CN108060209A - A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella - Google Patents
A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella Download PDFInfo
- Publication number
- CN108060209A CN108060209A CN201711427791.6A CN201711427791A CN108060209A CN 108060209 A CN108060209 A CN 108060209A CN 201711427791 A CN201711427791 A CN 201711427791A CN 108060209 A CN108060209 A CN 108060209A
- Authority
- CN
- China
- Prior art keywords
- colloidal gold
- salmonella
- constant
- detection
- recombinase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of kit of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella, the kit includes salmonella gene primer sets DNA, recombinase constant-temperature amplification kit, sterile distilled water and nucleic acid immunization colloidal gold strip.Kit of the present invention is easy to operate, is highly suitable for Site Detection；Testing cost is low, and entire detection process only needs 20min；Remolding sensitivity PCR combinations agarose gel electrophoresis detects 104 times of limit for height, therefore it can be widely used in the detection of various pathogenic bacteria, can it is accurate, sensitive, rapidly detect salmonella.
Description
Technical field
The invention belongs to technical field of food detection, the Salmeterol fluticasone propionate being related in food, especially a kind of recombinase
Polymerase constant-temperature amplification combination immunity colloidal gold test paper strip detects the kit of salmonella and detects sramana using the kit
The method of Salmonella.
Background technology
In recent years, taking place frequently due to food safety affair, the World Health Organization and consumer increasingly pay attention to food security
Problem.Salmonella is a kind of Gram-negative bacteria bacillus, widely distributed in nature, easily pollutes meat, marine product, egg
The numerous food products such as class, consumer eats will trigger food poisoning by salmonella-polluted food.In China, every year by bacterium
In caused food poisoning, salmonellal food poisoning ranks first, and accounts for 40%.The side of detection and identification salmonella
Method mainly includes traditional Bacteria Culture and biochemical identification and PCR method.Time-consuming for classical culture protocols, general 2-3 days, uncomfortable
Together in Site Detection.PCR method is complicated for operation, it is necessary to special operating personnel and expensive thermocirculator.Both detections
Method cannot meet food safety detection and require quick, portable, accurate, specific and high sensitivity requirement.
In order to be applicable in the requirement of Site Detection, constant-temperature amplification method is widely used, its biggest advantage is that can
To react at a constant temperature.Pathogenic bacteria are detected using isothermal amplification technology, simple number water-bath can substitute
Expensive PCR instrument, greatly reduces testing cost.Constant-temperature amplification method mainly includes ring mediated isothermal amplification (LAMP), rolling ring
Amplification (HDA), restructuring enzymatic amplification (RPA) based on amplification (RCA), unwindase.In these constant-temperature amplification methods, unwindase
(RPA) is expanded with easy to operate, reaction temperature low (37 DEG C -42 DEG C), amplification required time short (5-25min) and better than others
Constant-temperature amplification method.Recombinating enzymatic amplification is expanded by simulating in DNA bodies, utilizes recombinase, single strand binding protein and strand displacement
Archaeal dna polymerase, by billions of times of target DNA amplification.Recombinase can unlock double-stranded DNA not by heating.RPA reactions start
When, recombinase combination single stranded DNA (primer) forms nucleic acid-protein complex.These complexs can scan and primer sequence
Arrange complementary target double-stranded DNA.Then, nucleic acid-protein complex intrusion 5' ends site forms D shape rings.Single strand binding protein and quilt
Single-stranded combination is replaced to stabilize it.Meanwhile recombinase leaves the 3' ends of oligonucleotides, is utilized after degradation by archaeal dna polymerase.It
Afterwards, strand displacement archaeal dna polymerase is incorporated in the free 3' ends of nucleic acid-protein complex, carries out chain extension, forms new complementary strand.
During chain extension, the single-stranded and original, complementary chain newly synthesized matches.Above step Xun Huan carries out, it is possible to realize DNA's
Exponential increase.
The method of traditional detection constant-temperature amplification product is using agarose gel electrophoresis, and time-consuming for this method, and needs
Gel imaging system does not meet food-borne pathogens Site Detection simplicity, quickly requirement.Colloid gold immune test strip is
The novel in vitro diagnostic techniques that 20 worlds grow up the nineties.Test strips make simply, cheap, easy to use,
And long shelf-life.At present on test strips are combined with recombinase constant-temperature amplification detection salmonella analysis method not
It appears in the newspapers.The technical operation is simple and quick, as a result easily judges, without complicated instrument and technical professional, has very
Strong field application value.
By retrieval, patent publication us related with the present patent application is not yet found.
The content of the invention
It is immune it is an object of the invention in place of overcome the deficiencies in the prior art, provide a kind of recombinase constant-temperature amplification combination
The kit and the method using kit detection salmonella of colloidal gold strip detection salmonella, the kit have
High sensitivity, high specific, visualization, easy to operate, portable feature.
