CN108148894A - A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes - Google Patents
A kind of methods and applications of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes Download PDFInfo
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Abstract
The invention discloses a kind of method of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes, step is as follows：HlyA genes are chosen as detection target gene, the primer pair that there is specificity to Listeria monocytogenes is designed, using Listeria monocytogenes genomic DNA as template, carries out specific constant-temperature amplification, and application colloidal gold strip obtains testing result.This method is fast and convenient, high specificity, high sensitivity, repeatable high, this method constant-temperature amplification at 39 DEG C only needs 10 minutes, and colloidal gold strip testing result is within 5 minutes, testing result visually observes, entire reaction process does not need to expensive laboratory apparatus, only needs a thermostatical instrument, can be applied to that remote, natural resources shortage is regional and Site Detection.This method is expected to become easy conventional detection means, to ensureing that food security is significant.
Description
Technical field
The invention belongs to technical field of microbial detection, are related to quickly detecting skill for the constant-temperature amplification of Listeria monocytogenes
Art, especially a kind of recombinase polymerase constant-temperature amplification (RPA) is with reference to the methods and applications of ELISA test strip Listeria monocytogenes.
Background technology
Listeria monocytogenes (Listeria monocytogenes) are widely present in nature, meat easy to pollute, milk
Class, marine product and vegetables etc. since it easily forms biofilm, make it survive in food the longer time.World health group
It is woven in and 4% one 8% marine product, 5% one 10% milk and product and more than 30% meat is pointed out in being reported in relation to food poisoning
There are Listeria monocytogenes pollutions for product.Moreover, because in the food that the bacterium preserves in 4 DEG C of refrigerators also can growth and breeding, make to dive
Harmfulness further increase.It eats by the food that Listeria monocytogenes pollute in addition to will appear nausea, sepsis, diarrhea etc. one
As outside food posioning symptom, also result in myocarditis, meningitis, enterogastritis and pneumonia etc..In WHO and FDA in 1986
Listeria research center is set up, specially coordinates the research work such as aetology, epidemiology and the clinic of the bacterium, which is arranged
For one of four big food pathogenic nineties.In American-European, Japanese clinical disease and food pollution as caused by Listeria monocytogenes
Problem holds pride of place in food posioning.Therefore Listeria monocytogenes are all classified as food hygiene by countries in the world now
Legal detection project.
The detection method of Listeria monocytogenes mainly includes bacterium separation identification, immunological method, molecular biology at present
Method etc..Bacterium separation appraisal amount is big, and cost is relatively low, but detection cycle is long, and sensitivity is low；Immunological method easily intersects dirt
Dye, false positive is serious, and sensitivity is relatively low；Most widely used molecular biology method is PCR, but cumbersome time-consuming, it is necessary to
Expensive experimental facilities is relied on, is not easy to be promoted in base or Site Detection.
In recent years, recombinase polymerase (RPA) method attracts attention, and this method is a kind of novel nucleic acids amplification technique, quilt
It is known as to substitute the nucleic acid detection technique of PCR, after the advent of 2006, has been applied to agricultural, food security, medicine and has turned
The multiple fields such as genetic test.Can under constant temperature (37 DEG C~42 DEG C) realize to quick, the specific amplification of nucleic acid, and
Nucleic acid amplification to detectable level there can be good operability in 20min, it is considered to be closest to so-called " isothermal
Amplification ", has broad application prospects.The characteristics of colloidal gold strip (LF) is maximum is quick rapid, sensitive and accurate, safety letter
Just it is, of low cost and do not need to professional and instrument and equipment, the testing result that can be observed with the naked eye in usual a few minutes.
By retrieval, patent publication us related with the present patent application is not yet found.
Invention content
It is an object of the invention in place of overcome the deficiencies in the prior art, provide a kind of recombinase polymerase constant-temperature amplification knot
The methods and applications of ELISA test strip Listeria monocytogenes are closed, this method has high sensitivity, high specific, visualization, operation
Simply, portable feature.
The present invention solves its technical problem and following technical scheme is taken to realize：
A kind of method of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes, step are as follows：
HlyA genes are chosen as detection target gene, the primer pair that there is specificity to Listeria monocytogenes are designed, with list
It is template to increase Listeria genomic DNA, carries out specific constant-temperature amplification, and application colloidal gold strip obtains testing result.
