CN106434899A - Method, primers and kit for quickly detecting bacillus cereus at constant temperature - Google Patents
Method, primers and kit for quickly detecting bacillus cereus at constant temperature Download PDFInfo
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Abstract
The invention discloses a method, primer group and kit for quickly detecting bacillus cereus at constant temperature. The method comprises the steps that genomic DNA is extracted from a sample to be detected; a constant-temperature amplification reaction is conducted under an enzyme reaction system by taking the genomic DNA as a template and taking the primer group capable of amplifying specific sequences of the bacillus cereus as the primers; whether the bacillus cereus exists in the sample to be detected or not is determined by judging whether a reaction result is positive or not. The detection method has the advantages that the high sensitivity and the high specificity are achieved, the detection time is short, result determination is easy, operation is convenient and fast, the cost is low, and a wide application prospect is achieved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the method for a kind of fast constant temperature detection bacillus cereus, draw
Thing and kit.
Background technology
Bacillus cereus (Bacillus cereus) is distributed widely in nature, is that a kind of common food-borne causes
Sick bacterium.Clinically, the food poisoning that bacillus cereus causes can be divided into diarrhea-type and vomiting type two types, is respectively
Caused by the diarrhoea toxin being produced by this bacterium and vomitoxin, the former is sensitive to heat and trypsase, thus can prevent;And the latter couple
Thermally-stabilised, activity is not by pepsic interference, even if trace expression also can have bigger harm to human body, is difficult to take precautions against, Chang Yin
Play fulminant food poisoning.Therefore, for the prevention of this bacterium with detect extremely important.
Tradition bacillus cereus detection method is longer due to the detection cycle, operates relative complex, and detection efficiency is relatively low, difficult
With meet modern society for food-borne pathogens detection process high flux, high sensitivity, high specific, quick, want easily
Ask.Recently as the development of nucleic acid molecules detection technique, researcher have also been developed the detection means such as PCR, but the party
Method needs special detecting instrument, therefore, is not appropriate for being widely used in inside detection department of basic unit especially enterprise's production line
The real-time on-site detection carrying out.In order to ensure food security, be badly in need of quick, simple, accurately method detect the wax in food
Sample bacillus.
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.Bacillus cereus contains plasmid, therefore prior art such as patent of invention CN 101831493 B and CN
101402997 B are the design carrying out bacillus cereus LAMP primer based on target sequence on plasmid or chromosome respectively.But,
Owing to plasmid exists unstability on copy number and genetic structure, and for for bacillus cereus dyeing in prior art
The LAMP primer of the target sequence design on body there is also the inadequate problem of primer specificity, illustrates invention in table 1 of the present invention
This problem that patent CN 101402997 B exists.It is to say, the bacillus cereus used in art methods
Detection sequence actually not bacillus cereus institute is peculiar, i.e. be possible to non-bacillus cereus is regarded as wax mistakenly
Sample bacillus.Therefore, need one in industry badly and be able to ensure that specific bacillus cereus detection method, meet base simultaneously
Layer detection department, to quick, demand easily, can carry out real-time on-site detection easily inside enterprise's production 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 bacillus cereus, and forms Gao Ling on this basis
Sensitivity, high specific detection kit.The present invention based in GenBank database microbial genome data resource (by
On August 5th, 2013 data) carry out the design of bacillus cereus LAMP primer, provide a kind of fast constant temperature augmentation detection wax
The method of sample bacillus, primer sets and kit.Use the detection method detection bacillus cereus of the present invention, there is Gao Ling
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 quick detection Bacillus cereus strain, said method comprising the steps of:
(1) from testing sample, genomic DNA is extracted;
(2) with described genomic DNA as template, so that drawing of bacillus cereus genome specificity base sequence can be expanded
Thing group 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 bacillus cereus.
The method of Constant Temperature Detection Bacillus cereus strain of the present invention, extracts genomic DNA, which is from testing sample
Template, with bacillus cereus specificity amplification primer group as primer, carries out isothermal amplification reactions, then, by judging to react
Whether result is positive, determines in testing sample whether there is bacillus cereus.Wherein, described enzyme reaction system include but not
It is limited to DNA polymerase reaction system.
