CN106434887A - Method, primers and kit for rapid constant-temperature detection of staphylococcus aureus - Google Patents
Method, primers and kit for rapid constant-temperature detection of staphylococcus aureus Download PDFInfo
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Abstract
The invention discloses a method, primer set and kit for rapid constant-temperature detection of staphylococcus aureus. The method comprises the steps: extracting a genomic DNA from a to-be-tested sample; with the genomic DNA as a template, taking a primer set capable of amplifying a staphylococcus aureus specific sequence as primers, and carrying out constant-temperature amplification reaction in an enzyme reaction system; and by judging whether a reaction result is positive, determining whether the staphylococcus aureus exists in the to-be-tested sample. The detection method has the advantages of high sensitivity, high specificity, short detection time, simple judgment result, convenience in operation, low cost and wide application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of fast constant temperature detection staphylococcus aureus method,
Primer and kit.
Background technology
Staphylococcus aureus (Staphylococcus aureus) is under the jurisdiction of staphylococcus, and thalline is spherical, is a kind of
Gram-positive bacterium.Staphylococcus aureus is modal pathogen in mankind's suppurative infection, local can be caused to suppurate and feel
Dye, it is possible to cause pneumonia, pseudomembranous enteritis, pericarditis etc., the even general infection such as septicemia, pyemia.It produces simultaneously
Enterotoxin can contaminated food and cause food poisoning, bring very serious public health to bear for the mankind.Therefore, for this bacterium
Prevent and detect extremely important.
At present, the commonly used classical culture protocols of detection to staphylococcus aureus in the world, but the detection cycle is longer,
Operating relative complex, detection efficiency is relatively low, it is difficult to meet modern society for food-borne pathogens detection process high flux, Gao Ling
Sensitivity, high specific, quick, require easily.Recently as the development of nucleic acid molecules detection technique, researcher opens successively
Have issued the detection means of PCR and Fluorescence PCR assay, but both approaches needs special detecting instrument, therefore, and uncomfortable
Close the real-time on-site detection being widely used in carrying out inside detection department of basic unit especially enterprise's production line.For guaranteeing that food is pacified
Entirely, be badly in need of quick, simple, accurately method detect the staphylococcus aureus in food.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is in recent years
A kind of novel constant-temperature nucleic acid amplification method growing up, this method is for 4 specific primers of 6 region designs of target sequence
(including upstream and downstream outer primer F3 and B3 and upstream and downstream inner primer FIP and BIP, wherein FIP is made up of F1C and F2, and BIP is by B1C
With B2 composition), utilize a kind of archaeal dna polymerase with strand-displacement activity, be incubated about 60min at constant temperature, core can be completed
Acid amplified reaction, produces macroscopic byproduct of reaction-white magnesium pyrophosphate and precipitates (see document Notomi T, Okayama
H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T.Loop-mediated isothermal
amplification of DNA,Nucleic Acids Research,2000Jun 15；28(12):E63).This technology has
Not needing PCR instrument or quantitative real time PCR Instrument, can completing under constant temperature, naked eyes i.e. can determine whether reaction result, and highly sensitive,
High specificity, reaction time is short, simple operation, low cost and other advantages.
Design of primers is a step the most key in LAMP technology, and Normal practice is by the spy generally acknowledging of certain biology to be detected
Specific gene imports the online website (http of LAMP primer design://primerexplorer.jp/e), set relevant parameter raw
Become primer sets.It is to say, user is it is first necessary to guarantee the distinguished sequence that this target gene is species to be measured.With patent of invention CN
As a example by 101701252 B and CN 101880711 A, they are respectively directed to the special base of the staphylococcus aureus of document report
Because of nuc gene and clfA gene, LAMP technology is used to carry out staphylococcus aureus detection.But, the so-called " spy generally acknowledging
Specific gene " is often based upon delayed knowledge, and is not based on the renewal that ever-increasing microbial genome data carry out necessity,
The primer obtaining based on this target-gene sequence is caused not necessarily to can ensure that its versatility and/or specific in actual applications.This
Invention table 1 illustrates the problem that versatility present in prior art cannot ensure.It is to say, institute in art methods
The staphylococcus aureus detection sequence actually not staphylococcus aureus using is common, i.e. be possible to missing inspection golden yellow
Look staphylococcic part bacterial strain.Similar problem exists in specific confirmation, i.e. be possible to non-Staphylococcus aureus
Bacterium regards as staphylococcus aureus mistakenly.Therefore, need badly in industry and a kind of be able to ensure that the golden yellow of specific and versatility
Look staphylococcus detection method, meets detection department of basic unit to quick, demand easily simultaneously, can produce in enterprise easily
Carry out real-time on-site detection inside line.
