CN209412237U - A kind of bacterial identification kits based on DNA characteristics sequence - Google Patents

Abstract

The utility model relates to a kind of bacterial identification kits based on DNA characteristics sequence, including box body, box cover and liner, nucleic acid extracting reagent storage chamber is set in the box body, PCR reaction system storage chamber and PCR product purified reagent storage chamber, the liner is filled between box body and each storage chamber, lysozyme pipe is at least placed in nucleic acid extracting reagent storage chamber, sodium dodecyl sulfate solution pipe, cetyl trimethylammonium bromide pipe, TE buffering liquid pipe is at least placed in PCR product purified reagent storage chamber, sodium acetate solution pipe and sodium ethylene diamine tetracetate solution conduit, refrigeration storage chamber and off-duty storage chamber is still alternatively arranged in the kit.Kit described in the utility model can be used for bacterial pollutant from all operationss process to before nucleic acid sequencing is isolated and purified, and realize quick, the integrated identification of bacterial pollutant；The standard nucleic acid sequence database of the utility model building can be sequenced identification method for bacterial nucleic acid and provide objective, accurate judgment basis.

Description

A kind of bacterial identification kits based on DNA characteristics sequence

Technical field

The utility model relates to nucleic acid sequencing identification field more particularly to a kind of Bacteria Identifications based on DNA characteristics sequence Kit.

Background technique

Using Protocols in Molecular Biology to the dirt in medicine material, auxiliary material, water for pharmaceutical purposes, intermediate, finished product and environment Dye microorganism is identified, classified and is traced, be reinforce the control of drug production process quality, Improving The Quality of Products and safety, Reduce the effective means of drug risk.With traditional morphological observation, microscope inspection, physical and chemical identify and the technologies phases such as biochemical analysis It, can be from the inhereditary material level of essence to bacterial strain than, Protocols in Molecular Biology using biotinylated nucleic acid as target detection thing Kind and source are made explanations, and testing result is more accurate, and sensitivity is higher, and specificity is stronger, have been increasingly becoming state, interior at present The inexorable trend of outer drug standard development.Currently, " United States Pharmacopeia " (USP 40<1113>), " European Pharmacopoeia " (EP 9.0< 5.1.6>), " Japanese Pharmacopoeia " (JP 17<General Information G4>), receive in " Chinese Pharmacopoeia " (general rule 9204) Related Sections of the Protocols in Molecular Biology for the control of drug microbial quality are carried.

DNA characteristics sequence (DNA bar code) Molecular Identification utilizes one section of generally acknowledged, relatively short DNA in genome Sequence come carry out species identification molecular biology classify method.This method is logical by screening based on Nucleic acid sequencing techniques With DNA characteristics sequence, database and identification platform are established, with bioinformatics method analyses and comparison DNA data, and then to object Kind is identified, is to effective supplement of traditional biological identification method and important breakthrough, in recent years by domestic and international drug quality The extensive concern of standards setting organizations, and it is increasingly becoming the research hotspot of species identification and classification.

16S rRNA full length gene covers most comprehensive bacterial strain genetic evolution information, but is operated by method for nucleic acid sequencing Property, sequencing quality, sequencing cost, nucleic acid sequencing reaction length, particularly high throughput nucleic acid sequencing reaction read long limitation, it is most Document report still uses partial nucleic acid sequence (Partial Sequence) to be studied, and has the core of identification and category significance Heart district domain waits to discuss.

Currently, the microorganism identification method based on 16S rRNA Nucleic acid sequencing techniques is it has been established that and be applied to face The research of bed isolated strains, but its identification for being used for functions on common pollutant bacteria in drug quality control and pharmaceutical production environment also rarely has report Road, and the bacterial identification kits based on DNA characteristics sequence are also rarely reported.

Utility model content

The purpose of the utility model is to overcome defects in the prior art, by measuring 16S rRNA gene 5' It holds the nucleic acid sequence of variable region to carry out bacterial strain identification, provides a kind of bacterial identification kits based on DNA characteristics sequence, and provide A kind of easy, quick, standardization bacterial nucleic acid sequencing identification method based on DNA characteristics sequence.

To achieve the goals above, the technical solution that the utility model is taken includes:

First purpose of the utility model is to provide a kind of bacterial identification kits based on DNA characteristics sequence, including Box body, box cover and liner, the interior setting nucleic acid extracting reagent storage chamber of the box body, PCR reaction system storage chamber and PCR product are pure Change reagent storage chamber, the liner is filled between the box body and each storage chamber.

