CN106636387A - Salmonella nucleic acid rapid detection kit, test strip and detection method - Google Patents
Salmonella nucleic acid rapid detection kit, test strip and detection method Download PDFInfo
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- CN106636387A CN106636387A CN201611149351.4A CN201611149351A CN106636387A CN 106636387 A CN106636387 A CN 106636387A CN 201611149351 A CN201611149351 A CN 201611149351A CN 106636387 A CN106636387 A CN 106636387A
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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
- C12Q1/6804—Nucleic acid analysis using immunogens
Abstract
The invention relates to a salmonella nucleic acid rapid detection kit, a salmonella nucleic acid rapid detection test strip and a salmonella nucleic acid rapid detection method, and relates to design of salmonella specific primers, optimization of helicase isothermal amplification conditions and establishment of a nucleic acid film chromatography detection test strip, and visual observation of a nucleic acid detection result is achieved. Just one thermostat is adopted in a whole reaction process, and from pretreatment to detection completion on a single sample, the detection result can be obtained just by 1h; the method, on detecting salmonella, is 4.5*10<1>CFU/mL in sensitivity, and the method can avoid a cross reaction with other common food-borne pathogenic bacteria; and the detection test strip is simple and convenient to operate, high in sensitivity, strong in specificity and high in efficiency, and the detection strip is especially suitable for on-site detection and clinical diagnosis in grassroots units.
Description
Technical field
The present invention relates to the method for quick of nucleic acid constant-temperature amplification product, is examined using nucleic acid membrane chromatographic test strip
Target sequence is surveyed, is applied in the detection process of food-borne pathogens, and in particular to a kind of Salmonella unwindase constant-temperature amplification
And nucleic acid membrane chromatographic test strip.
Technical background
The food safety affair that analysis occurred in the past, the alimentary toxicosis situation occurrence frequency that Bacterial Contamination causes is high, shadow
The degree of sound is big, and coverage is wide.Food-borne pathogens cause the mankind and the sickness rate of zoogenetic infection disease still to remain high, common
Pathogen be mainly Salmonella, Escherichia coli O 157, staphylococcus aureuses, listeria spp etc..Salmonella conduct
Can result in zoonosiss pathogenic bacterium to be widely present in nature, at present identified Salmonella serogroup has more than 2500
Kind, wherein there is main serotype for enteritis, mouse typhuss and Salmonella choleraesuls three in the salmonellosiss for causing food-borne
Kind, endotoxin invasion and attack intestinal mucosa, nerve and the blood vessel discharged after cellular lysate, in causing headache, vomiting, diarrhoea, heating etc.
Toxication shape.As can be seen here food safety situation is still severe, and effective monitoring Bacterial Contamination problem becomes and avoids food pollution thing
The main solution route that part occurs.Therefore, quick, sensitive, special detection method is for clinical diagnosises and ensures food safety
It is requisite.The method that tradition culture is taken in conventional criteria detection, pathogenic bacterium to be measured need to be according to the detecting step list of regulation
Solely detected, detection cycle is long, complex operation, workload is big, and reviewer needs to have professional experiences, and testing result is easily received
Anthropic factor is affected so as to there is erroneous judgement.
The specific immunity association reaction of its antigen of the detection method Main Basiss of applied immunology ultimate principle and antibody.
Corresponding specific antibody is obtained by microorganism specific antigenic substance stimulating organism body itself.Wherein tried using enzyme linked immunological
Agent box and colloidal gold strip have evolved into quite ripe technology in terms of detection food-borne pathogens, are related to detection range wide
It is general.The features such as such product relies on its easy to operate, convenient and swift, cheap and accurate result, basic unit is stepped into
Detection and medical department are even applied to average family and independently detect, give people routine diagnosis disease bring it is many it is convenient it
Place.But immunological technique still suffers from place of some shortcomings, main reason is that Dan Ke in current conceptual phase and practical application
The preparation procedure of grand antibody is loaded down with trivial details, a large amount of manpowers and physical resources is expended, because many factors collective effect may cause
The antibody specificity for arriving is unsatisfactory, directly affects specificity and the sensitivity of testing result.During actually detected often
Generation false positive or false-negative report, show the method stability and need further raising.
