CN102517374B - Rapid identification method for homology of bacterial strains - Google Patents
Rapid identification method for homology of bacterial strains Download PDFInfo
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- CN102517374B CN102517374B CN201110391750.2A CN201110391750A CN102517374B CN 102517374 B CN102517374 B CN 102517374B CN 201110391750 A CN201110391750 A CN 201110391750A CN 102517374 B CN102517374 B CN 102517374B
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Abstract
The invention discloses a rapid identification method for the homology of bacterial strains. The rapid identification method comprises the following steps of: (I) identifying the families of bacteria according to the morphologies, the staining and the cultural characteristics of the bacteria; (II) identifying the genera or the species of the bacteria according to the biochemical reaction in which the bacteria utilize sugar and protein; (III) identifying the types of the bacteria by using serological antibodies; and (IV) determining the species and the contents of fatty acids of the bacteria by adopting a gas chromatography so as to obtain the fingerprint identification maps of the fatty acids of the bacteria, carrying out homology comparison, and identifying the strains of the bacteria. The method disclosed by the invention has the advantages of rapidness, simplicity and easiness, low cost, safety in experimenting process, and the like.
Description
Technical field
The present invention relates to a kind of homology discrimination method of bacterial strain, specifically a kind of on the basis of traditional division bacteria, discriminating, by the kind to the bacterium short chain fatty acid and content, detected, clearly determine the homology of bacterial strain, thereby bacterial strain is carried out to the method for Fast Classification and discriminating.
Background technology
The laboratory diagnosis of bacterial infection disease, relating to sex pheromone and learn the knowledge and skills of the aspects such as (containing medical science, agricultural, industry, environmental microorganism), epidemiology, medical jurisprudence, Nosocomiology, biological chemistry, is a multidisciplinary interpenetrative & ID Technology.The groundwork of microbiology laboratory is isolate pathogenic strains and accurately differentiate from sample.The bacterium homology is differentiated and can be understood in time kind, the Relationship between popular bacterial strain, its purpose is: the relation between clear and definite bacterial etiology diagnosis, differentiation bacterium and infection, quick, reliable treatment plan is provided, coaches for the epidemiology control in time.The homology that bacterium infects is differentiated as the monitoring of clinical diagnosis, treatment, prognosis and epidemiology survey and nosocomial infection, reliable experimental basis is provided.
At present, the homology that the microbiology laboratory bacterium infects is differentiated main rely on bacterium nucleotide sequencing or PCR hybridization etc., and because bacterium is prokaryotic organism,, than the virogene complexity, there are the shortcomings such as experimental cost is high, experimental period is long in the nucleotide gene sequence.Simultaneously, prokaryotic organism contain abundant sugar, protein (containing antibody, enzyme system) and lipid component.The sugar of bacterium and protein, through 100 years of researches, have formed traditional discrimination of bacteria method, according to the preliminary section that differentiates of form, dyeing, the cultural characters of bacterium; By biochemical reaction, utilize the enzyme system of bacterium uniqueness, thereby, to capacity of decomposition and the meta-bolites difference of substrate (sugar, protein), carry out different chemical reactions, can be by discrimination of bacteria to belonging to or planting with this; Recycling serology antibody arrives type by discrimination of bacteria.But traditional discrimination method can only be differentiated kind or type, and can not differentiate to strain.
Summary of the invention
Technical problem to be solved by this invention is to propose a kind of homology method for quick identification of quick, simple and easy, cost is low, experimentation is safe bacterial strain.
For solving the problems of the technologies described above, the homology method for quick identification of a kind of bacterial strain of the present invention comprises the steps: I. according to form, dyeing, the cultural characters of bacterium, tentatively differentiate; II. utilize bacterium to be belonged to biochemical reaction sugared, protein utilization or plant discriminating; III. utilize serology antibody that discrimination of bacteria is arrived to type; IV. adopt gas chromatographic technique, determine kind and the content of bacterium lipid acid, obtain this bacterium lipid acid fingerprint recognition collection of illustrative plates, carry out homology relatively, by discrimination of bacteria to strain.
