CN106367493A - Method for rapidly detecting salmonella at constant temperature, primer and applications of primer - Google Patents
Method for rapidly detecting salmonella at constant temperature, primer and applications of primer Download PDFInfo
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- CN106367493A CN106367493A CN201610767608.6A CN201610767608A CN106367493A CN 106367493 A CN106367493 A CN 106367493A CN 201610767608 A CN201610767608 A CN 201610767608A CN 106367493 A CN106367493 A CN 106367493A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a method for rapidly detecting salmonella at constant temperature, a primer group and a kit. The method comprises the following steps: extracting genome DNA from a to-be-detected sample; by taking the genome DNA as a template, and by taking the primer group capable of amplifying specific sequences of the salmonella as primers, carrying out isothermal amplification reaction under an enzyme reaction system; and determining that whether the salmonella exists in the to-be-detected sample or not by judging that whether the reaction result is positive or not. The detection method has high sensitivity and high specificity, the detection time is short, the result judgment is simple, the operation is convenient and rapid, the cost is low, and thus the detection method has a wide application prospect.
Description
Technical field
The invention belongs to biological technical field and in particular to a kind of fast constant temperature detect the method for Salmonella, primer and
Test kit.
Background technology
Salmonella (salmonella spp.) is a class Gram-negative bacillus, mainly inhabits humans and animals
Intestinal in, excreted by feces, and be propagated further by contaminated water and food, people's gastroenteritiss, typhoid fever can be caused
With symptoms such as paratyphoid fever.In recent years, the harm that Salmonella causes in the world is in continuous ascendant trend, is public health
The important pathogenic bacteria in field.Therefore, the detection for this bacterium and prevention are all significant.
Traditional Detection Methods of Salmonella is longer due to detection cycle, and operation is relative complex, detection efficiency relatively low it is difficult to full
Sufficient modern society for food-borne pathogens detection process high flux, high sensitivity, high specific, quick, easily require.Closely
With the development of nucleic acid molecules detection technique over year, research worker have developed pcr and the detection handss of fluorescence pcr technology successively
Section, but both approaches are required for special detecting instrument, therefore, are not appropriate for being widely used in basic unit's detection department especially
It is the real-time on-site detection carrying out inside enterprise's production line.In order to ensure food safety, it is badly in need of quick, simple, accurate method
To detect the Salmonella in food.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, lamp) is in recent years
Come a kind of novel constant-temperature nucleic acid amplification method to grow up, 4 specific primers are designed in 6 regions that this method is directed to target sequence
(include 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), using a kind of dna polymerase with strand-displacement activity, it is incubated about 60min in constant temperature, you can complete core
Sour 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,2000 jun 15；28(12):e63).This technology has
Have and do not need can complete under pcr instrument or fluorescent quantitation pcr instrument, constant temperature, naked eyes can determine whether reaction result, and sensitivity
Height, high specificity, response time is short, simple operation, low cost and other advantages.