The present invention solves its technical problem and following technical scheme is taken to realize：
A kind of kit of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella, the kit
Including salmonella gene primer sets DNA, recombinase polymerase constant-temperature amplification kit, sterile distilled water and nucleic acid immunization colloid
Gold test paper strip：
Wherein, the salmonella gene primer sets DNA is the forward primer and reverse primer of salmonella constant temperature amplification,
The forward primer sequence is SEQ1：5 '-digoxinCTACAAGCATGAAATGGCAGAACAGCGTCG-3 ', it is described reversely to draw
Object sequence is SEQ2：5’-biotin-CAACCAGATAGGTAGGTAATGGAATGACGA-3’；
The nucleic acid immunization colloidal gold strip includes PVC backboards, sample pad, bonding pad, nitrocellulose filter and water suction
Paper, the PVC backboards are horizontally disposed, be sequentially connected from left to right on the PVC backboards connect set sample pad, bonding pad,
Nitrocellulose filter and blotting paper, each several part are overlapped in adjacent, parallel in the horizontal direction on the nitrocellulose filter
Detection line and nature controlling line are arranged at intervals, the detection line and nature controlling line are longitudinally disposed, the nitrocellulose at the detection line
It is coated on film and Streptavidin is set, coating sets rabbit-anti mouse secondary antibody, the knot on the nitrocellulose filter at the nature controlling line
It closes coating on pad and digoxin antibody-colloid gold label object is set；
The nucleic acid immunization colloidal gold strip in use, if nature controlling line is without color, it was demonstrated that the nucleic acid immunization glue
Body gold test paper strip fails.
Moreover, the preparation method of the nucleic acid test strip is as follows：
(1) colloidal gold is prepared using trisodium citrate reduction method；
(2) the colloidal gold in taking 1ml steps (1) is added in sterile centrifugation tube, and the anti-digoxin monoclonal of 1mg/ml mouse is resisted
Body 10ul is added in above-mentioned colloidal gold, and anti digoxin antibody-colloid gold label object is made；
By step (2) in prepare anti digoxin antibody-colloid gold label object and be coated on bonding pad；
(4) Streptavidin is coated on the nitrocellulose filter of test strips to be coated with as detection line, and by rabbit-anti mouse secondary antibody
Nature controlling line is used as on nitrocellulose filter；During test strips use, if nature controlling line is without color, it was demonstrated that test strips fail；
(5) sample pad, bonding pad, nitrocellulose filter, blotting paper are pasted in order on PVC backboards to get nucleic acid immunization
Colloidal gold strip.
Moreover, the sample pad and nitrocellulose filter are purchased from Millipore companies of the U.S., the bonding pad, blotting paper
With PVC backboards purchased from Shanghai Jin Biao biotechnologies company, the Streptavidin is purchased from sigma companies, the anti-digoxin of mouse
Monoclonal antibody and rabbit-anti mouse polyclonal antibody are purchased from Abcam companies.
Moreover, the preparation method of the colloidal gold is as follows：
Colloidal gold is prepared using trisodium citrate reduction gold chloride method：
200ml deionized waters is first taken to pour into conical flask, in be heated on thermostatic electromagnetic power blender boiling one minute,
Conical flask is cleaned, outwells boiling water afterwards；99ml deionized waters are taken into conical flask, the mass concentration for taking 1ml is 1%
HAuCl4Into the deionized water of above-mentioned 99ml, it is heated to seething with excitement；After boiling, it is 1% to rapidly join 2.25ml mass concentrations
Citric acid three sodium solution, 2min are interior it is observed that apparent color change；When claret is presented in colloidal gold color and keeps
After constant, continue to heat 15min, be stored in after cooling down at room temperature afterwards in brown, wide-mouth bottle, 4 DEG C save backup；It prepares
Colloidal gold solution is limpid transparent, and surface is without floating material.
Moreover, the recombinase polymerase constant-temperature amplification kit is purchased from TwistDx Ltd.Babraham, UK companies.
Moreover, the recombinase polymerase constant-temperature amplification kit can augmentation detection nucleic acid molecules, the length of amplified fragments
It spends for 188bp, concentration >=20fg of target dna.
Moreover, the temperature of the recombinase polymerase constant-temperature amplification kit amplification is 39 DEG C, proliferation time 10min.
It, can be with the monoclonal anti-digoxin anticody knot that is marked on colloidal gold moreover, be connected with digoxin on the forward primer
It closes；Biotin is connected on reverse primer, can be combined with coated Streptavidin in test strips T line.
Utilize the kit of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip as described above detection salmonella
The method for detecting salmonella, step are as follows：
(1) 1ml salmonella bacterium solutions are taken, and 18000g centrifugation 5min abandon supernatant, are resuspended with PBS, incubated in 95 DEG C of water-baths
5min is educated, 5000g centrifugation 10min take supernatant, and extraction obtains DNA；
(2) using step, (1) the middle obtained DNA that extracts as template, takes 2.5ul to be added in PCR reaction tubes, uses recombinase
Polymerase constant-temperature amplification kit expands it, adds in Twist Basic buffer, the 10.5ul sterile waters of 29.5ul,
The forward primer of 2.4ul 10uM, the reverse primer 2.4ul of 2.4ul 10uM recombinate polymerase one, add in after mixing
2.5ul MgSO4, overall reaction system is 50ul, be placed in 39 DEG C of water-bath and react 10min, obtain amplified production；
(3) the amplified production 10ul in taking step (2) after being mixed with 90ul 0.01M PBS, is added drop-wise to nucleic acid immunization colloid
In the sample pad of gold test paper strip, visual results after 2~3min；
As a result interpretation：Visually direct interpretation：
1) it is negative (-)：Only there is a black gray expandable band in nature controlling line, occur in detection line without black gray expandable band, card
Bright detected sample is not salmonella-polluted；
2) it is positive (+)：There are two black gray expandable bands, one is located in detection line, and another is located in nature controlling line, it was demonstrated that
There are salmonella-polluted for the sample detected.