In order to advanced optimize mentioned reagent box, the technical measures that the utility model is taken further include:

Further, the several test tubes of setting in the nucleic acid extracting reagent storage chamber, the test tube include at least lysozyme Pipe, sodium dodecyl sulfate solution pipe, cetyl trimethylammonium bromide pipe；Further, the test tube further includes TE slow Fliud flushing pipe, sodium chloride solution pipe, saturation phenol chloroform-isoamyl alcohol pipe, dehydrated alcohol pipe, 70% ethanol solution pipe.

Further, the several test tubes of setting in the PCR reaction system storage chamber, the test tube include at least primer pipe, Archaeal dna polymerase pipe, PCR reaction solution pipe；Wherein, PCR buffer, magnesium ion solution and deoxidation are contained in the PCR reaction solution pipe Ribonucleoside triphosphote.

Further, the several test tubes of setting in the PCR product purified reagent storage chamber, the test tube are slow including at least TE Fliud flushing pipe, sodium acetate solution pipe and sodium ethylene diamine tetracetate solution conduit；Further, the test tube further includes sodium acetate solution Pipe, dehydrated alcohol pipe and 75% ethanol solution pipe.

Further, the off-duty storage chamber at least placing sample-loading buffer test tube is also set up in the box body；Further Ground, the intracavitary centrifuge tube for placing all kinds of specifications of the off-duty storage, semi-commercial (semiworks) production storage pipe etc..

Further, refrigeration storage chamber is also set up in the box body, the refrigeration storage chamber and the box body are detachable Installation；Further, the refrigeration storage chamber directly can be extracted out or be put into the box body, and the box body and the refrigeration are deposited It puts and cold insulation material is set between chamber.

Further, the refrigeration storage chamber includes refrigeration exocoel and refrigeration inner cavity chamber, and the refrigeration inner cavity chamber is used for The test tube containing the reagent that need to be stored under cryogenic is placed, is a sealing between the refrigeration exocoel and refrigeration inner cavity chamber Hollow structure, built-in cold-retaining agent.Further, the outer wall of the inner wall of the refrigeration exocoel and the refrigeration inner cavity chamber It is respectively provided with waterproof sealing layer, the cold-retaining agent includes one of gel or water；Further, the cold-retaining agent further includes protecting Ice medium and insulation particle, guarantor's ice medium are methylcellulose or acrylate copolymer, and the insulation particle is Poly Foam Material.

Further, a fastener is fixedly installed on the box body, the box cover setting one makes with fastener cooperation Convex block.

Further, the shape and size of the box body and each storage chamber are matched according to the shape and size of each test tube Setting.

Further, liner is fixedly installed in the inside of the box cover.

Second purpose of the utility model is to provide a kind of bacterium based on DNA characteristics sequence using mentioned reagent box Nucleic acid sequencing identification method comprising following steps:

Step 1: choosing bacterium bacterial strain to be identified, and extract and purify the genomic DNA of the bacterial strain；

Step 2: using the characteristic sequence fragment of the genomic DNA in amplimer PCR amplification step 1 after purification；

Step 3: the pcr amplification product obtained in extraction and purification step two；

Step 4: the sequence of the pcr amplification product in determination step two after purification, and it is soft using versatility sequence assembly Part removes guiding region, obtains the DNA sequence dna of respective segments；

Step 5: the DNA sequence dna of respective segments described in step 4, which is imported standard nucleic acid sequence database, carries out sequence It compares, carries out the identification of bacterium bacterial strain；

Wherein, in the step 1, the extraction and purifying of the genomic DNA of bacterial strain use lysozyme (Lysozyme), ten Sodium dialkyl sulfate, cetyl trimethylammonium bromide (Cetyltriethylammnonium Bromide, CTAB) carry out bacterium Body is broken；Using phenol chloroform-isoamyl alcohol, ethanol precipitation nucleic acid, the genomic DNA for meeting quality control requirement is obtained.

