CN106434891A - Method for rapidly detecting Shigella at constant temperature, primers used for method and kit - Google Patents
Method for rapidly detecting Shigella at constant temperature, primers used for method and kit Download PDFInfo
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Abstract
The invention discloses a method, a primer group and a kit for rapidly detecting Shigella at constant temperature. The method comprises the following steps: extracting genome DNA from a sample to be tested; by taking the genome DNA as a template and taking the primer group capable of amplifying the specific sequence of the bacillus cereus as primers, carrying out isothermal amplification reaction under the enzyme reaction system; and determining that whether Shigella exists in the sample to be tested or not by judging that whether the reaction result is positive or not. The detection method provided by the invention has the advantages that the sensitivity and specificity are high, the detection time is short, the result judgment is simple, the operation is convenient and rapid, the cost is low, and the application prospect is wide.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of fast constant temperature detection method of Shigella, primer and
Kit.
Background technology
The bacterium of Shigella (Shigella spp.) is a class Gram-negative enterobacteria, is human bacterial's property dysentery
The most commonly seen pathogen of disease, is generally called shigella dysenteriae.Will Hayes is belonged to bacterium and can be propagated by the water and the food that are contaminated.
The mankind have very high neurological susceptibility to Shigella bacterium, can cause acute toxic bacillary dysentery child, and the state of an illness is heavier, and the death rate is very
High.Therefore, carry out detecting to the Shigella in water or food accurately and in time and be not only prophylactic treatment and control water effectively
It or Food poisoning provides foundation, is also the necessary means ensureing people's health simultaneously.
Traditional Shigella method of detecting bacterium is longer due to the detection cycle, operates relative complex, and detection efficiency is relatively low,
It is difficult to meet modern society for food-borne pathogens detection process high flux, high sensitivity, high specific, quick, easily
Require.Recently as the development of nucleic acid molecules detection technique, researcher have also been developed the inspection such as PCR and real-time fluorescence PCR
Survey means, but both of which needs special detecting instrument, therefore, is not appropriate for being widely used in detection department of basic unit outstanding
It is the real-time on-site detection carrying out inside enterprise's production line.It in order to ensure food security, is badly in need of quick, simple, square accurately
Method detects the Shigella 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
As a example by ZL201010145716.2 and ZL201310737377.0, they are respectively directed to the special base of the Shigella of document report
Because of ipaH gene, LAMP technology is used to carry out Shigella detection.But, so-called " specific gene generally acknowledged " often base
It in delayed knowledge, and is not based on the renewal that ever-increasing microbial genome data carry out necessity, cause based on this target base
The primer obtaining because of sequence not necessarily can ensure that its versatility and/or specific in actual applications.Present invention table 1 illustrates
The problem that present in prior art, primer versatility cannot ensure.It is to say, the will used in art methods is congratulated
Salmonella detection sequence actually not Shigella is common, i.e. be possible to the part bacterial strain of missing inspection Shigella.Similar asks
Topic exists in the confirmation of versatility, i.e. be possible to non-Shigella is regarded as Shigella mistakenly.Therefore, in industry
Need badly and a kind of be able to ensure that specific and versatility Shigella detection method, meet simultaneously detection department of basic unit to quickly,
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 Shigella, and formed on this basis high sensitivity,
High specific detection kit.The present invention based on the microbial genome data resource in GenBank database (by 2013
August 5 data) carry out the design of Shigella LAMP primer, provide the side of a kind of fast constant temperature augmentation detection Shigella
Method, primer sets and kit.Use the detection method detection Shigella of the present invention, there is high sensitivity and high specific, inspection
The survey 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 Shigella 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 the primer sets of Shigella genome specificity base sequence can be expanded
For 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 Shigella.
The method of Constant Temperature Detection Shigella bacterial strain of the present invention, extracts genomic DNA, which is mould from testing sample
Plate, with Shigella specificity amplification primer group as primer, carries out isothermal amplification reactions, then, by judging that reaction result is
No is the positive, determines in testing sample whether there is Shigella.Wherein, described enzyme reaction system include but is not limited to DNA gather
Synthase reaction system.
