CN102517374A - Rapid identification method for homology of bacterial strains - Google Patents
Rapid identification method for homology of bacterial strains Download PDFInfo
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- CN102517374A CN102517374A CN2011103917502A CN201110391750A CN102517374A CN 102517374 A CN102517374 A CN 102517374A CN 2011103917502 A CN2011103917502 A CN 2011103917502A CN 201110391750 A CN201110391750 A CN 201110391750A CN 102517374 A CN102517374 A CN 102517374A
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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; Kind and content through to the bacterium short chain fatty acid detect; Clearly determine the homology of bacterial strain, thereby bacterial strain is carried out the method for Fast Classification and discriminating.
Background technology
The laboratory diagnosis of bacterial infection disease; The knowledge and skills that relates to aspects such as pathogeny microbiology (containing medical science, agricultural, industry, environmental microorganism), epidemiology, prudence, hospital infection, biological chemistry is a multidisciplinary interpenetrative comprehensive diagnos technology.The groundwork of microbiology laboratory is from sample, to isolate pathogenic strains and accurately differentiate.To the bacterium homology differentiate the kind can in time understand between the popular bacterial strain, belong between sibship; Its purpose is: clear and definite bacteria pathogeny is learned diagnosis, is differentiated the relation between bacterium and infection; Quick, reliable regimen is provided, in time coaches for the epidemiology control.The homology of infectation of bacteria is differentiated to the monitoring of clinical diagnosis, treatment, prognosis and epidemiology survey and nosocomial infection, reliable experimental evidence is provided.
At present, the homology of microbiology laboratory infectation of bacteria is differentiated main rely on bacterium nucleotide sequencing or PCR hybridization etc., because bacterium is prokaryotic organism, the nucleotide gene sequence is complicated than virogene, has shortcomings such as experimental cost is high, experimental period is long.Simultaneously, prokaryotic organism contain abundant sugar, protein (containing antibody, enzyme system) and lipid component.Sugar of bacterium and protein have formed the traditional bacteria discrimination method through 100 years of researches, promptly tentatively differentiate section according to form, dyeing, the cultural characters of bacterium; Through biochemical reaction, utilize the unique enzyme system of bacterium, thereby, carry out different chemical reactions the resolving power and the meta-bolites difference of substrate (sugar, protein), can bacterium be differentiated with this to belong to or plant; Utilize serology antibody that bacterium is differentiated type again.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 be to propose a kind of quick, simple and easy, cost is low, the homology method for quick identification of the bacterial strain of experimentation safety.
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. form, dyeing, cultural characters according to bacterium are tentatively differentiated; II. utilize bacterium that the biochemical reaction of sugar, protein utilization is belonged to or plant discriminating; III. utilize serology antibody that bacterium is differentiated type; IV. adopt gas chromatographic technique, determine the kind and the content of bacterium lipid acid, obtain this bacterium lipid acid fingerprint recognition collection of illustrative plates, carry out homology relatively, bacterium is differentiated to strain.
The homology method for quick identification of above-mentioned a kind of bacterial strain; Said homology relatively is to adopt the lipid acid standard substance to carry out homology relatively; The lipid acid standard substance that adopted 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 sfas.
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 of no 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 said bacterium colony comprises: be inoculated in bacterium on the TSBA flat board of fat-free composition by four districts setting-out method; 35 ℃, 24h cultivation; Get with transfering loop about the bacterium colony 40mg in the 3rd district, place aseptic, dry, screw-topped test tube bottom.
The homology method for quick identification of above-mentioned a kind of bacterial strain, said saponification process comprises: in test tube, add the saponification reagent of 1.Oml, cover tight test tube, concussion test tube 10s puts into 100 ℃ of boiling water water-baths, from boiling water, shifts out, cools off after the 5min, concussion 10s; With in the tube back water-bath, continue heating 25min again; Saponification is total to 30min in the boiling water, cooling.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the mixed solution that the reagent that is adopted in the said saponification process is made up 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 said process of methylating comprises: add the 2.0ml methylating reagent in every pipe, shake 10s behind the tight test tube lid of bolt, put 80 ℃ of heating in water bath 10 min; Be quickly cooled to room temperature with cold water, lipid acid is changed into methyl lipid acid.
The homology method for quick identification of above-mentioned a kind of bacterial strain, the mixed solution that used reagent is made up of 6.0 mol/L hydrochloric acid 325ml and methyl alcohol 275ml in the said process that methylates.
The homology method for quick identification of above-mentioned a kind of bacterial strain; Said extraction process comprises: every pipe adds the 1.25ml extraction solvent; Cover tight test tube lid, put the gentle mixing 10min of 28 ℃ of shaking tables, get clean suction pipe and draw upper organic phase; Put into another test tube, methyl lipid 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 the said 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, said washing process comprises: in test tube, add the 3.Oml alkaline detergent solution, 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 that adopts in the said washing process, the mixed solution of forming by sodium hydroxide 10.8g and water 900ml.
