CN106636387B - Kit, test paper and detection method for rapid detection of salmonella nucleic acid - Google Patents
Kit, test paper and detection method for rapid detection of salmonella nucleic acid Download PDFInfo
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- CN106636387B CN106636387B CN201611149351.4A CN201611149351A CN106636387B CN 106636387 B CN106636387 B CN 106636387B CN 201611149351 A CN201611149351 A CN 201611149351A CN 106636387 B CN106636387 B CN 106636387B
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- 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
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- 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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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6804—Nucleic acid analysis using immunogens
Abstract
The invention relates to a rapid detection kit, test paper and a detection method for salmonella nucleic acid, and relates to the design of a salmonella specific primer, the optimization of a helicase isothermal amplification condition, and the establishment of a nucleic acid thin film chromatography detection test paper strip, which can realize the visual observation of a nucleic acid detection result. The whole reaction process only needs one constant temperature device, and the single sample only needs 1 hour from pretreatment to detection, and the detection result can be obtained, and the sensitivity of the method for detecting the salmonella is 4.5 multiplied by 101CFU/mL, and has no cross reaction with other common food-borne pathogenic bacteria, the test strip of the invention has simple operation, high sensitivity, strong specificity and high efficiency, and is particularly suitable for field detection and clinical diagnosis of basic units.
Description
Technical Field
The invention relates to a rapid detection method of a nucleic acid isothermal amplification product, which is applied to the detection process of food-borne pathogenic bacteria by using a nucleic acid film chromatography detection test strip to detect a target sequence, and particularly relates to a salmonella helicase isothermal amplification and nucleic acid film chromatography detection test strip.
Technical Field
The analysis of the food safety events happened in the past has high frequency of food poisoning caused by bacterial contamination, large influence degree and wide range of involvement. The incidence of human and animal infectious diseases caused by food-borne pathogenic bacteria is still high, and common pathogens mainly comprise salmonella, escherichia coli O157, staphylococcus aureus, listeria and the like. Salmonella is widely existed in nature as pathogenic bacteria capable of causing human and livestock co-morbidity, and the currently identified salmonella serotypes are more than 2500, wherein the main serotypes causing food-borne salmonellosis are enteritis, typhimurium and swine cholera salmonella, and endotoxin released after thalli cracking attacks intestinal mucosa, nerves and blood vessels to cause poisoning symptoms such as headache, vomit, diarrhea, fever and the like. Therefore, the food safety situation is still severe, and the effective monitoring of the bacterial pollution problem becomes a main solution for avoiding the occurrence of food pollution events. Therefore, rapid, sensitive, specific detection methods are essential for clinical diagnosis and to ensure food safety. The conventional standard detection adopts a traditional culture method, pathogenic bacteria to be detected need to be detected independently according to specified detection steps, the detection period is long, the operation is complex, the workload is large, inspectors need professional experience, and the detection result is easily influenced by human factors, so that misjudgment occurs.
The detection method applying the immunological basic principle is mainly based on the specific immunological binding reaction of the antigen and the antibody. The microorganism itself specific antigen substance stimulates the organism to obtain corresponding specific antibody. The enzyme linked immunosorbent assay kit and the colloidal gold test strip are adopted to develop a rather mature technology in the aspect of detecting food-borne pathogenic bacteria, and the detection range is wide. The product has the characteristics of simple and convenient operation, convenience, rapidness, low price, accurate result and the like, gradually enters basic detection and medical departments, is even applied to common household autonomous detection, and brings a plurality of convenience for daily disease diagnosis of people. However, the immunological technology still has some disadvantages in the current research stage and practical application, mainly because the preparation procedure of the monoclonal antibody is complicated, a large amount of manpower and material resources are consumed, and the obtained antibody specificity is not ideal due to the combined action of various factors, and the specificity and the sensitivity of the detection result are directly influenced. Reports of false positives or false negatives frequently occur during the actual detection process, indicating that the stability of the method is to be further improved.