In the present invention, described bacillus cereus genome specificity base sequence be No. GI be 218895141 waxy
3193434~3194011bp bit sequence of bacillus.
In the present invention, the described primer sets that can expand bacillus cereus genome specificity base sequence is described gene
A part for the nucleotide sequence of 3193434~3194011bp position of group (No. GI is 218895141) or one of its complementary strand
Point.Wherein, described bacillus cereus genome specificity base sequence refers to that only bacillus cereus genome institute is peculiar
, and the base sequence that other microbial genome do not comprise.
Wherein, the described primer sets that can expand bacillus cereus genome specific base sequence includes but is not limited to primer
Group A, or be 55% and above primer sets with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence.
Primer sets A:
Upstream outer primer F3_A:5’-AGGAACTATTAGAAAACGCG-3’(SEQ ID NO:1);
Downstream outer primer B3_A:5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO:2);
Upstream inner primer FIP_A:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’(SEQ
ID NO:3);
Downstream inner primer BIP_A:5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-3’
(SEQ ID NO:4).
In the present invention, the described primer sets that can expand bacillus cereus genome specific base sequence can also include with
In aforementioned each primer sets sequence or its complementary strand sequence, wall scroll sequence homology is 55% and above primer sets, this primer sets bag
Include but be not limited to following primer sets B:
Primer sets B:
Upstream outer primer F3_B:5’-TAGAAAACGCGGGTATGA-3’(SEQ ID NO:5) (with primers F 3_A 5 '-
AGGAACTATTAGAAAACGCG-3 ' homology is 55%);
Downstream outer primer B3_B:5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO:6);
Upstream inner primer FIP_B:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’(SEQ ID
NO:7);
Downstream inner primer BIP_B:5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’
(SEQ ID NO:8).
In the inventive method, in one embodiment, the enzyme reaction system of described constant-temperature amplification is:1×Bst DNA
Polymeric enzyme reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L
FIP and BIP 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 and 0-
1.5mol/L glycine betaine.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer,
Comprise 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
Merging treatment.
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 that bacillus cereus is positive, containing waxy gemma
Bacillus;Band as stepped in electrophoretogram not expression characteristics, then testing sample is that bacillus cereus is negative.Described turbidity is examined
Surveying, being to detect by an unaided eye or transmissometer detection turbidity, detection pipe occurs substantially muddy, then testing sample is bacillus cereus sun
Property, containing bacillus cereus;As muddy in having no, then testing sample is that bacillus cereus is negative.Also can after centrifugal meat
Whether eye is observed has precipitation at the bottom of reaction tube, if there being precipitation at the bottom of reaction tube, then testing sample is that bacillus cereus is positive, containing wax
Sample bacillus;Do not precipitate as at the bottom of reaction tube, then testing sample is that bacillus cereus 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 bacillus cereus is negative;As after reaction, color is green
Look, then testing sample is that bacillus cereus is positive, containing bacillus cereus.When employing hydroxynaphthol blue is as developer
When, as after reaction, color is pansy, then testing sample is that bacillus cereus is negative;As after reaction, color is sky blue,
Then testing sample is that bacillus cereus is positive.Described color developing detection, in addition to above by visually observing reaction result, it is possible to
To carry out in real time or end point determination reaction result by detecting instrument, by the rational threshold value setting negative reaction, when to be measured
When the result of example reaction is less than or equal to this threshold value, then testing sample is that bacillus cereus is negative;When testing sample reaction
Result more than this threshold value when, then testing sample is that bacillus cereus is positive.Described detecting instrument includes but is not limited to fluorescence
Spectrophotometer, quantitative real time PCR Instrument, constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument and the inspection of Genie II isothermal duplication fluorescence
Examining 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 Bacillus cereus strain.Described primer includes
Can expand the primer sets of bacillus cereus genome specific base sequence, it includes but is not limited to, and the sequence of described primer is
No. GI be 218895141 bacillus cereus genome 3193434~3194011bp position nucleotide sequence a part or
A part for its complementary strand.