Further investigation and fast-developing, the cause of disease such as antibacterial body measurement side in practice examining recently as molecular biology
Method is via method such as polymerase chain reaction (PCR) skill of traditional single biochemical test horizontal transfer to molecular biology
The technologies such as art, gene chip, probe hybridization.Polymerase chain reaction is selectively carried out in-vitro simulated thin by specific primer
The method of intracellular portion DNA amplification or RNA fragments, the DNA replication dna method can be realized the specific gene in very small amount genome
Fragment is presented exponential amplification within of short duration a few houres, and significantly improve the sensitivity of detection has become most sensitive in current research level
Detection technique, the advantage of and high specificity high with degree of accuracy, in microorganism detection, the diagnosis of infectious and hereditary
And the numerous areas such as the detection of gene mutation play a significant role.However, round pcr needs accurate Thermal Cycling, limitation
Precision instrument and equipment must be relied in course of reaction, thus limit Site Detection that it lacks in facility and Countryside should
With.
At present both at home and abroad nucleic acid constant-temperature amplification technology is developed rapidly, be widely used have loop-mediated isothermal amplification,
Chain substitutes amplification, rolling circle amplification, nickase nucleic acid constant-temperature amplification, constant-temperature amplification of dependence unwindase etc..Its course of reaction is only needed
The specific amplification of DNA or RNA is carried out and can realized at a constant temperature, and sensitivity can reach round pcr
Detection level.
Patent of invention " can avoid false-negative salmonella constant temperature fluorescent detector primers group, test kit and detection method "
(publication number CN105219870A) and " strand displacement constant-temperature amplification (SDA) quick detection kit of Salmonella enteritidis and its inspection
Survey method " (publication number：CN102382884A) description is realized to Salmonella DNA according to different amplification principles with constant temperature
The amplification of nucleotide sequence, but such method still needs to attached gel electrophoresis and imaging system and carries out Treatment Analysis to experimental data, does not have
There is obvious reduced inspection operating procedure and its advantage cannot be embodied in Public Health Emergencies.
To be imported in amplified production using the upstream and downstream primer for being modified with particular marker respectively during constant-temperature amplification
Antibody or its part capture that two distinct types of nucleic acid markers can be specifically bound, Cleaning Principle is according to approximate Double
Antibody sandwich highly-pathogenic avian influenza method, the method is had been supplied in for various food-borne pathogens and viral gene
Constant-temperature amplification product detection in, with regard to the detection of nucleic acids of Salmonella, the patent of invention of Publication No. CN102329790A is " double
Labeling nucleic acid constant-temperature amplification method and test strip " provides the probe of a kind of utilization labelling biotin and Fluorescein isothiocyanate
Method quick detection is realized to LAMP product.But it is difficult because the amplification system needs a plurality of interior outer primer to participate in jointly
To avoid the formation of the formation of the non-specific amplification product such as primer dimer, therefore the type amplification is not suitable for test strips
Detection.
The content of the invention
Present invention aim to overcome that the deficiencies in the prior art part, there is provided one kind is using unwindase constant-temperature amplification detection tool
Have the Fast Detection Technique of hazardness food-borne pathogens-Salmonella (Salmonella), it is particularly a kind of quick, accurately,
Sensitive Salmonella nucleic acid rapid detection kit, reagent paper and detection method.
The present invention realizes that the technical scheme of purpose is as follows：
A kind of Salmonella nucleic acid rapid detection kit, including forward primer invA-F:Sequence 5 '-
ATTTCTATGTTCGTCATTCCATTACCTACC-3 ', reverse primer invA-R:Sequence：5’-
ATGTAGAACGACCCCATAAACACCAA-3 ', with Digoxin and 5 ' ends of biotin difference labelling upstream and downstream primer, Jin Biao
Antibody, the antibody of anti-gold labeling antibody kind, the ligand substance of the thing biotin that develops the color.
And, also including amplification system, the amplification system includes 10 × Annealing Buffer, dNTP, MgSO4、
NaCl、Enzyme Mix。
And, the gold labeling antibody is Digoxin and biotin.
And, the ligand substance streptavidin of the colour developing thing biotin.
And, the gold labeling antibody is Mus anti digoxin antibody, and anti-gold labeling antibody is anti-for sheep anti mouse two.
A kind of Salmonella nucleic acid quick detection test paper, including sample pad, gold standard pad, nitrocellulose filter and absorbent paper,
Sample pad one end is Chong Die with gold standard pad one end end, gold standard pad other end nitrocellulose filter Chong Die with nitrocellulose filter end
The other end is Chong Die with absorbent paper, is embedded with detection line and nature controlling line nitrocellulose filter is spaced and parallel, and detection line is in gold mark
Pad side, nature controlling line is between detection line and absorbent paper end；
Gold labeling antibody is sprayed in the gold standard pad, the ligand substance of coating colour developing thing biotin in detection line, on nature controlling line
It is coated with the antibody of anti-gold labeling antibody kind.