In recent years, with the intensive research and rapid development of molecular biology, the determination methods of pathogens such as bacteria in practical tests have been shifted to molecular biology methods such as Polymerase Chain Reaction (PCR), gene chip, probe hybridization, etc. through traditional single biochemical experiments. The polymerase chain reaction selectively carries out the method for amplifying DNA or RNA segments inside in vitro simulation cells through specific primers, the DNA replication method can realize that specific gene segments in a very small amount of genome are subjected to exponential amplification within a few hours, the detection sensitivity is obviously improved, the most sensitive detection technology on the current research level is provided, the method has the advantages of high accuracy and strong specificity, and plays an important role in the fields of microbial detection, diagnosis of infectious and hereditary diseases, detection of gene mutation and the like. However, the PCR technique requires a precise thermal cycle process, and is limited in that the reaction process must rely on a precision instrument and equipment, thereby limiting its application to on-site testing and in a grassroots area where facilities are scarce.
At present, nucleic acid isothermal amplification technology at home and abroad is rapidly developed, and the technology is widely applied to loop-mediated isothermal amplification, strand substitution amplification, rolling loop amplification, nicking enzyme nucleic acid isothermal amplification, helicase-dependent isothermal amplification and the like. The reaction process only needs to be carried out at a constant temperature, the specific amplification of DNA or RNA can be realized, and the sensitivity can reach the detection level of the PCR technology.
The invention discloses a salmonella constant-temperature fluorescence detection primer group, a kit and a detection method capable of avoiding false negative (publication No. CN105219870A) and a salmonella enteritidis strand displacement constant-temperature amplification (SDA) rapid detection kit and a detection method thereof (publication No. CN102382884A), which describe that amplification of a salmonella DNA nucleic acid sequence is realized under constant temperature conditions according to different amplification principles, but the method still needs to be combined with a gel electrophoresis and imaging system to process and analyze experimental data, does not obviously simplify the detection operation steps and cannot embody the advantages of the method in public health emergencies.
In the constant temperature amplification process, an upstream primer and a downstream primer which are respectively modified with specific markers are utilized to introduce two different types of nucleic acid markers into an amplification product and can be captured by an antibody or a ligand thereof which is specifically combined, the detection principle is based on an approximate double antibody sandwich method colloidal gold immunochromatography analysis method, the method is applied to the detection of the constant temperature amplification products aiming at various food-borne pathogenic bacteria and virus genes, and the invention patent 'double-marker nucleic acid constant temperature amplification method and detection test paper strip' with the publication number of CN102329790A provides a method for utilizing probes for marking biotin and fluorescein isothiocyanate to realize the rapid detection of LAMP reaction products. However, the amplification system needs a plurality of internal and external primers to participate together, so that the formation of non-specific amplification products such as primer dimers is difficult to avoid, and the amplification result of the type is not suitable for the detection of a test strip.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a rapid detection technology for detecting Salmonella (Salmonella) which is a harmful food-borne pathogenic bacterium by utilizing helicase for constant-temperature amplification, and particularly provides a rapid, accurate and sensitive Salmonella nucleic acid detection kit, test paper and detection method.
The technical scheme for realizing the purpose of the invention is as follows:
a kit for rapidly detecting salmonella nucleic acid comprises a forward primer invA-F sequence 5'-ATTTCTATGTTCGTCATTCCATTACCTACC-3' and a reverse primer invA-R sequence: 5'-ATGTAGAACGACCCCATAAACACCAA-3', labeling 5 ' ends of the upstream and downstream primers with digoxin and biotin, respectively, labeling the gold-labeled antibody, the antibody against the gold-labeled antibody species, and the ligand substance of the color-developing substance biotin.
Furthermore, the kit comprises an amplification system, wherein the amplification system comprises 10 x Annealing Buffer, dNTP and MgSO4、NaCl、Enzyme Mix。
Moreover, the gold-labeled antibody is digoxin and biotin.
Moreover, streptavidin is a ligand substance for the color former biotin.
Furthermore, the gold-labeled antibody is a mouse anti-digoxin antibody, and the anti-gold-labeled antibody is a goat anti-mouse secondary antibody.