Wherein, in the step 1, the specific steps of extraction and the purifying of the genomic DNA of the bacterial strain include: take it is suitable Thallus after amount isolates and purifies is placed in centrifuge tube, and TE buffer, lysozyme is added, and is mixed, heating water bath；Dodecyl is added Metabisulfite solution, heating water bath；Sodium chloride solution, cetyl trimethylammonium bromide solution, heating water bath is added；Saturation is added Phenol chloroform-isoamyl alcohol acutely shakes, is stored at room temperature, is centrifuged, take supernatant to be placed in new centrifuge tube, repetitive operation 1 time；Dehydrated alcohol is added, is stood under cryogenic conditions, is centrifuged, discards supernatant liquid；The washing of 70% ethanol solution is added, centrifugation is abandoned Supernatant is removed, is air-dried at room temperature complete to ethyl alcohol volatilization；TE buffer is added, nucleic acid extraction solution is used as after mixing.The step It can be replaced: the thallus after isolating and purifying in right amount being taken to be placed in centrifuge tube, lysozyme soln is added, mix, heating water bath；It is added Sodium dodecyl sulfate solution, heating water bath；Sodium chloride solution, cetyl trimethylammonium bromide solution, heating water bath is added； Cellular lysate liquid is transferred on suitable carrier, thallus and lysate are removed by way of centrifugation, nucleic acid is trapped in suitable On suitable carrier, saturation phenol chloroform-isoamyl alcohol is added, the washing such as ethanol solution is centrifuged, discards cleaning solution；Room Temperature air-dries complete to ethyl alcohol volatilization；Suitable buffer solution nucleic acid is added, centrifugation elutes it from suitable carrier, As nucleic acid extraction solution.In two kinds of interchangeable steps, used reagent dosage and concentration are all the same.

Wherein, in step 2, the nucleic acid sequence of the area the V1~V3 segment in the amplimer amplification 16S rRNA, the expansion The sequence for increasing primer is as follows:

Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3'；

Reverse primer: 5'-GTATTACCGCGGCTGCTGGC-3'；

The pcr amplification reaction system is using 25 μ l as reference, comprising: 10 × PCR buffer, 25mmol/L magnesium ion are molten 2.5 μ l, 2.5mmol/L dNTPs of liquid 2 μ l, each 0.5 μ l of 10 μm of ol/L primer pairs, 0.5 μ l, 5U/ μ l Taq DNA of template DNA 0.2 μ l of polymerase, adds aseptic double-distilled water to 25 μ l；Pcr amplification reaction program are as follows: 94 DEG C, initial denaturation 3~5 minutes；94 DEG C, become Property 30 seconds；It 55~60 DEG C, anneals 30 seconds；72 DEG C, extend 30~60 seconds, 28~32 circulations；72 DEG C, 3~5 minutes.

Wherein, in the step 3, the extraction of pcr amplification product and purification process include the following steps: to take suitable expansion Volume increase object and sample-loading buffer, which are blended in 1.5% Ago-Gel, carries out electrophoresis；It will be in the Ago-Gel that terminate electrophoresis Nucleic acid amplification product is cut, and TE buffer is added, and water-bath is completely dissolved；Sodium acetate solution is added and sodium ethylene diamine tetracetate is molten Liquid mixes；Dehydrated alcohol is added, stands, centrifugation, abandon supernatant；Ethanol washing is added, centrifugation abandons supernatant, ethyl alcohol volatilizees Completely；TE buffer solution is added, as the purification solution of nucleic acid amplification product after mixing.

The utility model by adopting the above technical scheme, compared with prior art, has the following technical effect that

The utility model establishes the nucleic acid sequencing identification method of common bacteria pollutant in pharmaceutical production and quality control, Quality verification is carried out to nucleic acid sequencing result, in a manner of the positioning of universal sequencing primer object, establishes bacterial nucleic acid sequencing identification of dna Characteristic sequence；Standard nucleic acid sequencing data storehouse on the basis of the utility model is established, can be the inspection result of drug microorganism Reliable judgment basis is provided.

The utility model is using the standard nucleic acid sequence constructed, according to unified generalized description and canonical code typing bacterium Nucleic acid sequencing standard of perfection GenBank can be sequenced identification method for bacterial nucleic acid and provide objective, accurate judgement Foundation；Kit described in the utility model can be used for bacterial pollutant from isolating and purifying all operationss mistake to before nucleic acid sequencing Journey realizes quick, the integrated identification of bacterial pollutant.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of the bacterial identification kits based on DNA characteristics sequence in an embodiment of the present invention.

Fig. 2 is the structural schematic diagram of the refrigeration storage chamber in another embodiment of the utility model in bacterial identification kits.