Design of primers is a most key step in lamp technology, and Normal practice is by certain biological generally acknowledged spy to be detected
Specific gene imports the online website (http://primerexplorer.jp/e) of lamp design of primers, sets relevant parameter life
Become primer sets.That is, user must assure that the distinguished sequence that this target gene is species to be measured first.With patent of invention
As a example zl201110026963.5 and zl201110433262.3, they are respectively directed to the special base of the Salmonella of document report
Because of fimy gene and inva gene, Salmeterol fluticasone propionate is carried out using lamp technology.However, so-called " generally acknowledged specificity
Gene " is often based upon delayed knowledge, and is not based on ever-increasing microbial genome data and carries out necessary renewal, leads to
Its versatility and/or specificity not necessarily be can ensure that in actual applications based on the primer that this target-gene sequence obtains.The present invention
Illustrate the problem that versatility present in prior art cannot ensure with table 1.That is, used in art methods
Salmeterol fluticasone propionate sequence actually and nonsalmonella common, i.e. be possible to the part bacterial strain of missing inspection Salmonella.Class
As problem exist in specific confirmation, i.e. be possible to mistakenly regard as nonsalmonella Salmonella.Therefore,
Need a kind of Detection Methods of Salmonella being able to ensure that specificity and versatility in industry badly, meet basic unit's detection department pair simultaneously
Quickly, easily demand, easily can carry out real-time on-site detection 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
The not enough defect with specificity, makes full use of abundant microbial genome sequence information in current common data resource and phase
The sequence analysis tools answered, are designed for the primer sets of specific recognition Salmonella, and on this basis formed high sensitivity,
High specific detection kit.The present invention is based on the microbial genome data resource in genbank data base (by 2013
August data on the 5th) carry out the design of Salmonella lamp primer, there is provided a kind of side of fast constant temperature augmentation detection Salmonella
Method, primer sets and test kit.Detection method using the present invention detects Salmonella, has high sensitivity and high specific, inspection
The survey time is short, and result judgement is simple, simple operation, the advantage of low cost.
The present invention proposes a kind of method of quick detection Salmonella strains, the method comprising the steps of:
(1) extract genome dna from testing sample；
(2) with described genome dna as template, so that the primer sets of salmonella gene group-specific base sequence can be expanded
For primer, under enzyme reaction system, carry out isothermal amplification reactions；
(3) pass through to judge whether reaction result is positive, determine and in testing sample, whether there is Salmonella.
The method of Constant Temperature Detection Salmonella strains of the present invention, extracts genome dna, with it as mould from testing sample
Plate, with Salmonella specificity amplification primer group as primer, carries out isothermal amplification reactions, then, by judging that reaction result is
No for the positive, determine and in testing sample, whether there is Salmonella.Wherein, described enzyme reaction system including but not limited to dna gathers
Synthase reaction system.
In the present invention, described salmonella gene group-specific base sequence is the Salmonella that No. gi is 194447306
4188946～4189180bp bit sequence.
In the present invention, the described primer sets that can expand salmonella gene group-specific base sequence are described genome
The part of the nucleotide sequence of 4188946～4189180bp position of (No. gi is 194447306) or a part for its complementary strand.
Wherein, described salmonella gene group-specific base sequence refers to only specific to salmonella gene group, and other micro-
The base sequence that biological genome does not comprise.
Wherein, the described primer sets that can expand salmonella gene group specific base sequence include but is not limited to primer sets a,
Or selected from being 73.7% with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence and the appointing of above primer sets
Anticipate one group.
Primer sets a:
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 ' (seq id no:1)；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 ' (seq id no:2)；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 ' (seq id
No:3)；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 ' (seq id
No:4).
In the present invention, the described primer sets that can expand salmonella gene group specific base sequence can also include with aforementioned
In each primer sets sequence or its complementary strand sequence, wall scroll sequence homology is 73.7% and above primer sets, and this primer sets includes
But it is not limited to following primer sets b:
Primer sets b:
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 ' (seq id no:5)；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 ' (seq id no:6) (with primer b3_a:5 '-
Taacgaacactaacagcag-3 ' homology 73.7%)；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 ' (seq id
No:7)；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 ' (seq
Id no:8).
In the inventive method, the described primer sets that can expand salmonella gene group-specific base sequence can comprise but
It is not limited to a ring primer.Preferably, described ring primer is one, including ring primer lf or lb.Described can expand Salmonella
The primer sets of genome specificity base sequence are selected from following primer sets a ', any one group of b '；Or with described primer sets a ', b '
In sequence or its complementary strand sequence, wall scroll sequence homology is 73.7% and any one group of above primer sets:
Primer sets a ':
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 '；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 '；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 '；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 '；Upper lantern
Primer lf_a:5 '-cggtaaaatcagcgtcctga-3 ' (seq id no:9)；
Primer sets b ':
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 '；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 '；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 '；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 '；
Lower lantern primer lb_b:5 '-ggcgaagcgaaaaaggaaga-3 ' (seq id no:10).