A rapid detection test paper for salmonella nucleic acid comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and absorbent paper, wherein one end of the sample pad is overlapped with one end of the gold-labeled pad, the other end of the gold-labeled pad is overlapped with the end of the nitrocellulose membrane, the other end of the nitrocellulose membrane is overlapped with the absorbent paper, detection lines and quality control lines are embedded in the nitrocellulose membrane at intervals in parallel, the detection lines are arranged on one side of the gold-labeled pad, and the quality control lines are arranged between the detection lines and the end of the absorbent paper;
the gold-labeled pad is sprayed with a gold-labeled antibody, the detection line is coated with a ligand substance of a chromogenic substance biotin, and the quality control line is coated with an antibody of an anti-gold-labeled antibody species.
And the gold-labeled pad is sprayed with colloidal gold labeled with digoxin antibody, a detection line is coated with streptavidin, the concentration of the streptavidin is 2mg/mL, and a quality control line is coated with goat-anti-mouse secondary antibody diluted by 80 times.
And the sample pad is made of glass fiber, the overlapped part of each part is 2-10mm, and a PVC back plate is arranged at the lower part of the detection test paper.
Furthermore, the quick test strip used the loading developing solution was PBS buffer pH7.4 with 0.1% BSA and 0.1% Tween 20 added.
A method for rapidly detecting salmonella nucleic acid comprises the following steps:
the method comprises the steps of adsorbing a monoclonal antibody such as a mouse anti-digoxin antibody as a gold-labeled antibody on colloidal gold particles of a chromogenic substance, fixing a combined body forming a stable structure on a gold-labeled pad, wherein the gold-labeled pad is a pad made of glass fibers;
streptavidin is used as a ligand substance of the marker biotin and is coated on the nitrocellulose membrane in a linear form to form a detection line;
selecting antibodies of anti-gold-labeled antibody species such as goat anti-mouse antibodies, coating the antibodies on the quality control line in a linear form, and combining excessive gold-labeled antibodies;
designing an upstream primer 5 'and a downstream primer 5' for specific constant-temperature amplification to respectively modify antigen or hapten substances such as digoxin and biotin, and carrying out amplification reaction on nucleic acid under a constant-temperature condition to obtain an amplification product simultaneously connected with two markers of digoxin-target fragment-biotin when a to-be-detected salmonella nucleic acid amplification product exists;
fifthly, combining the digoxin-target fragment-biotin generated in the step four with a digoxin antibody coated with colloidal gold particles on a gold label pad (glass fiber) to form a digoxin antibody-digoxin-target fragment-biotin color developing particle complex;
sixthly, moving the color developing particle compound obtained in the step I upwards along the cellulose membrane in a sample buffer solution through capillary action to a detection line to be captured by a receptor streptavidin to obtain a digoxin antibody-digoxin-target fragment-biotin-streptavidin complex, settling on the detection line to form a macroscopic color developing strip, combining a gold-labeled antibody coated on colloidal gold particles which are not combined with an amplification product with a goat anti-mouse antibody on a quality control line, and obtaining two color developing strips as a positive result;
or when no salmonella nucleic acid amplification product exists, the step three-sixteenth is not performed, the step three cannot be performed, the step three is performed.
Compared with other technologies, the invention has the following advantages:
the invention develops a more efficient molecular detection method for detecting salmonella by combining helicase constant-temperature amplification technology with nucleic acid thin-film chromatography detection test strips, the primers are designed and selected to have the most suitable amplification fragment position and length, so that the formation of nonspecific amplification can be effectively avoided, and the optimal development buffer solution is optimized to ensure that the nucleic acid test strips achieve the optimal color development effect. The detection method does not pollute a laboratory and the surrounding environment and does not need expensive instruments and equipment or professional operation skills, so that the detection method can be applied to the detection of large-scale samples under the condition of incomplete experimental conditions, and the detection method is characterized by comprising the following specific steps:
1. the nucleic acid thin film chromatography detection test strip for detecting the salmonella helicase isothermal amplification product has the advantages of strong specificity, high sensitivity, simple and convenient operation and strong stability;
2. the amplification process is carried out in a closed reaction system to effectively avoid the pollution of amplification products;
3. the reaction speed is high, and only about 1 hour is needed for completing all detection steps of the sample;
4. the detection process does not need complex and expensive instruments and equipment and professional technicians for operation;
5. is suitable for the preliminary judgment of the on-site rapid detection of the pathogenic bacteria of the food.