Content of the invention
The technical problem to be solved in the present invention is to overcome primer versatility present in existing LAMP technology design of primers
With specific not enough defect, make full use of microbial genome sequence information abundant in current common data resource and phase
The sequence analysis tools answered, is designed for the primer sets of specific recognition staphylococcus aureus, and forms height on this basis
Sensitivity, high specific detection kit.The present invention (cuts based on the microbial genome data resource in GenBank database
To data on August 5th, 2013) carry out the design of staphylococcus aureus LAMP primer, provide a kind of fast constant temperature amplification inspection
Survey method, primer sets and the kit of staphylococcus aureus.Use the detection method detection staphylococcus aureus of the present invention,
Having high sensitivity and high specific, the detection time is short, and result judges simple, simple operation, the advantage of low cost.
The present invention proposes a kind of method of fast detecting Staphylococcus aureus bacterial strain, said method comprising the steps of：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that staphylococcus aureus gene group-specific base sequence can be expanded
Primer sets is primer, under enzyme reaction system, carries out isothermal amplification reactions；
(3) by judging whether reaction result is positive, determine in testing sample whether there is staphylococcus aureus.
The method of Constant Temperature Detection staphylococcus aureus strains of the present invention, extracts genomic DNA, with it from testing sample
For template, with staphylococcus aureus specific amplimer group as primer, carry out isothermal amplification reactions, then, by judging
Whether reaction result is positive, determines in testing sample whether there is staphylococcus aureus.Wherein, described enzyme reaction system bag
Include but be not limited to DNA polymerase reaction system.
In the present invention, described staphylococcus aureus gene group-specific base sequence is the gold that No. GI is 148266447
2645869～2646117bp bit sequence of staphylococcus aureus.
In the present invention, the described primer sets that can expand staphylococcus aureus gene group-specific base sequence is described base
Because of the part of nucleotide sequence of 2645869～2646117bp position of group (No. GI is 148266447) or its complementary strand one
Point.Wherein, described staphylococcus aureus gene group-specific base sequence refers to only staphylococcus aureus gene group institute
Distinctive, and the base sequence that other microbial genome do not comprise.
Wherein, the described primer sets that can expand staphylococcus aureus gene group specific base sequence is including but not limited to drawn
Thing group A, or selected from being 68% and above primer sets with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence
Any one group.
Primer sets A：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’(SEQ ID
NO：3)；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’(SEQ
ID NO：4).
In the present invention, the described primer sets that can expand staphylococcus aureus gene group specific base sequence can also include
It is 68% and above primer sets with wall scroll sequence homology in aforementioned each primer sets sequence or its complementary strand sequence, this primer sets
Including but not limited to following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-ATGACTAAAGCCACATCCA-3’(SEQ ID NO：5) (with primers F 3_A 5 '-
CTAAAGCCACATCCAATATAGG-3 ' homology 68%)；
Downstream outer primer B3_B：5’-ATGCCTTACATTGATGCTG-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’(SEQ ID
NO：7)；
Downstream inner primer BIP_B：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’(SEQ
ID NO：8).
In the inventive method, the described primer sets that can expand staphylococcus aureus gene group-specific base sequence is permissible
Comprise or do not comprise ring primer.Described ring primer can be one or more, including primer LF and/or LB.Described energy amplification gold
The primer sets of staphylococcus aureus genome specificity base sequence is primer sets A '；Or selected from and described primer sets A ' sequence or
In its complementary strand sequence, wall scroll sequence homology is 68% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’；
Upper lantern primer LF_A：5’-TCCCTTTAATTAAGTAAACCC-3’(SEQ ID NO：9)；
And/or, lower lantern primer LB_A：5’-TAAGAACTAGTTAGTGACTA-3’(SEQ ID NO：10).
