CN101153329A - Primer, detection method and detection reagent kit for detecting staphylococcus aureus - Google Patents
Primer, detection method and detection reagent kit for detecting staphylococcus aureus 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 staphylococcus aureus can augment the specific base sequence of a target gene which is the nuc-GenBank (accession no. V01281) of the staphylococcus aureus, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 548-795loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the staphylococcus aureus, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the staphylococcus aureus, determines whether the staphylococcus aureusexists 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 streptococcus aureus specific gene fragment is had specific primer sets; Also relate to the detection method and the detection kit that described primer sets are detected streptococcus aureus with the ring mediated isothermal amplification method.
Background technology
The food origin disease sickness rate is higher, by Salmonellas, Shigellae, streptococcus aureus, Bacillus proteus, vibrio cholerae, Vibrio parahemolyticus and E.coli O157: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 streptococcus aureus are not arranged at present, and the report that streptococcus aureus specific gene fragment is had specific LAMP primer and primer sets.
Summary of the invention
One object of the present invention is, provides a kind of streptococcus aureus specific gene fragment is had specific primer.
Above-mentioned purpose is achieved by the following technical solution:
A kind of streptococcus aureus detects uses primer, it is characterized in that, the special base sequence of energy amplified target gene, described target gene is the nuc---GenBank number of landing of streptococcus aureus: V01281, described primer and described target gene 548--a part or its complementary strand complementation of-795 nucleotide sequence.
Another object of the present invention is, provides a kind of streptococcus aureus specific gene fragment is had specific primer sets.
Above-mentioned purpose is achieved by the following technical solution:
A kind of streptococcus aureus detects and uses primer sets, it is characterized in that described primer sets is made up of following primer:
Forward outer primer F3-nuc GTCAACCAATGACATTCAGAC
Reverse outer primer B3-nuc AACTTTAGCCAAGCCTTGA
Forward inner primer FIP-nuc ATGCACTTGCTTCAGGACCACACCTGAAACAAAGCATCC
Reverse inner primer BIP-nuc
GAAGTCGAGTTTGACAAAGGTCAAACGTTTACCATTTTTCCATCAGC
548 of nuc (V01281) gene order of streptococcus aureus can increase--a part or its complementary strand of-795 nucleotide sequence.
Another object of the present invention is, a kind of detection method of utilizing above-mentioned primer sets to detect streptococcus aureus based on the ring mediated isothermal amplification method is provided.
Above-mentioned purpose is achieved by the following technical solution:
The detection method of a kind of streptococcus aureus, this method is used for detecting sample and whether has streptococcus aureus, it is characterized in that, with 548 of nuc (V01281) gene order of streptococcus aureus--a part or its complementary strand of-795 nucleotide sequence are target, with the above-mentioned target region of above-mentioned 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 streptococcus aureus (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-nuc of the reverse inner primer BIP-nuc of forward inner primer FIP-nuc forward outer primer F3-nuc 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- |
| 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 190bp, 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 streptococcus aureus based on the ring mediated isothermal amplification method is provided.
Above-mentioned purpose is achieved by the following technical solution:
A kind of streptococcus aureus 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-nuc of the reverse inner primer BIP-nuc of forward inner primer FIP-nuc forward outer primer F3-nuc 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- |
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 8-16 activity unit.
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 staphylococcus aureus gene group DNA (1～100nM).
The present invention has specific primer sets by providing a kind of to streptococcus aureus specific gene fragment, and detect in the sample whether have streptococcus aureus specific gene fragment, and then whether there is streptococcus aureus in definite sample with the test kit that includes above-mentioned primer sets.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 primer sets 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 primer sets 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.
Fig. 3 primer sets of the present invention is carried out ring mediated isothermal amplification (LAMP) result's electrophorogram:
Among the figure, 1:100bp marker; 2: streptococcus aureus; 3, streptococcus aureus produces the enterotoxin A bacterial strain; 4-8 is respectively Salmonella typhimurium, salmonella typhi, Salmonella enteritidis, shigella dysenteriae and negative control, and above-mentioned bacterium is the reference culture bacterium.
Fig. 4 combination of primers of the present invention is carried out ring mediated isothermal amplification (LAMP) result's electrophorogram:
1:100bp marker among the figure; 2: streptococcus aureus produces the enterotoxin B bacterial strain; 3, streptococcus aureus produces enterotoxin C bacterial strain; 4-8 is respectively Escherichia coli O 157: H7, Bacillus proteus, Vibrio parahaemolyticus, vibrio cholerae, negative control, above-mentioned bacterium is reference culture.