Moreover, the primer pair is：
The nucleotide sequence SEQ1 of forward primer：GTAAGTGGGAAATCTGTCTCAGGTGATGTAG；
The nucleotide sequence SEQ2 of reverse primer：ACTCCTGGTGTTTCTCGATTAAAAGTAGCA.
Moreover, 5 ' ends of the forward primer are marked with digoxin digoxin, 5 ' end biotins of the reverse primer
Biotin is marked.
Moreover, the colloidal gold strip includes backboard, sample pad, gold-labelled pad, nitrocellulose filter and blotting paper, it is described
Backboard is horizontally disposed, be sequentially connected from left to right on the backboard connect setting sample pad, gold-labelled pad, nitrocellulose filter and
Blotting paper, each section are overlapped in adjacent (part), and the nitrocellulose filter upper edge horizontal direction parallel is arranged at intervals
Detection line and nature controlling line, the detection line and nature controlling line are longitudinally disposed, are coated on the nitrocellulose filter at the detection line
Streptavidin is set, coating setting sheep anti-mouse antibody on the nitrocellulose filter at the nature controlling line.
Moreover, the backboard is the liner with adhesive sticker.
Moreover, the preparation process of the gold-labelled pad is as follows：
(1) colloidal gold is prepared
The colloidal gold that colloidal gold method prepares 20nm is prepared according to reduction of sodium citrate method, method is as follows：
100mL ultra-pure waters will be added in conical flask containing rotor, heating keeps reject moisture after 3min after boiling；Take 99mL
Ultra-pure water and the filtered gold chloride that 1mL mass concentrations are 1% add to conical flask, are heated to adding in 2.25mL after boiling is boiled
The trisodium citrate that mass concentration is 1% is stirred continuously, and solution fades to claret, when color keep stabilization no longer changes, followed by
Continuous heating 15min；Solution is cooled to room temperature, and is restored with ultra-pure water to initial volume, obtains colloidal gold, and 4 DEG C are kept in dark place；
(2) colloidal gold labeled monoclonal antibody
The colloidal gold of 1mL steps (1) is taken, adds in 18 μ L 0.2mol/LK2CO3Colloidal gold is adjusted to pH=8.5,10 μ L is taken to resist
DigiTAb is added in colloidal gold solution, shakes 5min rapidly, and the solution after mixing stands 1h in 4 DEG C, is added dropwise molten after mixing
The mass concentration of liquid total volume 2% is 20% BSA solution, and the mass concentration of overall solution volume 1% is 20% PEG after mixing
20000 solution, after mixing, solution after must mixing, solution stands 30min in 4 DEG C after mixing, then 2000rpm, 4 DEG C, centrifugation
15min is taken in supernatant to clean centrifuge tube, 10000r/min, 4 DEG C, centrifuges 30min, reject supernatant, colloid gold particle
Precipitation is redissolved liquid with the gold mark of 5 times of volumes and is redissolved, and solution is sprayed on 30 μ L/cm on gold-labelled pad raw sheet, is dried in vacuum overnight,
Up to the gold-labelled pad for carrying colloidal gold labeled monoclonal antibody.
Moreover, it is as follows：
(1) RPA reaction systems：
10 μM of forward primers and each 29.5 μ L, 5ng sample moulds of 2.4 μ L, 1 × rehydration buffer of reverse primer
Plate DNA2.5 μ L, 10.7 μ L of distilled water, recombinase polymerase, 280mmol magnesium acetates, 2.5 μ L, 50 μ L of reaction total volume；
(2) RPA reaction systems expand：
Rehydration buffer, distilled water, forward primer, reverse primer, mould are sequentially added into sterile centrifugation tube
Plate DNA and recombinase polymerase, fully vibrate mixing, are eventually adding 280mmol/ μ L magnesium acetates, are sufficiently mixed, brief centrifugation,
39 DEG C of water-baths react 10min, take out reaction tube, obtain amplified production；
(3) ELISA test strip：
It takes in 100 μ L amplified productions to 900 μ L PBST and mixes, then mixed liquor is added drop-wise in test strips, it is quiet at room temperature
5min is put, amplification is judged by test strips colour developing situation；
When ELISA test strip line, nature controlling line have band appearance, illustrate the result positive, there are Listeria monocytogenes；If only
There is nature controlling line to have band, detection line position then illustrates result feminine gender without band, and there is no Listeria monocytogenes；If nature controlling line does not go out
Existing band, as a result in vain.
