CN101153331A - Primer, detection method and detection reagent kit for detecting bacillus coli O157:H7 - Google Patents
Primer, detection method and detection reagent kit for detecting bacillus coli O157:H7 Download PDFInfo
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Abstract
The invention relates a technique for fast detecting food-borne pathogens based on a loop-mediated isothermal amplification, LAMP technique. A primer for diction of colon bacillus O157:H7can augment the specific base sequence of a target gene which is the rfbE-GenBank (accession no. AE005429) of the colon bacillus O157:H7, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 7462-7471 loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the colon bacillus O157:H7, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the colon bacillus O157:H7, determines whether the colon bacillus O157:H7 exists in the specimen or not.
Description
Technical field
The present invention relates to a kind of based on ring mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) the food-borne causal agent Fast Detection Technique of technology.Be particularly related to Escherichia coli O 157: H7 specific gene fragment has specific primer and primer sets; Also relate to and use the ring mediated isothermal amplification method to detect detection method and the detection kit of Escherichia coli O 157: H7 described primer and primer sets.
Background technology
The food origin disease sickness rate is higher, by Salmonellas, Shigellae, streptococcus aureus, Bacillus proteus, vibrio cholerae, Vibrio parahemolyticus and Escherichia coli O 157: H7, the food poisoning that rotavirus and norovirus cause, its sickness rate accounts for very high ratio in China's food origin disease sickness rate, be a serious public health problem.
Detection to food-borne causal agent at present mainly relies on pathogen isolation method, immunological method and various PCR method.Though pathogen isolation is a gold standard, loaded down with trivial details time-consuming, generally need 5 day time, the longlyest need one month, and the specificity of immunological method and susceptibility are all lower.
Along with the development of Protocols in Molecular Biology, people adopt round pcr to be applied to the diagnosis of bacterium.The for example patent application of PCR Chinese patent publication number CN1526825 has disclosed a kind of specificity of utilizing Vibrio parahemolyticus PR72H sequence, detects the method for Vibrio parahemolyticus by DNA extraction, pcr amplification, electrophoresis observation equimolecular biological means.Though this method sensitivity, accurately, fast, owing to need the plant and instrument of costliness, higher testing cost and make it not be suitable for field quick detection and basic unit's popularization and application the higher technical requirements of testing staff.
Ring mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology (International Patent Publication No. WO 00/28082) is that Notomi in 2000 etc. develop a kind of nucleic acid amplification new technology, promptly at 4 special primers of 6 zone design of target gene (can also be added with ring primer to) if needed, utilize a kind of strand displacement archaeal dna polymerase (Bst DNA polymerase) to be incubated about 60 minutes at 65 ℃ of left and right sides constant temperatures, can finish nucleic acid amplification reaction, amplification can directly be judged by naked eyes amplification by product magnesium pyrophosphate precipitation or its turbidity be detected, also available fluorescence dye SYBR Green I dyeing in conjunction with double-stranded DNA is judged by naked eyes.2 pairs of primers that are used for LAMP technology amplification are at 6 sections of gene, thereby have the specificity higher than PCR, and possessing does not simultaneously need in thermal cycling, its unit time amplification efficiency higher and do not need advantage such as specific apparatus yet.But detection method and the detection kit of utilizing the ring mediated isothermal amplification method to detect Escherichia coli O 157: H7 are not arranged at present, and the specific rfbE gene fragment of Escherichia coli O 157: H7 is had specific LAMP primer sets report.
Summary of the invention
One object of the present invention is that provide a kind of to Escherichia coli O 157: H7 specific gene fragment has specific primer.
Above-mentioned purpose is achieved by the following technical solution:
A kind of Escherichia coli O 157: H7 detects and uses primer, it is characterized in that, the special base sequence of energy amplified target gene, described target gene is the rfbE---GenBank number of landing of Escherichia coli O 157: H7: AE005429, described primer and described target gene 7462--a part or its complementary strand complementation of-7671 nucleotide sequence.
Another object of the present invention is that provide a kind of to Escherichia coli O 157: H7 specific gene fragment has specific primer sets.