Embodiment
The present invention will be described below by concrete streptococcus aureus testing process, but the present invention is not limited to these embodiment.
The amplification of the nuc gene of a pair of known bacterial strain of embodiment
One) design of primer sets
By consulting document and filtering out the 548---795bp nucleotide sequence of staphylococcus aureus specific gene nuc with the BLAST software analysis, design LAMP primer and synthetic at these segmental six sites (these six sites difference: 548-568bp, 584-602bp, 624-643bp, 679-703bp, 742-763bp, 777-795bp), 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-nuc GTCAACCAATGACATTCAGAC
Reverse outer primer B3-nuc AACTTTAGCCAAGCCTTGA
Forward inner primer FIP-nuc
ATGCACTTGCTTCAGGACCACACCTGAAACAAAGCATCC
Reverse inner primer BIP-nuc
GAAGTCGAGTTTGACAAAGGTCAAACGTTTACCATTTTTCCATCAGC
The sequence in described 6 sites is respectively:
548-568bp：gtcaaccaatgacattcagac
584-602bp：cacctgaaacaaagcatcc
624-643bp tggtcctgaagcaagtg cat
679-703bp ga agtcgagtttgacaaaggtcaaa
742-763bp gctgatggaaaaatggtaaacg
777-795bp tcaaggcttggctaaagtt
Wherein,
Described forward outer primer F3: amplification starts from 548-568bp (gtcaaccaatgacattcagac);
Described forward inner primer FIP: amplification starts from complementary sequence and the 584-602bp (cacctgaaacaaagcatcc) of 624-643bp (tggtcctgaagcaagtgcat);
Described reverse outer primer B3: amplification starts from the complementary sequence of 777-795bp (tcaaggcttggctaaagtt);
Reverse inner primer BIP: amplification starts from the complementary sequence of 679-703bp (gaagtcgagtttgacaaaggtcaaa) and 742-763bp (gctgatggaaaaatggtaaacg).
The general character of each primer is in the above-mentioned primer sets: with the 548---795bp nucleic acid array complementation of staphylococcus aureus specific gene nuc.
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. 3 be arranged to consistent)
| Sequence number | Molecular weight standard product and strains tested | Have or not |
| 1 | |
|
| 2 | Streptococcus aureus type strain (CMCC-26003-25) | |
| 3 | Streptococcus aureus produces enterotoxin A bacterial strain (ATCC-13565) | |
| 4 | Salmonella enteritidis (ATCC-13076) | Cloudy |
| 5 | Salmonella typhimurtum (CMCC-50115) | Cloudy |
| 6 | Salmonella typhi (CMCC-50071) | Cloudy |
| 7 | Shigella dysenteriae (CMCC-51252) | Cloudy |
| 8 | Negative control | Cloudy |
Table two (for the ease of understanding, the applicant with number among following sequence number and Fig. 4 be arranged to consistent)
| 1 | |
|
| 2 | Streptococcus aureus produces enterotoxin B bacterial strain (ATCC-14458) | Sun |
| 3 | Streptococcus aureus produces enterotoxin C bacterial strain (ATCC-19095) | |
| 4 | Escherichia coli O 157: H7 (CMCC-44050-3) | |
| 5 | Vibrio parahemolyticus (VPL 4-90) | |
| 6 | Proteus mirabilis (CMCC-49005) | Cloudy |
| 7 | Vibrio cholerae (569B) | Cloudy |
| 8 | Negative control | 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 (except that streptococcus aureus) 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 TIANamp Bacteria DNA Kit) and boiled 5 minutes, directly get the 2ul supernatant liquor and make template DNA; The streptococcus aureus sample needs to increase the bacterium cultivation with the enteron aisle enrichment liquid and extracts DNA with genome DNA extracting reagent kit (TIANampBacteria 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-nuc BIP-nuc F3-nuc | 40uM 40uM 10uM | 2.0 1.0 1.0 0.5 | 1.6uM 1.6uM 0.2uM |
| B3-nuc betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 10uM 5M 10mM 150mM 10× 8U/ul | 0.5 5.0 3.5 0.6 2.5 1.0 7.4 | 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-nuc, the 1.4mM dNTP and the 1M trimethyl-glycine (betaine) that comprise forward outer primer F3-nuc, the 0.2uM of 10 * Bst DNA Polymerase Buffer reaction buffer, 3.6mM sal epsom, 1.6uM forward inner primer FIP-nuc, the reverse inner primer BIP-nuc of 1.6uM, 0.2uM
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.
