bacteria standard items mother liquor is diluted, is inoculated in plating medium, bacterium colony is counted through culture, with reference to dilute It releases multiple and calculates bacterial strain concentration in viable bacteria standard items mother liquor;Meanwhile bacterium ball is made in the viable bacteria standard items mother liquor after dilution, and will Bacterium ball is inoculated in plating medium, and bacterium colony is counted through culture, then compared with lyophilized preceding concentration, calculating is frozen Dry survival rate;

(4) according to lyophilized survival rate, the Quality Control viable bacteria standard items containing target bacterium number is prepared, are stored in -20 DEG C, are MALDI-TOF MS instant Quality Control viable bacteria standard items.

Further, the component of step (2) described freeze drying protectant includes sucrose, glucose sugar and bovine serum albumin(BSA).

Further, the preparation method of step (3) the bacterium ball is:The viable bacteria standard items mother liquor after dilution is taken with 20 μ L/ The amount of ball instill in the beaker containing liquid nitrogen carry out it is quick-frozen, be subsequently placed in freeze drier carry out lyophilized 14~18 it is small when, then It is put into the cillin bottle of 2mL, is covered what rubber plug was left unlocked or unlatched on cillin bottle, then carrying out vacuum with freeze drier rolls lid.

Further, the preparation method of step (3) the bacterium ball is:The viable bacteria standard items mother liquor after dilution is taken with 20 μ L/ The amount of ball instill in the beaker containing liquid nitrogen carry out it is quick-frozen, be subsequently placed in freeze drier carry out lyophilized 16 it is small when, place into It in the cillin bottle of 2mL, is covered what rubber plug was left unlocked or unlatched on cillin bottle, then carrying out vacuum with freeze drier rolls lid.

Compared with prior art, beneficial effects of the present invention are:

MALDI-TOF MS instants Quality Control viable bacteria standard items provided by the invention are for the bacterium for examining work common Kind, such as Escherichia coli, salmonella, Shigella, staphylococcus aureus, bacillus cereus strain, it is bright to select source Really, the bacterial strain that kind determines prepares Quality Control viable bacteria standard items, can immediately be used as positive control, so as to realizing to these bacterium The fast and reliable identification of kind.

MALDI-TOF MS instants Quality Control viable bacteria standard items provided by the invention, processing procedure is simple, i.e., only need by Viable bacteria standard items, which are dissolved in deionized water, directly to be used, can via MALDI-TOF MS acquisition collection of illustrative plates detections Reference strain as strain to be tested Species estimation.

The MALDI-TOF MS of the present invention with property control viable bacteria standard items, had both met MALDI-TOF MS bacterial strain kinds Identification accurately requirement, the drawbacks of when also avoiding positive control strain cultivation fee, so as to improve the timeliness of Micro biological Tests.

Description of the drawings

It in order to illustrate the technical solutions in the embodiments of the present application or in the prior art more clearly, below will be to institute in embodiment Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only one described in the present invention A little embodiments for those of ordinary skill in the art, can also be obtained according to these attached drawings other attached drawings.

Fig. 1 is the mass spectrogram of bacterium colony to be measured;

Fig. 2 is MALDI-TOF MS instants Quality Control staphylococcus aureus viable bacteria standard items provided in an embodiment of the present invention Mass spectrogram.

Specific embodiment

In order to which those skilled in the art is made to more fully understand technical scheme, below in conjunction with attached drawing to this hair It is bright to be further detailed.Bacterial strain of the present invention is using the staphylococcus aureus purchased from CMCC (CMCC26003)。

It is the common inspection project of daily Micro biological Tests that staphylococcus aureus, which is examined, which deposits extensively in nature , and there are many chance that food is contaminated, and such as common milk, meat, egg, fish and its product is all easily contaminated.It is in addition, golden Staphylococcus aureus are most common pathogens in mankind's suppurative infection, and numerous food is easily contaminated in addition, so to people There are grave dangers for class health.Suspicious bacterium colony is identified using MALDI-TOF MS, can be obtained in several minutes preliminary Qualification result.But generally require and further kind confirmation is carried out to suspicious bacterium colony, and the MALDI-TOF MS that will be prepared Instant staphylococcus aureus viable bacteria standard items are via foregoing deionized water dissolving and pre-treating method, by MALDI-TOF MS Collection of illustrative plates is gathered, so as to the reference as suspicious bacterium colony collection of illustrative plates, you can clear and definite to be carried out in several minutes to the kind of suspicious bacterium colony Judge, avoid subsequent biochemical identification and other operations, shorten Check-Out Time, can in several minutes to suspicious bacterium colony into Row precise Identification.