Above-mentioned purpose is achieved by the following technical solution:
A kind of Escherichia coli O 157: H7 detects and uses primer sets, it is characterized in that described primer sets is made up of following primer:
Forward outer primer F3-rfb GTACATTGGCATCGTGTG
Reverse outer primer B3-rfb CGAAAACGTGAAATTGCTGA
Forward inner primer FIP-rfb
GAGAGGAATTAAGGAATCACCTTGCGACAGGGTAAAAAACTGGC
Reverse inner primer BIP-rfb
TCTGCGGTCCTAGTTAGAATTGAAACAGTCTTGTACAAGTCCA
7462 of rfbE (AE005429) gene order of Escherichia coli O 157: H7 can increase--a part or its complementary strand of-7671 nucleotide sequence.
Another object of the present invention is, a kind of detection method based on ring mediated isothermal amplification method detection Escherichia coli O 157: H7 of utilizing above-mentioned primer sets is provided.
Above-mentioned purpose is achieved by the following technical solution:
The detection method of a kind of Escherichia coli O 157: H7, this method is used for detecting sample and whether has Escherichia coli O 157: H7 is characterized in that, with 7462 of rfbE (AE005429) gene order of Escherichia coli O 157: H7--a part or its complementary strand of-7671 nucleotide sequence are target, with above-mentioned primer or the above-mentioned target region of primer sets selective amplification, confirm whether to have amplified production by the LAMP method.
Concrete detection method is:
1) sample preparation and template extraction, the sample scope is applicable to samples such as food samples, ight soil, vomitus; Bacterium sample to be checked increases the bacterium cultivation after 8-12 hour with corresponding enteron aisle enrichment liquid, got 1.0ml enrichment liquid 10000rpm centrifugal 2 minutes, after abandoning its supernatant liquor, extract template DNA or add 20～30ul tri-distilled water with the DNA extraction test kit and boiled 5 minutes, get the 2ul supernatant liquor again and do template DNA to be checked;
2) ring mediated isothermal amplification of Escherichia coli O 157: H7 (LAMP)
Get amplification reaction solution, add template to be checked earlier, enzyme-added again, add distilled water at last, form the reaction system that following cumulative volume is 20ul～100ul, mixing point is incubated about 60-90 minute from back constant temperature under about 60-65 ℃ condition, places following 2 minutes inactivators of environment of 80 ℃ then.Reaction system be (reaction cumulative volume 20ul～100ul):
| Composition | Final concentration or amount | |
| Template to be checked | Nucleic acid-templated | 1-10ul |
| Amplification reaction solution | The reverse outer primer B3-rfb of the reverse inner primer BIP-rfb of forward inner primer FIP-rfb forward outer primer F3-rfb trimethyl-glycine betaine dNTP mgsO 4Reaction buffer Bst DNA Polymerase Buffer 10 * | 1.0-2.0uM 1.0-2.0uM 0.15-0.3uM 0.15-0.3uM 0.8-1.5M 1.0-1.6mM 2-6mM 1/10 reaction volume (ul) |
| Enzyme | Bst DNA Polymerase | 0.16-0.64U/ul |
| Distilled water | ddH 2O | Add to predetermined reaction volume (ul) |
3) detection of LAMP reaction product:
A) naked eyes detect: show relatively that with the negative control pipe detector tube occurs obviously muddy positive, do not see muddy negative; Or,
B) detect after adding dyestuff: the reaction tubes of every 25ul system adds 1000 * SYBR Green I (invitrogen) 1-2ul, 1-5 minute observations, and it is green positive that reaction solution turns, and keeps colourless or brown negative; Or,
C) electrophoresis detection: the 2-3% sepharose, the about 60-100 of 70V electrophoresis minute, the electrophoresis picture showed LAMP characteristic scalariform band, and minimal segment is about 160bp, and then the result is positive; As then the result is negative not have any band.
Another object of the present invention is, a kind of detection kit of utilizing above-mentioned primer or primer sets to detect Escherichia coli O 157: H7 based on the ring mediated isothermal amplification method is provided.