And, the gold colloidal of spraying mark anti digoxin antibody, is coated with streptavidin, chain in the gold standard pad in detection line
Mould avidin concentration is 2mg/mL, and the sheep anti mouse two of 80 times of coating dilution resists on nature controlling line.
And, the sample pad is glass fibre, and each several part lap is 2-10mm, is installed in Test paper bottom
PVC backboards.
And, quick detection test paper is the PBS bufferings for adding 0.1%BSA and 0.1%Tween 20 using loading developping solution
Liquid pH 7.4.
A kind of Salmonella nucleic acid method for quick, it comprises the steps：
(1) monoclonal antibody such as Mus anti digoxin antibody is adsorbed on substance that show color colloid gold particle as gold labeling antibody,
The coalition for forming rock-steady structure is fixed in gold standard pad, and gold standard pad is exactly to pad made by a glass fibre；
(2) Streptavidin is coated on nitrocellulose filter as the ligand substance of label biotin with string-like form
Form detection line；
(3) the antibody such as sheep anti-mouse antibody for selecting anti-gold labeling antibody kind is equally coated on knot on nature controlling line with string-like form
Close excessive gold labeling antibody；
(4) modified antigen or hapten thing are distinguished on the forward primer 5 ' and downstream primer 5 ' for designing specificity constant-temperature amplification
Matter such as Digoxin and biotin, when there is sramana's nucleic acid amplification product to be measured, the amplification that nucleic acid is carried out under constant temperature is anti-
Should obtain connecting the amplified production of two kinds of label Digoxin-target fragment-biotin simultaneously；
(5) Digoxin-target fragment-the biotin for (4) step being produced first with gold colloidal in gold standard pad (glass fibre)
The coated DigiTAb of granule is combined, and forms DigiTAb-Digoxin-target fragment-biotin colour developing particle composites；
(6) the colour developing particle composites that (5) step obtains in sample-loading buffer by capillarity along cellulose membrane to
On be moved to detection line by receptor streptavidin capture obtain DigiTAb-Digoxin-target fragment-biotin-strepto-
Avidin complex, the sedimentation in detection line forms the colour developing band of macroscopic, and the gold colloidal of uncombined amplified production
Coated gold labeling antibody is combined with the sheep anti-mouse antibody on nature controlling line on grain, two colour developing bands is occurred and is positive findingses；
Or, when there is no sramana's nucleic acid amplification product, do not occur above-mentioned steps (3)-(6), it is impossible to gold standard pad (glass
Fiber) on the coated DigiTAb of colloid gold particle combine, substance that show color colloid gold particle can not be coated with detection line
Material combines to form colour developing band, and the sheep anti-mouse antibody on nature controlling line is combined appearance with coated gold labeling antibody on colloid gold particle
One colour developing band is negative findings.
The present invention has the advantage that compared with other technologies：
The present invention is developed with unwindase isothermal amplification technology and combined nucleic acid membrane chromatographic test strip detection sramana
A kind of highly efficient molecular detecting method of Salmonella, the most suitable amplified fragments position of design alternative of primer and length can be effective
The formation of non-specific amplification, and optimization is avoided to obtain most preferably expansion buffer and make nucleic acid test strip reach optimal colour developing effect
Really.The detection method will not pollute laboratory and surrounding and not need expensive instrument and equipment or professional operation skill, from
And the detection of extensive sample can be tackled in the case of experiment condition is incomplete, following side is specifically embodied in a little
Face：
1st, the nucleic acid membrane chromatographic test strip of detection Salmonella unwindase constant-temperature amplification product of the present invention
High specificity, sensitivity is high, and easy to operate, stability is strong；
2nd, amplification procedure is effectively avoided the pollution of amplified production in the reaction system of closing；
3rd, response speed is fast, and completing whole detecting steps of sample only needs 1 hour or so；
4th, detection process does not need instrument and equipment and professional and technical personnel's operation of complex and expensive；
5th, it is adapted to the preliminary judgement of the field quick detection of food pathogenic.
