CN103868876A - Method for detecting pathogenic vibrios by MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization-Time-Of-Flight-Mass Spectrometry) - Google Patents

Abstract

The invention discloses a method for detecting pathogenic vibrios by MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization-Time-Of-Flight-Mass Spectrometry). The method comprises the following steps: acquiring MALDI-TOF-MS data information of a strain to be detected, and comparing the MALDI-TOF-MS data information with acquired information in an MALDI-TOF-MS database so as to obtain an identification result, wherein the MALDI-TOF-MS database is a database including MALDI-TOF-MS data information of selected microorganisms, and the selected microorganisms are microorganisms with strain numbers of 1-128 in a table 1. The invention also provides an MALDI-TOF-MS database, and a newly added base strain comprises 128 common pathogenic strains, which are wide in strain source; the constructed MALDI-TOF-MS strain database is large in amount of information. By using the constructed data, detection and identification of an unknown strain can be realized with high sensitivity, high specificity, high speed, high efficiency and high flux.

Description

MALDI-TOF-MS detects the method for kinds of pathogenic vibrio

Technical field

The present invention relates to the MALDI-TOF-MS detection method of Common Pathogenic vibrios in a kind of environment and food, relate in particular to the construction and application of MALDI-TOF-MS database.

Background technology

Substance assistant laser desorpted ionized flight time mass spectrum (Matrix assisted Laser Desorption Ionization Time of flight Mass Spectrometry, MALDI-TOF-MS) technology is a kind of new the food-borne pathogenic dientification of bacteria and the tracing technology growing up gradually in recent years, and this technology has two outstanding features: the accurate Rapid identification of pathogen and synchronously carry out pathogen protein somatotype and trace to the source.Except above-mentioned two outstanding features, MALDI-TOF-MS technology also has the advantages such as detection sensitivity is high, instrumental analysis automaticity is high, testing cost is low.Utilize before MALDI-TOF-MS identifies, only need carry out just can carrying out data acquisition after simple pre-service to bacterial strain, in subsequent process, can realize the high throughput identification in 96 holes and substantially realize automation equipment, the acquisition of each hole sample identification result is no more than 5min.Even with some authoritative method of generally acknowledging, as: the VITEK setting up on biochemical identification basis compares with ELISA method with the VIDAS method of setting up according to immunology principle, MALDI-TOF-MS technology is also not a halfpenny the worse, even guaranteeing under the prerequisite of accuracy, convenient more rapidly than them.

MALDI-TOF-MS technology is take bacterium surface albumen as detected object, and it must be based upon in the spectrum storehouse of containing enough known bacterial strains and retrieve the Classification Identification of unknown bacterium, just can realize coupling and the most true and reliable identification of strains result.At present, the spectrogram overwhelming majority in database is the data of external bacterial strain, the domestic research due to this technology and application are also at the early-stage, not yet set up the mass spectrogram database of various vibrios bacterial strains, pathogen identification data to kinds of pathogenic vibrio is still far from perfect, and country variant and regional bacterial strain may exist obvious geographical difference.Therefore, need to adopt the bacterial strain of domestic separation to set up as early as possible the spectrum library that is applicable to the various vibrios detections of China and identifies, the accuracy of this technology application of guarantee.The key control technology problem that detects and trace to the source for the main kinds of pathogenic vibrio of serious restriction China's aquatic products production and safe and sanitary, the method for foundation efficient special evaluation kinds of pathogenic vibrio from aquatic products; On this basis, application MALDI-TOF MS technology is carried out full cell analysis, the kinds of pathogenic vibrio separating is identified to kind level, according to bacterium source, whether be to produce the information such as strain, set up molecule parting and the authenticate database of main kinds of pathogenic vibrio, thereby realize the identification and analysis of tracing to the source to main kinds of pathogenic vibrio, set up MALDI-TOF-MS database and the tracing technology of main kinds of pathogenic vibrio in China's aquatic products and Food Poisoning Samples, improve microorganism health level and the safety in production of aquatic products, reduce the direct and indirect economic loss that produced thus.

Summary of the invention

The object of the present invention is to provide a kind of MALDI-TOF-MS to detect the method for kinds of pathogenic vibrio, the method should adapt to the demand that the various vibrios of China detect and identify effectively, and has enough specificitys and accuracy.