In the inventive method, in specific embodiments (primer containing ring), the enzyme reaction system of described constant-temperature amplification is:
1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+(mgso4Or mgcl2), 1.0-1.6mmol/l dntp,
Fip the and bip primer of 0.8-2.0 μm of ol/l, f3 the and b3 primer of 0.15-0.3 μm of ol/l, lf or lb of 0.4-1.0 μm of ol/l
Primer, 0.16-0.64u/ μ l bst dna polymerase and 0-1.5mol/l glycine betaine.In another specific embodiments (without ring
Primer) in, the enzyme reaction system of described constant-temperature amplification is: 1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+
(mgso4Or mgcl2), fip the and 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 dna polymerase and 0-1.5mol/l glycine betaine.Ring primer is favorably improved reaction
Efficiency.For example, 1 × bst dna polymeric enzyme reaction buffer 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.Mgso in 1 × bst dna polymeric enzyme reaction buffer4With the magnesium ion mg in enzyme reaction system2+Do merging treatment.
In the inventive method, the response procedures of described isothermal amplification reactions are 1. 60～65 DEG C incubation 10～90min, preferably
Ground is 10～60min；2. 80 DEG C of terminating 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 agarose gel or polyacrylamide gel.Electrophoresis detection
In result, band as stepped in electrophoretogram expression characteristicses, then testing sample is in that Salmonella is positive, containing Salmonella；As
The electrophoretogram not stepped band of expression characteristicses, then testing sample is in that Salmonella is negative.Described Turbidity measurement, is with the naked eye to see
Examine or transmissometer detection turbidity, substantially muddiness in detection pipe, then testing sample is in that Salmonella is positive, containing Salmonella；
As muddy in having no, then testing sample is that Salmonella is negative.Whether can also there is precipitation after centrifugation in perusal reaction tube bottom,
If there is precipitation at reaction tube bottom, testing sample is in that Salmonella is positive, containing Salmonella；As reaction tube bottom is not precipitated, then
Testing sample is in that Salmonella 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).When using calcein or sybr green i work
During for developer, after such as reacting, color is orange, then testing sample is that Salmonella is negative；As after reaction, color is green, then
Testing sample is that Salmonella is positive, containing Salmonella.When using hydroxynaphthol blue as developer, color after such as reacting
For pansy, then testing sample is that Salmonella is negative；As after reaction, color is sky blue, then testing sample is Salmonella
Positive.Described color developing detection, in addition to above by perusal 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 Salmonella is negative；When the result of testing sample reaction is more than this threshold value, then testing sample
Positive for Salmonella.Described detecting instrument includes but is not limited to spectrofluorophotometer, fluorescent quantitation pcr instrument, constant-temperature amplification
Micro-fluidic chip 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-in constant-temperature amplification
Should add it is also possible to add it is therefore preferable to add before isothermal amplification reactions after isothermal amplification reactions complete before, permissible
Effectively reduce the probability of 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 Salmonella strains.Described primer includes expanding
Increase the primer sets of salmonella gene group specific base sequence, it includes but is not limited to, and the sequence of described primer for No. gi is
A part for the nucleotide sequence of 4188946～4189180bp position for 194447306 salmonella gene group or its complementary strand
A part.
Wherein, the described primer sets that can expand salmonella gene group-specific base sequence be selected from following primer sets it
Any one group, or selected from wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence be 73.7% and more than
Arbitrary primer sets.Wherein, described primer sets include but is not limited to following primer sets a.Described with aforementioned primer sets sequence or its
In complementary strand sequence, wall scroll sequence homology is 73.7% and above primer sets include but is not limited to following primer sets b.
Primer sets a:
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 '；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 '；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 '；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 '；
Primer sets b:
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 '；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 '；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 '；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 '.