In a particular embodiment, for example, above-mentioned primer sets A ' in, can only comprise a upper lantern primer, or only wrap
Containing a lower lantern primer, or comprise a upper lantern primer and a lower lantern primer simultaneously.
In the inventive method, in a specific embodiments (primer containing ring), the enzyme reaction system of described constant-temperature amplification is：
1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L dNTP,
FIP and the BIP primer of 0.8-2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, the LF of 0.4-1.0 μm of ol/L and/or
LB primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.(do not contain in another specific embodiments
Ring primer) in, the enzyme reaction system of described constant-temperature amplification is：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+
(MgSO4Or MgCl2), FIP and the BIP primer of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L, 0.15-0.3 μm of ol/L's
F3 and B3 primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.Ring primer is favorably improved reaction
Efficiency.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer, comprises
20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%Triton X-100,
2mM MgSO4.MgSO in 1 × Bst DNA polymerase reaction buffer solution4With the magnesium ion Mg in enzyme reaction system2+Do and merge
Process.
In the inventive method, the response procedures of described isothermal amplification reactions hatches 10～90min, preferably for 1. 60～65 DEG C
Ground is 10～60min；2. 80 DEG C terminate reaction 2～20min.The present invention does not limit and realizes this by other suitable response procedures
Invention detection method.
In the inventive method, detection method includes but is not limited to electrophoresis detection, Turbidity measurement or color developing detection etc..Described electricity
Swimming detection, preferably gel electrophoresis assays, can be Ago-Gel, it is also possible to be polyacrylamide gel.Electrophoresis detection
In result, band as stepped in electrophoretogram expression characteristics, then testing sample is S. aureus-positive, containing golden yellow
Staphylococcus；Band as stepped in electrophoretogram not expression characteristics, then testing sample is that staphylococcus aureus is negative.Described turbid
Degree detection, is to detect by an unaided eye or transmissometer detection turbidity, and detection pipe occurs substantially muddy, then testing sample is in golden yellow grape
Coccus is positive, containing staphylococcus aureus；As muddy in having no, then testing sample is that staphylococcus aureus is negative.Also permissible
Visually observing whether have precipitation at the bottom of reaction tube after centrifugal, if there being precipitation at the bottom of reaction tube, then testing sample is Staphylococcus aureus
Bacterium is positive, containing staphylococcus aureus；Do not precipitate as at the bottom of reaction tube, then testing sample is that staphylococcus aureus is negative.
Described color developing detection, is addition developer, including but not limited to calcein (50 μM) or SYBR in reaction tube
Green I (30-50 ×), or hydroxynaphthol blue (i.e. HNB, 120-150 μM).Make when using calcein or SYBR Green I
During for developer, as after reaction, color is orange, then testing sample is that staphylococcus aureus is negative；As after reaction, color is green
Look, then testing sample is S. aureus-positive, containing staphylococcus aureus.When employing hydroxynaphthol blue is as colour developing
During agent, as after reaction, color is pansy, then testing sample is that staphylococcus aureus is negative；As after reaction, color is sky blue
Look, then testing sample is S. aureus-positive.Described color developing detection, except above by visually observing reaction result
Outward, it is also possible to carry out real-time or end point determination reaction result by detecting instrument, by the rational threshold value setting negative reaction,
When the result of testing sample reaction is less than or equal to this threshold value, then testing sample is that staphylococcus aureus is negative；When to be measured
When the result of example reaction is more than this threshold value, then testing sample is S. aureus-positive.Described detecting instrument include but
It is not limited to sepectrophotofluorometer, quantitative real time PCR Instrument, constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument and Genie II isothermal
Amplification fluorescent detecting system etc..
In described color developing detection, according to calcein or hydroxynaphthol blue as developer, can be anti-at constant-temperature amplification
Added before should, it is also possible to add after isothermal amplification reactions completes, it is therefore preferable to add before isothermal amplification reactions, permissible
The effective possibility reducing reaction pollution.According to SYBR Green I as developer, then complete in isothermal amplification reactions
Add afterwards.According to calcein as developer, then while adding 50 μM of calceins in enzyme reaction system, add
0.6-1mM[Mn2+], for example, the MnCl of 0.6-1mM2.