The advantages of present invention obtains and good effect are：
1st, kit of the present invention is easy to operate, is highly suitable for Site Detection；Testing cost is low, and entire detection process only needs
20min；Remolding sensitivity PCR combinations agarose gel electrophoresis detects limit for height 104Times, therefore it can be widely used in various pathogenic bacteria
Detection, can it is accurate, sensitive, rapidly detect salmonella.
2nd, since RPA constant-temperature amplification kits are easy to operate, without complicated reaction kit, recombinase, single-stranded knot are utilized
Hop protein, archaeal dna polymerase realize isothermal duplication under the conditions of 39 DEG C, therefore the kit use cost of the present invention is relatively low；This hair
The bright kit reaction time is rapid, and nucleic acid amplification can be completed in 10min, and ELISA test strip is obtained in 2min as a result, whole
A detection process includes：Boiling method extracts DNA, and RPA amplifications, ELISA test strip, entire detection process can be complete in 25min
Into.
3rd, the reaction result that kit of the present invention obtains is intuitively accurate, without carrying out complicated operation, although RPA amplification productions
Object can use agarose gel electrophoresis this time that detect, but need more complicated detecting instrument, time-consuming and sensitivity
It is low, if digoxin and biotin marks are carried out respectively at 5 ' ends of positive anti-primer, then using immunoassay test strip to expanding
Volume increase object is detected, and not only accurately can be carried out visual inspection to result but also can be improved the sensitivity of detection, and make it
It is more suitable for that Site Detection is quick, shirtsleeve operation requirement.
4th, kit of the present invention can quickly, sensitively detect salmonella, without expensive instrument, only need a number
Change water-bath and reaction can be completed, kit is easy to operate, and reaction result is easy to observe, and specificity is good, is highly suitable for food
Health and the Site Detection of livestock-raising field, are easy to promote the use of on a large scale.
5th, recombinase isothermal amplification technology is a kind of utilization recombinase, single strand binding protein and archaeal dna polymerase in constant temperature
Under degree (37 DEG C -42 DEG C), using forward and reverse primer, the new technology of realization target nucleic acid exponential amplification, this amplification is not required to
Complicated Thermal Cycling is wanted, without special instrument, the requirement to operating personnel is not also high, has simple, portable, quick etc.
Feature.Recombinase constant-temperature amplification product is detected using immunity colloidal gold test paper strip, and the composition of test strips includes sample pad, knot
Close pad, nitrocellulose filter, blotting paper and PVC backboards.Streptavidin and rabbit-anti are coated on nitrocellulose filter (NC films)
Mouse secondary antibody respectively constitutes detection line (T) and nature controlling line (C).
Description of the drawings
Fig. 1 is test strip operation principle, assemble method and the structure of amplifying nucleic acid immunity colloidal gold test paper strip of the present invention
Connection diagram；
Fig. 2 is optimization optimal reaction temperature and reaction time result figure in the present invention；Wherein, A is optimization optimum response temperature
Spend result figure；B is optimization optimum reacting time result figure；
Fig. 3 is detection limit figure of the constant-temperature amplification combination test strips in pure bacterium solution in the present invention：RPA amplification salmonellas
DNA expands 5min and 10min respectively with test strips and detected through gel electrophoresis amplified production, RPA amplified reaction times at 39 DEG C
For 5min, gel electrophoresis and the detection of test strips limit are respectively 20pg and 200pg, as shown in A, B in Fig. 3；During RPA amplified reactions
Between for 10min, gel electrophoresis and the detection of test strips limit are respectively 2pg and 20fg, such as C, D in Fig. 3；When template amount be 20fg,
Using the analysing amplified product of test strips, test strips T line color is substantially deeper than negative control test strips T line color, and therefore, 20fg is husky
Door Salmonella template DNA is the minimum detectability of RPA-LF；
Fig. 4 is the specificity verification figure of constant-temperature amplification combination test strips in the present invention：Under optimal amplification reaction condition,
Choose the specificity of 5 plants of salmonellas and 6 plants of nonsalmonella detection RPA-LF；Wherein, A detects salmonella strain for RPA-LF
Outer proof diagram, B detect proof diagram in salmonella strain for RPA-LF；
Fig. 5 is the inoculation 1.05 × 10 in milk, Fresh Grade Breast and egg respectively in the present invention0, 1.05 × 101, 1.05 ×
102The salmonella of CFU/mL or CFU/g increases bacterium 0-4h at 37 DEG C；Wherein, A, D, G are by the salmonella of various concentration
It is inoculated into milk sample, for the salmonella of various concentration is inoculated into Fresh Grade Breast sample, C, F, I are by difference by B, D, H
The salmonella of concentration is inoculated into egg sample；A, B, C are that sample is increased bacterium 0h, D, E, F to be by sample increasing bacterium 1h, G, H
Increase bacterium 2h, I to increase bacterium 4h.
Specific embodiment
With reference to embodiment, the present invention is further described；Following embodiments are illustrative, be not it is limited,
Protection scope of the present invention cannot be limited with following embodiments.
Raw material used in the present invention is conventional commercial product unless otherwise specified；Used in the present invention
Method is the conventional method of this field unless otherwise specified.