Drawings
FIG. 1 shows the Salmonella primer specificity verification. In the figure, M: DNA molecular weight marker DL 2000; 1: blank control; 2: salmonella enteritidis; 3: salmonella paratyphi type a; 4: salmonella enterica subspecies enterica; 5: salmonella choleraesuis; 6: salmonella enterica sub enteric typhi serotypes; 7: escherichia coli O157: H7; 8: e.coli (non-O157: H7); 9: shigella sonnei; 10: shigella flexneri; 11: enterobacter aerogenes; 12: listeria monocytogenes; 13: staphylococcus aureus bacteria; 14: campylobacter jejuni.
FIG. 2 shows a test strip structure for nucleic acid thin-film chromatography detection. In the figure 1: a sample pad; 2: a nitrocellulose membrane; 3: a back plate; 4: gold-labeled pads (glass fiber); 5: detecting lines; 6: a quality control line; 7: absorbent paper.
FIG. 3 shows the specificity verification of a test strip for Salmonella nucleic acid thin film chromatography detection. In the figure 1: salmonella enteritidis; 2: salmonella paratyphi type a; 3: salmonella enterica subspecies enterica; 4: salmonella choleraesuis; 5: salmonella enterica sub enteric typhi serotypes; 6: escherichia coli O157: H7; 7: e.coli (non-O157: H7); 8: shigella sonnei; 9: shigella flexneri; 10: enterobacter aerogenes; 11: listeria monocytogenes; 12: staphylococcus aureus bacteria; 13: campylobacter jejuni.
Detailed Description
For understanding the present invention, the present invention will be further described with reference to the following examples: the following examples are illustrative and not intended to be limiting, and are not intended to limit the scope of the invention.
The invention combines helicase constant temperature amplification technology and nucleic acid thin film chromatography detection technology to establish a method for quickly detecting salmonella nucleic acid. The invention aims to utilize the helicase constant-temperature amplification product detected by a nucleic acid thin film chromatography detection test strip so as to realize the rapid detection of nucleic acid. Specific primers of salmonella respectively labeled with biotin and digoxin are designed, target nucleic acid is amplified at constant temperature through helicase to form an amplification product modified with two markers, and the amplification product can be detected by a nucleic acid film chromatography detection test strip coated with a ligand and an antibody which are specifically identified. The detection of the target nucleic acid can be realized by applying the immunological detection principle that the nucleic acid molecule marked by the antigen or the hapten biological micromolecule can be recognized by the specific antibody or the ligand thereof. The commonly used nucleic acid markers, biotin, are capable of specifically binding to its ligand streptavidin, while digoxin readily makes available its specific antibody. Therefore, the two substances were selected as primer markers.
1. The invention provides two specific amplification primers of salmonella, and a forward primer invA-F is designed by selecting a specific invA gene of the salmonella, wherein the sequence is 5'-ATTTCTATGTTCGTCATTCCATTACCTACC-3', and the sequence is as follows: 5'-ATGTAGAACGACCCCATAAACACCAA-3', marking digoxin and biotin on the 5 ' ends of the upstream primer and the downstream primer respectively, and obtaining a DNA fragment with the size of 101bp through helicase isothermal amplification when target nucleic acid exists; if no target nucleic acid is present, no amplification product is formed, as shown in FIG. 1.
2. The invention provides a helicase constant-temperature amplification method of salmonella, which comprises the following steps: the helicase isothermal amplification reaction system of the salmonella comprises a forward primer, a reverse primer (75nM), 10 × Annealing Buffer (7 μ L), dNTP (3.5 μ L), MgSO4(2. mu.L), NaCl (4. mu.L), Enzyme Mix (3.5. mu.L), sterile ultrapure waterMake up to 50. mu.L. And (3) adding 1 mu L of extracted salmonella DNA serving as a template of a positive control and equivalent amount of sterile ultrapure water serving as a negative control into a PCR (polymerase chain reaction) tube containing the salmonella helicase isothermal amplification reaction liquid, and placing the reaction system at 68 ℃ for reaction for 90min to perform the salmonella isothermal amplification.