In the present invention, described Shigella genome specificity base sequence is the Shigella that No. GI is 110804074
1921404～1921811bp bit sequence.
In the present invention, the described primer sets that can expand Shigella genome specificity base sequence is described genome
A part for the nucleotide sequence of 1921404～1921811bp position of (No. GI is 110804074) or a part for its complementary strand.
Wherein, described Shigella genome specificity base sequence refers to only specific to Shigella genome, and other are micro-
The base sequence that biological genome does not comprises.
Wherein, the described primer sets that can expand Shigella genome specific base sequence includes but is not limited to primer sets A,
Or selected from being 75% with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence and above primer sets is arbitrarily
One group.
Primer sets A：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’(SEQ ID
NO：3)；
Downstream inner primer BIP_A：5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3’(SEQ
ID NO：4).
In the present invention, the described primer sets that can expand Shigella genome specific base sequence can also include with aforementioned
In each primer sets sequence or its complementary strand sequence, wall scroll sequence homology is 75% and above primer sets, this primer sets include but
It is not limited to following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’(SEQ ID NO：6) (with primer B3_A5 '-
AAGAGTGATTTCCTGGCCTG-3 ' homology is 75%)
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’(SEQ ID
NO：7)；
Downstream inner primer BIP_B：5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’(SEQ
ID NO：8).
In the inventive method, the described primer sets that can expand Shigella genome specificity base sequence can comprise but
It is not limited to a ring primer.Preferably, described ring primer is one, is ring primer LB.Described can expand Shigella genome
The primer sets of specific base sequence is selected from following primer sets A ', any one group of B '；Or be selected from and described primer sets A ', B ' sequence
In row or its complementary strand sequence, wall scroll sequence homology is 75% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’；
Downstream inner primer BIP_A：5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3’；
Lower lantern primer LB_A：5’-GTGTGTCGGGTATGATGATGCCG-3’(SEQ ID NO：9)；
Primer sets B '：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’；
Downstream inner primer BIP_B：
5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’；
Lower lantern primer LB_B：5’-GTGTGTCGGGTATGATGATGCCG-3’(SEQ ID NO：10).
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 LB primer of 0.4-1.0 μm of ol/L,
0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.Another specific embodiments (not containing ring primer)
In, the enzyme reaction system of described constant-temperature amplification is：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+(MgSO4
Or MgCl2), FIP and the BIP primer of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L, the F3 of 0.15-0.3 μm of ol/L 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 effect
Rate.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer, comprises 20mmol/L
Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%Triton X-100,2mM MgSO4.1×
MgSO in 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 Shigella is positive, containing Shigella；As
The stepped band of electrophoretogram not expression characteristics, then testing sample is that Shigella is negative.Described Turbidity measurement, is with the naked eye to see
Examining or transmissometer detection turbidity, detection pipe becomes turbid, then testing sample is that Shigella is positive, containing Shigella；As not
See muddiness, then testing sample is that Shigella is negative.Also can visually observe whether have precipitation at the bottom of reaction tube after centrifugal, if instead
Should have precipitation at the bottom of pipe, then testing sample is that Shigella is positive, containing Shigella；Do not precipitate as at the bottom of reaction tube, then to be measured
Sample is that Shigella 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 Shigella is negative；As after reaction, color is green, then
Testing sample is that Shigella is positive, containing Shigella.When using hydroxynaphthol blue as developer, such as color after reaction
For pansy, then testing sample is that Shigella is negative；As after reaction, color is sky blue, then testing sample is Shigella
Positive.Described color developing detection, in addition to above by visually observing reaction result, it is also possible to by detecting instrument carry out in real time or
End point determination reaction result, by the rational threshold value setting negative reaction, when the result of testing sample reaction is less than or equal to
During this threshold value, then testing sample is that Shigella is negative；When the result of testing sample reaction is more than this threshold value, then testing sample
Positive for Shigella.Described detecting instrument includes but 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 duplication fluorescence 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 Shigella bacterial strain.Described primer includes expanding
Increasing the primer sets of Shigella genome specific base sequence, it includes but is not limited to, and the sequence of described primer is No. GI and is
A part for the nucleotide sequence of 1921404～1921811bp position of the Shigella genome of 110804074 or its complementary strand
A part.