Above-mentioned purpose is achieved by the following technical solution:
A kind of Escherichia coli O 157: H7 detects and uses test kit, it is characterized in that described test kit comprises amplification reaction solution, the enzyme that contains above-mentioned primer or primer sets at least.
Comprise following reagent in the described amplification reaction solution:
| Composition | Final concentration or amount | |
| Amplification reaction solution | The reverse outer primer B3-rfb of the reverse inner primer BIP-rfb of forward inner primer FIP-rfb forward outer primer F3-rfb trimethyl-glycine betaine dNTP mgsO 4Reaction buffer Bst DNA Polymerase Buffer 10 * | 1.0-2.0uM 1.0-2.0uM 0.15-0.3uM 0.15-0.3uM 0.8-1.5M 1.0-1.6mM 2-6mM 1/10 predetermined reaction volume (ul) |
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-Hcl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH8.8;
Described enzyme is the Bst DNA enzyme that every microlitre contains 8 activity units.
Described test kit also comprises feminine gender and positive control template, and described negative control template is a distilled water; Described positive control template is an Escherichia coli O 157: H7 genomic dna (1～100nM).
The present invention is by providing a kind of to Escherichia coli O 157: H7 specific gene fragment has specific primer or primer sets, and with whether there being Escherichia coli O 157 in the test kit detection sample that includes above-mentioned primer sets: H7 specific gene fragment, and then whether have Escherichia coli O 157: H7 in definite sample.Detection reagent of the present invention and detection method have susceptibility height, high specificity, convenient and swift, do not need specific apparatus, advantage such as applied widely, can solve field quick detection and basic unit's popularization and application difficult problem of food-borne causal agent; Particularly on-the-spot detect and wartime field application.Simultaneously, compare with existing P CR method have high specificity, susceptibility than PCR high or quite, than PCR save time about 2 hours, do not need specific apparatus advantages such as (amplified reaction can be finished) in water-bath.
Description of drawings
Fig. 1 combination of primers of the present invention can be by macroscopic result after carrying out ring mediated isothermal amplification (LAMP).Legend: 1, positive amplification; 2, negative amplification.
Fig. 2 combination of primers of the present invention is carried out ring mediated isothermal amplification (LAMP) back and is added the result that dyestuff is observed.Legend: 1, negative amplification; 2,3, positive amplification.
The described combination of primers of Fig. 3 is carried out ring mediated isothermal amplification (LAMP) result's electrophorogram;
Among the figure, 1:100bp marker; 2: Escherichia coli O 157: H7; 3-13 is respectively streptococcus aureus, streptococcus aureus produces enterotoxin A bacterial strain, streptococcus aureus product enterotoxin B bacterial strain, streptococcus aureus product enterotoxin C bacterial strain, Salmonella typhimurium, salmonella typhi, Salmonella enteritidis, shigella dysenteriae, Vibrio parahemolyticus, Proteus mirabilis, vibrio cholerae, and above-mentioned bacterium is reference culture.
Embodiment
Below by concrete Escherichia coli O 157: the H7 testing process comes that the present invention will be described, but the present invention is not limited to these embodiment.
The amplification of the rfb gene of a pair of known bacterial strain of embodiment
One) design of primer sets
By consulting document and filtering out Escherichia coli O 157: the 7462---7671bp nucleotide sequence of H7 specific gene rfbE with the BLAST software analysis, design LAMP primer and synthetic at these segmental six sites (these six sites difference: 7462-7479bp, 7480-7498bp, 7520-7544bp, 7550-7572bp, 7613-7632bp, 7652-7671bp), obtain following primer; Design of primers is finished by LAMP primer special design software binding molecule biological analysis software Advance Vector NTI.