Wherein, the appended drawing reference in figure are as follows: box body 1；Fastener 11；Box cover 2；Convex block 12；Off-duty storage chamber 3；Nucleic acid mentions Take reagent storage chamber 4；PCR reaction system puts storage chamber 5；PCR product purified reagent storage chamber 6；Refrigerate storage chamber 7；Refrigeration is outer Chamber 71；Refrigerate inner cavity chamber 72.

Specific embodiment

The utility model provides a kind of bacterial identification kits based on DNA characteristics sequence, including box body, box cover and interior Lining, the interior setting nucleic acid extracting reagent storage chamber of the box body, PCR reaction system storage chamber and PCR product purification reagent storage Chamber fills the liner between the box body and each storage chamber；And using mentioned reagent box based on DNA characteristics sequence Bacterial nucleic acid be sequenced identification method.

With reference to the accompanying drawings and examples, specific embodiment of the present utility model is further described.Implement below Example is only used for clearly illustrating the technical solution of the utility model, and cannot be used as a limitation the protection model of limitation the utility model It encloses.

The bacterial identification kits based on DNA characteristics sequence of mono- preferred versions of embodiment 1-

As shown in Figure 1, bacterial identification kits described in the present embodiment include box body 1, box cover 2 and liner, the box body 1 It is realized at a side with box cover 2 collapsible hinged；In the hinged place opposite side, fixation is respectively corresponded on box body 1 and box cover 2 and is set The fastener 11 and convex block 12 being used in conjunction with each other are set, to realize the closure of the kit, while fixation is set in the inside of box cover 2 Liner is set, to effectively avoid the big displacement of the test tube of its internal storage under kit closed state mobile.

Nucleic acid extracting reagent storage chamber 4, PCR reaction system storage chamber are set in the box body 1 of above-mentioned bacterial identification kits 5, PCR product purified reagent storage chamber 6, in the box body 1 and nucleic acid extracting reagent storage chamber 4, PCR reaction system storage chamber 5, the liner is filled between PCR product purified reagent storage chamber 6；Wherein nucleic acid extracting reagent storage chamber 4, PCR reactant It is storage chamber 5, is respectively provided with several test tubes in PCR product purified reagent storage chamber 6, nucleic acid extracting reagent storage chamber 4 is at least placed Lysozyme pipe, sodium dodecyl sulfate solution pipe, cetyl trimethylammonium bromide pipe；PCR reaction system storage chamber 5 is at least put Primer pipe, DNA polymerase pipe, PCR reaction solution pipe are set, contains PCR buffer, magnesium ion solution and deoxidation in PCR reaction solution pipe Ribonucleoside triphosphote；PCR product purified reagent storage chamber 6 at least places TE buffering liquid pipe, sodium acetate solution pipe and ethylenediamine tetrem Acid sodium solution pipe.

In above-mentioned nucleic acid extracting reagent storage chamber 4, PCR reaction system storage chamber 5, PCR product purified reagent storage chamber 6 In, nucleic acid extracting reagent storage chamber 4 also optionally places TE buffering liquid pipe, sodium chloride solution pipe, saturation phenol chloroform- Isoamyl alcohol pipe, dehydrated alcohol pipe, 70% ethanol solution pipe；PCR product purified reagent storage chamber 6 is also optionally placed Sodium acetate solution pipe, dehydrated alcohol pipe and 75% ethanol solution pipe.

In the present embodiment, it is not shown in the figure, the cross sectional shape of each storage chamber is circle, and wherein nucleic acid extracting reagent is deposited Chamber, PCR reaction system storage chamber, the setting of PCR product purified reagent storage chamber same column are put, each test tube is cylindrical cuvette, respectively A rack for test tube is fixed at the middle part of storage chamber, and each test tube can be embedded in the through-hole of the rack for test tube.

The bacterial identification kits based on DNA characteristics sequence of another preferred versions of embodiment 2-

The present embodiment is for the alternative embodiment 1 that is embodiment 1, from embodiment 1 only with flowering structure, there are different:

As shown in Figure 1, the present embodiment is compared with Example 1, refrigeration storage chamber 7 and extremely is also set up in the box body of the kit Less place sample-loading buffer test tube off-duty storage chamber 3, can be placed in the off-duty storage chamber 3 all kinds of specifications centrifuge tube, in Between reagent storage pipe, with the extraction and purifying for nucleic acid and pcr amplification product.