A kind of kit of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella, feature exist
In：The kit includes salmonella gene primer sets DNA, recombinase polymerase constant-temperature amplification kit (TwistAmp
Basic), sterile distilled water and nucleic acid immunization colloidal gold strip：
Wherein, the salmonella gene primer sets DNA is the forward primer and reverse primer of salmonella constant temperature amplification,
The forward primer sequence is SEQ1：5 '-digoxinCTACAAGCATGAAATGGCAGAACAGCGTCG-3 ', it is described reversely to draw
Object sequence is SEQ2：5’-biotin-CAACCAGATAGGTAGGTAATGGAATGACGA-3’；
The nucleic acid immunization colloidal gold strip includes PVC backboards, sample pad, bonding pad, nitrocellulose filter and water suction
Paper, the PVC backboards are horizontally disposed, be sequentially connected from left to right on the PVC backboards connect set sample pad, bonding pad,
Nitrocellulose filter and blotting paper, each several part is overlapped in adjacent (part), along level side on the nitrocellulose filter
Detection line and nature controlling line are set to parallel interval, the detection line and nature controlling line are longitudinally disposed, the nitric acid at the detection line
It being coated on cellulose membrane and Streptavidin is set, coating sets rabbit-anti mouse secondary antibody on the nitrocellulose filter at the nature controlling line,
Coating sets digoxin antibody-colloid gold label object on the bonding pad；
The nucleic acid immunization colloidal gold strip in use, if nature controlling line is without color, it was demonstrated that the nucleic acid immunization glue
Body gold test paper strip fails.
More preferably, the preparation method of the nucleic acid test strip is as follows：
(1) colloidal gold is prepared using trisodium citrate reduction method；
(2) the colloidal gold in taking 1ml steps (1) is added in sterile centrifugation tube, and the anti-digoxin monoclonal of 1mg/ml mouse is resisted
Body 10ul is added in above-mentioned colloidal gold, and anti digoxin antibody-colloid gold label object is made；
By step (2) in prepare anti digoxin antibody-colloid gold label object and be coated on bonding pad；
(4) Streptavidin is coated on the nitrocellulose filter of test strips to be coated with as detection line, and by rabbit-anti mouse secondary antibody
Nature controlling line is used as on nitrocellulose filter；During test strips use, if nature controlling line is without color, it was demonstrated that test strips fail；
(5) sample pad, bonding pad, nitrocellulose filter, blotting paper are pasted in order on PVC backboards to get nucleic acid immunization
Colloidal gold strip.
More preferably, the sample pad and nitrocellulose filter are purchased from Millipore companies of the U.S., the bonding pad, water suction
Purchased from Shanghai Jin Biao biotechnologies company, the Streptavidin is purchased from sigma companies for paper and PVC backboards, and the anti-ground of mouse is high
Pungent monoclonal antibody and rabbit-anti mouse polyclonal antibody are purchased from Abcam companies.
More preferably, the preparation method of the colloidal gold is as follows：
Colloidal gold is prepared using trisodium citrate reduction gold chloride method：
200ml deionized waters is first taken to pour into conical flask, in be heated on thermostatic electromagnetic power blender boiling one minute,
Conical flask is cleaned, outwells boiling water afterwards；99ml deionized waters are taken into conical flask, the mass concentration for taking 1ml is 1%
HAuCl4Into the deionized water of above-mentioned 99ml, it is heated to seething with excitement；After boiling, it is 1% to rapidly join 2.25ml mass concentrations
Citric acid three sodium solution, 2min are interior it is observed that apparent color change；When claret is presented in colloidal gold color and keeps
After constant, continue to heat 15min, be stored in after cooling down at room temperature afterwards in brown, wide-mouth bottle, 4 DEG C save backup；It prepares
Colloidal gold solution is limpid transparent, and surface is without floating material.
More preferably, the recombinase polymerase constant-temperature amplification kit (TwistAmp Basic) is purchased from TwistDx
Ltd.Babraham, UK company.
More preferably, the recombinase polymerase constant-temperature amplification kit (TwistAmp Basic) being capable of augmentation detection nucleic acid
Molecule, the length of amplified fragments is 188bp, concentration >=20fg of target dna.
More preferably, the temperature of recombinase polymerase constant-temperature amplification kit (TwistAmp Basic) amplification is 39
DEG C, proliferation time 10min.
More preferably, digoxin is connected on the forward primer, it can be with the monoclonal anti-digoxin anticody that is marked on colloidal gold
With reference to；Biotin is connected on reverse primer, can be combined with coated Streptavidin in test strips T line.
Utilize the kit of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip as described above detection salmonella
The method for detecting salmonella, step are as follows：
(1) 1ml salmonella bacterium solutions are taken, and 18000g centrifugation 5min abandon supernatant, are resuspended with PBS, incubated in 95 DEG C of water-baths
5min is educated, 5000g centrifugation 10min take supernatant, and extraction obtains DNA；
(2) using step, (1) the middle obtained DNA that extracts as template, takes 2.5ul to be added in PCR reaction tubes, uses recombinase
Polymerase constant-temperature amplification kit (TwistAmp Basic) expands it, adds in the Twist Basic of 29.5ul
Buffer, 10.5ul sterile water, the forward primer of 2.4ul 10uM, the reverse primer 2.4ul of 2.4ul 10uM recombinate polymerase
One, 2.5ul MgSO are added in after mixing4, overall reaction system is 50ul, be placed in 39 DEG C of water-bath and react
10min obtains amplified production；
(3) the amplified production 10ul in taking step (2) after being mixed with 90ul 0.01M PBS, is added drop-wise to nucleic acid immunization colloid
In the sample pad of gold test paper strip, visual results after 2~3min；
As a result interpretation：Visually direct interpretation：
1) it is negative (-)：Only there is a black gray expandable band in nature controlling line, occur in detection line without black gray expandable band, card
Bright detected sample is not salmonella-polluted；
2) it is positive (+)：There are two black gray expandable bands, one is located in detection line, and another is located in nature controlling line, it was demonstrated that
There are salmonella-polluted for the sample detected.