Present invention also offers for the primer in the method for Constant Temperature Detection staphylococcus aureus strains.Described primer bag
Including the primer sets that can expand staphylococcus aureus gene group specific base sequence, it includes but is not limited to, the sequence of described primer
Be classified as 2645869～2646117bp position of the staphylococcus aureus gene group that No. GI is 148266447 nucleotide sequence one
Part or a part for its complementary strand.
Wherein, the described primer sets that can expand staphylococcus aureus gene group-specific base sequence is drawn selected from following
Any one group of thing group, or selected from wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence be 68% and
Above arbitrary primer sets.Wherein, described primer sets includes but is not limited to primer sets A.Described with aforementioned primer sets sequence or its
In complementary strand sequence, wall scroll sequence homology is 68% and above primer sets includes but is not limited to following primer sets B.
Primer sets A：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’；
Primer sets B：
Upstream outer primer F3_B：5’-ATGACTAAAGCCACATCCA-3’；
Downstream outer primer B3_B：5’-ATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_B：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_B：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’.
The present invention is in the primer in described Constant Temperature Detection staphylococcus aureus method, and described energy expands golden yellow Portugal
The primer sets of grape coccus genome specificity base sequence can also comprise or not comprise one or more ring primer；Described ring draws
Thing is LF and/or LB.The described primer sets that can expand staphylococcus aureus gene group-specific base sequence is primer sets A '；
Or selected from and described primer sets A ' in sequence or its complementary strand sequence wall scroll sequence homology be 68% and above primer sets it
Any one group：
Primer sets A '：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’；
Upper lantern primer LF_A：5’-TCCCTTTAATTAAGTAAACCC-3’；
And/or, lower lantern primer LB_A：5’-TAAGAACTAGTTAGTGACTA-3’.
In a particular embodiment, above-mentioned primer sets A ' in, can only comprise a upper lantern primer, or only comprise one
Lower lantern primer, or comprise a upper lantern primer and a lower lantern primer simultaneously.In one embodiment, described draw
Thing is respectively the primer shown in FIP, BIP, F3, B3, LF and LB or draws with wall scroll in aforementioned primer sequence or its complementary strand sequence
Thing homology is 68% and above primer.
The present invention also provides a kind of for the kit in above-mentioned Constant Temperature Detection staphylococcus aureus strains method, its bag
Include the described primer sets that can expand staphylococcus aureus gene group specific base sequence.In kit of the present invention, described energy expands
Increase the primer sets of staphylococcus aureus gene group-specific base sequence, including but not limited to genome (No. GI:
148266447) part for nucleotide sequence for 2645869～2646117bp position or a part for its complementary strand are as described
Primer sequence；Described primer includes but is not limited to described primer sets A.Also including but not limited to with aforementioned primer sequence or it is mutual
In benefit chain-ordering, wall scroll sequence homology is 68% and above primer sets is as primer；Including but not limited to primer sets B.
In kit of the present invention, the described primer sets that can expand staphylococcus aureus gene group-specific base sequence can
To comprise or not comprise one or more ring primer；Ring primer is as optional component.Described ring primer is LF and/or LB.Comprise
The primer sets of ring primer LF and/or LB includes but is not limited to primer sets A '.In a particular embodiment, can in kit of the present invention
To comprise LF and/or the LB ring primer of 0.4-1.0 μm of ol/L.In one embodiment, the sequence of primer sets is respectively
Primer shown in FIP, BIP, F3, B3, LF and LB or be 68% with foregoing sequences or its complementary strand sequence wall scroll primer homology
And above primer.
In kit of the present invention, also include Bst DNA polymerase buffer liquid, Bst archaeal dna polymerase, dNTP solution, Mg2+
(MgSO4Or MgCl2) and glycine betaine in one or more.In one embodiment, kit enzyme reaction system of the present invention
Comprise 1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L
FIP and the BIP primer of dNTP, 0.8-2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst
Archaeal dna polymerase and the glycine betaine of 0-1.5mol/L.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 ×
Thermopol reaction buffer, comprises 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%Triton X-100,2mM MgSO4.MgSO in 1 × Bst DNA polymerase reaction buffer solution4With enzyme reaction body
Magnesium ion Mg in system2+Do merging treatment.