As shown in Fig. 2, refrigeration storage chamber 7 described in the present embodiment directly can be extracted out or be put into box body 1,1 He of box body Cold insulation material is set between refrigeration storage chamber 7, one waterproof layer is set on the surface of cold insulation material, to prevent the vapor in air Cold insulation material surface condensation and drench cold insulation material, thus play extend cold insulation material service life；Refrigerate storage chamber 7 Including refrigeration exocoel 71 and refrigeration inner cavity chamber 72, refrigeration inner cavity chamber 72 is used to place the examination containing that need to store under cryogenic The test tube of agent, low-temp storage reagent such as low-temp storage reagent nucleic acid extracts solution (4 DEG C preservation) and nucleic acid amplification product is pure Change solution (4 DEG C of preservations).It refrigerates exocoel 71 and refrigerates between the Room 72 of inner cavity as the hollow structure of a sealing, built-in cold insulation The outer wall of agent, the inner wall and refrigeration inner cavity chamber 72 that refrigerate exocoel 71 is respectively provided with waterproof sealing layer, to prevent the leakage of cold-retaining agent, The cold-retaining agent includes gel or water and guarantor's ice medium and insulation particle, wherein protecting ice medium is methylcellulose or acrylic acid Polymer, insulation particle are Poly Foam material, can effectively be extended, be met the use of long-time reagent refrigerating using above-mentioned cold-retaining agent Demand.

In the present embodiment, the cross sectional shape of off-duty storage chamber 3 and refrigeration storage chamber 7 is rectangle, and the two same column is arranged, And be arranged with nucleic acid extracting reagent storage chamber 4, PCR reaction system storage chamber 5, PCR product purified reagent storage chamber 6 with row, respectively The cross sectional shape of storage chamber 3~7 is rectangle, and each test tube is cylindrical cuvette (not shown), is all provided in each storage chamber The bottom plate (not shown) of embeddable each test tube is set, in favor of the stability of test tube storage.

Refrigeration storage chamber 7 described in the present embodiment, which needs to be placed on before use, to carry out being freezed in refrigerator, to be frozen Retrieval is placed in box body after good.

Embodiment 3- carries out bacterial nucleic acid using embodiment 1 or kit as described in example 2 and identification method is sequenced

Described in the present embodiment based on DNA characteristics sequence bacterial nucleic acid sequencing identification method the following steps are included:

(1) extraction and purification of bacterial genomes DNA

It takes the thallus after isolating and purifying in right amount to be placed in centrifuge tube, 450 μ l of TE buffer, lysozyme (10 mg/ml) is added 25 μ l, mix, 37 DEG C heating water bath 30 minutes；5% (w/v) sodium dodecyl sulfate solution, 50 μ l, 37 DEG C of heating water baths are added 15 minutes；It is added 1% sodium chloride solution, 80 μ l, 5% (w/v) cetyl trimethylammonium bromide (CTAB) solution 70 μ l, 65 DEG C Heating water bath 15 minutes；Saturation 350 μ l of phenol chloroform-isoamyl alcohol (25:24:1, v/v/v) solution is added, acutely shakes, room temperature 5 minutes are stood, centrifugation (revolving speed is 12000 turns per minute) 10 minutes takes supernatant to be placed in new centrifuge tube, repetitive operation 1 It is secondary；2 times of volume dehydrated alcohols are added, -20 DEG C stand no less than 30 minutes, 10 points of centrifugation (revolving speed is 12000 turns per minute) Clock discards supernatant liquid；Appropriate 75% ethyl alcohol (v/v) solution washing is added, is centrifuged (revolving speed is 12000 turns per minute) 10 minutes, Liquid is discarded supernatant, is air-dried at room temperature complete to ethyl alcohol volatilization；The TE buffer of appropriate volume is added, nucleic acid extraction is used as after mixing Solution (template DNA) is set in 4 DEG C of refrigerators or is refrigerated and is spare in storage chamber.Using the dense of determined by ultraviolet spectrophotometry template DNA Degree and purity, should be able to meet the requirement of follow-up test, nucleic acid concentration is preferably not less than 10ng/ μ l, A260/A280 ratio and preferably exists Between 1.8~2.0；

(2) amplification and detection of DNA of bacteria characteristic sequence fragment

1. 16S rRNA gene order amplimer:

Forward primer (16SV1F): 5'-AGAGTTTGATCCTGGCTCAG-3'；

Reverse primer (16SV3R): 5'-GTATTACCGCGGCTGCTGGC-3'.