More preferably, the specific method using kit of the present invention detection salmonella is as follows：
1st, using boiling method extraction salmonella gene group DNA：
Specific method is：1ml thalline are taken, 1,8000g centrifugation 5min removes supernatant, is resuspended afterwards with the PBS buffer solution of 1ml,
5min is incubated in 95 DEG C of water-baths, 5000g centrifugation 10min take supernatant spare into clean centrifuge tube；
2nd, the primer sets needed for RPA amplifications are as follows：
Forward primer sequence is：5 '-digoxinCTACAAGCATGAAATGGCAGAACAGCGTCG-3 ',
Reverse primer sequences are：5’-biotin-CAACCAGATAGGTAGGTAATGGAATGACGA-3’；
The RPA constant-temperature amplification kits of the detection salmonella are purchased from TwistDx Ltd. (Babraham, UK) public affairs
Department；
The RPA nucleic acid test strip kits of the detection salmonella, also comprising sterile distilled water, salmonella gene
Group DNA, further includes the nucleic acid test strip prepared in the content of the invention；
The method of the RPA nucleic acid test strips kit detection salmonella of the detection salmonella, comprising following
Step：
(1) RPA reaction systems are prepared, the Buffer of addition 29.5ul into the PCR pipe of 200ul, 10.5ul sterile waters, just
To primer 2 .4ul (10uM), reverse primer 2.4ul (10uM), template DNA 2.5ul recombinate polymerase 1, are added in after mixing
2.5ul MgSO4, overall reaction system is 50ul, is placed in 39 DEG C of water-bath and reacts 10min；
(2) the RPA amplified productions of 10ul steps (1) are taken, with the PBS buffer solution (0.01M) of 90ul after mixing, with system
The nucleic acid test strip got ready detects, and result is observed after 3min；
(3) result interpretation：Visually direct interpretation：
1) it is negative (-)：Only there is a black gray expandable band in quality control region (C), without black gray expandable band in detection zone (T)
Occur, it was demonstrated that the sample detected is not salmonella-polluted；
2) it is positive (+)：There are two black gray expandable bands, one is located in detection zone (T), and another is located at quality control region (C)
It is interior, it was demonstrated that there are salmonella-polluted for the sample detected；
(4) judgement of sensitivity：The DNA of RPA amplification salmonellas expands 5min and 10min at 39 DEG C and uses test paper respectively
Item and detected through gel electrophoresis amplified production, RPA amplified reaction times are 5min, and gel electrophoresis and the detection of test strips limit are respectively
20pg and 200pg, such as attached drawing 3A, shown in B.The RPA amplified reaction times are 10min, gel electrophoresis and the detection of test strips limit point
Not Wei 2pg and 20fg, such as Fig. 3 C, D.When template amount is 20fg, using the analysing amplified product of test strips, test strips T line color is bright
Aobvious deeper than negative control test strips T line color, therefore, 20fg salmonella template DNAs are the minimum detectabilities of RPA-LF.
The minimum detection limit experiment of salmonella total plate count is then carried out, it is dilute that the pure bacterium solution of salmonella is carried out series
It releases, it is respectively 1.05 × 10 to obtain clump count0To 1.05 × 108The pure bacterium solution of CFU/mL discharges thalline using boiling water bath 15min
DNA, 10000g centrifuging and taking supernatant as the template of RPA amplified reactions, are analyzed amplified production using test strips, simultaneously
Using PCR combination test strips to 1.05 × 100To 1.05 × 108The pure bacterium solution of salmonella of CFU/mL is detected, and compares RPA
With the sensitivity of PCR, as shown in figure 4, RPA-LF is limited to 1.05 × 10 to the detection of pure bacterium solution1CFU/mL, and the inspection of PCR-LF
Rising limit is 1.05 × 105CFU/mL。
Optimize optimal reaction temperature and the time of RPA reactions in the present invention：
In order to determine the optimum temperature of RPA reactions, amplification temperature is chosen as 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, 41
DEG C, the time of amplified reaction chooses 10min, and the product of amplified reaction is analyzed by test strips, as shown in A in Fig. 2,
It is expanded at 36 DEG C, 37 DEG C and 38 DEG C, test strips paler colour, the amplified reaction at 39 DEG C, 40 DEG C and 41 DEG C, test strips face
Therefore color, selects 39 DEG C of reaction temperatures as subsequent experimental without marked difference.Then examine the amplified reaction time anti-to expanding
The influence answered, the amplified reaction time of selection is respectively 2.5,5,10,15,20 and 25min, as shown in B in Fig. 2, from 2.5min
To 10min, with the extension of amplified reaction time, the brightness of test strips is gradually deepened.The amplified reaction time for 10min or
15min, the brightness of test paper band is without marked difference.It is 20 or 25min when the amplified reaction time, although test paper band is bright
Degree is deepened, but the RPA reaction time is long, easily generates the false positive generated due to non-specific amplification.Therefore, it is of the invention
In, the optimal proliferation time selected is 10min.