MALDI-TOF-MS of the present invention detects the method for kinds of pathogenic vibrio, it is the MALDI-TOF-MS data message that gathers bacterial strain to be detected, and the information of including in itself and MALDI-TOF-MS database is compared, thereby obtain the method for qualification result, it is characterized in that, described MALDI-TOF-MS database is the database that has comprised selected microorganism MALDI-TOF-MS data message, and described selected microorganism is the microorganism that in table 1, strain number is 1～128.

Table 1 kinds of pathogenic vibrio is built storehouse bacterial strain information

The present invention's object on the other hand, is to build to be applicable to the MALDI-TOF-MS database that above-mentioned purpose of the present invention is used, and this database builds by the following method:

A. selected microorganism is carried out to pre-service: the culture of selecting microorganism with aseptic swab stick picking 6～10mg is put in 1.5mLEppendorf pipe, adding 300 μ L pure water mixes, add again 900 μ L absolute ethyl alcohols to mix, 12,000r/min is centrifugal, and 2min discards supernatant, in Eppendorf pipe, adds 50 μ L70% formic acid and 50 μ L acetonitriles, 12, the centrifugal 2min of 000r/min, gets supernatant and transfers to new Eppendorf and manage and obtain pretreated detection sample;

Described selected microorganism is the microorganism that in table 1, strain number is 1～128;

Described culture be the selected microorganism of preservation take 2%NaCl brain heart leachate agar as nutrient culture media at 36 ℃ of cultures of cultivating 24h gained;

B. the detection sample after pretreatment of step a gained 1 μ L point is added on the special sample target of MALDI-TOF-MS: MSP96target ground steel^tM600-μ m sample target, dries at ambient temperature naturally, drips 1 μ L matrix solution and cover on it, naturally dries rear for subsequent use;

C. sample detection: carry out detecting after instrumental correction with reference culture Escherichia coli ATCC8739; Detecting instrument parameter is: nitrogen LASER Light Source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20.00kv, postpone to extract voltage 18.25kv, set mass range 2,000～2,0000Da, Laser emission voltage 2,500mv, laser frequency 20Hz;

D. data acquisition: the data acquisition software Flexcontrol that adopts MALDI-TOF-MS^tM3.0 couples of step c detect the result obtaining and carry out sample data collection, parallel 10 sample wells of every strain bacterium, and every hole is got 10 diverse locations and is carried out laser click;

E. data analysis: the evaluation software Biotyper that adopts MALDI-TOF-MS^tM2.0 pairs form collection of illustrative plates and carry out the matching analysis of data and obtain qualification result and score value, and score value is greater than 1.7 and is less than 2.0 to be judged to be genus level credible, and being greater than 2.0, to be judged to be kind of level credible, and being greater than 2.3, to be judged to be kind of level height credible;

F. Database: select the chart adding of score value between 2.0-3.0 in step e, then, in BioTyper software, call in selected spectral data, become the reference culture mass spectrogram in database, build up MALDI-TOF-MS database.

The present invention's MALDI-TOF-MS kinds of pathogenic vibrio Test database that increases, newly increase and build storehouse kinds of pathogenic vibrio bacterial strain and comprised main kinds of pathogenic vibrio, comprise malicious type vibrio parahemolyticus 34 strains of non-product, produce malicious type vibrio parahemolyticus 10 strains, non-O139 group cholera vibrio 6 strains of non-O1/, O1 group cholera vibrio 24 strains (producing strain 4 strains), O139 group cholera vibrio 3 strains (producing strain 1 strain), vibrio alginolyticus 23 strains, 7 strains of Maxwell vibrios, Vibrio furnissii 4 strains, vibrio mimicus 3 strains, Vibrio flurialis 4 strains, Aeromonas hydrophila 3 strains and Vibrio damsela, Vibrio vulnificus, shark vibrio, Vibrio anguillarum, the each strain of Vibrio campbellii and Vibrio hollisae, bacterium source is extensive, guarantee that constructed MALDI-TOF-MS bacterial strain database information amount is large.Utilize constructed data, can realize unknown strains high sensitivity, high specific, rapidly and efficiently high-throughout detection and evaluation.

Accompanying drawing explanation

By the description to embodiment of carrying out below in conjunction with accompanying drawing, above-mentioned and/or other object of the present invention and advantage will become apparent, wherein:

Fig. 1 is Escherichia coli ATCC8739 mass spectrogram.