The homology method for quick identification of above-mentioned a kind of bacterial strain, said stratographic analysis process comprises: extract is moved to sample flasket, draw upper organic phase with clean suction pipe, transfer to clean chromatographic sample bottle, put on the 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 an Agilent 19091B-102 type, and detector is a hydrogen flame ionization detector; After the calibration of lipid acid standard substance is passed through, add bacteria sample to be checked, adopt gas chromatographic technique, determine bacterium lipid acid kind and relative content, obtain 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 said homology comparison is: if two kinds of bacteriums are under the identical situation of said I to III step results; There is notable difference like bacterium lipid acid chromatogram peak position, then points out bacterium to belong to together of the same race but homology not; If lipid acid chromatogram peak position is identical, peak height is close, then points out bacterium not only to belong to together of the same race but also homology.
Because bacterium lipid comprises SUV, lipid acid, mycolic acids, phosphatide, quinone etc.; And lipid acid mainly is present in the organoid as the fundamental component of cell, and 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, so contained abundant systematic bacteriology information in the component of bacterium lipid acid and the content.The present invention combines bacterium to sugar, protein utilization characteristic; (kind and the content of C5～C20) detect to the bacterium short chain fatty acid; 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 bacterium evaluator; Promptly under the identical situation of bacterium dyeing microscopic examination, biochemical reaction, serological reaction; Based on the homology of sugar, protein and fatty acid analysis technology discriminating infectation of bacteria, be extension to traditional bacteriology discriminating, somatotype, be again a kind of novel method after bacterium Nucleotide somatotype; For bacteriological somatotype, discriminating, relatively new way is provided, have quick, simple and easy, advantages such as cost is low, experimentation safety.
Description of drawings
Fig. 1 is the operating process synoptic 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 gc 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. form, dyeing, cultural characters according to bacterium differentiate that tentatively this method can be differentiated section with bacterium.
II. utilize bacterium that the biochemical reaction of sugar, protein utilization is differentiated, this method can be differentiated bacterium and belong to or kind.
III. serology is differentiated: go to detect unknown bacterium with containing known differential protein antibody, this method can be differentiated bacterium to planting or type.
IV. bacterium to be measured is seeded on the substratum of no 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 dull and stereotyped by the TSBA that setting-out method in four districts is inoculated in fat-free composition, and 35 ℃, 24h are cultivated, and get with transfering loop about the bacterium colony 40mg in the 3rd district, and putting aseptic, drying has bottom the screw capped tube.
2, bacterium lipid acid extracting:
(1) saponification: in test tube, add the saponification reagent of 1.Oml, cover tight test tube, concussion test tube 10s puts into 100 ℃ of boiling water water-baths, from boiling water, shifts out, cools off after 5 min, concussion 10s.With in the tube back water-bath, continue heating 25 min again.Saponification is total to 30min in the boiling water, cooling.Saponification reagent can adopt the mixture of being made up of sodium hydroxide 45g, methyl alcohol 150ml and water 150ml.
(2) methylate: lipid acid is changed into methyl lipid acid, and every pipe adds the 2.0ml methylating reagent, shakes 10s behind the tight lid of bolt, puts 80 ℃ of heating in water bath 10 min, is quickly cooled to room temperature with cold water.Methylating reagent can adopt the mixture of being made up of 6.0 mol/L hydrochloric acid 325ml and methyl alcohol 275ml.
(3) extraction: methyl lipid acid is transferred to organic phase from water.Every pipe adds the 1.25ml extraction solvent, covers tight test tube lid, puts the gentle mixing 10min of 28 ℃ of shaking tables, gets clean suction pipe and draws upper organic phase, puts into another test tube.Extraction solvent can adopt the mixture of being made up of hexane 200ml and methyl tert-butyl ether 200ml.
(4) washing: in test tube, add the 3.Oml alkaline detergent solution, the tight lid of bolt, concussion test tube 5min, static several minutes, layering was clear.Alkaline detergent solution can adopt the mixture of being made up of sodium hydroxide 10.8g and water 900ml.
V. bacterium lipid acid stratographic analysis: adopt gas chromatographic technique, determine lipid acid kind and the relative content of bacterium, reproduce bacterium lipid acid finger printing, can bacterium be differentiated to strain.
1, the instrument that adopted of bacterium lipid acid stratographic analysis is a U.S. Agilent HP6890 type gas chromatograph.
2, the lipid acid standard substance that adopted 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 sfas; Reagent is available from U.S. MIDI company, lot number MIDI 601230-A.The significant parameter table of lipid acid standard substance is seen Fig. 2, Fig. 3.
3, mensuration process: extract is moved to sample flasket, draw upper organic phase, transfer to clean chromatographic sample bottle, put on the gas chromatograph with clean suction pipe.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 an Agilent 19091B-102 type, and detector is a hydrogen flame ionization detector.After the calibration of lipid acid standard substance is passed through, add bacteria sample to be checked, adopt gas chromatographic technique, determine bacterium lipid acid kind and relative content, obtain bacterium lipid acid fingerprint recognition collection of illustrative plates (seeing Fig. 4, Fig. 5).