In kit of the present invention, also comprise positive control template.In one embodiment, described positive control template
Include but is not limited to complete genome DNA, portion gene group DNA of staphylococcus aureus, or it is complete to comprise staphylococcus aureus
Genomic DNA or the carrier of portion gene group DNA.
In kit of the present invention, also comprising negative control template, described negative control template includes but is not limited to distilled water.
In kit of the present invention, also comprising developer, developer includes but is not limited to calcein, SYBR Green I or
Hydroxynaphthol blue.When developer is calcein, kit also comprises [Mn2+], for example, MnCl2.
In kit of the present invention, also comprise distilled water.
In kit of the present invention, also comprise nucleic acid extraction reagent.
The invention allows for a kind of carrier, described carrier comprises selected from primer sets A, B, A ' any one group of primer.Should
Carrier has the DNA sequence dna of staphylococcus aureus specific owing to containing, and therefore can be applicable to microbial taxonomy, compares
The research field such as genomics, evolution, and the application such as microorganism detection.This carrier can be but not limited to plasmid vector
(such as pBR322, pUC18, pUC19, pBluescript M13, Ti-plasmids etc.), viral vectors (such as bacteriophage lambda etc.) and artificial dye
Colour solid carrier (such as Bacterial artificial chromosome BAC, yeast artificial chromosome YAC etc.).For example, any one of primer sets A is comprised
The carrier pBR322-A of primer, any one the primer comprising primer sets B carrier pBR322-B, comprise primer sets A ' arbitrarily
Article one, the carrier pBR322-A ' of primer.Carrier bacteriophage lambda-the A of any one the primer comprising primer sets A, comprise primer sets B
Any one primer carrier bacteriophage lambda-B, comprise primer sets A ' the carrier bacteriophage lambda-A ' etc. of any one primer.
The invention allows for selected from primer sets A, B, A ' the primer of any one group at Constant Temperature Detection Staphylococcus aureus
Application in bacterium.
The invention allows for application in Constant Temperature Detection staphylococcus aureus for the described kit.
The invention allows for application in Constant Temperature Detection staphylococcus aureus for the described carrier.
The present invention is that technical field of food safety detection provides a kind of simple and quick sensitive detection Staphylococcus aureus
The method of bacterium, primer/primer sets, detection reagent/kit, have greater significance to the food security of China.The present invention is beneficial
Effect includes：Use detection methods of staphylococcus aureus of the present invention have high specificity, highly sensitive, the detection time is short, knot
Fruit judges simple, simple operation, low cost and other advantages.Compared with conventional at present detection method, the constant-temperature amplification that the present invention uses
Method, can be carried out under constant temperature, only need to use simple thermostat, it is not necessary to the expensive instrument in PCR experiment, be not required to
To carry out the steps such as electrophoresis detection to amplified production, thus, it is very suitable for being widely used in various circles of society and include basic unit's food peace
Full detection department promotes the use of, even if also can be abundant in the environment of molecular biology professional knowledge and skills base relative deficiency
Application.Above-mentioned each optimum condition can be combined based on common sense in the field, all belong to scope.
Brief description
Fig. 1 shows the specific of the embodiment of the present invention 7 staphylococcus aureus Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 detection methods of staphylococcus aureus.
Detailed description of the invention
Being combined to lower specific embodiments and the drawings, the present invention is described in further detail, the protection content of the present invention
It is not limited to following example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and with appending claims as protection domain.Implement the present invention process,
Condition, reagent, experimental technique etc., outside the lower content mentioned specially, be universal knowledege and the common knowledge of this area,
The present invention is not particularly limited content.
Embodiment 1-6 staphylococcus aureus isothermal reaction system and detection method
Detect according to following (1)～(3) step：
(1) extraction of genomic DNA
Staphylococcus aureus bacterial classification for detection derives from Chinese industrial Microbiological Culture Collection administrative center, numbering
CICC21600(ATCC27217).Taking 1mL bacterial cultures uses the bacterial nucleic acid of Beijing Tian Gen bio-engineering corporation to extract examination
Agent box extracts genomic DNA, DNA OD260/OD280Being 1.8, concentration is 50.4ng/ μ L.