Description of the drawings
Fig. 1 is the checking of Salmonella primer specificity.M in figure：DNA molecular amount labelling DL2000；1：Blank；2：Intestinal
Scorching Salmonella；3：Salmonella paratyphi A；4：Intestinal Salmonella intestinal subspecies；5：Salmonella choleraesuls；6：Intestinal sramana
Salmonella intestinal subspecies antityphoid sera type；7：Escherichia coli O 157:H7；8：Escherichia coli (non-O157:H7)；9：Song's Nei Shi will Hayes
Bacterium；10：Shigella flexneri；11：Clostridium perfringen；12：Listeria monocytogenes；13：Staphylococcus aureuses；14：Jejunum is curved
Curved bar bacterium.
Fig. 2 is nucleic acid membrane chromatographic test strip structure.1 in figure：Sample pad；2：Nitrocellulose filter；3：Backboard；4：
Gold standard pad (glass fibre)；5：Detection line；6：Nature controlling line；7：Absorbent paper.
Fig. 3 is Salmonella nucleic acid membrane chromatographic test strip specificity verification.1 in figure：Salmonella enteritidis；2：First
Type salmonella paratyphi；3：Intestinal Salmonella intestinal subspecies；4：Salmonella choleraesuls；5：Intestinal Salmonella intestinal subspecies typhoid fever blood
Clear type；6：Escherichia coli O 157:H7；7：Escherichia coli (non-O157:H7)；8：Bacillus ceylonensis A；9：Shigella flexneri；
10：Clostridium perfringen；11：Listeria monocytogenes；12：Staphylococcus aureuses；13：Campylobacter jejuni.
Specific embodiment
In order to understand the present invention, with reference to embodiment, the invention will be further described：Following embodiments are illustrative
, it is not determinate, it is impossible to limit protection scope of the present invention with following embodiments.
The present invention combines unwindase isothermal amplification technology and nucleic acid membrane chromatographic detection technique, sets up Salmonella nucleic acid fast
The method of speed detection.It is contemplated that the unwindase constant-temperature amplification product detected using nucleic acid membrane chromatographic test strip, from
And realize the quick detection of nucleic acid.The specific primer of the Salmonella of design difference labelling biotin and Digoxin, target core
Acid forms the amplified production for being modified with two kinds of labels through unwindase constant-temperature amplification, and the amplified production coated can have specifically
Property identification part and antibody nucleic acids membrane chromatographic test strip detection.Using antigen or hapten biological micromolecule labelling
Nucleic acid molecules can be realized the detection to target nucleic acid by the antibody of its specificity or part identification immunology detection principle.It is conventional
Nucleic acid markers in biotin can specifically bind its part streptavidin, and Digoxin is easily obtained its specificity and resists
Body.Therefore above two material is selected as primer mark thing.
1st, the present invention provides the specificity amplification primer of two Salmonellas, selects Salmonella specificity invA genes to set
Meter forward primer invA-F:- the ATTTCTATGTTCGTCATTCCATTACCTACC-3 ' of sequence 5 ', reverse primer invA-R:Sequence
Row：5 ' ends of 5 '-ATGTAGAACGACCCCATAAACACCAA-3 ', Digoxin and biotin difference labelling upstream and downstream primer,
In the presence of target nucleic acid, through unwindase constant-temperature amplification the DNA fragmentation that size is 101bp can be obtained；If there is no target core
In the presence of acid, then formed without amplified production, as shown in Figure 1.
2nd, the present invention provides the unwindase constant-temperature amplification method of Salmonella：The unwindase isothermal amplification reactions of Salmonella
System includes forward direction, reverse primer (75nM), 10 × Annealing Buffer (7 μ L), dNTP (3.5 μ L), MgSO4(2μL)、
NaCl (4 μ L), Enzyme Mix (3.5 μ L), with aseptic ultra-pure water 50 μ L are added to.1 μ L Salmonellas DNA that extraction is obtained
Used as the template of positive control, the aseptic ultra-pure water of equivalent is added as negative control and contains Salmonella unwindase constant-temperature amplification
In the PCR pipe of reactant liquor, above-mentioned reaction system is placed in into reaction 90min at 68 DEG C carries out the constant-temperature amplification of Salmonella.
3rd, the detection method of the Salmonella nucleic acid membrane chromatographic test strip that the present invention is provided, it comprises the steps
(principle is as shown in Figure 2)：
(1) monoclonal antibody such as Mus anti digoxin antibody is adsorbed on substance that show color colloid gold particle as gold labeling antibody,
The coalition for forming rock-steady structure is fixed in gold standard pad (being exactly a fiberglass packing).