A kind of method of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes as described above exists
Application in the detection of Listeria monocytogenes.
The advantages of present invention obtains and good effect are：
1st, this method is fast and convenient, high specificity, high sensitivity, and repeatability is high, this method constant-temperature amplification at 39 DEG C
It only needs 10 minutes, within 5 minutes, testing result visually observes colloidal gold strip testing result, and entire reaction process does not need to
Expensive laboratory apparatus only needs a thermostatical instrument, can be applied to that remote, natural resources shortage is regional and Site Detection.This method has
Hoping becomes easy conventional detection means, to ensureing that food security is significant.
2nd, the method for the quick detection Listeria monocytogenes provided by the invention based on molecular biology, can realize to list
Increase Listeria and carry out quick, sensitive, easy quick detection, this method can in terms of inspection and quarantine, food industry portion and
Safe and healthy portion has broad application prospects.
3rd, the method for the present invention is easy, quickly, detection can be completed in 20min, as a result can detect by an unaided eye；Entire amplification
Process only needs 39 DEG C of constant temperature, does not need to complicated instrument, and use scope is extensive.
Description of the drawings
Fig. 1 is test strip operation principle, assemble method and the structure connection signal of colloidal gold strip in the present invention
Figure；
Fig. 2 is the result figure that the method for the present invention optimizes temperature and time；Wherein, the 1-8 schemed in a represents 25 successively respectively
DEG C, 30 DEG C, 35 DEG C, 37 DEG C, 39 DEG C, 42 DEG C, 45 DEG C and negative control, the 1-6 schemed in b represent 5min, 10min successively respectively,
15min, 20min, 25min and negative control；
Fig. 3 optimizes sheep anti mouse secondary antibody and Streptavidin concentration map for the method for the present invention；Wherein, scheme 1-4 in a to distinguish successively
Represent 0.1,0.5,0.8, the different sheep anti mouse secondary antibody concentration of 1mg/mL, scheme 1-4 in b represent 0.5 successively respectively, 1.0,1.5,
The different Streptavidin concentration of 2mg/mL；
Fig. 4 is the specific outcome figure that the method for the present invention detects Listeria monocytogenes；
Fig. 5 is the sensitivity results figure that the method for the present invention detects Listeria monocytogenes.
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 method of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes, step are as follows：
According to Listeria monocytogenes conserved region hlyA (GenBank:AF253320.1 specific primer sequence) is designed, is chosen
HlyA genes design the primer pair for having specificity to Listeria monocytogenes, with Listeria monocytogenes base as detection target gene
Because group DNA is template, specific constant-temperature amplification is carried out, and application colloidal gold strip obtains testing result.
Specifically, the primer pair is：
The nucleotide sequence SEQ1 of forward primer：GTAAGTGGGAAATCTGTCTCAGGTGATGTAG；
The nucleotide sequence SEQ2 of reverse primer：ACTCCTGGTGTTTCTCGATTAAAAGTAGCA.
Specifically, 5 ' ends of the forward primer are marked with digoxin digoxin, 5 ' the ends biology of the reverse primer
Plain biotin labels.
Specifically, the colloidal gold strip includes backboard, sample pad, gold-labelled pad, nitrocellulose filter and blotting paper, institute
It is horizontally disposed to state backboard, is sequentially connected from left to right on the backboard and connects setting sample pad, gold-labelled pad, nitrocellulose filter
And blotting paper, each section partly overlap setting in adjacent, the nitrocellulose filter upper edge horizontal direction parallel is arranged at intervals
Detection line and nature controlling line, the detection line and nature controlling line are longitudinally disposed, are coated on the nitrocellulose filter at the detection line
Streptavidin is set, coating setting sheep anti-mouse antibody on the nitrocellulose filter at the nature controlling line.
Specifically, the backboard is the liner with adhesive sticker.