The present invention is used in the primer in described Constant Temperature Detection Salmonella method, described can expand salmonella gene group
The primer sets of specific base sequence can also be including but not limited to a ring primer；Preferably, described ring primer is one, bag
Include lf or lb.The described primer sets that can expand salmonella gene group-specific base sequence are selected from following primer sets a ', b ' it
Any one group；Or selected from and described primer sets a ', in b ' sequence or its complementary strand sequence wall scroll sequence homology be 73.7% and
Any one group of above primer sets:
Primer sets a ':
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 '；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 '；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 '；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 '；
Upper lantern primer lf_a:5 '-cggtaaaatcagcgtcctga-3 '；
Primer sets b ':
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 '；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 '；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 '；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 '；
Lower lantern primer lb_b:5 '-ggcgaagcgaaaaaggaaga-3 '.
The present invention also provide a kind of for the test kit in above-mentioned Constant Temperature Detection Salmonella strains method, it includes described
The primer sets of salmonella gene group specific base sequence can be expanded.In test kit of the present invention, described can expand Salmonella base
Because of the primer sets of group-specific base sequence, including but not limited to genome (No. gi: 4,188,946 194447306)～
A part for a part for the nucleotide sequence of 4189180bp position or its complementary strand is as described primer sequence；Described primer includes
But it is not limited to described primer sets a etc..Also including but not limited to with wall scroll sequence in aforementioned primer sequence or its complementary strand sequence
Homology is 73.7% and above primer sets are as primer；Including but not limited to primer sets b etc..
In test kit of the present invention, the described primer sets that can expand salmonella gene group-specific 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, including lf or lb.Comprise
The primer sets of ring primer lf or lb include but is not limited to primer sets a ', b ' etc..In a particular embodiment, in test kit of the present invention
Lf the or lb ring primer of 0.4-1.0 μm of ol/l can be comprised.In one embodiment, the sequence of primer sets be respectively fip,
Primer shown in bip, f3, b3, lf or fip, bip, f3, b3, lb or same with foregoing sequences or its complementary strand sequence wall scroll primer
Source property is 73.7% and above primer.
In test kit of the present invention, also include bst dna polymerase buffer, bst dna polymerase, dntp solution, mg2+
(mgso4Or mgcl2One or more of) and glycine betaine.In one embodiment, test kit enzyme reaction system of the present invention
Comprise 1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+(mgso4Or mgcl2), 1.0-1.6mmol/l
Fip the and bip primer of dntp, 0.8-2.0 μm of ol/l, f3 the and b3 primer of 0.15-0.3 μm of ol/l, 0.16-0.64u/ μ l bst
Dna polymerase and the glycine betaine of 0-1.5mol/l.For example, 1 × bst dna polymeric enzyme reaction buffer can from 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 polymeric enzyme reaction buffer4With enzyme reaction body
Magnesium ion mg in system2+Do merging treatment.
In test kit of the present invention, also comprise positive control template.In one embodiment, described positive control template
The including but not limited to full-length genome dna of Salmonella, portion gene group dna, or comprise Salmonella full-length genome dna or portion
Divide the carrier of genome dna.
In test kit of the present invention, also comprise negative control template, described negative control template includes but is not limited to distilled water.
In test kit of the present invention, also comprise developer, developer includes but is not limited to calcein, sybr green i or
Hydroxynaphthol blue.When developer is for calcein, in test kit, also comprise [mn2+], for example, mncl2.
In test kit of the present invention, also comprise distilled water.
In test kit of the present invention, also comprise nucleic acid extraction reagent.
The invention allows for a kind of carrier, described carrier comprise selected from primer sets a, b, a ', b ' any one group of primer.
This carrier, due to containing with Salmonella specific dna sequence, therefore can be applicable to microbial taxonomy, icp gene
Group such as learns, evolves at the research field, 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 plasmid etc.), viral vector (as bacteriophage lambda etc.) and artificial coloring
Body carrier (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 of any one primer comprising primer sets b ... comprise primer sets b ' appoint
Carrier pbr322-b ' of one primer of meaning etc..Carrier bacteriophage lambda-a of any one primer comprising primer sets a, comprise primer
Group b the carrier bacteriophage lambda-b of any one primer ... comprise primer sets b ' any one primer carrier bacteriophage lambda-
B ' etc..