(2) Streptavidin is coated on nitrocellulose filter as the ligand substance of label biotin with string-like form
Form detection line.
(3) the antibody such as sheep anti-mouse antibody for selecting anti-gold labeling antibody kind is equally coated on knot on nature controlling line with string-like form
Close excessive gold labeling antibody.
(4) modified antigen or hapten thing are distinguished on the forward primer 5 ' and downstream primer 5 ' for designing specificity constant-temperature amplification
Matter such as Digoxin and biotin, when there is sramana's nucleic acid amplification product to be measured, the amplification that nucleic acid is carried out under constant temperature is anti-
Should obtain connecting the amplified production of two kinds of label Digoxin-target fragment-biotin simultaneously.
(5) Digoxin-target fragment-the biotin for (4) step being produced first with gold colloidal in gold standard pad (glass fibre)
The coated DigiTAb of granule is combined, and forms DigiTAb-Digoxin-target fragment-biotin colour developing particle composites.
(6) the colour developing particle composites that (5) step obtains in sample-loading buffer by capillarity along cellulose membrane to
On be moved to detection line by receptor streptavidin capture obtain DigiTAb-Digoxin-target fragment-biotin-strepto-
Avidin complex, the sedimentation in detection line forms the colour developing band of macroscopic, and the gold colloidal of uncombined amplified production
Coated gold labeling antibody is combined with the sheep anti-mouse antibody on nature controlling line on grain, two colour developing bands is occurred and is positive findingses；
Or, when there is no sramana's nucleic acid amplification product, do not occur above-mentioned steps (3)-(6), it is impossible to gold standard pad (glass
Fiber) on the coated DigiTAb of colloid gold particle combine, substance that show color colloid gold particle can not be coated with detection line
Material combines to form colour developing band, and the sheep anti-mouse antibody on nature controlling line is combined appearance with coated gold labeling antibody on colloid gold particle
One colour developing band is negative findings.
4th, the present invention also offer Salmonella nucleic acid membrane chromatographic test strip structure is as shown in Figure 2：It includes that PVC is carried on the back
Sample pad, pad, nitrocellulose filter (NC) and absorbent paper are followed successively by plate, each part mentioned above lap at least retains
2mm, is respectively equipped with detection line (ligand substance comprising label biotin) and nature controlling line (comprising strepto- on nitrocellulose filter
Avidin), all of material need at 37 DEG C dried overnight.
5th, the present invention optimizes to Salmonella nucleic acid membrane chromatographic test strip：First to Salmonella unwindase constant temperature
The reaction condition of amplification is optimized respectively, including primer concentration, MgSO4Concentration, dNTP concentration and reaction temperature, obtain
Optimal reaction condition makes amplified reaction reach optimal effect.Optimize the design of colloidal gold colloidal gold detection test paper strip simultaneously, including
The type of nitrocellulose filter, detection line and nature controlling line coating concentration and loading developping solution, make test strips that optimal colour developing is presented
Effect.
Experimental technique in following examples, if no special instructions, is conventional method.Examination used in following examples
Agent material, if no special instructions, is routine biochemistry reagent suppliers and is commercially available.
Embodiment 1
The optimization of Salmonella unwindase constant-temperature amplification system
With primer concentration, MgSO4, dNTP and reaction temperature carry out orthogonal test, primer concentration for variable to Salmonella
Respectively 75nm, 80nm and 85nm；MgSO4Concentration is respectively 3mM, 3.5mM and 4mM；DNTP additions distinguish 2.5 μ L, 3.5 μ L
With 4.5 μ L；Reaction temperature is respectively 63 DEG C, 64 DEG C and 65 DEG C.Its amplified production is Jing after agarose gel electrophoresiies amplification using solidifying
Glue imager observes expanding effect, and empirical tests show that Salmonella expands 50 μ L overall reaction systems, including the μ L of DNA profiling 1,
Primer concentration is 80nM, MgSO4Concentration is 3.5mM, and dNTP additions are 4.5 μ L, finally add 3.5 μ L Enzyme Mix, are put
60min is reacted at a temperature of 64 DEG C.