Wherein, the described primer sets that can expand Shigella genome specificity base sequence selected from following primer sets it
Any one group, or selected from being 75% and above with wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence
Arbitrary primer sets.Wherein, described primer sets includes but is not limited to following primer sets A.Described and aforementioned primer sets sequence or it is mutual
In benefit chain-ordering, wall scroll sequence homology is 75% and above primer sets includes but is not limited to following primer sets B.
Primer sets A：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’；
Downstream inner primer BIP_A：
5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3’；
Primer sets B：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’；
Downstream inner primer BIP_B：
5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’.
The present invention in the primer in described Constant Temperature Detection Shigella method, described can expand Shigella genome
The primer sets of specific base sequence can also be including but not limited to a ring primer；Preferably, described ring primer is one, is
LB.The described primer sets that can expand Shigella genome specificity base sequence is selected from following primer sets A ', B's ' is any one
Group；Or be selected from and described primer sets A ', in B ' sequence or its complementary strand sequence, wall scroll sequence homology is 75% and above drawing
Any one group of thing group：
Primer sets A '：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’；
Downstream inner primer BIP_A：
5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3’；
Lower lantern primer LB_A：5’-GTGTGTCGGGTATGATGATGCCG-3’；
Primer sets B '：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’；
Downstream inner primer BIP_B：
5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’；
Lower lantern primer LB_B：5’-GTGTGTCGGGTATGATGATGCCG-3’.
The present invention also provides a kind of for the kit in above-mentioned Constant Temperature Detection Shigella bacterial strain method, and it includes described
The primer sets of Shigella genome specific base sequence can be expanded.In kit of the present invention, described can expand Shigella base
Because of the primer sets of group-specific base sequence, including but not limited to genome (No. GI:110804074) 1921404～
A part for a part for the nucleotide sequence of 1921811bp position or its complementary strand is as described primer sequence；Described primer includes
But it is not limited to described primer sets A.Also including but not limited to with wall scroll sequence in aforementioned primer sequence or its complementary strand sequence together
Source property is 75% 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 Shigella genome specificity base sequence can comprise
But it is not limited to a ring primer；Ring primer is as optional component.Preferably, described ring primer is one, is LB.Comprise ring primer
The primer sets of LB includes but is not limited to primer sets A ', B ' etc..In a particular embodiment, kit of the present invention can comprise
The LB ring primer of 0.4-1.0 μm of ol/L.In one embodiment, the sequence of primer sets is respectively FIP, BIP, F3, B3, LB
Shown primer or be 75% and above primer with foregoing sequences or its complementary strand sequence wall scroll primer homology.
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 Shigella, or comprise Shigella complete genome DNA or portion
Divide the carrier of genomic 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 of B '.
This carrier has the specific DNA sequence dna of Shigella owing to containing, and therefore can be applicable to microbial taxonomy, icp gene
Organize the applications such as the research field such as, evolution, and 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, any one the primer comprising primer sets B carrier pBR322-B, comprise primer sets A ' any one
The carrier pBR322-A ' of bar primer, comprise primer sets B ' the carrier pBR322-B ' of any one primer.Comprise primer sets A it
Carrier bacteriophage lambda-the A of any one primer, any one the primer comprising primer sets B carrier bacteriophage lambda-B, comprise primer
Carrier bacteriophage lambda-the A ' of any one the primer of group A ', comprise primer sets B ' the carrier bacteriophage lambda-B ' of any one primer
Deng.
The invention allows for selected from primer sets A, B, A ', the primer of any one group of B ' is in Constant Temperature Detection Shigella
Application.
The invention allows for application in Constant Temperature Detection Shigella for the described kit.
The invention allows for application in Constant Temperature Detection Shigella for the described carrier.