Fig. 2 is that Maxwell vibrios repeatability detects mass spectrogram.

Embodiment

The invention provides the method that MALDI-TOF-MS detects kinds of pathogenic vibrio, it is the MALDI-TOF-MS data message that gathers bacterial strain to be detected, and the information of including in itself and MALDI-TOF-MS database is compared, thereby obtain the method for qualification result, described MALDI-TOF-MS database is the database that has comprised selected microorganism MALDI-TOF-MS data message, and described selected microorganism is the microorganism that in table 1, strain number is 1～128.

These data itself, are also one of objects of the present invention, and described database is set up by the following method:

A. detection sample 1 μ L point after pretreatment described selected microorganism is added on the special sample target of MALDI-TOF-MS: MSP96target ground steel^tM600-μ m sample target(Bruker Daltonics GmbH, Germany), naturally dry at ambient temperature (approximately needing 3min), on it, drip 1 μ L matrix solution and cover, naturally dry rear for subsequent use; After drying, sample supernatant should put as early as possible matrix solution.

B. sample detection: in order to guarantee in 2,000Da～2, the accuracy detecting within the scope of 0000Da, with reference culture Escherichia coli ATCC8739(Fig. 1) carry out detecting after instrumental correction; The statistical parameter mass-to-charge ratio error obtaining after proofreading and correct will be less than 500ppm.Detecting instrument parameter is: nitrogen LASER Light Source, optical maser wavelength 377nm, linear positive ion (LP-BioTyper) operator scheme, accelerating potential 20.00kv, postpone to extract voltage 18.25kv, set mass range 2,000～2,0000Da, Laser emission voltage 2,500mv, laser frequency 20Hz.

C. data acquisition: the data acquisition software Flexcontrol that adopts MALDI-TOF-MS^tM3.0 couples of step a detect the result obtaining and carry out sample data collection, parallel 10 sample wells of every strain bacterium, and every hole is got 10 diverse locations and is carried out laser click; That is: the click of the laser of each bacterial strain is 10000 effects time (100Hz × 100 time);

D. data analysis: the evaluation software Biotyper that adopts MALDI-TOF-MS^tM2.0 pairs form the matching analysis that collection of illustrative plates carries out data and (mate with database resource, before this law amplification, database comprises 21 kinds of pathogenic vibrio reference spectrums) and obtain qualification result and score value, score value is greater than 1.7 and is less than 2.0 to be judged to be genus level credible, being greater than 2.0, to be judged to be kind of level credible, is greater than 2.3 and is judged to be kind of level height credible (judgment criteria of this criterion based on manufacturer);

Meanwhile, utilize software to carry out the cluster analysis (Dendrogram) based on MALDI-TOF-MS.

E. Database: select the chart adding of score value between 2.0-3.0 in steps d, then in BioTyper software, call in selected spectral data, become the reference culture mass spectrogram in database, build up MALDI-TOF-MS database, i.e. Biotyper database after amplification.

Because the quantity of mass spectra peak and intensity have determined the matching degree of mass spectrogram and database resource, finally affect qualification result and score value, test determines that the suitableeest bacterium amount is 6mg～10mg: bacterium amount is below 6mg time, and qualification result is inaccurate, identify that score value is below 2.0 points, with a low credibility; Bacterium amount is in the time of 6mg～10mg, and qualification result is accurate, identifies that score value is more than 2.3 points, with a high credibility.

The condition of culture of microorganism not only affects growth conditions and the biochemical characteristic of microorganism self, also affects the final mass spectrogram forming that detects by substance assistant laser desorpted ionized ionization time of flight (MALDI-TOF-MS).Need to be tested and appraised the comparison of result and evaluation score value, weigh the matching degree of the data obtained and MALDI-TOF-MS data, decide the appropriate incubation condition of the microorganism of ginseng test.

Based on the consideration of above factor, in one of embodiment, the preprocess method of stating selected microorganism comprises:

1) conserving microorganism, comprises selected microorganism for building storehouse and unknown microorganism to be detected, needs first to cultivate 24h take 2%NaCl brain heart leachate agar as nutrient culture media at 36 ℃, and the culture of gained is as detected object.