Specifically, the preparation process of the gold-labelled pad is as follows：
(1) colloidal gold is prepared
The colloidal gold that colloidal gold method prepares 20nm is prepared according to reduction of sodium citrate method, method is as follows：
100mL ultra-pure waters will be added in conical flask containing rotor, heating keeps reject moisture after 3min after boiling；Take 99mL
Ultra-pure water and the filtered gold chloride that 1mL mass concentrations are 1% add to conical flask, are heated to adding in 2.25mL after boiling is boiled
The trisodium citrate that mass concentration is 1% is stirred continuously, and solution fades to claret, and color keep is stablized to be further continued for when no longer changing
Heat 15min；Solution is cooled to room temperature, and is restored with ultra-pure water to initial volume, obtains colloidal gold, and 4 DEG C are kept in dark place；
(2) colloidal gold labeled monoclonal antibody
The colloidal gold of 1mL steps (1) is taken, adds in 18 μ L 0.2mol/LK2CO3Colloidal gold is adjusted to pH=8.5,10 μ L is taken to resist
DigiTAb is added in colloidal gold solution, shakes 5min rapidly, and the solution after mixing stands 1h in 4 DEG C, is added dropwise molten after mixing
The mass concentration of liquid total volume 2% is 20% BSA solution, and the mass concentration of overall solution volume 1% is 20% PEG after mixing
20000 solution, after mixing, solution after must mixing, solution stands 30min in 4 DEG C after mixing, then 2000rpm, 4 DEG C, centrifugation
15min is taken in supernatant to clean centrifuge tube, 10000r/min, 4 DEG C, centrifuges 30min, reject supernatant, colloid gold particle
Precipitation is redissolved liquid with the gold mark of 5 times of volumes and is redissolved, and solution is sprayed on 30 μ L/cm in gold-labelled pad, is dried in vacuum overnight.
More preferably, the method for the recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes, body
Step is as follows：
(1) RPA reaction systems：
10 μM of forward primers and each 29.5 μ L, 5ng sample moulds of 2.4 μ L, 1 × rehydration buffer of reverse primer
Plate DNA2.5 μ L, 10.7 μ L of distilled water, recombinase polymerase, 280mmol magnesium acetates, 2.5 μ L, 50 μ L of reaction total volume；
(2) RPA reaction systems expand：
Rehydration buffer, distilled water, forward primer, reverse primer, mould are sequentially added into sterile centrifugation tube
Plate DNA and recombinase polymerase, fully vibrate mixing, are eventually adding 280mmol/ μ L magnesium acetates, are sufficiently mixed, brief centrifugation,
39 DEG C of water-baths react 10min, take out reaction tube, obtain amplified production；
(3) ELISA test strip：
It takes in 100 μ L amplified productions to 900 μ L PBST and mixes, then mixed liquor is added drop-wise in test strips, it is quiet at room temperature
5min is put, amplification is judged by test strips colour developing situation；
When ELISA test strip line, nature controlling line have band appearance, illustrate the result positive, there are Listeria monocytogenes；If only
There is nature controlling line to have band, detection line position then illustrates result feminine gender without band, and there is no Listeria monocytogenes；If nature controlling line does not go out
Existing band, as a result in vain.
The method of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes of the present invention can be with
It applies in the detection of Listeria monocytogenes.
The optimization time of the method for the present invention and the optimization of temperature：
(1) DNA is extracted according to DNA extraction kit specification；
Inoculum 1mL, 10000rpm, 1min are taken, supernatant is abandoned, adds in the final concentration of 20mg/mL lysozymes of 180 μ L,
37 DEG C of processing more than 30min；Add in 20 μ L Proteinase K Solution mixings；220 μ L buffer solution GB are added in, shake 15s, 70 DEG C of placements
10min, solution become limpid, and brief centrifugation removes tube wall droplet；220 μ L absolute ethyl alcohols are added in, shake 15s, brief centrifugation removes
Tube wall droplet；Previous step acquired solution and precipitation are moved in adsorption column CB3,12000r/min, 30s abandon supernatant, by adsorption column
CB3 is put into collecting pipe；500 μ L buffer solution GB, 12000r/min, 30s are added in into adsorption column, supernatant are abandoned, by adsorption column CB3
It puts back in collecting pipe；600 μ L rinsing liquid PW, 12000r/min, 30s are added in into adsorption column, supernatant is abandoned, adsorption column CB3 is put
It recycles in collector；It is repeated twice.Adsorption column CB3 is put back in collecting pipe, is added in 500 μ L buffer solution GB, 12000r/min, 30s, is abandoned
Supernatant puts back to adsorption column CB3 in collecting pipe, and 200 μ L elution buffer TE are added dropwise to adsorbed film centre position, are placed at room temperature for
5min, 12000r/min, 2min are collected into centrifuge tube, -20 DEG C of preservations.The DNA of extraction detects its OD value with microplate reader, sentences
Quality (the OD of disconnected DN A260There are notable absorption peak, OD260/OD280Value is between 1.7-1.9).