The present invention is the side that technical field of food safety detection provides a kind of simple and quick sensitive detection Shigella
Method, primer/primer sets, detection reagent/kit, have greater significance to the food security of China.Beneficial effect bag of the present invention
Include：Use that Shigella detection method of the present invention has high specificity, highly sensitive, the detection time is short, result judges simple, behaviour
Make convenient, low cost and other advantages.Compared with conventional at present detection method, the constant-temperature amplification method that the present invention uses, can be at constant temperature
Under the conditions of carry out, only need to use simple thermostat, it is not necessary to the expensive instrument in PCR experiment, it is not necessary to amplified production
Carry out the steps such as electrophoresis detection, thus, it is very suitable for being widely used in various circles of society and include that food safety detection department of basic unit pushes away
Wide use, even if also can fully apply in the environment of molecular biology professional knowledge and skills base relative deficiency.Based on this
Above-mentioned each optimum condition can be combined by field general knowledge, all belongs to scope.
Brief description
Fig. 1 shows the specific of the embodiment of the present invention 7 Shigella Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 Shigella 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 Shigella isothermal reaction system and detection method
Detect according to following (1)～(3) step：
(1) extraction of genomic DNA
Shigella (shigella flexneri) bacterial classification for detection derives from China General Microbiological DSMZ,
Numbering CGMCC 1.1868.Taking 1mL bacterial cultures uses the bacterial nucleic acid of Beijing Tian Gen bio-engineering corporation to extract kit
Extract genomic DNA, DNA OD260/OD280Being 1.8, concentration is 400ng/ μ L.
(2) it with Shigella genomic DNA to be measured as template, is respectively adopted the kit (being shown in Table 2, table 3) of autogamy, and presses
According to condition described in table 3, prepare reaction system, with Shigella specificity amplification primer group as primer, carry out constant-temperature amplification anti-
Should.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
Shigella specific fragment carries out expanding and obtains testing result.Additionally, when using detector to detect, shorten reaction
Time is to also there being good Detection results (such as embodiment 6) during 10min.Therefore, present invention could apply to detection sample in be
No containing Shigella.
Embodiment 7 Shigella specific detection
Collect non-Shigella 26 strain (in table 4 and Fig. 1 1～13,17～29), by these bacterial strains and Shigella bacterial strain
(in table 4 and Fig. 1 14～16) cultivate respectively, take 1mL bacterium solution, use kit IA, extract 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. 1,1～13 is respectively staphylococcus aureus, Staphylococcus aureus
The golden yellow subspecies of bacterium, MRSE, Rhodococcus equi, bacillus cereus, gill fungus sample bacillus, listeria monocytogenes,
Ying Nuoke Listeria, listeria ivanovii, intestines salmonella intestines subspecies, Bacterium enteritidis, salmonella typhimurium, B-mode
Salmonella paratyphi, 17～29 are respectively ETEC (gene containing Clostridium botulinum type A), pathogenic E
Bacterium, Diarrheogenil Escherichia coli, product enterotoxin ETEC, enterotoxigenic ETEC, hemorrhagic large intestine angstrom are wished
Salmonella, the rugged Cronobacter sakazakii of slope, yersinia enterocolitica, artificial tuberculosis yersinia genus, Vibrio vulnificus, secondary haemolysis arc
Bacterium, Freund vibrios and comma bacillus, NTC：Negative control, 14～16 are respectively shigella dysenteriae, Song Shi Shigella and good fortune
Family name Shigella.In Fig. 1, only the product after Shigella bacterial strain amplified reaction is rendered as bright green, is positive findings, and such as the
Shown in 14～No. 16 pipes.And the product after other non-Shigella bacterial strains and negative control amplified reaction is all rendered as orange, is
Negative findings, as the 1st～No. 13,17～No. 29 is managed and shown in 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 Shigella bacterium
Strain is specific, i.e. the only Shigella bacterial strain amplification positive, and other non-Shigella bacterial strains are feminine gender.
Preparation detection kit, the primer using in kit is respectively primer sets B, primer sets A ', B ', by above-mentioned special
Property detection method, respectively obtains same testing result, i.e. the product after non-Shigella bacterial strain and negative control amplified reaction
For negative findings, the product after Shigella bacterial strain amplified reaction is positive findings.