(2) with staphylococcus aureus gene group DNA to be measured as template, the kit being respectively adopted autogamy (is shown in Table 2, table
3), and according to condition described in table 3, prepare reaction system, with staphylococcus aureus specific amplimer group as primer, enter
Row isothermal amplification reactions.Primer in embodiment 1～6 is respectively primer sets A, A ' (2 ring primer), A ' (1 ring primer), A '
(1 ring primer), B, B.
(3) according to condition described in table 3, by electrophoresis detection, Turbidity measurement or color developing detection, amplification is carried out true
Recognize.
As can be seen from Table 3, detection method and the primer sets being used and reaction system thereof can be right well
Staphylococcus aureus specific fragment carries out expanding and obtains testing result.Additionally, when using detector to detect, contracting
The short reaction time is to also there being good Detection results (such as embodiment 6) during 10min.Therefore, present invention could apply to detect sample
Whether staphylococcus aureus is contained in Ben.
Embodiment 7 staphylococcus aureus specific detects
Collect non-staphylococcus aureus 27 strain (in table 4 and Fig. 1 3～29), by these bacterial strains and Staphylococcus aureus
Bacteria strain (in table 4 and Fig. 11 and 2) is cultivated respectively, takes 1mL bacterium solution, uses kit IA, extracts DNA of bacteria, and joins
According to reaction system and the condition of embodiment 1, carry out LAMP amplification (primer sets is A) respectively and add developer to observe.
Its testing result is as shown in table 4 and Fig. 1, and in Fig. 1,3～29 are respectively MRSE, Rhodococcus equi, waxy
Bacillus, gill fungus sample bacillus, listeria monocytogenes, Ying Nuoke Listeria, listeria ivanovii, intestines salmonella
Intestines subspecies, Bacterium enteritidis, salmonella typhimurium, moscow' paratyphi B, shigella dysenteriae, Boydii will Hayes
Large intestine angstrom is rushed down in bacterium, shigella flexneri, ETEC (gene containing Clostridium botulinum type A), pathogenic ETEC, cause
Uncommon Salmonella, product enterotoxin ETEC, enterotoxigenic ETEC, hemorrhagic ETEC, the rugged Crow of slope
Promise bacillus, yersinia enterocolitica, artificial tuberculosis yersinia genus, Vibrio vulnificus, vibrio parahaemolytious, Freund vibrios and suddenly
Random vibrios, NTC：Negative control, 1～2 is respectively staphylococcus aureus, the golden yellow subspecies of staphylococcus aureus.In Fig. 1, only
There is the product after staphylococcus aureus strains amplified reaction to be rendered as bright green, be positive findings, as shown in the 1st～No. 2 pipe.
It and the product after other non-staphylococcus aureus strains and negative control amplified reaction is all rendered as orange, is negative findings,
As shown in the 3rd～No. 29 pipe and NTC negative control pipe.
By Fig. 1 and Biao 4 result it can be seen that detection kit of the present invention and detection method have good golden yellow grape
Meningitidis strains is specific, i.e. the only staphylococcus aureus strains amplification positive, and other non-staphylococcus aureus strains are the moon
Property.
Preparation detection kit, in kit use primer be respectively primer sets B, primer sets A ', by above-mentioned specifically
Detection method, respectively obtains same testing result, i.e. after non-staphylococcus aureus strains and negative control amplified reaction
Product is negative findings, and the product after staphylococcus aureus strains amplified reaction is positive findings.
Additionally, according to method described in table 1, respectively to primer sets A～B, primer sets A ' specifically carry out theory analysis,
It was found that in the case that each bar primer at most allows three mispairing, each primer sets is up to a primer comparison to non-gold
On staphylococcus aureus, show the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
As described in Example 1 extract bacterium CICC 21600 DNA, use kit IB, and according to 10ng, 1ng,
100pg, 10pg, 1pg, 100fg, 10fg DNA ladder degree adds reaction system, and other reaction conditions are with reference to the side of table 3 embodiment 1
Method carries out LAMP amplification (primer sets is A) respectively and adds developer to observe.As in figure 2 it is shown, 1-7 be respectively 10ng, 1ng,
100pg, 10pg, 1pg, 100fg, 10fg, NTC：Negative control.The product that in Fig. 2,10ng, 1ng, 100pg are processed presents
For bright green, being positive findings, 10pg, 1pg, 100fg, 10fg are processed and the product of negative control is rendered as orange, are
Negative findings.Testing result shows, minimum in each reaction tube (is approximately equivalent to 3 × 10 containing 100pg4Individual bacterium) DNA when can
It is detected.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B, primer sets A respectively ', in each reaction tube
The DNA of as little as 100pg～1pg still can be detected.