2) processing to above-mentioned culture: put in 1.5mL Eppendorf pipe with aseptic swab stick picking 6～10mg culture, adding 300 μ L pure water mixes, add again 900 μ L absolute ethyl alcohols to mix, 12,000r/min is centrifugal, and 2min discards supernatant, in Eppendorf pipe, adds 50 μ L70% formic acid and 50 μ L acetonitriles, 12, the centrifugal 2min of 000r/min, gets supernatant and transfers to new Eppendorf and manage and obtain pretreated detection sample;

According to the thinking of object of the present invention and method foundation, in the specific embodiment of the present invention, all identical with preprocess method, the MALDI-TOF-MS data information acquisition method of selected microorganism to the preprocess method of bacterial strain to be detected.

In embodiment, the MALDI-TOF-MS data information acquisition method of described selected microorganism and microorganism to be detected is:

A. detection sample 1 μ L point after pretreatment described selected microorganism is added on the special sample target of MALDI-TOF-MS: MSP96target ground steel^tM600-μ m sample target, dries at ambient temperature naturally, drips 1 μ L matrix solution and cover on it, naturally dries rear for subsequent use;

In this detecting step, the configuration store method of matrix solution is: 50% acetonitrile (Acetonitrile, ACN), 2.5% trifluoroacetic acid (Trifluoroacetic acid, the mixed solution of TFA) and 47.5% ultrapure water, add CHCA powder until it forms saturated solution, 4 ℃ save backup.The optimal proportions of matrix solution and sample is 1:1(v/v), under this condition, matrix is scattered in sample molecule uniformly, and has given full play to macroion, transfer laser energy, maldi ion and the polymeric effect of molion.So, in the time that matrix and sample ratio are 1:1, be a best critical point, fully desorbed molecule ion does not cause again the waste of matrix.

B. sample detection: carry out detecting after instrumental correction with reference culture Escherichia coli ATCC8739; Detecting instrument parameter is: nitrogen LASER Light Source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20.00kv, postpone to extract voltage 18.25kv, set mass range 2,000～2,0000Da, Laser emission voltage 2,500mv, laser frequency 20Hz;

C. data acquisition: the data acquisition software Flexcontrol that adopts MALDI-TOF-MS^tM3.0 couples of step b detect the result obtaining and carry out sample data collection, parallel 10 sample wells of every strain bacterium, and every hole is got 10 diverse locations and is carried out laser click;

D. data analysis: the evaluation software Biotyper that adopts MALDI-TOF-MS^tM2.0 pairs form collection of illustrative plates and carry out the matching analysis of data and obtain qualification result and score value;

E1. the data message of the selected microorganism gathering by above method is for Database: select the chart adding of steps d score value between 2.0-3.0, then in BioTyper software, call in selected spectral data, become the reference culture mass spectrogram in database, build up MALDI-TOF-MS database.

E2. the data message of the unknown microorganism gathering by above method is directed in the MALDI-TOF-MS database of having set up by e1 compares, obtain result, and judge according to following principle: score value is greater than 1.7 and is less than 2.0 to be judged to be genus level credible, being greater than 2.0, to be judged to be kind of level credible, and being greater than 2.3, to be judged to be kind of level height credible.

Be specific embodiments of the invention below, its foundation and application thereof to technical solution of the present invention is further described, but does not limit in any form content of the present invention.

If without specified otherwise, biological sample used is tested from Dandong Entry-Exit Inspection and Quarantine Bureau microorganism keeping center in this part.

Matrix: α-cyanogen-4-hydroxycinnamic acid (α-Cyano-4-hydroxy-cinnamic acid, CHCA) is purchased from Brooker company;

Bacteria culture media is purchased from U.S. company BD;

The screw cap test tube of 50ml is purchased from U.S. Corning;

Vibrio cholerae O 1 group multivalence diagnostic serum, Ogawa, Inaba diagnostic serum and O139 group's diagnostic serum are Japanese Sheng Yan biological products company and produce.

Instrument and equipment: substance assistant laser desorpted ionized time of-flight mass spectrometer (Brooker dalton company).