Wherein, the described primer sets that can expand bacillus cereus genome specificity base sequence is selected from following primer
Any one group of group, or selected from wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence be 55% and with
On arbitrary primer sets.Wherein, described primer sets includes but is not limited to following primer sets A.Described with aforementioned primer sets sequence or
In its complementary strand sequence, wall scroll sequence homology is 55% and above primer sets includes but is not limited to following primer sets B.
Primer sets A:
Upstream outer primer F3_A:5’-AGGAACTATTAGAAAACGCG-3’;
Downstream outer primer B3_A:5’-CCGGTTTAGATAATTCACGT-3’;
Upstream inner primer FIP_A:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’;
Downstream inner primer BIP_A:5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-3’;
Primer sets B:
Upstream outer primer F3_B:5’-TAGAAAACGCGGGTATGA-3’;
Downstream outer primer B3_B:5’-CCGGTTTAGATAATTCACGT-3’;
Upstream inner primer FIP_B:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’;
Downstream inner primer BIP_B:5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’.
The present invention also provides a kind of for the kit in above-mentioned Constant Temperature Detection Bacillus cereus strain method, and it includes
The described primer sets that can expand bacillus cereus genome specific base sequence.In kit of the present invention, described can expand wax
The primer sets of sample bacillus gene group-specific base sequence, including but not limited to genome (No. GI:218895141)
A part for a part for the nucleotide sequence of 3193434~3194011bp position or its complementary strand is as described primer sequence;Described
Primer includes but is not limited to described primer sets A.Also including but not limited to single with aforementioned primer sequence or its complementary strand sequence
Bar sequence homology is 55% and above primer sets is as primer;Including but not limited to primer sets B.
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 bacillus cereus genomic DNA, or comprise waxy gemma bar
Bacterium genomic DNA complete genome 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 any one group of primer selected from primer sets A, B.This carrier
Owing to containing, there is the specific DNA sequence dna of bacillus cereus, therefore can be applicable to microbial taxonomy, Comparative genomic strategy
The research fields such as, evolution, and the application such as microorganism detection.This carrier can be but not limited to plasmid vector (as
PBR322, pUC18, pUC19, pBluescript M13, Ti-plasmids etc.), viral vectors (such as bacteriophage lambda etc.) and artificial coloring
Body carrier (such as Bacterial artificial chromosome BAC, yeast artificial chromosome YAC etc.).For example, any one that comprises primer sets A is drawn
The carrier pBR322-A of thing, the carrier pBR322-B etc. comprising any one primer of primer sets B.Comprise primer sets A arbitrarily
Article one, carrier bacteriophage lambda-A, the carrier bacteriophage lambda-B etc. comprising any one primer of primer sets B of primer.
The invention allows for selected from primer sets A, the primer of any one group of B in Constant Temperature Detection bacillus cereus
Application.
The invention allows for application in Constant Temperature Detection bacillus cereus for the described kit.
The invention allows for application in Constant Temperature Detection bacillus cereus for the described carrier.
The present invention is that technical field of food safety detection provides a kind of simple and quick sensitive detection bacillus cereus
Method, primer/primer sets, detection reagent/kit, greater significance is had to the food security of China.The beneficial effect of the present invention
Fruit includes:Use that bacillus cereus detection method of the present invention has high specificity, highly sensitive, the detection time is short, result is sentenced
Calmly simply, simple operation, low cost and other advantages.Compared with conventional at present detection method, the constant-temperature amplification method that the present invention uses, can
To carry out under constant temperature, only simple thermostat need to be used, it is not necessary to the expensive instrument in PCR experiment, it is not necessary to right
Amplified production carries out the steps such as electrophoresis detection, thus, it is very suitable for being widely used in various circles of society and include basic unit's food security inspection
Survey department promotes the use of, even if also can fully answer in the environment of molecular biology professional knowledge and skills base relative deficiency
With.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 bacillus cereus Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 bacillus cereus detection method.
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 bacillus cereus isothermal reaction system and detection method
Detect according to following (1)~(3) step:
(1) extraction of genomic DNA
Bacillus cereus bacterial classification for detection derives from China General Microbiological DSMZ, numbering CGMCC
1.3760 (=ATCC 14579).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 5.36ng/ μ L.