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 190bp, and then the result is positive; As then the result is negative not have any band.(referring to Fig. 3,4)
Through detection, show that above-mentioned primer sets is to streptococcus aureus, streptococcus aureus product enterotoxin A bacterial strain to amplified production; Streptococcus aureus produces the enterotoxin B bacterial strain; Streptococcus aureus produces enterotoxin C bacterial strain and the LAMP amplified reaction occurs, and amplified reaction does not appear in the contrast bacterium, has shown good specificity.
6) remolding sensitivity is:
With golden yellow grape ball standard bacterium 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-nuc, B3-nuc) to detect respectively, detected result shows that the LAMP reaction sensibility reaches 10
1CFU/mL; The PCR reaction sensibility reaches 10
2CFU/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-nuc BIP-nuc F3-nuc B3-nuc betaine dNTP | 25uM 25uM 7.5uM 7.5uM 4M 10mM | 1.0 1.0 1.0 0.5 0.5 5.0 2.5 | 1.0uM 1.0uM 0.15uM 0.15uM 0.8M 1.0mM |
| mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 100mM 10× 8U/ul | 0.5 2.5 0.5 10.0 | 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-nuc, the 1.0mM dNTP and the 0.8M trimethyl-glycine (betaine) that comprise forward outer primer F3-nuc, the 0.15uM of 10 * Bst DNA Polymerase Buffer reaction buffer, 2mM sal epsom, 1.0uM forward inner primer FIP-nuc, the reverse inner primer BIP-nuc of 1.0uM, 0.15uM;
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 65 ℃ 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-nuc BIP-nuc | 50uM 50uM | 1.0 1.0 1.0 | 2.0uM 2.0uM |
| F3-nuc B3-nuc betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 15uM 15uM 7.5M 10mM 150mM 10× 16U/ul | 0.5 0.5 5.0 4.0 1.0 2.5 1.0 7.5 | 0.3uM 0.3uM 1.5M 1.6mM 6.0mM 0.64U/ul |
Except that nucleic acid-templated, above-mentioned anti-0 system of answering can be reduced to amplification reaction solution, enzyme and distilled water.
Amplification reaction solution A: reverse outer primer B3-nuc, the 1.6mM dNTP and the 1.5M trimethyl-glycine (betaine) that comprise forward outer primer F3-nuc, the 0.3uM of 10 * Bst DNA Polymerase Buffer reaction buffer, 6mM sal epsom, 2.0uM forward inner primer FIP-nuc, the reverse inner primer BIP-nuc of 2.0uM, 0.3uM;
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 16U activity unit).
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 | 10.0 |
| FIP-nuc BIP-nuc F3-nuc B3-nuc betaine dNTP mgsO 4 Bst DNA Polymerase Buffer Bst DNA Polymerase ddH 2O | 40uM 40uM 10uM 10uM 5M 10mM 150mM 10× 8U/ul | 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-nuc BIP-nuc F3-nuc B3-nuc 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, increase the bacterium cultivation after 8-12 hour, extract DNA with genome DNA extracting reagent kit (TIANamp Bacteria DNA Kit) with corresponding enteron aisle enrichment liquid.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 VITEC32 full automatic microorganism analyser in addition.
The gene DNA of said extracted and the embodiment one same LAMP method of passing through is carried out nucleic acid amplification and amplified production detects.Amplification and the comparison of VITEC qualification result are as following table two.
| The bacterial strain sequence number | Have or not amplification | Qualification result |
| |
Have | Streptococcus aureus |
| |
Do not have | Intestinal bacteria |
Evaluation based on VITEC 32 full automatic microorganism analysers
As shown in Table 2, qualification result shows that strain separated contains streptococcus aureus and intestinal bacteria, qualification result based on VITEC 32 full automatic microorganism analysers is consistent with the result who uses the primer sets amplification, only observes the amplification that above-mentioned primer sets produces in being accredited as the sample of streptococcus aureus.