The homology method for quick identification of above-mentioned a kind of bacterial strain, described homology is relatively to adopt the lipid acid standard substance to carry out homology relatively, the lipid acid standard substance that adopt comprise fatty acid methyl ester admixture liquid, contain C5～C20 linear saturated fatty acids methyl esters and 2-hydroxyl-10 carbon, 3-hydroxyl-10 carbon, 2-hydroxyl-14 carbon, 2-hydroxyl-16 carbon saturated fatty acid.
The homology method for quick identification of above-mentioned a kind of bacterial strain, before adopting gas chromatographic technique to measure the kind and content of bacterium lipid acid, bacterium to be measured is seeded on the substratum without lipid component and cultivates, collect bacterium colony, through saponification, methylate, extraction, washing process, bacterium lipid acid is transferred to organic phase from water.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the collection process of described bacterium colony comprises: bacterium is inoculated on the TSBA flat board of fat-free composition by four district's setting-out methods, 35 ℃, 24h cultivation, with the bacterium colony 40mg left and right in transfering loop Qu 3rd district, be placed in aseptic, dry, screw-topped test tube bottom.
The homology method for quick identification of above-mentioned a kind of bacterial strain, described saponification process comprises: add the saponification reagent of 1.Oml in test tube, cover tightly test tube, concussion test tube 10s, put into 100 ℃ of boiling water water-baths, shifts out, cooling concussion 10s after 5min from boiling water; By in the tube back water-bath, continue heating 25min again; In boiling water, saponification is total to 30min, cooling.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the mixed solution that the reagent adopted in described saponification process is comprised of sodium hydroxide 45g, methyl alcohol 150ml and water 150ml.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the described process of methylating comprises: add the 2.0ml methylating reagent in every pipe, 10s is swung in the after shock of the tight test tube lid of bolt, puts 80 ℃ of heating in water bath 10 min, be quickly cooled to room temperature with cold water, lipid acid is changed into to methylfatty acid.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the mixed solution that in the described process that methylates, reagent used is comprised of 6.0 mol/L hydrochloric acid 325ml and methyl alcohol 275ml.
The homology method for quick identification of above-mentioned a kind of bacterial strain, described extraction process comprises: every pipe adds the 1.25ml extraction solvent, cover tightly the test tube lid, put 28 ℃ of shaking table gentlenesses and mix 10min, get clean suction pipe and draw upper organic phase, put into another test tube, methylfatty acid is transferred to organic phase from water.
The homology method for quick identification of above-mentioned a kind of bacterial strain, solvent used in described extraction process is mixed with volume 1:1 by hexane and methyl tert-butyl ether.
The homology method for quick identification of above-mentioned a kind of bacterial strain, described washing process comprises: add the 3.Oml alkaline detergent solution in test tube, the tight lid of bolt, concussion test tube 5min, static several minutes, layering was clear.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the alkaline detergent solution adopted in described washing process, the mixed solution formed by sodium hydroxide 10.8g and water 900ml.
The homology method for quick identification of above-mentioned a kind of bacterial strain, described stratographic analysis process comprises: extract is moved to sample flasket, with clean suction pipe, draw upper organic phase, transfer to clean chromatographic sample bottle, put on gas chromatograph; Chromatographic condition: 40 ℃ of furnace temperature, 250 ℃ of injection ports, 300 ℃ of detectors; Hydrogen flowing quantity 30mL/min, air flow quantity 400mL/min, nitrogen flow 30mL/min; Hydrogen pressure 10.6psi; Chromatographic column is Agilent 19091B-102 type, and detector is hydrogen flame ionization detector; The calibration of lipid acid standard substance adds bacteria sample to be checked after passing through, and adopts gas chromatographic technique, determines bacterium lipid acid kind and relative content, obtains bacterium lipid acid fingerprint recognition collection of illustrative plates.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the method of discrimination of described homology comparison is: if two kinds of bacteriums are in the situation that described I is identical to the III step results, as there is notable difference in bacterium lipid acid chromatogram peak position, point out bacterium to belong to of the same race but homology not together; If lipid acid chromatogram peak position is identical, peak height is close, points out bacterium not only to belong to of the same race but also homology together.