The invention allows for selected from primer sets a, b, a ', any one group of b ' of primer in Constant Temperature Detection Salmonella
Application.
The invention allows for application in Constant Temperature Detection Salmonella for the described test kit.
The invention allows for application in Constant Temperature Detection Salmonella for the described carrier.
The present invention provides a kind of side of simple and quick sensitive detection Salmonella for technical field of food safety detection
Method, primer/primer sets, detectable/test kit, the food safety to China has greater significance.Beneficial effect bag of the present invention
Include: high specificity is had using Detection Methods of Salmonella of the present invention, sensitivity is high, detection time is short, result judgement is simple, behaviour
Make convenient, low cost and other advantages.Compared with commonly using detection method at present, the constant-temperature amplification method that the present invention adopts, can be in constant temperature
Under the conditions of carry out, only need to be using simple thermostat it is not necessary to expensive instrument in pcr experiment be 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 including food safety detection department of basic unit pushing 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 carried out combination in any by field general knowledge, all belong to the scope of the present invention.
Brief description
Fig. 1 shows the specificity of the embodiment of the present invention 7 salmonella constant temperature detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 Detection Methods of Salmonella.
Specific embodiment
In conjunction with specific examples below and accompanying drawing, the present invention is described in further detail, the protection content of the present invention
It is not limited to following examples.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.The process of the enforcement present invention,
Condition, reagent, experimental technique etc., in addition to the following content specially referring to, are universal knowledege and the common knowledge of this area,
The present invention is not particularly limited content.
Embodiment 1-6 salmonella constant temperature reaction system and detection method
Detected according to following (1)～(3) step:
(1) extraction of genome dna
For detection Salmonella strain source in Chinese industrial Microbiological Culture Collection administrative center, number
cicc10420.1ml bacterial culturess are taken to use the bacterial nucleic acid extracts kit of Beijing Tiangeng bio-engineering corporation to extract gene
Group dna, dna od260/od280For 1.8, concentration is 408ng/ μ l.
(2) with salmonella gene group dna to be measured as template, it is respectively adopted the test kit (being shown in Table 2, table 3) of autogamy, and press
According to condition described in table 3, prepare reaction system, with Salmonella 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, carry out amplification true
Recognize.
As can be seen from Table 3, detection method and its primer sets being adopted and reaction system can be right well
Salmonella specific fragment is expanded and is obtained testing result.Additionally, when being detected using detector, shortening reaction
Time is to also there being good Detection results (as embodiment 6) during 10min.Therefore, present invention could apply in detection sample being
No containing Salmonella.
Embodiment 7 Salmonella specific detection
Collect 26 plants of nonsalmonella (1～9 in table 4 and Fig. 1 and 14～30), by these bacterial strains and Salmonella strains
(in table 4 and Fig. 1 10～13) are cultivated respectively, take 1ml bacterium solution, using test kit ia, extract antibacterial dna, and with reference to real
Apply reaction system and the condition of example 1, carry out lamp amplification (primer sets are a) respectively and add developer to observe.