Sequence table
＜110〉Zhuhai Disease Prevention And Control Center
＜120〉streptococcus aureus detects with primer, detection method, detection kit
<160>5
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>1
GTCAACCAATGACATTCAGAC 21
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>2
AACTTTAGCCAAGCCTTGA 19
<210>3
<211>38
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>3
ATGCACTTGCTTCAGGACCACACCTGAAACAAAGCATCC 39
<210>4
<211>47
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
GAAGTCGAGTTTGACAAAGGTCAAACGTTTACCATTTTTCCATCAGC 47
<210>5
<211>248
<212>DNA
＜213〉streptococcus aureus
<400>5
548 gtc aaccaatgac attcagacta ttattggttg atacacctga aacaaagcat
601 cctaaaaaag gtgtagagaa atatggtcct gaagcaagtg catttacgaa aaaaatggta
661 gaaaatgcaa agaaaattga agtcgagttt gacaaaggtc aaagaactga taaatatgga
721 cgtggcttag cgtatattta tgctgatgga aaaatggtaa acgaagcttt agttcgtcaa
781 ggcttggcta aagtt
Claims (10)
1. a streptococcus aureus detects and uses primer, it is characterized in that, the special base sequence of energy amplified target gene, described target gene is the nuc---GenBank number of landing of streptococcus aureus: V01281, described primer and described target gene 548--a part or its complementary strand complementation of-795 nucleotide sequence.
2. a streptococcus aureus detects and uses primer sets, it is characterized in that described primer sets is made up of following primer:
Forward outer primer F3-nuc GTCAACCAATGACATTCAGAC
Reverse outer primer B3-nuc AACTTTAGCCAAGCCTTGA
Forward inner primer FIP-nuc ATGCACTTGCTTCAGGACCACACCTGAAACAAAGCATCC
Reverse inner primer BIP-nuc
GAAGTCGAGTTTGACAAAGGTCAAACGTTTACCATTTTTCCATCAGC
548 of nuc gene order of streptococcus aureus can increase--a part or its complementary strand of-795 nucleotide sequence.
3. a kind of streptococcus aureus according to claim 2 detects and uses primer sets, it is characterized in that described primer sets can increase following segment or the complementary strand of streptococcus aureus:
Described forward outer primer F3: amplification starts from 548-568bp;
Described forward inner primer FIP: amplification starts from complementary sequence and the 584-602bp of 624-643bp;
Described reverse outer primer B3: amplification starts from the complementary sequence of 777-795bp;
Reverse inner primer BIP: amplification starts from the complementary sequence of 679-703bp and 742-763bp.
4. the detection method of a streptococcus aureus, this method is used for detecting sample and whether has streptococcus aureus, it is characterized in that, with 548 of the nuc gene order of streptococcus aureus--a part or its complementary strand of-795 nucleotide sequence are target, with the above-mentioned target region of above-mentioned primer sets selective amplification, confirm whether to have amplified production by the LAMP method.
5. according to the detection method of right 4 described streptococcus aureuses, it is characterized in that described detection method is specially:
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 streptococcus aureus
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 190bp, 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 streptococcus aureuses, it is characterized in that when the reaction cumulative volume was 25ul, described reaction system was specially:
7. a streptococcus aureus detects and uses test kit, it is characterized in that described test kit comprises amplification reaction solution, the enzyme that contains described primer of claim 1 or the described primer sets of claim 2 at least.
8. streptococcus aureus according to claim 7 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. streptococcus aureus according to claim 8 detects and uses test kit, it is characterized in that,
Amplification reaction solution: reverse outer primer B3-nuc, 1.4mM dNTP and the 1M trimethyl-glycine of forward outer primer F3-nuc, 0.2uM that comprises 10 * Bst DNAPolymerase Buffer reaction buffer, 3.6mM sal epsom, 1.6uM forward inner primer FIP-nuc, the reverse inner primer BIP-nuc of 1.6uM, the 0.2uM of 1/10 predetermined reaction volume;
Enzyme: enzyme is that every microlitre contains 8 activity units, the Bst DNA enzyme of final concentration 0.32U/ul.
10. use test kit according to claim 7 or 8 or 9 described streptococcus aureuses detections, it is characterized in that described test kit also comprises feminine gender and positive control template, described negative control template is a distilled water; The staphylococcus aureus gene group DNA that described positive control template is 1～100nM.
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| CN1161060A (en) * | 1994-09-12 | 1997-10-01 | M·G·伯杰龙 | Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis Lab. |
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