Embodiment 2
It is prepared by Salmonella nucleic acid membrane chromatographic test strip
1st, the preparation of colloidal gold labeled monoclonal antibody
(1) the 10nm colloidal gold solutions for taking 1mL are placed in the clean centrifuge tubes of 1.5mL, a certain amount of K of Deca2CO3Solution is adjusted
Section colloidal gold solution is to optimum pH；
(2) the concentration for taking 15 μ L is added drop-wise in colloidal gold solution for 1 μ g/ μ L antibody, and 5min is shaken rapidly, molten after mixing
Liquid stands 1h under the conditions of 4 DEG C；
(3) the BSA solution of the μ L 20% of Deca 20, the solution of PEG 20000 of 10 μ L 20%, the solution after mixing is in 4 DEG C of bars
30min is stood under part；
(4) in 2000rpm, under 4 DEG C of cryogenic conditions, 15min is centrifuged, carefully pipettes supernatant into clean centrifuge tube, abandoned
Remove unlabelled gold colloidal precipitate；
(5) by supernatant in 10000rpm, under 4 DEG C of cryogenic conditions, 30min is centrifuged, abandons supernatant, retain the colloid of labelling
Gold grain is precipitated, and redissolves 5 times with gold mark working solution, 4 DEG C of preservations.
2nd, the optimization of Salmonella nucleic acid membrane chromatographic test strip working condition
(1) choose the nitrocellulose filter AE 98, Milipore HF90s of four kinds of different models, Milipore HF135s,
Milipore HF180s, verify test strips color developing effect；
Detection line coating streptavidin concentration choose 0.5 respectively, 1.0,1.5,2mg/mL, the coated goat-anti of nature controlling line
Mus two are anti-to choose respectively 60,80,100,120 times of dilution, verifies test strips color developing effect；
(3) loading developping solution is respectively adopted PBS (pH 7.4), the PBS (pH 7.4) containing 0.1%BSA,
The PBS (pH 7.4) and Tris EDTA (TE, pH 8.0) of 0.1%Tween 20, verifies test strips color developing effect；
Wherein select the nitric acid nitrocellulose filter of model Milipore HF 135s, detection line coating streptavidin
Concentration 2mg/mL, the sheep anti mouse two of 80 times of nature controlling line coating dilution resists, using reagent paper under conditions of Tris EDTA sample-loading buffers
Bar color developing effect is optimal.
3rd, Salmonella nucleic acid membrane chromatographic test strip assembling
(1) it is room in 83mm × 3mm gold standard pads colloidal gold labeled monoclonal antibody to be dropwise uniformly coated on into specification according to 30 μ L/cm
Warm vacuum overnight dry for standby.
(2) detection line (T lines) is coated with streptavidin, and concentration is 2mg/mL, and nature controlling line (C lines) coating sheep anti mouse two resists, dilute
Release 100 times, C, T line is marked according to 1 μ L/cm on nitrocellulose filter (NC films) using film instrument is drawn, 37 DEG C of overnight dry for standby.
(3) equally sample pad and absorbent paper are overnight dried at 37 DEG C, with gold standard pad, NC films, absorbent paper is assembled into reagent paper
Bar, using cutting machine the test strips that specification is 60mm × 3.7mm are cut into, and are fixed in cabinet, sealed under room temperature, are kept away
Light, kept dry.
Embodiment 3
The foundation of Salmonella nucleic acid membrane chromatographic test strip detection method
(1) the PCR containing Salmonella unwindase isothermal amplification reactions system is added as template using 1 μ L Salmonellas DNA
Guan Zhong, while using the aseptic double-distilled water of equivalent as negative control, 60min is reacted at above-mentioned reaction system is placed in into 68 DEG C to be carried out
The unwindase constant-temperature amplification of Salmonella.
(2) select the PBS (pH 7.4) of 0.1%BSA and 0.1%Tween 20 as loading developping solution, take 10 μ L
Amplified production is added drop-wise on colloidal gold strip Jing after loading developping solution dilutes 10 times and is detected, test is needed in triplicate, 5 points
Testing result is observed after clock.Be positive result detection line and nature controlling line of the amplified production of Salmonella shows redness；Blank
There was only the aobvious redness of nature controlling line into negative.
Embodiment 4
Salmonella nucleic acid membrane chromatographic test strip specificity experiments
Detect that other common food-borne pathogens include Escherichia coli O 157 according to the method described in example 3:H7, will are congratulated
Salmonella, clostridium perfringen, Listeria monocytogenes, staphylococcus aureuses and jejunum campylobacter bar, and made with the aseptic ultra-pure water of equivalent
For negative control, except five plants of Salmonella results are into the positive, other experimental strain results into feminine gender, experimental result such as Fig. 3 institutes
Show, show that the detection technique possesses high specific.