Embodiment 9 versatility detects
According to method described in table 1, respectively to primer sets A～B, primer sets A ' versatility carry out theory analysis, result
Discovery, the primer region of each primer sets and the mating completely of 43 strain staphylococcus aureuses in database, may be used in theory
The detection of above-mentioned 43 strain staphylococcus aureus strains, shows that the versatility of each primer sets is all preferable.
The versatility of primer and specifically analysis in the existing detection method of table 1 staphylococcus aureus
Note：A) 43 full-length genome of the sequence between primers F in patent 3 and B3 and staphylococcus aureus are carried out
Bowtie comparison, determines detection position in No. GI 148266447 genomes for the region.B) regional sequence will be detected at public number
According to carrying out Blast comparison in base resource, it is good that primer region is mated completely for versatility.This table only lists at three genomes
On carry out the result of General Use Analysis.C) detection regional sequence is carried out in common data base resource Blast comparison, guiding region
Territory matching degree is higher, specifically poorer；Can not simultaneously comparison on non-staphylococcus aureus, it is specifically good to show.
The kit species of table 2 Constant Temperature Detection staphylococcus aureus and mainly comprise composition
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention staphylococcus aureus and testing result
Table 4 tests bacterial strain uses therefor and testing result
Note：a)CGMCC：China General Microbiological DSMZ, CICC：Chinese industrial Microbiological Culture Collection manages
Center, CMCC：Chinese medicine bacteria culture preservation administrative center.b)+：Positive findings ,-：Negative findings.
Claims (19)
1. the method for a fast constant temperature detection staphylococcus aureus, it is characterised in that comprise the following steps：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that the primer of staphylococcus aureus gene group-specific base sequence can be expanded
Group, as primer, carries out isothermal amplification reactions under enzyme reaction system；
(3) by judging whether reaction result is positive, determine in testing sample whether there is staphylococcus aureus；
Wherein, described staphylococcus aureus gene group-specific base sequence is the golden yellow grape that No. GI is 148266447
2645869～2646117bp bit sequence of coccus genome.
2. the method for claim 1, it is characterised in that described can expand staphylococcus aureus gene group-specific alkali
The primer sets sequence of basic sequence is staphylococcus aureus gene group 2645869～2646117bp position that No. GI is 148266447
The part of nucleotide sequence or the part of its complementary strand.
3. method as claimed in claim 2, it is characterised in that described can expand staphylococcus aureus gene group-specific alkali
The primer sets of basic sequence is primer sets A；Or be selected from and wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence
It is 68% and any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’(SEQ ID NO：
3)；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’(SEQ ID
NO：4).
4. method as claimed in claim 3, it is characterised in that with wall scroll in described primer sets A sequence or its complementary strand sequence
Sequence homology is 68% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-ATGACTAAAGCCACATCCA-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-ATGCCTTACATTGATGCTG-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’(SEQ ID NO：
7)；
Downstream inner primer BIP_B：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’(SEQ ID
NO：8).
5. method as claimed in claim 2, it is characterised in that described can expand staphylococcus aureus gene group-specific alkali
The primer sets of basic sequence also comprises one or more ring primer；Described ring primer is LF and/or LB.
6. method as claimed in claim 5, it is characterised in that described can expand staphylococcus aureus gene group-specific alkali
The primer sets of basic sequence is primer sets A '；Or selected from and described primer sets A ' wall scroll sequence homology in sequence or its complementary strand sequence
Property is 68% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’；
Upper lantern primer LF_A：5’-TCCCTTTAATTAAGTAAACCC-3’(SEQ ID NO：9)；
And/or, lower lantern primer LB_A：5’-TAAGAACTAGTTAGTGACTA-3’(SEQ ID NO：10).
7. the method for claim 1, it is characterised in that in step (2), described enzyme reaction system includes：1×Bst
DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, FIP and BIP of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L
Primer, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst archaeal dna polymerase, the beet of 0-1.5mol/L
Alkali, including or do not include LF and/or the LB primer of 0.4-1.0 μm of ol/L.