2. PCR reaction system:

Using 25 μ l as reference, comprising: 10 × PCR buffer (containing trishydroxymethylaminomethane (10mmol/L, pH 8.3~ 8.8), 2.5 μ l of magnesium ion solution (25mmol/L), deoxynucleoside triphosphate (dNTPs, 2.5 mmol/L) 2 μ l, primer pair (10 μ Mol/L) each 0.5 μ l, template DNA 0.5 μ l, high-fidelity Taq DNA polymerase (5U/ μ l) 0.2 μ l, adds aseptic double-distilled water to 25 μl.Suitable PCR amplification premixed liquid or kit operation can also be used.

3. 16S rRNA gene order amplification program:

94 DEG C 3~5 minutes (initial denaturation)；94 DEG C 30 seconds (denaturation), 60 DEG C 30 seconds (annealing), 72 DEG C (are prolonged for 30~60 seconds Stretch), 28~32 circulations；72 DEG C 3~5 minutes.By the way of grads PCR.

4. test result

Take 5 μ l of pcr amplification product, 1 μ l of sample-loading buffer, loading after mixing, the electrophoresis under 100~150V voltage, bromine phenol Blue Streak band, which is moved at the 1/2~2/3 of gel film, terminates electrophoresis.Gel film is taken to inspect in ultraviolet gel imager, nucleic acid expands Increase production object and a purpose band occurs in the position of about 500bp.

(3) purifying of DNA of bacteria characteristic sequence fragment

Nucleic acid amplification product in Ago-Gel is cut, is placed in centrifuge tube, the TE buffering of appropriate volume is added Liquid, 65 DEG C of water-bath to gel pieces are completely dissolved；It is separately added into 1/10 volumes of acetic acid sodium solution (3mol/L, pH5.2) and ethylenediamine Tetrem acid sodium solution (125mmol/L, pH 8.0) mixes；2 times of volume dehydrated alcohols are added, -20 DEG C stand 30 minutes；Centrifugation (revolving speed is 12000 turns per minute) 10 minutes, discards supernatant liquid；Appropriate 75% ethyl alcohol (v/v) solution washing is added, centrifugation (turns Speed is 12000 turns per minute) 10 minutes, liquid is discarded supernatant, is air-dried at room temperature complete to ethyl alcohol volatilization；The TE that appropriate volume is added is slow Solution is rushed, as the purification solution of nucleic acid amplification product after mixing, sets in 4 DEG C of refrigerators or refrigerates and is spare in storage chamber.

Take 5 μ l of PCR amplification purified product, 1 μ l of sample-loading buffer, loading after mixing, the electrophoresis under 100~150V voltage, Bromophenol blue band, which is moved at the 1/2~2/3 of gel film, terminates electrophoresis.Gel film is taken to inspect in ultraviolet gel imager, core There is a purpose band in the position of about 500bp in sour amplified production.

(4) foundation of nucleic acid sequencing and standard nucleic acid sequence

The result of nucleic acid sequencing should carry out sequence assembly and quality evaluation, and when sequence assembly should use versatility sequence assembly Software removes guiding region, obtains the DNA sequence dna of respective segments；For the reliability for ensuring standard nucleic acid sequence, when sequence assembly, Sequencing quality need to be assessed, remove the low quality part at sequencing result both ends.It sequence direction should be with PCR amplification forward primer Direction is consistent.The average Q value of sequencing result should be greater than or be equal to 30.After sequence assembly and quality verification, it is consistent to obtain length Standard nucleic acid sequence, according to generalized description and unified coding, typing standard nucleic acid sequence database can be the mirror of this method Determine result and objective, accurate judgment basis is provided.

(5) identification of angstrom uncommon bacterium DNA characteristics sequence

Collect domestic and international microbial strains collection angstrom uncommon bacterium and totally 10 plants of enterobacteriaceae other standards bacterial strain, packet Include 2 plants of the source ATCC, 2 plants of the source CMCC, 6 plants of the source CICC.Be respectively adopted Morphological Identification, biochemical identification method to bacterial strain Carry out hereditary information confirmation；Then according to the method described above step extract genomic DNA, PCR, nucleic acid sequencing, to reference culture into Row nucleic acid sequencing establishes standard nucleic acid sequence.