Sequence numbering 1
Forward outer primer F3-rfb GTACATTGGCATCGTGTG
Sequence numbering 2
Reverse outer primer B3-rfb CGAAAACGTGAAATTGCTGA
Sequence numbering 3
Forward inner primer FIP-rfb
GAGAGGAATTAAGGAATCACCTTGCGACAGGGTAAAAAACTGGC
Sequence numbering 4
Reverse inner primer BIP-rfb
TCTGCGGTCCTAGTTAGAATTGAAACAGTCTTGTACAAGTCCA
The sequence in described 6 sites is respectively:
7462-7479bp：gtacattggcatcgtgtg
7480-7498bp gacagggtaaaaaactggc
7520-7544bp g caaggtgattccttaattcc tctc
7550-7572bp t ctgcggtcctagttagaattga
7613-7632bp tggacttgtacaagactgtt
7652-7671bp tcagcaatttcacgttttcg
Wherein,
Described forward outer primer F3: amplification starts from 7462-7479bp (gtacattggcatcgtgtg);
Described forward inner primer FIP: amplification starts from complementary sequence and the 7480-7498bp (gacagggtaaaaaactggc) of 7520-7544bp (gcaaggtgattccttaattcctctc);
Described reverse outer primer B3: amplification starts from the complementary sequence of 7652-7671bp (tcagcaatttcacgttttcg);
Described reverse inner primer BIP: amplification starts from the complementary sequence of 7550-7572bp (tctgcggtcctagttagaattga) and 7613-7632 (tggacttgtacaagactgtt).
The general character of each primer is in the above-mentioned primer sets: with Escherichia coli O 157: the 7462--7671bp nucleic acid array complementation of H7 specific gene rfbE.
Two) bacterial strain of present embodiment experiment and the amplification such as the following table one of each bacterial strain
Table one (for the ease of understanding, the applicant with number among following sequence number and Fig. 1 be arranged to consistent)
| Sequence number | Molecular weight standard product and strains tested | Have or not amplification |
| 1 | 100bp marker | |
| 2 | Escherichia coli O 157: H7 (CMCC-44050-3) | Sun |
| 3 | Streptococcus aureus (CMCC-26003-25) | Cloudy |
| 4 | Streptococcus aureus produces enterotoxin A bacterial strain (ATCC-13565) | Cloudy |
| 5 | Streptococcus aureus produces enterotoxin B bacterial strain (ATCC-14458) | Cloudy |
| 6 | Streptococcus aureus produces enterotoxin C bacterial strain (ATCC-19095) | Cloudy |
| 7 | Salmonella enteritidis (ATCC-13076) | Cloudy |
| 8 | Salmonella typhimurtum (CMCC-50115) | Cloudy |
| 9 | Salmonella typhi (CMCC-50071) | Cloudy |
| 10 | Shigella dysenteriae (CMCC-51252) | Cloudy |
| 11 | Vibrio parahemolyticus (VPL 4-90) | Cloudy |
| 12 | Proteus mirabilis (CMCC-49005) | Cloudy |
| 13 | Vibrio cholerae (569B) | Cloudy |
Above-mentioned strains tested is reference culture, available from Chinese medicine microbial strains preservation administrative center.The no amplified reaction of " the moon " expression, " sun " expression has amplified reaction.
Three) extraction of above-mentioned each strain gene DNA
Bacterium sample to be checked increases the bacterium cultivation after 8-12 hour with corresponding enteron aisle enrichment liquid, got 1.0ml enrichment liquid 10000rpm centrifugal 2 minutes, after abandoning its supernatant liquor, extract template DNA or add the 30ul tri-distilled water with DNA extraction test kit (as TIANAampBacteria DNA Kit) and boiled 5 minutes, directly get the 2ul supernatant liquor and make template DNA; Streptococcus aureus sample to be checked needs to increase the bacterium cultivation with the enteron aisle enrichment liquid and extracts DNA with genome DNA extracting reagent kit (TIANAamp Bacteria DNA Kit) after 8-12 hour.
Four) based on the gene amplification of LAMP method
Get amplification reaction solution 14.6ul, add 2.0ul template to be checked, add the 1ul enzyme, add 7.4ul ddH again
2O, forming as the following table cumulative volume is the reaction system of 25ul, is that 65 ℃ thermostat water bath is incubated about 60 minutes in temperature, places 80 ℃, 2 minutes inactivators then.