Additionally, according to method described in table 1, respectively to primer sets A, primer sets B, primer sets A ', B ' is specifically carried 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 ratio
To on non-Shigella, show the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
The DNA of extraction shigella flexneri CGMCC 1.1868 as described in Example 2, employing kit IIB, and according to
10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10fg DNA ladder degree adds reaction system, and other reaction conditions are real with reference to table 3
The method executing example 2 carries out LAMP amplification (primer sets is A) respectively and adds developer to observe.As in figure 2 it is shown, 1～7 is respectively
10ng, 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, NTC：Negative control.The product that in Fig. 2,10ng and 1ng is processed
Being rendered as bright green, being positive findings, 100pg, 10pg, 1pg, 100fg, 10fg are processed and the product of negative control presents
It for orange, is negative findings.Testing result shows, minimum in each reaction tube (is approximately equivalent to 2 × 10 containing 1ng5Individual bacterium)
Still can be detected during DNA.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B, primer sets A respectively ', B ', each reaction
In pipe, the DNA of as little as 1ng～1pg still can be detected.
Embodiment 9 versatility detects
According to the method for embodiment 1, to shigella dysenteriae, Song Shi Shigella and shigella flexneri (table 4 and Fig. 1
In 14～16) carry out cultivating and extracting DNA respectively, and carry out LAMP amplification (primer sets is A), testing result such as table 4 and Fig. 1
Shown in, the product after three kinds of Shigella strain amplified reactions is all rendered as bright green, is positive findings, shows the logical of this primer sets
Preferable by property.
Preparation detection kit, the primer using in kit is respectively primer sets B, primer sets A ', primer sets B ', by upper
State versatility detection method, respectively obtain same testing result, the i.e. three kinds Shigella strain amplification positives, show each primer sets
Versatility preferable.
According to method described in table 1, respectively to primer sets A, primer sets B, primer sets A ', the versatility of B ' carries out theoretical point
Analysis, it was found that the primer region of each primer sets and 8 strain Shigellas (No. GI is respectively 30061571,74310614,
82542618,110804074,187730020,344915202,377520096 and 384541581) coupling completely, with No. GI
Be 82775382 shigella dysenteriae have 1～2 mispairing at guiding region (B3 and/or B2), may be used for above-mentioned 9 in theory
The detection of strain Shigella bacterial strain, 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 Shigella
Note：A) by 9 genomes of the sequence between primers F in patent 3 and B3 and Shigella, (No. GI is respectively
110804074,82775382,30061571,74310614,82542618,187730020,344915202,377520096 Hes
384541581) carry out Bowtie comparison, determine detection position in No. GI 110804074 genomes for the region.B) by detection zone
Territory sequence carries out Blast comparison in common data base resource, and it is good that primer region is mated completely for versatility.C) region will be detected
Sequence carries out Blast comparison in common data base resource, and primer region matching degree is higher, specifically poorer；If primer is not
Can simultaneously comparison in non-Shigella strain, it is specifically good to show.
The kit species of table 2 Constant Temperature Detection Shigella and mainly comprise composition
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention Shigella 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 Shigella, it is characterised in that comprise the following steps：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, using can expand the primer sets of Shigella genome specificity base sequence 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 Shigella；
Wherein, described Shigella genome specificity base sequence is the Shigella genome that No. GI is 110804074
1921404～1921811bp bit sequence.
2. the method for claim 1, it is characterised in that described can expand Shigella genome specificity base sequence
Primer sets sequence be the nucleotide sequence of Shigella genome 1921404～1921811 bp position that No. GI is 110804074
A part or the part of its complementary strand.
3. method as claimed in claim 2, it is characterised in that described can expand Shigella genome specificity base sequence
Primer sets be primer sets A；Or selected from being 75% with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence
And above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’(SEQ ID NO：
3)；
Downstream inner primer BIP_A：5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-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 75% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’(SEQ ID NO：
7)；
Downstream inner primer BIP_B：5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’(SEQ ID
NO：8).
5. method as claimed in claim 2, it is characterised in that described can expand Shigella genome specificity base sequence
Primer sets also comprise a ring primer；Described ring primer is LB.