(2) RPA reaction systems
System includes 2.4 each primers of μ L (10 μM), 29.5 μ L1 × fluid infusion buffer solution, 10.7 μ LddH2O, 2.5 μ LDNA, weight
Group enzymatic polymerization enzyme, rapid oscillation mixing are eventually adding 2.5 μ L magnesium acetates, are put into water-bath and react.It is 25 to set reaction temperature
DEG C, 30 DEG C, 35 DEG C, 37 DEG C, 39 DEG C, 42 DEG C and 45 DEG C, the reaction time is respectively 5min, 10min, 15min, 20min and
25min.100 μ LRPA products are taken, by the two mixing, 1000 μ L mixtures are dripped by 900 μ LPBS (PBS+0.05%Tween 20)
It is added in test strips, stands 5min at room temperature, observe result.
Forward primer：5’—digoxin—GTAAGTGGGAAATCTGTCTCAGGTGATGTAG—3’
Reverse primer：5’—biotin—ACTCCTGGTGTTTCTCGATTAAAAGTAGCA—3’
All primers are synthesized by Jiangsu Jin Weizhi companies.
Negative control：Aseptic double-distilled water.
The reaction time and temperature of RPA are optimized, as a result show that amplification optimum temperature is 39 DEG C, the reaction time only needs
10min is i.e. detectable, as shown in Fig. 2, the 1-8 in Fig. 2 a represents 25 DEG C, 30 DEG C, 35 DEG C, 37 DEG C, 39 DEG C, 42 DEG C, 45 DEG C respectively
And negative control, the 1-6 in Fig. 2 b represent 5min, 10min, 15min, 20min, 25min and negative control respectively.Above-mentioned implementation
1 result of example illustrates that entirely detection only needs 15min or so that can just complete with the method for the present invention, greatly shortens detection time.
More preferably, the method for the invention for preparing colloidal gold strip can be as follows：
1) colloidal gold is prepared
The colloidal gold that colloidal gold method prepares 20nm is prepared according to reduction of sodium citrate method, method is as follows：It will be containing rotor
100mL ultra-pure waters are added in conical flask, heating keeps reject moisture after 3min after boiling.Take 99mL ultra-pure waters and 1mL mass dense
The filtered gold chloride spent for 1% adds to conical flask, is heated to adding in the lemon that 2.25mL mass concentrations are 1% after boiling is boiled
Lemon acid trisodium is stirred continuously, and solution fades to claret, when color keep stabilization no longer changes, is further continued for heating 15min.Solution is cold
But to room temperature, restored with ultra-pure water to initial volume, 4 DEG C are kept in dark place.
2) colloidal gold labeled monoclonal antibody
The colloidal gold that 1mL is above-mentioned is taken, adds in 18 μ L 0.2mol/LK2CO3Colloidal gold is adjusted to pH=8.5, takes 10 μ L anti-ly
Digoxin antibody is added in colloidal gold solution, shakes 5min rapidly, and the solution after mixing stands 1h in 4 DEG C, and solution after mixing is added dropwise
The PEG20000 of the mass concentration 20% of overall solution volume 1% after the BSA solution of the mass concentration 20% of total volume 2%, mixing
Solution, after mixing, solution after must mixing, the solution after mixing stands 30min in 4 DEG C；2000rpm, centrifuges 15min, takes by 4 DEG C
In supernatant to clean centrifuge tube, 10000r/min, centrifuges 30min, reject supernatant by 4 DEG C, and colloid gold particle is precipitated with 5
The gold mark of times volume redissolves liquid and redissolves, and solution is sprayed on 30 μ L/cm in gold-labelled pad, is dried in vacuum overnight.