VI. homology relatively: two kinds of bacteriums if there is notable difference in bacterium lipid acid chromatogram peak position (Peak Name), then point out bacterium to belong to (kind), homology (strain) not together under the identical situation of biochemical reaction; If lipid acid chromatogram peak position is identical, (Percent) is close for peak height, then 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 (10)
1. the homology method for quick identification of a bacterial strain comprises the steps: I. form, dyeing, cultural characters according to bacterium are tentatively differentiated; II. utilize bacterium that the biochemical reaction of sugar, protein utilization is belonged to or plant discriminating; III. utilize serology antibody that bacterium is differentiated type; It is characterized in that it also comprises the steps: IV. adopt gas chromatographic technique, determine the kind and the content of bacterium lipid acid, obtain this bacterium lipid acid fingerprint recognition collection of illustrative plates, carry out homology relatively, bacterium is differentiated to strain.
2. the homology method for quick identification of a kind of bacterial strain as claimed in claim 1; It is characterized in that; Said homology relatively is to adopt the lipid acid standard substance to carry out homology relatively; The lipid acid standard substance that adopted 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 sfas.
3. according to claim 1 or claim 2 a kind of homology method for quick identification of bacterial strain; It is characterized in that; Before adopting gas chromatographic technique to measure the kind and content of bacterium lipid acid, bacterium to be measured is seeded on the substratum of no lipid component and cultivates the collection bacterium colony; Through saponification, methylate, extraction, washing process, bacterium lipid acid is transferred to organic phase from water.
4. the homology method for quick identification of a kind of bacterial strain as claimed in claim 3; It is characterized in that; The collection process of said bacterium colony comprises: be inoculated in bacterium on the TSBA flat board of fat-free composition by four districts setting-out method; 35 ℃, 24h cultivation are got with transfering loop about the bacterium colony 40mg in the 3rd district, place aseptic, dry, screw-topped test tube bottom.
5. the homology method for quick identification of a kind of bacterial strain as claimed in claim 3; It is characterized in that said saponification process comprises: in test tube, add the saponification reagent of 1.Oml, cover tight test tube; Concussion test tube 10s; Put into 100 ℃ of boiling water water-baths, from boiling water, shift out, cool off after the 5min, concussion 10s; With in the tube back water-bath, continue heating 25min again; Saponification is total to 30min in the boiling water, cooling; The mixed solution that said saponification reagent is made up of sodium hydroxide 45g, methyl alcohol 150ml and water 150ml.
6. the homology method for quick identification of a kind of bacterial strain as claimed in claim 3; It is characterized in that; The said process of methylating comprises: add the 2.0ml methylating reagent in every pipe, shake 10s behind the tight test tube lid of bolt, put 80 ℃ of heating in water bath 10 min; Be quickly cooled to room temperature with cold water, lipid acid is changed into methyl lipid acid; The mixed solution that said methylating reagent is made up of 6.0 mol/L hydrochloric acid 325ml and methyl alcohol 275ml.
7. the homology method for quick identification of a kind of bacterial strain as claimed in claim 3; It is characterized in that said extraction process comprises: every pipe adds the 1.25ml extraction solvent, covers tight test tube lid; Put the gentle mixing 10min of 28 ℃ of shaking tables; Get clean suction pipe and draw upper organic phase, put into another test tube, methyl lipid acid is transferred to organic phase from water; Said extraction solvent is mixed with volume 1:1 by hexane and methyl tert-butyl ether.
8. the homology method for quick identification of a kind of bacterial strain as claimed in claim 3 is characterized in that, said washing process comprises: in test tube, add the 3.Oml alkaline detergent solution, and the tight lid of bolt, concussion test tube 5min, static several minutes, layering was clear; The mixed solution that said alkaline detergent solution is made up of sodium hydroxide 10.8g and water 900ml.
9. the homology method for quick identification of a kind of bacterial strain as claimed in claim 3; It is characterized in that said stratographic analysis process comprises: extract is moved to sample flasket, draw upper organic phase with clean suction pipe; Transfer to clean chromatographic sample bottle, put on the 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 an Agilent 19091B-102 type, and detector is a hydrogen flame ionization detector; After the calibration of lipid acid standard substance is passed through, add bacteria sample to be checked, adopt gas chromatographic technique, determine bacterium lipid acid kind and relative content, obtain bacterium lipid acid fingerprint recognition collection of illustrative plates.
10. according to claim 1 or claim 2 a kind of homology method for quick identification of bacterial strain; It is characterized in that; The method of discrimination of said homology comparison is: if two kinds of bacteriums are under the identical situation of said I to III step results; There is notable difference like bacterium lipid acid chromatogram peak position, then points out bacterium to belong to together of the same race but homology not; If lipid acid chromatogram peak position is identical, peak height is close, then points out bacterium not only to belong to together of the same race but also homology.
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| CN110146638A (en) * | 2019-05-22 | 2019-08-20 | 遵义医科大学 | A method of fatty acid species in measurement actinomyces novel species M8JJ-5 |
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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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| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110146638A (en) * | 2019-05-22 | 2019-08-20 | 遵义医科大学 | A method of fatty acid species in measurement actinomyces novel species M8JJ-5 |
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