6. method as claimed in claim 5, it is characterised in that described can expand Shigella genome specificity base sequence
Primer sets be selected from following primer sets A ', any one group of B '；Or be selected from and described primer sets A ', B ' sequence or its complementary strand sequence
In row, wall scroll sequence homology is 75% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’；
Downstream inner primer BIP_A：
5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3’；
Lower lantern primer LB_A：5’-GTGTGTCGGGTATGATGATGCCG-3’(SEQ ID NO：9)；
Primer sets B '：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’；
Downstream inner primer BIP_B：
5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’；
Lower lantern primer LB_B：5’-GTGTGTCGGGTATGATGATGCCG-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 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 Shigella method as claimed in claim 1, it is characterised in that described primer includes
Can expand the primer sets of Shigella genome specificity base sequence, its sequence is the Shigella that No. GI is 110804074
A part for the nucleotide sequence of 1921404～1921811bp position of genome or a part for its complementary strand.
10. primer as claimed in claim 9, it is characterised in that described can expand Shigella genome specificity base sequence
The primer sets of row is primer sets A；Or with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence be 75% and
Above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’；
Downstream inner primer BIP_A：
5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-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 75% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’；
Downstream inner primer BIP_B：
5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’.
12. primers as claimed in claim 9, it is characterised in that described can expand Shigella genome specificity base sequence
The primer sets of row also comprises a ring primer；Described ring primer is LB.
13. primers as claimed in claim 12, it is characterised in that described can expand Shigella genome specificity base sequence
The primer sets of row is selected from following primer sets A ', any one group of B '；Or be selected from and described primer sets A ', B ' sequence or its complementary strand
In sequence, wall scroll sequence homology is 75% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_A：5’-AAGAGTGATTTCCTGGCCTG-3’；
Upstream inner primer FIP_A：5’-ACGCCCTGAGACAACAGAACCGGGTGTGATAAATGGGGCC-3’；
Downstream inner primer BIP_A：
5’-GGATGCTGAGCACCCCTTCAAACGTGAATATTCCGTTGCTGGC-3’；
Lower lantern primer LB_A：5’-GTGTGTCGGGTATGATGATGCCG-3’；
Primer sets B '：
Upstream outer primer F3_B：5’-AGTCAGTCTGGATATGGGCC-3’；
Downstream outer primer B3_B：5’-TGATTTCCTGGCCTGGGTAA-3’；
Upstream inner primer FIP_B：5’-ACGCCCTGAGACAACAGAACCCGGGTGTGATAAATGGGGC-3’；
Downstream inner primer BIP_B：
5’-GGATGCTGAGCACCCCTTCAAACCTGCCAGGTGAATATTCCGT-3’；
Lower lantern primer LB_B：5’-GTGTGTCGGGTATGATGATGCCG-3’.
14. 1 kinds of kits for Constant Temperature Detection Shigella, it is characterised in that described kit includes such as claim 9
Primer described in any one of～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 Shigella, it is characterised in that the enzyme reaction system bag of described kit
Include：1 × BstDNA polymeric enzyme reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L's
FIP and BIP primer, F3 and the B3 primer of 0.15-0.3 μm of ol/L, including or do not include the LB primer of 0.4-1.0 μm of ol/L,
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 Shigella for 18. primers, it is characterised in that described primer for as claim 9～13 it
Primer described in any one.
19. kits as described in any one of claim 14～16 or carrier as claimed in claim 17 are congratulated in constant temperature will
Application in Salmonella.
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| CN202010036148.6A CN111020048B (en) | 2015-09-02 | 2016-08-30 | Rapid constant-temperature detection method, primer group and kit for shigella |
| CN202010035812.5A CN111073989B (en) | 2015-09-02 | 2016-08-30 | Rapid constant-temperature detection method and application of shigella nucleic acid |
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| CN202010035812.5A Division CN111073989B (en) | 2015-09-02 | 2016-08-30 | Rapid constant-temperature detection method and application of shigella nucleic acid |
| CN202010036148.6A Division CN111020048B (en) | 2015-09-02 | 2016-08-30 | Rapid constant-temperature detection method, primer group and kit for shigella |
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