3) NC films coating nature controlling line and detection line antibody
The test strips are included on the liner with adhesive sticker is equipped with sample pad, colloidal gold conjugate pad and water suction in order
Filter paper pads, each part mentioned above partly overlap in adjacent, and tunica fibrosa is equipped with detection line and nature controlling line, there is strepto- parent in detection line
It is coated with element, has sheep anti-mouse antibody on nature controlling line.Determine the sheep anti-mouse antibody on coating C lines be diluted to 0.1,0.5,0.8,1mg/
ML and the Streptavidin concentration for being coated on T lines, with PB buffer solutions dilute its concentration gradient be respectively 0.5,1.0,1.5,2mg/
ML marks C, T line using film instrument is drawn on NC films according to 1 μ L/cm, 37 DEG C be dried overnight it is spare.According to colour developing interpretation of result not
The factors such as the color intensity of its detection line, stability, developing time, cost-effective are combined with diluting condition, are selected most suitable
Sheep anti mouse secondary antibody and Streptavidin concentration.As a result show that 0.1mg/mL sheep anti mouses secondary antibody and 2mg/mL Streptavidins are made respectively
During for C lines and T lines, band is clear, and stability is good.As shown in figure 3, in Fig. 3 a 1-4 represent 0.1 respectively, 0.5,0.8,1mg/mL
Different sheep anti mouse secondary antibody concentration, in Fig. 3 b 1-4 represent 0.5 respectively, 1.0,1.5, the different Streptavidin concentration of 2mg/mL.
The method of the present invention detects the specificity of Listeria monocytogenes：
According to the method for the present invention detection Listeria monocytogenes ATCC 19115, Listera grayi CICC 21494, Si Shi
Li Site bacterium CICC 10867, Listeria monocytogenes ATCC 25923, Escherichia coli ATCC 10305, clostridium perfringen CICC
10293, cholera salmonella CICC 21494 and negative control, as a result showing the detection method of the present invention has well specifically
Property, except Listeria monocytogenes ATCC 19115, other identifications are feminine gender.As shown in figure 4,1-8 distinguishes table successively in figure
Show Listeria monocytogenes ATCC 19115, Listera grayi CICC 21494, listeria seeligeri CICC 10867, it is single to increase Lee
This special bacterium ATCC 25923, Escherichia coli ATCC 10305, clostridium perfringen CICC 10293, cholera salmonella CICC
21494 and negative control.
The method of the present invention detects the sensitivity of Listeria monocytogenes：
Listeria monocytogenes genomic DNA is quantified, is diluted to concentration 3ng, 300pg, 30pg, 3pg respectively,
300fg, 30fg take the method for the present invention to determine the sensitivity of detection Listeria monocytogenes.As a result as figure 5 illustrates, 1-7 points in figure
Do not represent 3ng, 300pg, 30pg successively, 3pg, 300fg, the experimental result of 30fg and negative control, it can be found that this method is examined
Survey is limited to 300fg, has very high sensitivity, can meet the requirement of quick detection Listeria monocytogenes.
Claims (8)
- A kind of 1. method of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes, it is characterised in that：Step It is rapid as follows：HlyA genes are chosen as detection target gene, the primer pair that there is specificity to Listeria monocytogenes is designed, increases Lee with single This special bacterium genomic DNA is template, carries out specific constant-temperature amplification, and application colloidal gold strip obtains testing result.
- 2. the side of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes according to claim 1 Method, it is characterised in that：The primer pair is：The nucleotide sequence SEQ1 of forward primer：GTAAGTGGGAAATCTGTCTCAGGTGATGTAG；The nucleotide sequence SEQ2 of reverse primer：ACTCCTGGTGTTTCTCGATTAAAAGTAGCA.
- 3. the side of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes according to claim 2 Method, it is characterised in that：5 ' ends of the forward primer are marked with digoxin digoxin, 5 ' the ends biology of the reverse primer Plain biotin labels.
- 4. the side of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes according to claim 1 Method, it is characterised in that：The colloidal gold strip includes backboard, sample pad, gold-labelled pad, nitrocellulose filter and blotting paper, institute It is horizontally disposed to state backboard, is sequentially connected from left to right on the backboard and connects setting sample pad, gold-labelled pad, nitrocellulose filter And blotting paper, each section are overlapped in adjacent, the nitrocellulose filter upper edge horizontal direction parallel is arranged at intervals detection Line and nature controlling line, the detection line and nature controlling line are longitudinally disposed, and setting is coated on the nitrocellulose filter at the detection line Streptavidin, coating setting sheep anti-mouse antibody on the nitrocellulose filter at the nature controlling line.
- 5. the side of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes according to claim 4 Method, it is characterised in that：The backboard is the liner with adhesive sticker.