Reaction system is: (the reaction cumulative volume is 25ul)
| Composition | Storage liquid concentration | Amount (ul) | Final concentration |
| Nucleic acid-templated FIP-rfb BIP-rfb F3-rfb B3-rfb betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 40uM 40uM 10uM 10uM 5M 10mM 150mM 10× 8U/ul | 2.0 1.0 1.0 0.5 0.5 5.0 3.5 0.6 2.5 1.0 7.4 | 1.6uM 1.6uM 0.2uM 0.2uM 1M 1.4mM 3.6mM 0.32U/ul |
Except that nucleic acid-templated, above-mentioned reaction system can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution: reverse outer primer B3-rfb, 1.4mM dNTP and the 1M trimethyl-glycine (betaine) of forward outer primer F3-rfb, 0.2uM that comprises 10 * Bst DNAPolymerase Buffer reaction buffer, 3.6mM sal epsom, 1.6uM forward inner primer FIP-rfb, the reverse inner primer BIP-rfb of 1.6uM, the 0.2uM of 1/10 predetermined reaction volume;
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-Hcl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH8.8;
Enzyme: BstDNA polysaccharase (every microlitre contains 8 activity units).
The adding principle of distilled water is after the amount of other reagent is determined, to add distilled water and make reaction volume arrive predetermined reaction volume.
Five) detection of amplified production
Along with the carrying out of amplified reaction, the magnesium ion that exists pyrophosphate ion of separating out from dNTP and the reaction solution will form the magnesium pyrophosphate precipitation.Therefore, only just white opacity can appear in the reaction solution that nucleic acid amplification takes place.Can judge in the following way like this.
A) naked eyes detect: show relatively that with the negative control pipe detector tube occurs obviously muddy positive, do not see muddy negative; (referring to Fig. 1) or,
B) detect after adding dyestuff: the reaction tubes of every 25ul system adds 1000 * SYBR Green I (invitrogen) 1-2ul, 1-5 minute observations, and it is green positive that reaction solution turns, and keeps colourless or brown negative; (referring to Fig. 2) or,
C) electrophoresis detection: the 2-3% sepharose, the about 60-100 of 70V electrophoresis minute, the electrophoresis picture showed LAMP characteristic scalariform band, and minimal segment is about 160bp, and then the result is positive; As then the result is negative not have any band.(referring to Fig. 3)
Detection through to amplified production shows that above-mentioned primer sets the LAMP amplified reaction occurs to Escherichia coli O 157: H7, and amplified reaction does not appear in the contrast bacterium, has shown good specificity.
6) remolding sensitivity is:
With Escherichia coli O 157: H7 as detecting bacterium, with 1 * 10
6The bacterium liquid of CFU/mL is done a series of dilutions: 10
6, 10
5, 10
4, 10
3, 10
2, 10
1, 10
0, use LAMP method and PCR method (the upstream and downstream primer is respectively F3-rfb, B3-rfb) to detect respectively, detected result shows that the LAMP reaction sensibility reaches 10
0CFU/mL; The PCR reaction sensibility reaches 10
1CFU/mL; The result shows that the LAMP reaction sensibility is than responsive 10 times of PCR reaction.
Embodiment two
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is:
Reaction system is: (the reaction cumulative volume is 25ul)
| Composition | Storage liquid concentration | Amount (ul) | Final concentration |
| Nucleic acid-templated FIP-rfb BIP-rfb F3-rfb B3-rfb betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 25uM 25uM 7.5uM 7.5uM 4M 10mM 100mM 10× 8U/ul | 1.0 1.0 1.0 0.5 0.5 5.0 2.5 0.5 2.5 0.5 10.0 | 1.0uM 1.0uM 0.15uM 0.15uM 0.8M 1.0mM 2.0mM 0.16U/ul |
Except that nucleic acid-templated, above-mentioned reaction system can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution: reverse outer primer B3-rfb, 1.0mM dNTP and the 0.8M trimethyl-glycine (betaine) of forward outer primer F3-rfb, 0.15uM that comprises 10 * Bst DNA Polymerase Buffer reaction buffer, 2mM sal epsom, 1.0uM forward inner primer FIP-rfb, the reverse inner primer BIP-rfb of 1.0uM, the 0.15uM of 1/10 predetermined reaction volume;
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-Hcl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH8.8;
Enzyme: Bst archaeal dna polymerase (every microlitre contains 8 activity units).