Embodiment 1, MALDI-TOF-MS detect vibrio parahemolyticus

Adopt definite appropriate media and incubation time: 2%NaCl brain heart leachate agar, cultivate after 29 strains (strain number 1-29) experimental strain 24h for 36 ℃, each 8 ± 2mg bacterium colony that weighs, the pre-service of mixed solvent facture adopts the definite condition of above-mentioned research contents to carry out analysis of the accuracy to experimental strain after making sample.By the data acquisition software Flexcontrol of MALDI-TOF-MS^tM3.0 pairs of sample wells carry out data acquisition, then by the evaluation software Biotyper of MALDI-TOF-MS^tM2.0 pairs of each bacterial strain mass spectrograms that obtain are analyzed, and mate with the resource in database.Carry out the comparison of MALDI-TOF-MS qualification result and biochemical identification result.

Obtain qualification result and identify score value and compare (table 2) with biochemical identification result.The MALDI-TOF-MS qualification result of experimental strain is vibrio parahemolyticus, identifies that score value is all more than 2.3, good with data bank matching.The biochemical identification result of the qualification result of MALDI-TOF-MS and bacterial strain is in full accord, with a high credibility.

Table 2MALDI-TOF-MS and the comparison of biochemical identification result

Strain number	MALDI-TOF-MS qualification result	Identify score value	Biochemical identification result a
				1	Vibrio?parahaemolyticus	2.33	V.parahaemolyticus
2	Vibrio?parahaemolyticus	2.46	V.parahaemolyticus
				3	Vibrio?parahaemolyticus	2.54	V.parahaemolyticus
4	Vibrio?parahaemolyticus	2.37	V.parahaemolyticus
				5	Vibrio?parahaemolyticus	2.44	V.parahaemolyticus
6	Vibrio?parahaemolyticus	2.35	V.parahaemolyticus
				7	Vibrio?parahaemolyticus	2.41	V.parahaemolyticus
8	Vibrio?parahaemolyticus	2.57	V.parahaemolyticus
				9	Vibrio?parahaemolyticus	2.38	V.parahaemolyticus
10	Vibrio?parahaemolyticus	2.36	V.parahaemolyticus
				11	Vibrio?parahaemolyticus	2.55	V.parahaemolyticus
12	Vibrio?parahaemolyticus	2.36	V.parahaemolyticus
				13	Vibrio?parahaemolyticus	2.55	V.parahaemolyticus
14	Vibrio?parahaemolyticus	2.45	V.parahaemolyticus
				15	Vibrio?parahaemolyticus	2.43	V.parahaemolyticus
16	Vibrio?parahaemolyticus	2.35	V.parahaemolyticus
				17	Vibrio?parahaemolyticus	2.44	V.parahaemolyticus
18	Vibrio?parahaemolyticus	2.47	V.parahaemolyticus
				19	Vibrio?parahaemolyticus	2.54	V.parahaemolyticus

20	Vibrio?parahaemolyticus	2.48	V.parahaemolyticus
				21	Vibrio?parahaemolyticus	2.39	V.parahaemolyticus
22	Vibrio?parahaemolyticus	2.42	V.parahaemolyticus
				23	Vibrio?parahaemolyticus	2.47	V.parahaemolyticus
24	Vibrio?parahaemolyticus	2.65	V.parahaemolyticus
				25	Vibrio?parahaemolyticus	2.43	V.parahaemolyticus
26	Vibrio?parahaemolyticus	2.39	V.parahaemolyticus
				27	Vibrio?parahaemolyticus	2.52	V.parahaemolyticus
28	Vibrio?parahaemolyticus	2.47	V.parahaemolyticus
				29	Vibrio?parahaemolyticus	2.62	V.parahaemolyticus

^a.routine biochemistry qualification result and BD biochemical identification result

Embodiment 2.MALDI-TOF-MS detects Maxwell vibrios

Adopt the method that the present invention sets up to carry out 6 repeatability analyses to Maxwell Protein Profiles of Vibrio Sp (ATCC700040).All bacterial strains carry out, after MALDI-TOF-MS detection, carrying out according to the operation steps of summary of the invention, 6 mass spectrograms that more every strain bacterial strain obtains respectively, and identify score value and qualification result.6 mass spectrograms that bacterial strain obtains all extremely approach (see figure 2), and qualification result is in full accord, identify that score value is all more than 2.3 points.Demonstrate MALDI-TOF-MS detection method of the present invention and there is desirable repeatability.