- 6. the side of recombinase polymerase constant-temperature amplification combination ELISA test strip Listeria monocytogenes according to claim 4 Method, it is characterised in that：The preparation process of the gold-labelled pad is as follows：(1) colloidal gold is preparedThe colloidal gold that colloidal gold method prepares 20nm is prepared according to reduction of sodium citrate method, method is as follows：100mL ultra-pure waters will be added in conical flask containing rotor, heating keeps reject moisture after 3min after boiling；Take 99mL ultrapure Water and the filtered gold chloride that 1mL mass concentrations are 1% add to conical flask, are heated to adding in 2.25mL mass after boiling is boiled A concentration of 1% trisodium citrate is stirred continuously, and solution fades to claret, when color keep stabilization no longer changes, is further continued for adding Hot 15min；Solution is cooled to room temperature, and is restored with ultra-pure water to initial volume, obtains colloidal gold, and 4 DEG C are kept in dark place；(2) colloidal gold labeled monoclonal antibodyThe colloidal gold of 1mL steps (1) is taken, adds in 18 μ L 0.2mol/LK2CO3Colloidal gold is adjusted to pH=8.5, takes the anti-ground of 10 μ L high Pungent antibody is added in colloidal gold solution, shakes 5min rapidly, and the solution after mixing stands 1h in 4 DEG C, and solution is total after mixing is added dropwise The mass concentration of volume 2% is 20% BSA solution, and the mass concentration of overall solution volume 1% is 20% PEG after mixing 20000 solution, after mixing, solution after must mixing, solution stands 30min in 4 DEG C after mixing, then 2000rpm, 4 DEG C, centrifugation 15min is taken in supernatant to clean centrifuge tube, 10000r/min, 4 DEG C, centrifuges 30min, reject supernatant, colloid gold particle Precipitation is redissolved liquid with the gold mark of 5 times of volumes and is redissolved, and solution is sprayed on 30 μ L/cm on gold-labelled pad raw sheet, is dried in vacuum overnight, Up to the gold-labelled pad for carrying colloidal gold labeled monoclonal antibody.
- 7. recombinase polymerase constant-temperature amplification combination ELISA test strip list according to any one of claims 1 to 6 increases Li Si The method of special bacterium, it is characterised in that：It is as follows：(1) RPA reaction systems：10 μM of forward primers and each 29.5 μ L, 5ng sample templates of 2.4 μ L, 1 × rehydration buffer of reverse primer DNA2.5 μ L, 10.7 μ L of distilled water, recombinase polymerase, 280mmol magnesium acetates, 2.5 μ L, 50 μ L of reaction total volume；(2) RPA reaction systems expand：Rehydration buffer, distilled water, forward primer, reverse primer, template DNA are sequentially added into sterile centrifugation tube With recombinase polymerase, mixing is fully vibrated, 280mmol/ μ L magnesium acetates is eventually adding, is sufficiently mixed, brief centrifugation, 39 DEG C of water Bath reacts 10min, takes out reaction tube, obtains amplified production；(3) ELISA test strip：It takes in 100 μ L amplified productions to 900 μ L PBST and mixes, then mixed liquor is added drop-wise in test strips, is stood at room temperature 5min judges amplification by test strips colour developing situation；When ELISA test strip line, nature controlling line have band appearance, illustrate the result positive, there are Listeria monocytogenes；If only matter Control line has band, and detection line position then illustrates result feminine gender without band, and there is no Listeria monocytogenes；If nature controlling line does not occur item Band, as a result in vain.
- 8. a kind of recombinase polymerase constant-temperature amplification combination ELISA test strip list as described in any one of claim 1 to 7 increases Lee Application of the method for this special bacterium in the detection of Listeria monocytogenes.
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| CN112553355A (en) * | 2020-12-29 | 2021-03-26 | 上海海关动植物与食品检验检疫技术中心 | Primer probe combination, kit and method for detecting listeria monocytogenes based on RAA technology |
| CN113981119A (en) * | 2021-12-21 | 2022-01-28 | 合肥工业大学 | Method for detecting Listeria monocytogenes in cheese |
| CN113981119B (en) * | 2021-12-21 | 2023-08-22 | 合肥工业大学 | Detection method of listeria monocytogenes in cheese |
| CN115060904A (en) * | 2022-08-16 | 2022-09-16 | 山东康华生物医疗科技股份有限公司 | Preparation method of colloidal gold solution for hepatitis B surface antigen detection kit, reagent strip and kit |
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