By hereditary information confirmation angstrom uncommon bacterium and enterobacteriaceae other standards bacterial strain DNA characteristics Sequence clustering, building NJ into Change tree.The nucleic acid sequence of escherichia coli is polymerized to cluster, and it is preferable to illustrate that the level of escherichia coli dna characteristic sequence " kind " has Identification and category significance；Other bacterial strains of Escherichia are accurately distinguished with enterobacteriaceae clostridium perfringen, Klebsiella Pneumoniae, Illustrate that angstrom uncommon bacterium reference culture DNA characteristics sequence has preferable identification and category significance in the level of " category ".

(6) verifying of bacillus DNA characteristics Sequence Identification method

Totally 5 plants of bacillus reference culture for collecting domestic and international microbial strains collection, including 2 plants of the source ATCC, 3 plants of the source CICC.Morphological Identification is respectively adopted, the method for biochemical identification carries out hereditary information confirmation to bacterial strain；Then according to Above method step extracts genomic DNA, PCR, nucleic acid sequencing, carries out nucleic acid sequencing to reference culture, establishes standard nucleic acid sequence Column.

The DNA characteristics sequence that qualification result is confirmed as bacillus is subjected to sequence alignment analysis, and using clostridium as external source Control group constructs NJ chadogram, and as the result is shown: bacillus subtilis DNA characteristics sequence can be distinguished clearly with other kinds.

As can be seen from the above embodiments, the bacterial identification kits described in the utility model based on DNA characteristics sequence are available In bacterial pollutant from all operationss process to before nucleic acid sequencing is isolated and purified, the quick, integrated of bacterial pollutant is realized Identification.

Specific embodiment of the utility model is described in detail above, but it is merely an example, this is practical new Type is not restricted to particular embodiments described above.To those skilled in the art, any pair of the utility model carries out Equivalent modifications and substitution also all among the scope of the utility model.Therefore, in the spirit and model for not departing from the utility model Lower made equal transformation and modification are enclosed, should all be covered in the scope of the utility model.

Claims (10)

1. a kind of bacterial identification kits based on DNA characteristics sequence, including box body (1), box cover (2) and liner, feature exist In setting nucleic acid extracting reagent storage chamber (4), PCR reaction system storage chamber (5), PCR product purifying examination in the box body (1) Agent storage chamber (6), in the box body (1) and nucleic acid extracting reagent storage chamber (4), PCR reaction system storage chamber (5), PCR product Purified reagent storage chamber fills the liner between (6).

2. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 1, which is characterized in that described The several test tubes of setting in nucleic acid extracting reagent storage chamber (4), it is molten that the test tube includes at least lysozyme pipe, lauryl sodium sulfate Liquid pipe, cetyl trimethylammonium bromide pipe.

3. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 1, which is characterized in that described The several test tubes of setting in PCR reaction system storage chamber (5), the test tube include at least primer pipe, archaeal dna polymerase pipe, PCR reaction Liquid pipe；Wherein, PCR buffer, magnesium ion solution and deoxynucleoside triphosphate are contained in the PCR reaction solution pipe.

4. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 1, which is characterized in that described The several test tubes of setting in PCR product purified reagent storage chamber (6), the test tube include at least TE and buffer liquid pipe, sodium acetate solution Pipe and sodium ethylene diamine tetracetate solution conduit.

5. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 1, which is characterized in that described The off-duty storage chamber (3) at least placing loading buffer liquid pipe is also set up in box body (1).

6. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 1, which is characterized in that described Refrigeration storage chamber (7) is also set up in box body (1), the refrigeration storage chamber and the box body (1) are detachable installation.

7. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 6, which is characterized in that described Refrigerating storage chamber (7) includes refrigeration exocoel (71) and refrigeration inner cavity chamber (72), and the refrigeration inner cavity chamber (72) contains for placing There is the test tube for the reagent that need to be stored under cryogenic, it is close for one between the refrigeration exocoel (71) and refrigeration inner cavity chamber (72) The hollow structure of envelope, built-in cold-retaining agent.

8. a kind of bacterial identification kits based on DNA characteristics sequence according to claim 1, which is characterized in that described It is fixedly installed on box body (1) fastener (11), the convex block that box cover (2) setting one is used cooperatively with the fastener (11) (12)。

9. a kind of bacterial identification kits based on DNA characteristics sequence described according to claim 1~any one of 8, special Sign is that the shape and size of the box body and each storage chamber carry out matching setting according to the shape and size of each test tube.

10. a kind of bacterial identification kits based on DNA characteristics sequence described according to claim 1~any one of 8, special Sign is that liner is fixedly installed in the inside of the box cover (2).