Embodiment 5
Salmonella nucleic acid thin film testing test strips pure culture sensitivity experiment
(1) pure bacterium solution sensitivity technique Salmonella, in being inoculated in 10mL LB fluid mediums, 37 DEG C are overnight trained
Support；
(2) respectively taking 1mL carries out count of bacteria, 10 times of gradient dilutions is carried out to pure bacterium solution with 0.9% physiological saline solution, respectively
Take 1mL for DNA extraction as doing experiment pattern；
(3) tested according to isothermal amplification reactions system, take 10 μ L amplified production loading developping solutions and be diluted to 100 μ L,
It is added drop-wise in test strips sample pad, takes test in triplicate, testing result is observed after 5min, determines that the method is detecting pure bacterium solution
Sensitivity.
The pure training liquid concentration of Salmonella is supported into gradient dilution to 4.5 × 101CFU/mL, Jing after unwindase isothermal amplification reactions
Deca can show positive findingses in test strips, and the sensitivity for showing Salmonella nucleic acid membrane chromatographic test strips is 4.5
×101CFU/mL。
Embodiment 6
Salmonella nucleic acid thin film test strips actual sample sensitivity experiment
1st, solid sample sensitivity experiment
(1) aseptically weigh 25g solid samples to be placed in aseptic homogenizing bag, homogenizing 3min.
(2) in injecting a sample into 225mL SC culture fluid, will cultivate to concentration is 108The Salmonella of CFU/mL, uses
After 0.9% 10 times of physiological saline solution gradient dilution, the bacterium solution for adding 1mL is placed in sample enrichment liquid so as to final concentration of
101CFU/mL,
(3), at 37 DEG C, cultivate under the conditions of 200r/min.Period gathers a culture sample every 2h,
(4) two parallel laboratory test group heat treating process of per part of setting extract DNA, while the sample for being not added with bacterium is right as blank
According to, tested according to isothermal amplification reactions system,
(5) take 10 μ L amplified production loading developping solutions and be diluted to 100 μ L, be added drop-wise in test strips sample pad, test repeats
Three times, testing result is observed after 5min, it is determined that the sensitivity of detection solid kind sample the method.
2nd, liquid sample sensitivity experiment
(1) 25mL liquid or semi-solid sample are aseptically measured, in injecting a sample into 225mL SC culture fluid；
(2) it is 10 that will cultivate to concentration8The Salmonella of CFU/mL, with 0.9% physiological saline solution, 10 times of gradient dilutions
Afterwards, the bacterium solution for adding 1mL is placed in sample enrichment liquid so as to final concentration of 101CFU/mL；
(3), at 37 DEG C, cultivate under the conditions of 200r/min.Period gathers a culture sample every 2h；
(4) two parallel laboratory test group heat treating process of per part of setting extract DNA, while the sample for being not added with bacterium is right as blank
According to being tested according to isothermal amplification reactions system；
(5) take 10 μ L amplified productions and be diluted to 100 μ L with the loading developping solution for having optimized, be added drop-wise in test strips sample pad,
Test in triplicate, observes testing result after 5min, it is determined that the sensitivity of detection liquid state sample the method.
Concentration is added with for 101The actual sample of the Salmonella of CFU/mL, can detect, while not adding bacterium after bacterium 2h is increased
Sample do not detect, as a result show that the method can be applied in the quick detection of actual sample.
Claims (10)
1. a kind of Salmonella nucleic acid rapid detection kit, it is characterised in that：Including forward primer invA-F:Sequence 5 '-
ATTTCTATGTTCGTCATTCCATTACCTACC-3 ', reverse primer invA-R:Sequence：5’-
ATGTAGAACGACCCCATAAACACCAA-3 ', with Digoxin and 5 ' ends of biotin difference labelling upstream and downstream primer, Jin Biao
Antibody, the antibody of anti-gold labeling antibody kind, the ligand substance of the thing biotin that develops the color.
2. Salmonella nucleic acid rapid detection kit according to claim 1, it is characterised in that：Also include amplification body
System, the amplification system includes 10 × Annealing Buffer, dNTP, MgSO4、NaCl、Enzyme Mix。
3. Salmonella nucleic acid rapid detection kit according to claim 1, it is characterised in that：The gold labeling antibody is
Digoxin and biotin.