MALDI-TOF MS instant Quality Control staphylococcus aureus viable bacteria standard items described in the embodiment of the present invention, pass through Following steps are prepared:

(1) the second-generation bacterial strain that purpose obtains single bacterium colony source is cultivated, by biochemical identification, confirms that kind is correct;

(2) fresh second-generation bacterial strain to be collected, is suspended in freeze drying protectant, vortex makes second-generation bacterial strain be dispersed into single bacterium colony, And be filtered through 5 μm of filter membranes, removing fails to be dispersed into single bacterium colony cell mass, and viable bacteria standard items are made and prepare mother liquor;

(3) viable bacteria standard items mother liquor is diluted, is inoculated in plating medium, bacterium colony is counted through culture, with reference to dilute It releases multiple and calculates bacterial strain concentration in viable bacteria standard items mother liquor;Meanwhile bacterium ball is made in the viable bacteria standard items mother liquor after dilution, and will Bacterium ball is inoculated in plating medium, and bacterium colony is counted through culture, then compared with lyophilized preceding concentration, calculating is frozen Dry survival rate;

(4) according to lyophilized survival rate, the Quality Control viable bacteria standard items containing target bacterium number is prepared, are stored in -20 DEG C, are MALDI-TOF MS instant Quality Control staphylococcus aureus viable bacteria standard items.

Wherein, the component of step (2) described freeze drying protectant includes sucrose, glucose sugar and bovine serum albumin(BSA).

Wherein, the preparation method of step (3) the bacterium ball is:The viable bacteria standard items mother liquor after dilution is taken with 20 μ L/ balls Amount instill in the beaker containing liquid nitrogen carry out it is quick-frozen, be subsequently placed in freeze drier carry out lyophilized 16 it is small when, place into 2mL's It in cillin bottle, is covered what rubber plug was left unlocked or unlatched on cillin bottle, then carrying out vacuum with freeze drier rolls lid.

In use, with 100ul deionized water dissolving MALDI-TOF MS instant Quality Control staphylococcus aureus viable bacteria marks Quasi- product go supernatant to remove impurity, clean 3 times, add 70% formic acid 50ul and dissolve and add in through centrifuging (8000rpm, 3min) Positive control strain is can serve as after isometric acetonitrile, control strain that can at any time as bacterium to be checked.

Dissolved MALDI-TOF MS instants Quality Control staphylococcus aureus viable bacteria is gathered using MALDI-TOF MS Standard items and the collection of illustrative plates of bacterium to be checked, as a result referring to the mass spectrogram of Fig. 1 bacterium colonies to be measured and Fig. 2 MALDI-TOF MS instants Quality Control gold Shown in the mass spectrogram of staphylococcus aureus viable bacteria standard items, the peak position of the two is identical, so as to quickly judge that bacterium colony to be measured is Staphylococcus aureus.

MALDI-TOF MS instants Quality Control viable bacteria standard items provided by the invention, can immediately be used as positive control, And processing procedure is simple, i.e., only needs viable bacteria standard items being dissolved in deionized water and can directly use, via MALDI-TOF MS acquisition collection of illustrative plates detections, can be as the reference strain of strain to be tested Species estimation.The MALDI-TOF of the present invention MS with property control viable bacteria standard items, had both met MALDI-TOF MS bacterial strain Species estimations and had accurately required, and also avoided sun The drawbacks of during property control strain cultivation fee, so as to improve the timeliness of Micro biological Tests.