Embodiment 3, MALDI-TOF-MS detection method specificity analyses

The method and the condition that adopt the present invention to set up are carried out the evaluation of MALDI-TOF-MS to vibrio parahemolyticus (ATCC17802), Vibrio vulnificus (ATCC27562), vibrio mimicus (ATCC33653), Maxwell vibrios (ATCC700040) and other strains A TCC25922, ATCC25923, ATCC13076, ATCC19111.By the qualification result of acquisition and the specificity of score value and mass spectrogram evaluation MALDI-TOF-MS detection method.

MALDI-TOF-MS precise Identification 4 strains belong to the reference culture (ATCC17802, ATCC27562, ATCC33653 and ATCC700040) of vibrio and the reference culture (ATCC25922, ATCC25923, ATCC13076, ATCC19111) of the different Pseudomonas of 4 strain.Obtain mass spectrogram by observation, belong to vibrio parahemolyticus, Vibrio vulnificus, vibrio mimicus and the Maxwell vibrios 4 strain reference cultures of vibrio in mass-to-charge ratio (m/z) 3,000～8, the difference within the scope of 000Da is obvious, can effectively distinguish.The mass spectrogram of Escherichia coli, staphylococcus aureus, Bacterium enteritidis, single increasing listeria spp and the vibrio parahemolyticus 4 strain reference cultures of different Pseudomonas is in mass-to-charge ratio (m/z) 2,000～12, difference within the scope of 000Da is very obvious, has shown the good specificity of MALDI-TOF-MS detection method.The MALDI-TOF-MS qualification result of above-mentioned reference culture is in table 3.

Table 3MALDI-TOF-MS detection method specificity analyses result

Reference culture number	Strain name	MALDI-TOF-MS qualification result/score value
			ATCC17802	Vibrio?parahaemolyticus	Vibrio?parahaemolyticus/2.73
ATCC27562	Vibrio?vulnificus	Vibrio?vulnificus/2.66
			ATCC33653	Vibrio?mimicus	Vibrio?mimicus/2.58
ATCC700040	Vibrio?metschnikovii	Vibrio?metschnikovii/2.49
			ATCC25922	E.coil	E.coil/2.40
ATCC25923	Staphylococcus?aureus	Staphylococcus?aureus/2.56
			ATCC13076	Salmonella?enteritidis	Salmonella?enteritidis/2.47
ATCC19111	Listeria?monocytogenes	Listeria?monocytogenes/2.65

Claims (6)

1.MALDI-TOF-MS detects the method for kinds of pathogenic vibrio, it is the MALDI-TOF-MS data message that gathers bacterial strain to be detected, and the information of including in itself and MALDI-TOF-MS database is compared, thereby obtain the method for qualification result, it is characterized in that, described MALDI-TOF-MS database is the database that has comprised selected microorganism MALDI-TOF-MS data message, and described selected microorganism is the microorganism that in table 1, strain number is 1～128.

2. MALDI-TOF-MS according to claim 1 detects the method for kinds of pathogenic vibrio, it is characterized in that, builds by the following method MALDI-TOF-MS database:

A. detection sample 1 μ L point after pretreatment described selected microorganism is added on the special sample target of MALDI-TOF-MS: MSP96target ground steel^tM600-μ m sample target, dries at ambient temperature naturally, drips 1 μ L matrix solution and cover on it, naturally dries rear for subsequent use;

B. sample detection: carry out detecting after instrumental correction with reference culture Escherichia coli ATCC8739; Detecting instrument parameter is: nitrogen LASER Light Source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20.00kv, postpone to extract voltage 18.25kv, set mass range 2,000～2,0000Da, Laser emission voltage 2,500mv, laser frequency 20Hz;

C. data acquisition: the data acquisition software Flexcontrol that adopts MALDI-TOF-MS^tM3.0 couples of step b detect the result obtaining and carry out sample data collection, parallel 10 sample wells of every strain bacterium, and every hole is got 10 diverse locations and is carried out laser click;

D. data analysis: the evaluation software Biotyper that adopts MALDI-TOF-MS^tM2.0 pairs form collection of illustrative plates and carry out the matching analysis of data and obtain qualification result and score value, and score value is greater than 1.7 and is less than 2.0 to be judged to be genus level credible, and being greater than 2.0, to be judged to be kind of level credible, and being greater than 2.3, to be judged to be kind of level height credible;

E. Database: select the chart adding of score value between 2.0-3.0 in steps d, then, in BioTyper software, call in selected spectral data, become the reference culture mass spectrogram in database, build up MALDI-TOF-MS database.