(2) it with bacillus cereus genomic DNA to be measured as template, is respectively adopted the kit (being shown in Table 2, table 3) of autogamy,
And according to condition described in table 3, prepare reaction system, with bacillus cereus specificity amplification primer group as primer, carry out perseverance
Isothermal amplification reaction.Primer in embodiment 1~6 is respectively primer sets A, A, A, B, 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
Bacillus cereus specific fragment carries out expanding and obtains testing result.Therefore, present invention could apply in detection sample
Whether contain bacillus cereus.
Embodiment 7 bacillus cereus specific detection
Collect non-bacillus cereus 28 strain (in table 4 and Fig. 1 1~4,6~29), by these bacterial strains and waxy gemma bar
Bacteria strain (in table 4 and Fig. 1 5) is cultivated respectively, takes 1mL bacterium solution, uses kit IA, extracts DNA of bacteria, and with reference in fact
Execute reaction system and the condition of example 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. 1,1~4 is respectively staphylococcus aureus, Staphylococcus aureus
The golden yellow subspecies of bacterium, MRSE, Rhodococcus equi, 6~29 are respectively gill fungus sample bacillus, listeria monocytogenes, English
Promise gram Listeria, listeria ivanovii, intestines salmonella intestines subspecies, Bacterium enteritidis, salmonella typhimurium, B-mode pair
Salmonella typhi, shigella dysenteriae, Shigella bogdii, shigella flexneri, ETEC (A containing clostridium botulinum
Type gene), pathogenic ETEC, Diarrheogenil Escherichia coli, product enterotoxin ETEC, enterotoxigenic large intestine
Escherichia, hemorrhagic ETEC, yersinia enterocolitica, artificial tuberculosis yersinia genus, Vibrio vulnificus, pair
Hemolysis vibrion, Freund vibrios and bacillus cereus, NTC:Negative control, 5:Bacillus cereus.In Fig. 1, only waxy bud
Product after spore bacillus strain amplified reaction is rendered as bright green, is positive findings, as shown in No. 5 pipe.And other are non-waxy
Product after Bacillus strain and negative control amplified reaction is all rendered as orange, be negative findings, as the 1st~No. 4,6~
Shown in No. 29 pipes 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 waxy gemma bar
Bacteria strain is specific, i.e. the only Bacillus cereus strain amplification positive, and other non-Bacillus cereus strain are feminine gender.
Preparation detection kit, the primer using in kit is primer sets B, by above-mentioned method for detecting specificity, obtains
Same testing result, i.e. the product after non-Bacillus cereus strain and negative control amplified reaction is negative findings, waxy
Product after Bacillus strain amplified reaction is positive findings.
Additionally, according to method described in table 1, respectively theory analysis is specifically carried out to primer sets A~B, it was found that
In the case that each bar primer at most allows three mispairing, each primer sets is up to 1 primer comparison to non-bacillus cereus
On, show the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
As described in Example 2 extract bacterium CGMCC 1.3760 DNA, use kit IIB, and according to 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 2
Method carries out LAMP amplification (primer sets is A) respectively and adds developer to observe.As in figure 2 it is shown, 1-6 be respectively 1ng, 100pg,
10pg, 1pg, 100fg and 10fg, NTC:Negative control.The product that in Fig. 2,1ng, 100pg and 10pg are processed is rendered as bright
Green, is positive findings, and 1pg, 100fg and 10fg are processed and the product of negative control is rendered as orange, is negative findings.
Testing result shows, still can be detected in each reaction tube during minimum DNA containing 10pg (being approximately equivalent to 1700 bacteriums).
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B, the DNA of as little as 10pg in each reaction tube
Still can be detected.
The specific analysis of primer in the existing detection method of table 1 bacillus cereus
Note:The detection regional sequence of primer in patent CN 101402997 B is carried out in common data base resource
Blast comparison, determine this detection region respectively with bacillus cereus and each bar primer region mispairing of non-bacillus cereus
Base number.If primer region and non-bacillus cereus (including artificial tuberculosis yersinia genus and Yersinia pestis) bacterial strain
Base mismatch number is fewer, shows that its matching degree is higher, specifically poorer.