The adding principle of distilled water is after the amount of other reagent is determined, to add distilled water and make reaction volume arrive predetermined reaction volume.
Reaction conditions: in temperature is that 60 ℃ thermostat water bath is incubated about 60 minutes, places 80 ℃, 2 minutes inactivators then.
Embodiment three
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is:
Reaction system is: (the reaction cumulative volume is 25ul)
| Composition | Storage liquid concentration | Amount (ul) | Final concentration |
| Nucleic acid-templated FIP-rfb BIP-rfb F3-rfb B3-rfb betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 50uM 50uM 15uM 15uM 7.5M 10mM 150mM 10× 16U/ul | 1.0 1.0 1.0 0.5 0.5 5.0 4.0 1.0 2.5 1.0 7.5 | 2.0uM 2.0uM 0.3uM 0.3uM 1.5M 1.6mM 6.0mM 0.64U/ul |
Except that nucleic acid-templated, above-mentioned reaction system can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution A: reverse outer primer B3-rfb, 1.6mM dNTP and the 1.5M trimethyl-glycine (betaine) of forward outer primer F3-rfb, 0.3uM that comprises 10 * Bst DNA PolymeraseBuffer reaction buffer, 6mM sal epsom, 2.0uM forward inner primer FIP-rfb, the reverse inner primer BIP-rfb of 2.0uM, the 0.3uM of 1/10 predetermined reaction volume;
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid (Tris-Hcl), 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH8.8;
Enzyme: Bst archaeal dna polymerase (every microlitre contains 16 activity units).
The adding principle of distilled water is after the amount of other reagent is determined, to add distilled water and make reaction volume arrive predetermined reaction volume.
Reaction conditions: in temperature is that 65 ℃ thermostat water bath is incubated about 60 minutes, places 80 ℃, 2 minutes inactivators then.
Embodiment four
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is: (the reaction cumulative volume is 100ul)
| Composition | Storage liquid concentration | Amount (ul) | Final concentration |
| Nucleic acid-templated FIP-rfb BIP-rfb F3-rfb B3-rfb betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 40uM 40uM 10uM 10uM 5M 10mM 150mM 10× 8U/ul | 10.0 4.0 4.0 2.0 2.0 20.0 14.0 2.4 10.0 4.0 27.6 | 1.6uM 1.6uM 0.2uM 0.2uM 1M 1.4mM 3.6mM 0.32U/ul |
Embodiment five
The difference of present embodiment and embodiment one is that in the present embodiment, the reaction system of using based on the gene amplification of LAMP method is: (the reaction cumulative volume is 20ul)
| Composition | Storage liquid concentration | Amount (ul) | Final concentration |
| Nucleic acid-templated FIP-rfb BIP-rfb F3-rfb B3-rfb betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 40uM 40uM 10uM 10uM 5M 10mM 150mM 10× 8U/ul | 2.0 0.8 0.8 0.4 0.4 4.0 2.8 0.48 2.0 0.8 5.52 | 1.6uM 1.6uM 0.2uM 0.2uM 1M 1.4mM 3.6mM 0.32U/ul |
The detection of embodiment six strain separated from vomitus
The extraction of strain gene DNA and amplification, detection
Get food poisoning person's vomitus 25g, after increasing bacterium and cultivate 8-12 hour with the enteron aisle enrichment liquid, got 1.0ml enrichment liquid 10000rpm centrifugal 2 minutes, abandon its supernatant liquor after, extract template DNA or add 20～30ul tri-distilled water with the DNA extraction test kit and boiled 5 minutes, get the 2ul supernatant liquor again and do template DNA to be checked.With aforementioned enteron aisle enrichment culture medium inoculation separation and Culture flat board, bacterium colony is with carrying out the evaluation of strain isolated with biology-Mei Liai GNI+ card and VITEC 32 full automatic microorganism analysers in addition.