As shown in table 4 and Fig. 1, in Fig. 1,1～9 is respectively staphylococcus aureuses, Staphylococcus aureus to its testing result
The golden yellow subspecies of bacterium, staphylococcus epidermidiss, Rhodococcus equi, Bacillus cercuses, bacillus mycoides, listeria monocytogenes,
Ying Nuoke Listerella, listeria ivanovii, 14～30 are respectively shigella dysenteriae, Shigella bogdii, Fu Shi will Hayes
Bacterium, colon bacillus (the type gene of a containing bacillus botulinuss), pathogenic colon bacillus, Diarrheogenil Escherichia coli, product intestinal poison
Plain colon bacillus, enterotoxigenic colon bacillus, hemorrhagic colon bacillus, slope rugged Cronobacter enterobacteria, small intestinal
Colitis yersinia, artificial tuberculosis yersinia genus, Vibrio vulnificus, vibrio parahaemolytious, Freund vibrio, vibrio cholera o1 group and
Song Shi shigella, ntc: negative control, 10～13 are respectively intestinal Salmonella intestinal subspecies, Salmonella enteritidis, mouse typhuss sand
Door Salmonella and moscow' paratyphi B.In Fig. 1, only the product after Salmonella strains amplified reaction is rendered as viride nitens
Color, is positive findingses, as shown in the 10th～No. 13 pipe.And the product after other nonsalmonella bacterial strains and negative control amplified reaction
Thing is all rendered as orange, is negative findings, as the 1st～9 and 14～No. 30 manage and ntc negative control pipe shown in.
Detection kit of the present invention be can be seen that by Fig. 1 and Biao 4 result and detection method has good Salmonella bacterium
Strain specificity, i.e. only Salmonella strains amplification is positive, other nonsalmonella bacterial strains are feminine gender.
Prepare detection kit, the primer adopting in test kit is respectively primer sets b, primer sets a ', primer sets b ', by upper
State method for detecting specificity, respectively obtain same testing result, i.e. after nonsalmonella bacterial strain and negative control amplified reaction
Product be negative findings, the product after Salmonella strains amplified reaction be positive findingses.
Additionally, according to method described in table 1, respectively to primer sets a～b, primer sets a ' specificity of～b ' carries out theory
Analysis, it was found that in the case that each bar primer at most allows 2 mispairing, each primer sets at most have two primer ratios simultaneously
To on nonsalmonella bacterium, show that the specificity of each primer sets is all preferable.
Embodiment 8 sensitivity technique
As described in Example 2 extract antibacterial cicc10420 dna, using test kit iib, and according to 50pg, 5pg,
500fg, 50fg and 5fg dna gradient adds reaction system, and other reaction conditions are carried out respectively with reference to the method for table 3 embodiment 2
Lamp amplification (primer sets are a) and addition developer are observed.As shown in Fig. 2 1-5 be respectively 50pg, 5pg, 500fg, 50fg and
5fg, ntc: negative control.In Fig. 2 50pg process product be rendered as bright green, be positive findingses, 5pg, 500fg,
50fg and 5fg is processed and the product of negative control is rendered as orange, is negative findings.Testing result shows, each reaction
Minimum in pipe (it is approximately equivalent to 10 containing 50pg4Individual antibacterial) dna when can be detected.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets b, primer sets a respectively '～b ', each reaction
In pipe, as little as the dna of 50pg～500fg can be detected.
Embodiment 9 versatility detects
According to the method for embodiment 7, to intestinal Salmonella intestinal subspecies, Salmonella enteritidis, Salmonella typhimurium and second
Type salmonella paratyphi (in table 4 and Fig. 1 10～13) is cultivated respectively and is extracted dna, and carries out lamp amplification (primer
Organize as a), as shown in table 4 and Fig. 1, the product after four kinds of salmonella strain amplified reactions is all rendered as bright green to testing result, is
Positive findingses, show that the versatility of this primer sets is preferable.
Prepare detection kit, the primer adopting in test kit is respectively primer sets b, primer sets a ', primer sets b ', by upper
State versatility detection method, respectively obtain same testing result, that is, four kinds of salmonella strain amplifications are positive, show each primer sets
Versatility preferable.
According to method described in table 1, respectively to primer sets a～b, primer sets a ' versatility of～b ' carries out theory analysis,
It was found that mating completely of the primer region of each primer sets and 38 Salmonella strains, can be used for above-mentioned 38 in theory
The detection of individual Salmonella strains, shows that the versatility of each primer sets is all preferable.