The kit species of table 2 Constant Temperature Detection bacillus cereus and mainly comprise composition
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention bacillus cereus 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 (15)
1. the method for a fast constant temperature detection bacillus cereus, 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 sets of bacillus cereus genome specificity base sequence can be expanded
As primer, under enzyme reaction system, carry out isothermal amplification reactions;
(3) by judging whether reaction result is positive, determine in testing sample whether there is bacillus cereus;
Wherein, described bacillus cereus genome specificity base sequence is the bacillus cereus that No. GI is 218895141
3193434~3194011bp bit sequence of genome.
2. the method for claim 1, it is characterised in that described can expand bacillus cereus genome specificity base
The primer sets sequence of sequence is the core of bacillus cereus genome 3193434~3194011bp position that No. GI is 218895141
A part for acid sequence or a part for its complementary strand.
3. method as claimed in claim 2, it is characterised in that described can expand bacillus cereus genome specificity base
The primer sets of sequence is primer sets A;Or be 55% with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence
And any one group of above primer sets;
Primer sets A:
Upstream outer primer F3_A:5’-AGGAACTATTAGAAAACGCG-3’(SEQ ID NO:1);
Downstream outer primer B3_A:5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO:2);
Upstream inner primer FIP_A:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’(SEQ ID
NO:3);
Downstream inner primer BIP_A:5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-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 55% and above primer sets includes following primer sets B:
Primer sets B:
Upstream outer primer F3_B:5’-TAGAAAACGCGGGTATGA-3’(SEQ ID NO:5);
Downstream outer primer B3_B:5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO:6);
Upstream inner primer FIP_B:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’(SEQ ID NO:
7);
Downstream inner primer BIP_B:5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’(SEQ
ID NO:8).
5. 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.
6. 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.
7. for the primer in Constant Temperature Detection bacillus cereus method as claimed in claim 1, it is characterised in that described primer
Including the primer sets of bacillus cereus genome specificity base sequence can be expanded, it is 218895141 that its sequence is No. GI
A part for the nucleotide sequence of 3193434~3194011bp position of bacillus cereus genome or a part for its complementary strand.
8. primer as claimed in claim 7, it is characterised in that described can expand bacillus cereus genome specificity base
The primer sets of sequence is primer sets A;Or be 55% with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence
And any one group of above primer sets;
Primer sets A:
Upstream outer primer F3_A:5’-AGGAACTATTAGAAAACGCG-3’;
Downstream outer primer B3_A:5’-CCGGTTTAGATAATTCACGT-3’;
Upstream inner primer FIP_A:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’;
Downstream inner primer BIP_A:5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-3’.
9. primer as claimed in claim 8, it is characterised in that with wall scroll in described primer sets A sequence or its complementary strand sequence
Sequence homology is 55% and above primer sets includes primer sets B:
Primer sets B:
Upstream outer primer F3_B:5’-TAGAAAACGCGGGTATGA-3’;
Downstream outer primer B3_B:5’-CCGGTTTAGATAATTCACGT-3’;
Upstream inner primer FIP_B:5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’;
Downstream inner primer BIP_B:5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’.
10. the kit for Constant Temperature Detection bacillus cereus, it is characterised in that described kit includes that right such as is wanted
Seek the primer described in any one of 7~9.
11. kits as claimed in claim 10, 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.
12. 1 kinds of kits for Constant Temperature Detection bacillus cereus, it is characterised in that the enzyme reaction system of described kit
Including:1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/
FIP and the BIP primer of 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 0-
The glycine betaine of 1.5mol/L.
13. 1 kinds of carriers, it is characterised in that described carrier comprises the primer as described in any one of claim 7~9.
Application in Constant Temperature Detection bacillus cereus for 14. primers, it is characterised in that described primer is for such as claim 7~9
The primer described in any one.
15. kits as described in any one of claim 10~12 or carrier as claimed in claim 13 are in Constant Temperature Detection
Application in bacillus cereus.
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