Above some exemplary embodiments that the present invention is only described by way of explanation, undoubtedly, for ability The those of ordinary skill in domain, without departing from the spirit and scope of the present invention, can be with a variety of modes to institute The embodiment of description is modified.Therefore, above-mentioned attached drawing and description are regarded as illustrative in nature, and should not be construed as to the present invention The limitation of claims.

Claims (4)

1.MALDI-TOF MS instant Quality Control viable bacteria standard items, which is characterized in that be prepared by following steps:
(1) the second-generation bacterial strain that purpose obtains single bacterium colony source is cultivated, by biochemical identification, confirms that kind is correct;
(2) fresh second-generation bacterial strain to be collected, is suspended in freeze drying protectant, vortex makes second-generation bacterial strain be dispersed into single bacterium colony, and through 5 μm filter membrane is filtered, and removing fails to be dispersed into single bacterium colony cell mass, and viable bacteria standard items mother liquor is made;
(3) viable bacteria standard items mother liquor is diluted, is inoculated in plating medium, bacterium colony is counted through culture, with reference to dilution times Number calculates bacterial strain concentration in viable bacteria standard items mother liquor;Meanwhile the viable bacteria standard items mother liquor after dilution is made bacterium ball, and by bacterium ball It is inoculated in plating medium, bacterium colony is counted through culture, then compared with lyophilized preceding concentration, calculate and obtain lyophilized deposits Motility rate;
(4) according to lyophilized survival rate, the Quality Control viable bacteria standard items containing target bacterium number is prepared, are stored in -20 DEG C, be MALDI- TOF MS instant Quality Control viable bacteria standard items.
2. MALDI-TOF MS instants Quality Control viable bacteria standard items according to claim 1, which is characterized in that step (2) The component of the freeze drying protectant includes sucrose, glucose sugar and bovine serum albumin(BSA).
3. MALDI-TOF MS instants Quality Control viable bacteria standard items according to claim 1, which is characterized in that step (3) The preparation method of the bacterium ball is:The viable bacteria standard items mother liquor after dilution is taken to instill the beaker containing liquid nitrogen with the amount of 20 μ L/ balls It is middle carry out it is quick-frozen, be subsequently placed in freeze drier carry out lyophilized 14~18 it is small when, place into the cillin bottle of 2mL, by rubber plug That is left unlocked or unlatched covers on cillin bottle, and then carrying out vacuum with freeze drier rolls lid.
4. MALDI-TOF MS instants Quality Control viable bacteria standard items according to claim 1, which is characterized in that step (3) The preparation method of the bacterium ball is:The viable bacteria standard items mother liquor after dilution is taken to instill the beaker containing liquid nitrogen with the amount of 20 μ L/ balls It is middle carry out it is quick-frozen, be subsequently placed in freeze drier carry out lyophilized 16 it is small when, place into the cillin bottle of 2mL, rubber plug be left unlocked or unlatched Cover on cillin bottle, then carrying out vacuum with freeze drier rolls lid.
CN201810018888.XA 2018-01-09 2018-01-09 MALDI-TOFMS instant Quality Control viable bacteria standard items CN108102966A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104931572A (en) * 2015-05-14 2015-09-23 中国疾病预防控制中心传染病预防控制所 Mass spectrometer molecular weight calibration standard for microbiological assay, and preparation method and application thereof
CN105219737A (en) * 2015-10-29 2016-01-06 广州呼研所生物技术有限公司 The preparation of quality control product, application and test kit is marked in Escherichia coli M13 phage
CN106635802A (en) * 2016-11-22 2017-05-10 浙江泰林生物技术股份有限公司 Solid-state freeze-dried product of quantitative strain and preparation method and using method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104931572A (en) * 2015-05-14 2015-09-23 中国疾病预防控制中心传染病预防控制所 Mass spectrometer molecular weight calibration standard for microbiological assay, and preparation method and application thereof
CN105219737A (en) * 2015-10-29 2016-01-06 广州呼研所生物技术有限公司 The preparation of quality control product, application and test kit is marked in Escherichia coli M13 phage
CN106635802A (en) * 2016-11-22 2017-05-10 浙江泰林生物技术股份有限公司 Solid-state freeze-dried product of quantitative strain and preparation method and using method thereof

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