The versatility of primer and specificity analyses in the existing detection method of table 1 Salmonella
Note: a) whole genome sequence of the sequence between primer f3 and b3 in patent and 38 gi bacterial strains of Salmonella is entered
Row bowtie compares, and determines position in genome for the detection zone；Detection zone sequence is entered in common data base resource
Row blast compares, and primer region is mated good for versatility completely.B) detection zone sequence is carried out in common data base resource
Blast compares, and primer region matching degree is higher, and specificity is poorer；If primer can not compare in nonsalmonella strain simultaneously,
Show that specificity is good.
The test kit species of table 2 Constant Temperature Detection Salmonella and main constituents
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention Salmonella and testing result
Table 4 test 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 findingses ,-: negative findings.
Claims (19)
1. a kind of fast constant temperature detects the method for Salmonella it is characterised in that comprising the following steps:
(1) extract genome dna from testing sample；
(2) with described genome dna as template, using can expand the primer sets of salmonella gene group-specific base sequence as
Primer, carries out isothermal amplification reactions under enzyme reaction system；
(3) pass through to judge whether reaction result is positive, determine and in testing sample, whether there is Salmonella；
Wherein, described salmonella gene group-specific base sequence is the salmonella gene group that No. gi is 194447306
4188946～4189180bp bit sequence.
2. the method for claim 1 is it is characterised in that described can expand salmonella gene group-specific base sequence
Primer sets sequence be the nucleotide sequence of salmonella gene group 4188946～4189180bp position that No. gi is 194447306
A part or a part for its complementary strand.
3. method as claimed in claim 2 is it is characterised in that described can expand salmonella gene group-specific base sequence
Primer sets be primer sets a；Or selected from wall scroll sequence homology in described primer sets a sequence or its complementary strand sequence being
73.7% and any one group of above primer sets；
Primer sets a:
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 ' (seq id no:1)；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 ' (seq id no:2)；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 ' (seq id no:
3)；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-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 73.7% and above primer sets include following primer sets b:
Primer sets b:
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 ' (seq id no:5)；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 ' (seq id no:6)；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 ' (seq id no:
7)；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 ' (seq id
No:8).
5. method as claimed in claim 2 is it is characterised in that described can expand salmonella gene group-specific base sequence
Primer sets also comprise a ring primer；Described ring primer is lf or lb.
6. method as claimed in claim 5 is it is characterised in that described can expand salmonella gene group-specific base sequence
Primer sets be selected from following primer sets a ', any one group of b '；Or with described primer sets a ', in b ' sequence or its complementary strand sequence
Wall scroll sequence homology is 73.7% and any one group of above primer sets:
Primer sets a ':
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 '；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 '；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 '；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 '；
Upper lantern primer lf_a:5 '-cggtaaaatcagcgtcctga-3 ' (seq id no:9)；
Primer sets b ':
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 '；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 '；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 '；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 '；
Lower lantern primer lb_b:5 '-ggcgaagcgaaaaaggaaga-3 ' (seq id no:10).
7. the method for claim 1 is it is characterised in that in step (2), described enzyme reaction system includes: 1 × bst
Dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+, fip and bip of 1.0-1.6mmol/l dntp, 0.8-2.0 μm of ol/l
Primer, f3 the and b3 primer of 0.15-0.3 μm of ol/l, 0.16-0.64u/ μ l bst dna polymerase, the Radix Betae of 0-1.5mol/l
Alkali, including or do not include lf the or lb primer of 0.4-1.0 μm of ol/l.
8. the method for claim 1 is it is characterised in that the response procedures of described isothermal amplification reactions are: 1. 60～65
DEG C incubation 10～90min；2. 80 DEG C of terminating reaction 2～20min.
9. for the primer in Constant Temperature Detection Salmonella method as claimed in claim 1 it is characterised in that described primer includes
The primer sets of salmonella gene group-specific base sequence can be expanded, its sequence is the Salmonella that No. gi is 194447306
A part for the nucleotide sequence of 4188946～4189180bp position for genome or a part for its complementary strand.