4. Salmonella nucleic acid rapid detection kit according to claim 1, it is characterised in that：The colour developing thing is biological
The ligand substance streptavidin of element.
5. Salmonella nucleic acid rapid detection kit according to claim 1, it is characterised in that：The gold labeling antibody is
Mus anti digoxin antibody, anti-gold labeling antibody is anti-for sheep anti mouse two.
6. a kind of Salmonella nucleic acid quick detection test paper, it is characterised in that：Including sample pad, gold standard pad, nitrocellulose filter
And absorbent paper, sample pad one end is Chong Die with gold standard pad one end end, gold standard pad other end nitre Chong Die with nitrocellulose filter end
Acid cellulose film the other end is Chong Die with absorbent paper, is embedded with detection line and nature controlling line nitrocellulose filter is spaced and parallel, inspection
, in gold standard pad side, nature controlling line is between detection line and absorbent paper end for survey line；
Gold labeling antibody is sprayed in the gold standard pad, the ligand substance of coating colour developing thing biotin, is coated with nature controlling line in detection line
The antibody of anti-gold labeling antibody kind.
7. Salmonella nucleic acid quick detection test paper according to claim 6, it is characterised in that：Spray in the gold standard pad
The gold colloidal of mark anti digoxin antibody, is coated with streptavidin in detection line, streptavidin concentration is 2mg/mL, on nature controlling line
Coating sheep anti mouse two resists.
8. Salmonella nucleic acid quick detection test paper according to claim 6, it is characterised in that：The sample pad is glass
Fiber, each several part lap is 2-10mm, and in Test paper bottom PVC backboards are installed.
9. Salmonella nucleic acid quick detection test paper according to claim 6, it is characterised in that：Quick detection test paper is used
Loading developping solution is the PBS pH7.4 for adding 0.1%BSA and 0.1%Tween 20.
10. a kind of Salmonella nucleic acid method for quick, it is characterised in that：It comprises the steps：
(1) monoclonal antibody such as Mus anti digoxin antibody is adsorbed on substance that show color colloid gold particle as gold labeling antibody, is formed
The coalition of rock-steady structure is fixed in gold standard pad；
(2) Streptavidin is coated on nitrocellulose filter with string-like form as the ligand substance of label biotin and is formed
Detection line；
(3) select the antibody of anti-gold labeling antibody kind such as sheep anti-mouse antibody to be equally coated on nature controlling line with string-like form to combine
Many gold labeling antibodies；
(4) design on the forward primer 5 ' and downstream primer 5 ' of specificity constant-temperature amplification respectively that modified antigen or hapten material be such as
Digoxin and biotin, when there is sramana's nucleic acid amplification product to be measured, the amplified reaction that nucleic acid is carried out under constant temperature is obtained
To the amplified production for connecting two kinds of label Digoxin-target fragment-biotin simultaneously；
(5) Digoxin-target fragment-the biotin for (4) step being produced first with colloid gold particle in gold standard pad (glass fibre)
Coated DigiTAb is combined, and forms DigiTAb-Digoxin-target fragment-biotin colour developing particle composites；
(6) the colour developing particle composites that (5) step obtains are moved upwards by capillarity in sample-loading buffer along cellulose membrane
Move to detection line and DigiTAb-Digoxin-target fragment-biotin-strepto- affinity is obtained by the capture of receptor streptavidin
Plain complex, the sedimentation in detection line is formed on the colour developing band of macroscopic, and the colloid gold particle of uncombined amplified production
Coated gold labeling antibody is combined with the sheep anti-mouse antibody on nature controlling line, two colour developing bands is occurred and is positive findingses；
Or, when there is no sramana's nucleic acid amplification product, do not occur above-mentioned steps (3)-(6), it is impossible to gold standard pad (glass fibers
Dimension) on the coated DigiTAb of colloid gold particle combine, substance that show color colloid gold particle can not be with coated thing in detection line
Matter combines to form colour developing band, and the sheep anti-mouse antibody on nature controlling line is combined appearance one with coated gold labeling antibody on colloid gold particle
Bar colour developing band is negative findings.
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| CN111235223A (en) * | 2020-02-18 | 2020-06-05 | 华中农业大学 | Primer and test strip detection method for small fragment deletion typing detection |
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| CN111647642B (en) * | 2020-05-19 | 2023-10-20 | 深圳市众循精准医学研究院 | Nucleic acid detection method and nucleic acid detection kit based on detection test paper display |
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