Use the related test results of the specificity of kit of the present invention detection salmonella：
Under optimal amplification reaction condition, choose 5 plants of salmonellas and 6 plants of nonsalmonella detect the special of RPA-LF
Property.As shown in figure 4,5 plants of salmonellas, after RPA amplified reactions, test strips T line color is brighter, 6 plants of nonsalmonella test paper
Bar T lines do not develop the color, therefore, it can be proved that RPA-LF has preferable specificity.
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>A kind of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detects the kit of salmonella and utilizes and is somebody's turn to do
The method that kit detects salmonella
<130> 2017-12-22
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Forward primer sequence is SEQ1 ()
<400> 1
ctacaagcat gaaatggcag aacagcgtcg 30
<210> 2
<211> 30
<212> DNA
<213>Reverse primer sequences are SEQ2 ()
<400> 2
caaccagata ggtaggtaat ggaatgacga 30
Claims (9)
1. a kind of kit of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella, it is characterised in that：
The kit includes salmonella gene primer sets DNA, recombinase polymerase constant-temperature amplification kit, sterile distilled water and core
Sour immunity colloidal gold test paper strip：
Wherein, the salmonella gene primer sets DNA is the forward primer and reverse primer of salmonella constant temperature amplification, described
Forward primer sequence is SEQ1：5 '-digoxinCTACAAGCATGAAATGGCAGAACAGCGTCG-3 ', the reverse primer sequence
It is classified as SEQ2：5’-biotin-CAACCAGATAGGTAGGTAATGGAATGACGA-3’；
The nucleic acid immunization colloidal gold strip includes PVC backboards, sample pad, bonding pad, nitrocellulose filter and blotting paper, institute
It is horizontally disposed to state PVC backboards, is sequentially connected from left to right on the PVC backboards and connects setting sample pad, bonding pad, nitric acid fibre
The plain film of dimension and blotting paper, each several part are overlapped in adjacent, and parallel interval is set in the horizontal direction on the nitrocellulose filter
Detection line and nature controlling line are put, the detection line and nature controlling line are longitudinally disposed, are wrapped on the nitrocellulose filter at the detection line
It is set Streptavidin, coating sets rabbit-anti mouse secondary antibody on the nitrocellulose filter at the nature controlling line, on the bonding pad
Coating sets digoxin antibody-colloid gold label object；
The nucleic acid immunization colloidal gold strip in use, if nature controlling line is without color, it was demonstrated that the nucleic acid immunization colloidal gold
Test strips fail.
2. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 1
Box, it is characterised in that：The preparation method of the nucleic acid test strip is as follows：
(1) colloidal gold is prepared using trisodium citrate reduction method；
(2) the colloidal gold in taking 1ml steps (1) is added in sterile centrifugation tube, by 1mg/ml mouse monoclonal anti-digoxin anticodies
10ul is added in above-mentioned colloidal gold, and anti digoxin antibody-colloid gold label object is made；
By step (2) in prepare anti digoxin antibody-colloid gold label object and be coated on bonding pad；
(4) Streptavidin is coated on the nitrocellulose filter of test strips and is coated on nitre as detection line, and by rabbit-anti mouse secondary antibody
Nature controlling line is used as on acid cellulose film；During test strips use, if nature controlling line is without color, it was demonstrated that test strips fail；
(5) sample pad, bonding pad, nitrocellulose filter, blotting paper are pasted in order on PVC backboards to get nucleic acid immunization colloid
Gold test paper strip.
3. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 2
Box, it is characterised in that：The sample pad and nitrocellulose filter are purchased from Millipore companies of the U.S., the bonding pad, blotting paper
With PVC backboards purchased from Shanghai Jin Biao biotechnologies company, the Streptavidin is purchased from sigma companies, the anti-digoxin of mouse
Monoclonal antibody and rabbit-anti mouse polyclonal antibody are purchased from Abcam companies.
4. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 2
Box, it is characterised in that：The preparation method of the colloidal gold is as follows：
Colloidal gold is prepared using trisodium citrate reduction gold chloride method：
200ml deionized waters is first taken to pour into conical flask, in being heated to boiling one minute on thermostatic electromagnetic power blender, are cleaned
Conical flask outwells boiling water afterwards；99ml deionized waters are taken into conical flask, the HAuCl that the mass concentration for taking 1ml is 1%4
Into the deionized water of above-mentioned 99ml, it is heated to seething with excitement；After boiling, the citric acid that 2.25ml mass concentrations are 1% is rapidly joined
Three sodium solutions, 2min are interior it is observed that apparent color change；After claret is presented in colloidal gold color and remains unchanged,
Continue to heat 15min, be stored in after cooling down at room temperature afterwards in brown, wide-mouth bottle, 4 DEG C save backup；The colloidal gold prepared is molten
Liquid is limpid transparent, and surface is without floating material.
5. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 1
Box, it is characterised in that：The recombinase polymerase constant-temperature amplification kit is purchased from TwistDx Ltd.Babraham, UK companies.
6. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 1
Box, it is characterised in that：The recombinase polymerase constant-temperature amplification kit can augmentation detection nucleic acid molecules, the length of amplified fragments
It spends for 188bp, concentration >=20fg of target dna.
7. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 1
Box, it is characterised in that：The temperature of the recombinase polymerase constant-temperature amplification kit amplification is 39 DEG C, proliferation time 10min.
8. the reagent of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella according to claim 1
Box, it is characterised in that：Digoxin is connected on the forward primer, it can be with the monoclonal anti-digoxin anticody knot that is marked on colloidal gold
It closes；Biotin is connected on reverse primer, can be combined with coated Streptavidin in test strips T line.
9. using such as claim 1 to 8 any one of them recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection sand
The method of the kit detection salmonella of door Salmonella, it is characterised in that：Step is as follows：
(1) 1ml salmonella bacterium solutions are taken, and 18000g centrifugation 5min abandon supernatant, are resuspended with PBS, are incubated in 95 DEG C of water-baths
5min, 5000g centrifuge 10min, take supernatant, and extraction obtains DNA；
(2) using step, (1) the middle obtained DNA that extracts as template, takes 2.5ul to be added in PCR reaction tubes, uses restructuring enzymatic polymerization
Enzyme constant-temperature amplification kit expands it, adds in Twist Basic buffer, the 10.5ul sterile waters of 29.5ul,
The forward primer of 2.4ul 10uM, the reverse primer 2.4ul of 2.4ul 10uM recombinate polymerase one, add in after mixing
2.5ul MgSO4, overall reaction system is 50ul, be placed in 39 DEG C of water-bath and react 10min, obtain amplified production；
(3) the amplified production 10ul in taking step (2) after being mixed with 90ul 0.01M PBS, is added drop-wise to the examination of nucleic acid immunization colloidal gold
In the sample pad of paper slip, visual results after 2~3min；
As a result interpretation：Visually direct interpretation：
1) it is negative (-)：Only there is a black gray expandable band in nature controlling line, occur in detection line without black gray expandable band, it was demonstrated that institute
The sample of detection is not salmonella-polluted；
2) it is positive (+)：There are two black gray expandable bands, one is located in detection line, and another is located in nature controlling line, it was demonstrated that is examined
There are salmonella-polluted for the sample of survey.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711427791.6A CN108060209A (en) | 2017-12-26 | 2017-12-26 | A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711427791.6A CN108060209A (en) | 2017-12-26 | 2017-12-26 | A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108060209A true CN108060209A (en) | 2018-05-22 |
Family
ID=62140220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711427791.6A Pending CN108060209A (en) | 2017-12-26 | 2017-12-26 | A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060209A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957622A (en) * | 2019-03-27 | 2019-07-02 | 华南农业大学 | It is a kind of detect duck infectious serositis RPA-LFD visualizing agent box and its application |
| CN110241021A (en) * | 2019-05-23 | 2019-09-17 | 广州普世利华科技有限公司 | One kind exempting from instrument nucleic acid on-site quick detection kit |
| CN110794130A (en) * | 2019-10-22 | 2020-02-14 | 中科佑隆（杭州）食安标准科技有限公司 | Nucleic acid two-in-one immune gold-labeled rapid detection card, preparation method and detection method thereof |
| CN110982914A (en) * | 2019-12-18 | 2020-04-10 | 江苏海洋大学 | Specific forward and reverse primers and probe for salmonella, detection kit and application of specific forward and reverse primers and probe |
| CN111505275A (en) * | 2020-03-20 | 2020-08-07 | 浙江工业大学 | Cas9 nucleic acid isothermal amplification-based immunochromatography multiple gene detection method |
| CN111560449A (en) * | 2020-05-20 | 2020-08-21 | 中国农业科学院农业质量标准与检测技术研究所 | Kit for early and rapid diagnosis of salmonella pullorum |
| CN112779358A (en) * | 2021-01-27 | 2021-05-11 | 重庆威斯腾前沿生物研究院有限责任公司 | Immunochromatography method for detecting HPV16 type E6 gene mediated by dcas9 |
| CN114525352A (en) * | 2022-03-24 | 2022-05-24 | 郑州轻工业大学 | Method for detecting pathogenic bacteria at high flux by combining multiple PCR and colloidal gold test strip |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331710A (en) * | 2015-11-20 | 2016-02-17 | 浙江省疾病预防控制中心 | Nucleic acid isothermal amplification detection kit for Salmonella and detection method |
| CN106636387A (en) * | 2016-12-14 | 2017-05-10 | 天津科技大学 | Salmonella nucleic acid rapid detection kit, test strip and detection method |
-
2017
- 2017-12-26 CN CN201711427791.6A patent/CN108060209A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331710A (en) * | 2015-11-20 | 2016-02-17 | 浙江省疾病预防控制中心 | Nucleic acid isothermal amplification detection kit for Salmonella and detection method |
| CN106636387A (en) * | 2016-12-14 | 2017-05-10 | 天津科技大学 | Salmonella nucleic acid rapid detection kit, test strip and detection method |
Non-Patent Citations (2)
| Title |
|---|