To the gene DNA of said extracted and embodiment one same pass through the detection that the LAMP method is carried out nucleic acid amplification and amplified production.Amplification and the comparison of VITEC qualification result are as following table two.
| The bacterial strain sequence number | Have or not amplification | Qualification result |
| Bacterial strain 1 | Have | Escherichia coli O 157: H7 |
| Bacterial strain 2 | Do not have | Colibacillus of excrement |
Evaluation based on VITEC 32 full automatic microorganism analysers
As shown in Table 2, qualification result shows that strain separated contains Escherichia coli O 157: the bacterial strain of H7 and colibacillus of excrement, qualification result based on VITEC 32 full automatic microorganism analysers is consistent with the result who uses the primer sets amplification, only is being accredited as Escherichia coli O 157: observe the amplification that above-mentioned primer sets produces in the sample of H7.
Sequence table
＜110〉Zhuhai Disease Prevention And Control Center
＜120〉Escherichia coli O 157: H7 detects with primer, detection method, detection kit
<160>5
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>1
GTACATTGGCATCGTGTG 18
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
CGAAAACGTGAAATTGCTGA 20
<210>3
<211>44
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
GAGAGGAATTAAGGAATCACCTTGCGACAGGGTAAAAAACTGGC 44
<210>4
<211>43
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
TCTGCGGTCCTAGTTAGAATTGAAACAGTCTTGTACAAGTCCA 43
<210>5
<211>210
<212>DNA
＜213〉Escherichia coli O 157: H7
<400>5
7462 gtacattgg catcgtgtgg acagggtaaa aaactggcct
7501 tgtttcgatg agtttatctg caaggtgatt ccttaattcc tctctttcct ctgcggtcct
7561 agttagaatt gagaccatcc aataagtgtg aaaaacatct ttactttcct tgtggacttg
7621 tacaagactg ttgatatttt ttttataaat atcagcaatt tcacgttttc g
Claims (10)
1. Escherichia coli O 157 a: H7 detects and uses primer, it is characterized in that, the special base sequence of energy amplified target gene, described target gene is the rfbE---GenBank number of landing of Escherichia coli O 157: H7: AE005429, described primer and described target gene 7462--a part or its complementary strand complementation of-7671 nucleotide sequence.
2. Escherichia coli O 157 a: H7 detects and uses primer sets, it is characterized in that described primer sets is made up of following primer:
Forward outer primer F3-rfb GTACATTGGCATCGTGTG
Reverse outer primer B3-rfb CGAAAACGTGAAATTGCTGA
Forward inner primer FIP-rfb
GAGAGGAATTAAGGAATCACCTTGCGACAGGGTAAAAAACTGGC
Reverse inner primer BIP-rfb
A part or its complementary strand of can increase 7462 of rfbE gene order of Escherichia coli O 157: H7 of TCTGCGGTCCTAGTTAGAATTGAAACAGTCTTGTACAAGTCCA---7671 nucleotide sequence.
3. Escherichia coli O 157 according to claim 2: H7 detects and to use primer sets, it is characterized in that described primer sets can increase following segment or the complementary strand of Escherichia coli O 157: H7:
Described forward outer primer F3: amplification starts from 7462-7479bp;
Described forward inner primer FIP: amplification starts from complementary sequence and the 7480-7498bp of 7520-7544bp;
Described reverse outer primer B3: amplification starts from the complementary sequence of 7652-7671bp;
Described reverse inner primer BIP: amplification starts from the complementary sequence of 7550-7572bp and 7613-7632.
4. the detection method of Escherichia coli O 157 a: H7, this method is used for detecting sample and whether has Escherichia coli O 157: H7, it is characterized in that, with 7462 of the rfbE gene order of Escherichia coli O 157: H7--a part or its complementary strand of-7671 nucleotide sequence are target, with above-mentioned primer or the above-mentioned target region of primer sets selective amplification, confirm whether to have amplified production by the LAMP method.