8. the method for claim 1, it is characterised in that the response procedures of described isothermal amplification reactions is：1. 60～65
DEG C hatch 10～90min；2. 80 DEG C terminate reaction 2～20min.
9. for the primer in Constant Temperature Detection staphylococcus aureus method as claimed in claim 1, it is characterised in that described draw
Thing includes the primer sets that can expand staphylococcus aureus gene group-specific base sequence, and its sequence is No. GI and is
A part for the nucleotide sequence of 2645869～2646117bp position of the staphylococcus aureus gene group of 148266447 or it is mutual
Mend a part for chain.
10. primer as claimed in claim 9, it is characterised in that described can expand staphylococcus aureus gene group-specific
The primer sets of base sequence is primer sets A；Or be selected from and wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence
Property is 68% and any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’.
11. primers as claimed in claim 10, it is characterised in that single with described primer sets A sequence or its complementary strand sequence
Bar sequence homology is 68% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-ATGACTAAAGCCACATCCA-3’；
Downstream outer primer B3_B：5’-ATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_B：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_B：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’.
12. primers as claimed in claim 9, it is characterised in that described can expand staphylococcus aureus gene group-specific
The primer sets of base sequence also comprises one or more ring primer；Described ring primer is LF and/or LB.
13. primers as claimed in claim 12, it is characterised in that described can expand staphylococcus aureus gene group-specific
The primer sets of base sequence is primer sets A '；Or selected from and described primer sets A ' in sequence or its complementary strand sequence wall scroll sequence with
Source property is 68% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-CTAAAGCCACATCCAATATAGG-3’；
Downstream outer primer B3_A：5’-TATGCCTTACATTGATGCTG-3’；
Upstream inner primer FIP_A：5’-TCGTTGGTGAGCAATTGAGGAGTATCGTTCCAGATTTGTG-3’；
Downstream inner primer BIP_A：5’-GACTAGGGGTAGTAATCATTGGCCACTTTGCTATGGAACGTAA-3’；
Upper lantern primer LF_A：5’-TCCCTTTAATTAAGTAAACCC-3’；
And/or, lower lantern primer LB_A：5’-TAAGAACTAGTTAGTGACTA-3’.
14. 1 kinds of kits for Constant Temperature Detection staphylococcus aureus, it is characterised in that described kit includes such as right
Require the primer described in any one of 9～13.
15. kits as claimed in claim 14, it is characterised in that its also include Bst DNA polymerase reaction buffer solution,
Bst archaeal dna polymerase, dNTP solution, Mg2+, one or more in glycine betaine.
16. 1 kinds of kits for Constant Temperature Detection staphylococcus aureus, it is characterised in that the enzyme reaction body of described kit
System includes：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 0.8-2.0 μ
FIP and the BIP primer of mol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, including or do not include the LF of 0.4-1.0 μm of ol/L
And/or LB primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase, and the glycine betaine of 0-1.5mol/L.
17. 1 kinds of carriers, it is characterised in that described carrier comprises the primer as described in any one of claim 9～13.
Application in Constant Temperature Detection staphylococcus aureus for 18. primers, it is characterised in that described primer is for such as claim 9
Primer described in any one of～13.
19. kits as described in any one of claim 14～16 or carrier as claimed in claim 17 are in Constant Temperature Detection
Application in staphylococcus aureus.
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| CN110195121A (en) * | 2019-07-08 | 2019-09-03 | 华南理工大学 | A kind of CPA primer detecting Methicillin-resistant Staphylococcus aureus and kit and detection method |
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| CN105861702A (en) * | 2016-05-16 | 2016-08-17 | 昆明理工大学 | Specific gene of staphylococcus aureus and loop-mediated isothermal amplification kit |
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| CN106987644A (en) * | 2017-05-05 | 2017-07-28 | 宁夏出入境检验检疫局检验检疫综合技术中心 | A kind of LAMP detections primer sets and detection method for being used to detect staphylococcus aureus |
| CN110195121A (en) * | 2019-07-08 | 2019-09-03 | 华南理工大学 | A kind of CPA primer detecting Methicillin-resistant Staphylococcus aureus and kit and detection method |
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