Because bacterium lipid comprises cholesterol, lipid acid, mycolic acids, phosphatide, quinone etc., and lipid acid is as the fundamental component of cell, mainly be present in organoid, its component is relatively stable, bacterium not of the same race, as to belong to, the composition of its lipid acid and content show difference in various degree, and this essence and heredity with bacterium is relevant, therefore contained abundant systematic bacteriology information in the component of bacterium lipid acid and content.The present invention in conjunction with bacterium to sugar, the protein utilization characteristic, kind and content to bacterium short chain fatty acid (C5～C20) are detected, according to the kind of bacterium lipid acid and the difference of content, the clear homology that determines bacterial strain, thus carry out classification and the discriminating of bacterium.It is on the basis of traditional discrimination of bacteria program, in the situation that Bacterial stain microscopy, biochemical reaction, serological reaction are identical, the homology infected based on sugar, protein and fatty acid analysis technology discriminating bacteria, it is the extension to traditional bacteriology discriminating, somatotype, it is again a kind of novel method after bacterium Nucleotide somatotype, for bacteriological somatotype, discriminating, relatively provide new way, have quick, simple and easy, cost is low, experimentation waits safely advantage.
The accompanying drawing explanation
Fig. 1 is the operating process schematic diagram of the inventive method;
Fig. 2 is the significant parameter of the gas chromatogram of bacterium lipid acid standard substance (lot number MIDI 601230-A);
Fig. 3 is bacterium lipid acid standard substance (lot number MIDI 601230-A) gas chromatograms;
Fig. 4 is the lipid acid gas-chromatography parameter of Vibrio vulnificus 1.758 strains;
Fig. 5 is the lipid acid gas chromatogram of Vibrio vulnificus 1.758 strains.
Embodiment
As shown in Figure 1, the homology method for quick identification of a kind of bacterial strain of the present invention comprises the steps:
I. according to form, dyeing, the cultural characters of bacterium, tentatively differentiate, this method can be by discrimination of bacteria to section.
II. utilize bacterium to be differentiated the biochemical reaction of sugar, protein utilization, this method can be by discrimination of bacteria to belonging to or planting.
III. serology is differentiated: with containing known differential protein antibody, going to detect unknown bacterium, this method can be by discrimination of bacteria to planting or type.
IV. bacterium to be measured is seeded on the substratum without lipid component and cultivates, collect bacterium colony, through saponification, methylate, extraction, washing process, bacterium lipid acid is transferred to organic phase from water.Specifically comprise following process:
1, bacterium colony obtains: bacterium is inoculated in to the TSBA flat board of fat-free composition by four district's setting-out methods, 35 ℃, 24h are cultivated, and with the bacterium colony 40mg left and right in transfering loop Qu 3rd district, putting aseptic, drying has the screw capped tube bottom.
2, bacterium lipid acid extracting:
(1) saponification: add the saponification reagent of 1.Oml in test tube, cover tightly test tube, concussion test tube 10s, put into 100 ℃ of boiling water water-baths, shifts out, cooling concussion 10s after 5 min from boiling water.By in the tube back water-bath, continue heating 25 min again.In boiling water, saponification is total to 30min, cooling.Saponification reagent can adopt the mixture be comprised of sodium hydroxide 45g, methyl alcohol 150ml and water 150ml.
(2) methylate: lipid acid is changed into to methylfatty acid, and every pipe adds the 2.0ml methylating reagent, after the tight lid of bolt, shakes 10s, puts 80 ℃ of heating in water bath 10 min, with cold water, is quickly cooled to room temperature.Methylating reagent can adopt the mixture be comprised of 6.0 mol/L hydrochloric acid 325ml and methyl alcohol 275ml.
(3) extraction: methylfatty acid is transferred to organic phase from water.Every pipe adds the 1.25ml extraction solvent, covers tightly the test tube lid, puts 28 ℃ of shaking table gentlenesses and mixes 10min, gets clean suction pipe and draws upper organic phase, puts into another test tube.Extraction solvent can adopt the mixture be comprised of hexane 200ml and methyl tert-butyl ether 200ml.