| HAIBIN LIU等: "Development of an isothermal amplification-based assay for the rapid visual detection of salmonella bacteria", 《JOURNAL OF DAIRY SCIENCE》 * |
| 吕世静等: "《临床免疫学检验》", 31 August 2015, 中国医药科技出版社 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109957622A (en) * | 2019-03-27 | 2019-07-02 | 华南农业大学 | It is a kind of detect duck infectious serositis RPA-LFD visualizing agent box and its application |
| CN110241021A (en) * | 2019-05-23 | 2019-09-17 | 广州普世利华科技有限公司 | One kind exempting from instrument nucleic acid on-site quick detection kit |
| CN110241021B (en) * | 2019-05-23 | 2022-04-12 | 广州普世利华科技有限公司 | Instrument-free nucleic acid on-site rapid detection kit |
| CN110794130A (en) * | 2019-10-22 | 2020-02-14 | 中科佑隆（杭州）食安标准科技有限公司 | Nucleic acid two-in-one immune gold-labeled rapid detection card, preparation method and detection method thereof |
| CN110982914A (en) * | 2019-12-18 | 2020-04-10 | 江苏海洋大学 | Specific forward and reverse primers and probe for salmonella, detection kit and application of specific forward and reverse primers and probe |
| CN111505275A (en) * | 2020-03-20 | 2020-08-07 | 浙江工业大学 | Cas9 nucleic acid isothermal amplification-based immunochromatography multiple gene detection method |
| CN111505275B (en) * | 2020-03-20 | 2023-03-31 | 浙江工业大学 | Cas9 nucleic acid isothermal amplification-based immunochromatography multiple gene detection method |
| CN111560449A (en) * | 2020-05-20 | 2020-08-21 | 中国农业科学院农业质量标准与检测技术研究所 | Kit for early and rapid diagnosis of salmonella pullorum |
| CN112779358A (en) * | 2021-01-27 | 2021-05-11 | 重庆威斯腾前沿生物研究院有限责任公司 | Immunochromatography method for detecting HPV16 type E6 gene mediated by dcas9 |
| CN112779358B (en) * | 2021-01-27 | 2023-06-09 | 重庆威斯腾前沿生物研究院有限责任公司 | Immunochromatography method for detecting HPV16 type E6 gene by non-diagnostic dcas9 mediation |
| CN114525352A (en) * | 2022-03-24 | 2022-05-24 | 郑州轻工业大学 | Method for detecting pathogenic bacteria at high flux by combining multiple PCR and colloidal gold test strip |
| CN114525352B (en) * | 2022-03-24 | 2024-05-17 | 郑州轻工业大学 | Method for detecting pathogenic bacteria by combining multiple PCR and colloidal gold test strip with high flux |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108060209A (en) | A kind of kit and detection method of recombinase constant-temperature amplification combination immunity colloidal gold test paper strip detection salmonella | |
| Churchill et al. | Detection of Listeria monocytogenes and the toxin listeriolysin O in food | |
| CN110551846B (en) | Cpf1 kit for quickly detecting African swine fever virus nucleic acid and detection method thereof | |
| Wang et al. | Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Listeria monocytogenes | |
| CN104862420B (en) | Primer, probe and kit for detecting foot-and-mouth disease virus aerosol by RPA-lateral flow chromatography | |
| CN104946795B (en) | Primer, probe and kit for Site Detection various serotype foot and mouth disease virus | |
| CN110804670A (en) | Specific primer pair and kit for rapidly detecting vibrio parahaemolyticus based on RPA-LFS and application | |
| CN110004250A (en) | A kind of African swine fever virus LAMP visual detection kit | |
| CN106868152A (en) | A kind of detection method of food-borne pathogens salmonella | |
| JP2023530205A (en) | Typhoid test kit, its manufacturing method and its use | |
| CN110283935A (en) | A kind of African swine fever virus LAMP detection kit and its application | |
| CN108148894A (en) | A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes | |
| CN110004240A (en) | The real-time fluorescence detection kit of chicken virus mycoplasma based on RPA, test strips detection kit and application thereof | |
| CN110283936A (en) | A kind of African swine fever virus LAMP-HNB Visual retrieval kit | |
| Li et al. | Development of a recombinase-aided amplification combined with lateral flow dipstick assay for the rapid detection of the African swine fever virus | |
| EP3902929A1 (en) | Fast and portable microfluidic detection system as an alternative to salmonella's classical culture method | |
| CN105603123A (en) | Real-time fluorescence RPA reagent kit and test strip RPA reagent kit for rapidly detecting porcine parvovirus and application of reagent kits | |
| CN106834432B (en) | Cross primer amplification primer group for detecting haemophilus parasuis, kit and application | |
| CN103740803A (en) | Food borne pathogenic organism nucleic acid lateral flow test strip detection kit and application thereof | |
| CN110438260A (en) | A kind of African swine fever virus nucleic acid test strips detection kit | |
| CN109337995A (en) | The PCR detection method and kit of bacillus tularense and its subspecies | |
| CN108441542A (en) | Streptococcusagalactiae visualization kit for detecting nucleic acid based on recombinase polymeric enzymatic amplification technology and method | |
| CN108070638A (en) | A kind of recombinase polymerase constant-temperature amplification method, its primer special and probe and purposes for detecting Orientia Tsutsugamushi | |
| CN111518935A (en) | Kit, primer pair, probe and method for detecting enterobacter sakazakii | |
| CN108048586A (en) | A kind of detection method of ox kind Brucella sp attenuated vaccine strain S19 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180522 |