3. MALDI-TOF-MS according to claim 2 detects the method for kinds of pathogenic vibrio, it is characterized in that, the preprocess method of described selected microorganism is: put in 1.5mL Eppendorf pipe with aseptic swab stick picking 6～10mg culture, adding 300 μ L pure water mixes, add again 900 μ L absolute ethyl alcohols to mix, 12,000r/min is centrifugal, and 2min discards supernatant, in Eppendorf pipe, add 50 μ L70% formic acid and 50 μ L acetonitriles, 12, the centrifugal 2min of 000r/min, gets supernatant and transfers to new Eppendorf and manage and obtain pretreated detection sample;

Described culture be the selected microorganism of preservation take 2%NaCl brain heart leachate agar as nutrient culture media at 36 ℃ of cultures of cultivating 24h gained.

4. MALDI-TOF-MS according to claim 3 detects the method for kinds of pathogenic vibrio, it is characterized in that, the preprocess method of described bacterial strain to be detected is identical with the preprocess method of selected microorganism.

5. MALDI-TOF-MS according to claim 2 detects the method for kinds of pathogenic vibrio, it is characterized in that, adopt the method identical with the MALDI-TOF-MS data information acquisition method of selected microorganism to gather the MALDI-TOF-MS data message of bacterial strain to be measured, the matching analysis of the Biotyper that employing has comprised MALDI-TOF-MS database to the data obtained, analysis result score value is greater than 1.7 and is less than 2.0 to be judged to be genus level credible, being greater than 2.0, to be judged to be kind of level credible, and being greater than 2.3, to be judged to be kind of level height credible.

6.MALDI-TOF-MS database, is characterized in that, this database builds by the following method:

A. selected microorganism is carried out to pre-service: the culture of selecting microorganism with aseptic swab stick picking 6～10mg is put in 1.5mLEppendorf pipe, adding 300 μ L pure water mixes, add again 900 μ L absolute ethyl alcohols to mix, 12,000r/min is centrifugal, and 2min discards supernatant, in Eppendorf pipe, adds 50 μ L70% formic acid and 50 μ L acetonitriles, 12, the centrifugal 2min of 000r/min, gets supernatant and transfers to new Eppendorf and manage and obtain pretreated detection sample;

Described selected microorganism is the microorganism that in table 1, strain number is 1～128;

Described culture be the selected microorganism of preservation take 2%NaCl brain heart leachate agar as nutrient culture media at 36 ℃ of cultures of cultivating 24h gained;

B. the detection sample after pretreatment of step a gained 1 μ L point is added on the special sample target of MALDI-TOF-MS: MSP96target ground steel^tM600-μ m sample target, dries at ambient temperature naturally, drips 1 μ L matrix solution and cover on it, naturally dries rear for subsequent use;

C. sample detection: carry out detecting after instrumental correction with reference culture Escherichia coli ATCC8739; Detecting instrument parameter is: nitrogen LASER Light Source, optical maser wavelength 377nm, linear positive ion operator scheme, accelerating potential 20.00kv, postpone to extract voltage 18.25kv, set mass range 2,000～2,0000Da, Laser emission voltage 2,500mv, laser frequency 20Hz;

D. data acquisition: the data acquisition software Flexcontrol that adopts MALDI-TOF-MS^tM3.0 couples of step c detect the result obtaining and carry out sample data collection, parallel 10 sample wells of every strain bacterium, and every hole is got 10 diverse locations and is carried out laser click;

E. data analysis: the evaluation software Biotyper that adopts MALDI-TOF-MS^tM2.0 pairs form collection of illustrative plates and carry out the matching analysis of data and obtain qualification result and score value, and score value is greater than 1.7 and is less than 2.0 to be judged to be genus level credible, and being greater than 2.0, to be judged to be kind of level credible, and being greater than 2.3, to be judged to be kind of level height credible;

F. Database: select the chart adding of score value between 2.0-3.0 in step e, then, in BioTyper software, call in selected spectral data, become the reference culture mass spectrogram in database, build up MALDI-TOF-MS database.

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