(4) washing: add the 3.Oml alkaline detergent solution in test tube, the tight lid of bolt, concussion test tube 5min, static several minutes, layering was clear.Alkaline detergent solution can adopt the mixture be comprised of sodium hydroxide 10.8g and water 900ml.
V. the stratographic analysis of bacterium lipid acid: adopt gas chromatographic technique, determine lipid acid kind and the relative content of bacterium, reproduce bacterium lipid acid finger printing, can be by discrimination of bacteria to strain.
1, the instrument that the stratographic analysis of bacterium lipid acid adopts is U.S. Agilent HP6890 type gas chromatograph.
2, the lipid acid standard substance that adopt in the homology comparison procedure comprise fatty acid methyl ester admixture liquid, contain C5～C20 linear saturated fatty acids methyl esters and 2-hydroxyl-10 carbon, 3-hydroxyl-10 carbon, 2-hydroxyl-14 carbon, 2-hydroxyl-16 carbon saturated fatty acid, reagent is purchased from U.S. MIDI company, lot number MIDI 601230-A.The significant parameter table of lipid acid standard substance is shown in Fig. 2, Fig. 3.
3, mensuration process: extract is moved to sample flasket, with clean suction pipe, draw upper organic phase, transfer to clean chromatographic sample bottle, put on gas chromatograph.Chromatographic condition: 40 ℃ of furnace temperature, 250 ℃ of injection ports, 300 ℃ of detectors; Hydrogen flowing quantity 30mL/min, air flow quantity 400mL/min, nitrogen flow 30mL/min; Hydrogen pressure 10.6psi.Chromatographic column is Agilent 19091B-102 type, and detector is hydrogen flame ionization detector.The calibration of lipid acid standard substance adds bacteria sample to be checked after passing through, and adopts gas chromatographic technique, determines bacterium lipid acid kind and relative content, obtains bacterium lipid acid fingerprint recognition collection of illustrative plates (seeing Fig. 4, Fig. 5).
VI. homology relatively: two kinds of bacteriums are in the situation that biochemical reaction is identical, if there is notable difference in bacterium lipid acid chromatogram peak position (Peak Name), point out bacterium to belong to (kind), homology (strain) not together; If lipid acid chromatogram peak position is identical, (Percent) is close for peak height, points out bacterium both to belong to (kind) together, again homology (strain).In the present embodiment, the bacterial strain of differentiation is Vibrio vulnificus 1.758 strains.
Claims (3)
1. the homology method for quick identification of a bacterial strain, comprise the steps: I. according to form, dyeing, the cultural characters of bacterium, tentatively differentiate; II. utilize bacterium to be belonged to biochemical reaction sugared, protein utilization or plant discriminating; III. utilize serology antibody that discrimination of bacteria is arrived to type; It is characterized in that, it also comprises the steps: IV. bacterium to be measured is seeded on the substratum without lipid component and cultivates, collect bacterium colony, through saponification, methylate, extraction, washing process, bacterium lipid acid is transferred to organic phase from water; The collection process of described bacterium colony comprises: bacterium is inoculated on the TSBA flat board of fat-free composition by four district's setting-out methods, and 35 ℃, 24h cultivation, with the bacterium colony 40mg left and right in transfering loop Qu 3rd district, be placed in aseptic, dry, screw-topped test tube bottom; Described saponification process comprises: add the saponification reagent of 1.Oml in test tube, cover tightly test tube, concussion test tube 10s, put into 100 ℃ of boiling water water-baths, shifts out, cooling concussion 10s after 5min from boiling water; By in the tube back water-bath, continue heating 25min again; In boiling water, saponification is total to 30min, cooling; The mixed solution that described saponification reagent is comprised of sodium hydroxide 45g, methyl alcohol 150ml and water 150ml; The described process of methylating comprises: add the 2.0ml methylating reagent in every pipe, 10s is swung in the after shock of the tight test tube lid of bolt, puts 80 ℃ of heating in water bath 10 min, with cold water, is quickly cooled to room temperature, and lipid acid is changed into to methylfatty acid; The mixed