5. a kind of Escherichia coli O 157 according to claim 4: the H7 detection method is characterized in that concrete detection method is:
1) sample preparation and template extraction, the sample scope is applicable to samples such as food samples, ight soil, vomitus; Bacterium sample to be checked increases the bacterium cultivation after 8-12 hour with corresponding enteron aisle enrichment liquid, got 1.0ml enrichment liquid 10000rpm centrifugal 2 minutes, after abandoning its supernatant liquor, extract template DNA or add 20～30ul tri-distilled water with the DNA extraction test kit and boiled 5 minutes, get the 2ul supernatant liquor again and do template DNA to be checked;
2) ring mediated isothermal amplification of Escherichia coli O 157: H7
Get amplification reaction solution, add template to be checked earlier, enzyme-added again, add distilled water at last, form the reaction system that following cumulative volume is 20ul～100ul, mixing point is incubated about 60-90 minute from back constant temperature under about 60-65 ℃ condition, places following 2 minutes inactivators of environment of 80 ℃ then.
The reaction system of reaction cumulative volume 20ul～100ul:
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid, 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH 8.8;
Described enzyme is the Bst DNA enzyme that every microlitre contains 8-16 activity unit.
3) detection of LAMP reaction product:
A) naked eyes detect: show relatively that with the negative control pipe detector tube occurs obviously muddy positive, do not see muddy negative; Or,
B) detect after adding dyestuff: the reaction tubes of every 25ul system adds 1000 * SYBR Green I invitrogen1-2ul, 1-5 minute observations, and it is green positive that reaction solution turns, and keeps colourless or brown negative; Or,
C) electrophoresis detection: the 2-3% sepharose, the about 60-100 of 70V electrophoresis minute, the electrophoresis picture showed LAMP characteristic scalariform band, and minimal segment is about 160bp, and then the result is positive; As then the result is negative not have any band.
6. according to the detection method of right 5 described Escherichia coli O 157: H7, it is characterized in that when the reaction cumulative volume was 25ul, described reaction system was specially:
7. Escherichia coli O 157 a: H7 detects and uses test kit, it is characterized in that described test kit comprises amplification reaction solution, the enzyme that contains have the right requirement 1 described primer or the described primer sets of claim 2 at least.
8. a kind of Escherichia coli O 157 according to claim 7: H7 detects and uses test kit, it is characterized in that, comprises following reagent in the described amplification reaction solution:
Wherein 10 * Bst DNA Polymerase Buffer reaction buffer contains trihydroxy-methionine(Met) methane-hydrochloric acid, 100mM Repone K, 100mM ammonium sulfate, 20mM sal epsom and 1% triton x-100 of 200mM pH 8.8;
Described enzyme is that every microlitre contains 8-16 activity unit, the Bst DNA enzyme of final concentration 0.16-0.64U/ul.
9. a kind of Escherichia coli O 157 according to claim 8: H7 detects and uses test kit, it is characterized in that amplification reaction solution: reverse outer primer B3-rfb, 1.4mM dNTP and the 1M trimethyl-glycine of forward outer primer F3-rfb, 0.2uM that comprises 10 * Bst DNA PolymeraseBuffer reaction buffer, 3.6mM sal epsom, 1.6uM forward inner primer FIP-rfb, the reverse inner primer BIP-rfb of 1.6uM, the 0.2uM of 1/10 predetermined reaction volume;
Described enzyme is that every microlitre contains 8 activity units, the Bst DNA enzyme of final concentration 0.32U/ul.
According to Claim 8 or 9 described a kind of Escherichia coli O 157: H7 detect and to use test kit, it is characterized in that described test kit also comprises feminine gender and positive control template, described negative control template is a distilled water; Described positive control template is 1～100nM Escherichia coli O 157: the H7 genomic dna.
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| CN103866030B (en) * | 2014-04-02 | 2015-10-28 | 江苏省农业科学院 | The LAMP detection primer of Escherichia coli O 157: H7 and detection kit |
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