10. primer as claimed in claim 9 is it is characterised in that described can expand salmonella gene group-specific base sequence
The primer sets of row are primer sets a；Or selected from wall scroll sequence homology in described primer sets a sequence or its complementary strand sequence being
73.7% and any one group of above primer sets；
Primer sets a:
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 '；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 '；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 '；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 '.
11. primers as claimed in claim 10 are it is characterised in that single with described primer sets a sequence or its complementary strand sequence
Bar sequence homology is 73.7% and above primer sets include following primer sets b:
Primer sets b:
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 '；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 '；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 '；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 '.
12. primers as claimed in claim 9 are it is characterised in that described can expand salmonella gene group-specific base sequence
The primer sets of row also comprise a ring primer；Described ring primer is lf or lb.
13. primers as claimed in claim 12 are it is characterised in that described can expand salmonella gene group-specific base sequence
The primer sets of row are selected from following primer sets a ', any one group of b '；Or selected from and described primer sets a ', b ' sequence or its complementary strand
In sequence, wall scroll sequence homology is 73.7% and any one group of above primer sets:
Primer sets a ':
Upstream outer primer f3_a:5 '-aggctttaaaaaggccatg-3 '；
Downstream outer primer b3_a:5 '-taacgaacactaacagcag-3 '；
Upstream inner primer fip_a:5 '-ttttcgcttcgccttgcttaatgccaaacaggataaaacc-3 '；
Downstream inner primer bip_a:5 '-aggaagacgctaaaagccaacgctaaaaccgatatcaaac-3 '；
Upper lantern primer lf_a:5 '-cggtaaaatcagcgtcctga-3 '；
Primer sets b ':
Upstream outer primer f3_b:5 '-aaaaactcggttccatcg-3 '；
Downstream outer primer b3_b:5 '-aacactaacagcagttcg-3 '；
Upstream inner primer fip_b:5 '-gactggttttatcctgtttggcgcgtctatcaaaggcttt-3 '；
Downstream inner primer bip_b:5 '-gctaaatctatcgcggataagcaacggattatacctgctctt-3 '；
Lower lantern primer lb_b:5 '-ggcgaagcgaaaaaggaaga-3 '.
A kind of 14. test kits for Constant Temperature Detection Salmonella are it is characterised in that described test kit includes such as claim 9
Primer described in～13 any one.
15. test kits as claimed in claim 14 it is characterised in that its also include bst dna polymeric enzyme reaction buffer,
Bst dna polymerase, dntp solution, mg2+, one or more of glycine betaine.
A kind of 16. test kits for Constant Temperature Detection Salmonella are it is characterised in that the enzyme reaction system bag of described test kit
Include: 1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+, 1.0-1.6mmol/l dntp, 0.8-2.0 μm of ol/l
Fip and bip primer, f3 the and b3 primer of 0.15-0.3 μm of ol/l, including or do not include lf or lb of 0.4-1.0 μm of ol/l and draw
Thing, 0.16-0.64u/ μ l bst dna polymerase, and the glycine betaine of 0-1.5mol/l.
A kind of 17. carriers are it is characterised in that described carrier comprises the primer as described in any one of claim 9～13.
Application in Constant Temperature Detection Salmonella for 18. primers it is characterised in that described primer be as claim 9～13 it
Primer described in any one.
19. test kits as described in any one of claim 14～16 or carrier as claimed in claim 17 are in Constant Temperature Detection
Application in Salmonella.
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| CN106978503A (en) * | 2017-05-03 | 2017-07-25 | 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 salmonella |
| CN107022638A (en) * | 2017-06-05 | 2017-08-08 | 温和心 | The LAMP primer group of quick detection salmonella and its application |
| CN109402271A (en) * | 2017-08-14 | 2019-03-01 | 中国科学院微生物研究所 | For detecting the primer system and kit and their application and the method for detecting salmonella of salmonella |
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