solution that described methylating reagent is comprised of 6.0 mol/L hydrochloric acid 325ml and methyl alcohol 275ml; Described extraction process comprises: every pipe adds the 1.25ml extraction solvent, covers tightly the test tube lid, puts 28 ℃ of shaking table gentlenesses and mixes 10min, gets clean suction pipe and draws upper organic phase, puts into another test tube, and methylfatty acid is transferred to organic phase from water; Described extraction solvent is mixed with volume 1:1 by hexane and methyl tert-butyl ether; Described washing process comprises: add the 3.Oml alkaline detergent solution in test tube, and the tight lid of bolt, concussion test tube 5min, static several minutes, layering was clear; The mixed solution that described alkaline detergent solution is comprised of sodium hydroxide 10.8g and water 900ml; V. adopt gas chromatographic technique, determine kind and the content of bacterium lipid acid, obtain this bacterium lipid acid fingerprint recognition collection of illustrative plates; VI. adopt the lipid acid standard substance to carry out homology relatively, by discrimination of bacteria to strain; The method of discrimination of described homology comparison is: if two kinds of bacteriums, in the situation that described I is identical to the III step results, as there is notable difference in bacterium lipid acid chromatogram peak position, point out bacterium to belong to of the same race but homology not together; If lipid acid chromatogram peak position is identical, peak height is close, points out bacterium not only to belong to of the same race but also homology together.
2. the homology method for quick identification of a kind of bacterial strain as claimed in claim 1, it is characterized in that, described lipid acid standard substance comprise fatty acid methyl ester admixture liquid, contain C5～C20 linear saturated fatty acids methyl esters and 2-hydroxyl-10 carbon, 3-hydroxyl-10 carbon, 2-hydroxyl-14 carbon, 2-hydroxyl-16 carbon saturated fatty acid.
3. the homology method for quick identification of a kind of bacterial strain as claimed in claim 1 or 2, it is characterized in that, described stratographic analysis process comprises: extract is moved to sample flasket, with clean suction pipe, draw upper organic phase, transfer to clean chromatographic sample bottle, put on gas chromatograph; Chromatographic condition: 40 ℃ of furnace temperature, 250 ℃ of injection ports, 300 ℃ of detectors; Hydrogen flowing quantity 30mL/min, air flow quantity 400mL/min, nitrogen flow 30mL/min; Hydrogen pressure 10.6psi; Chromatographic column is Agilent 19091B-102 type, and detector is hydrogen flame ionization detector; The calibration of lipid acid standard substance adds bacteria sample to be checked after passing through, and adopts gas chromatographic technique, determines bacterium lipid acid kind and relative content, obtains bacterium lipid acid fingerprint recognition collection of illustrative plates.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588048A (en) * | 2004-07-09 | 2005-03-02 | 中国科学院生态环境研究中心 | Analyzing soil microbial community diversity using lipoid fatty acid atlas technology |
| CN101020921A (en) * | 2006-12-22 | 2007-08-22 | 福建省农业科学院生物技术研究所 | Process of analyzing descending differentiated fatty acid type of Ralstonia solanacearum |
| CN101936960A (en) * | 2009-07-02 | 2011-01-05 | 湖北出入境检验检疫局检验检疫技术中心 | Analytical method of components of fatty acid contained in listeria cells |
| CN102033114A (en) * | 2010-10-12 | 2011-04-27 | 四川大学 | Method for quickly and simply analyzing microbial community structure of yeast for traditional brewage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1588048A (en) * | 2004-07-09 | 2005-03-02 | 中国科学院生态环境研究中心 | Analyzing soil microbial community diversity using lipoid fatty acid atlas technology |
| CN101020921A (en) * | 2006-12-22 | 2007-08-22 | 福建省农业科学院生物技术研究所 | Process of analyzing descending differentiated fatty acid type of Ralstonia solanacearum |
| CN101936960A (en) * | 2009-07-02 | 2011-01-05 | 湖北出入境检验检疫局检验检疫技术中心 | Analytical method of components of fatty acid contained in listeria cells |
| CN102033114A (en) * | 2010-10-12 | 2011-04-27 | 四川大学 | Method for quickly and simply analyzing microbial community structure of yeast for traditional brewage |
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