3. The detection method of the salmonella nucleic acid thin film chromatography detection test strip provided by the invention comprises the following steps (the principle is shown in figure 2):
the method comprises the steps of adsorbing a monoclonal antibody such as a mouse anti-digoxin antibody as a gold-labeled antibody on a coloring matter colloidal gold particle, and fixing a combined body forming a stable structure on a gold-labeled pad (namely a glass fiber pad).
And streptavidin is used as a ligand substance of the marker biotin and is coated on the nitrocellulose membrane in a linear form to form a detection line.
Selecting antibodies of anti-gold-labeled antibody species such as goat anti-mouse antibodies, and coating the antibodies on the quality control line in a linear form to bind excessive gold-labeled antibodies.
Fourthly, antigen or hapten substances such as digoxin and biotin are respectively modified on an upstream primer 5 'and a downstream primer 5' of the specific constant-temperature amplification, and when a to-be-detected salmonella nucleic acid amplification product exists, the nucleic acid amplification reaction is carried out under the constant-temperature condition to obtain an amplification product which is simultaneously connected with two markers of digoxin-target fragment-biotin.
Fifthly, the digoxin-target fragment-biotin generated in the step four is firstly combined with the digoxin antibody coated on the gold particles on the gold label pad (glass fiber) to form a digoxin antibody-digoxin-target fragment-biotin color developing particle complex.
Sixthly, moving the color developing particle compound obtained in the step I upwards along the cellulose membrane in a sample buffer solution through capillary action to a detection line to be captured by a receptor streptavidin to obtain a digoxin antibody-digoxin-target fragment-biotin-streptavidin complex, settling on the detection line to form a macroscopic color developing strip, combining a gold-labeled antibody coated on colloidal gold particles which are not combined with an amplification product with a goat anti-mouse antibody on a quality control line, and obtaining two color developing strips as a positive result;
or when no salmonella nucleic acid amplification product exists, the step three-sixteenth is not performed, the step three cannot be performed, the step three is performed.
4. The invention also provides a salmonella nucleic acid thin film chromatography detection test strip structure as shown in figure 2: the detection device comprises a sample pad, a combination pad, a nitrocellulose membrane (NC) and absorbent paper which are sequentially arranged on a PVC (polyvinyl chloride) backboard, wherein at least 2mm of overlapped parts of the above parts are reserved, a detection line (containing a ligand substance of a marker biotin) and a quality control line (containing streptavidin) are respectively arranged on the nitrocellulose membrane, and all materials are required to be dried overnight at 37 ℃.
5. The invention optimizes the salmonella nucleic acid thin film chromatography detection test strip: firstly, respectively optimizing the reaction conditions of constant-temperature amplification of salmonella helicase, wherein the reaction conditions comprise primer concentration and MgSO4Concentration, dNTP concentration and reaction temperature to obtain optimal reaction conditions so that the amplification reaction achieves the optimal effect. Meanwhile, the design of the colloidal gold detection test strip is optimized, and the test strip comprises the type of the nitrocellulose membrane, the coating concentration of the detection line and the quality control line and a sample loading developing solution, so that the test strip presents the optimal color development effect.
The experimental procedures in the following examples are conventional unless otherwise specified. The reagent materials used in the following examples were purchased from conventional biochemical reagent suppliers, unless otherwise specified.
Example 1
Optimization of salmonella helicase isothermal amplification system
In primer concentration, MgSO4Performing orthogonal test on salmonella by using dNTP and reaction temperature as variables, wherein the primer concentrations are 75nm, 80nm and 85nm respectively; MgSO (MgSO)4Concentrations of 3mM, 3.5mM and 4mM, respectively; the addition amounts of dNTP are respectively 2.5 muL, 3.5 muL and 4.5 muL; the reaction temperatures were 63 deg.C, 64 deg.C and 65 deg.C, respectively. It expandsThe amplification product is subjected to agarose gel electrophoresis amplification, and then the amplification effect is observed by using a gel imager, so that a total reaction system of 50 mu L of salmonella amplification is obtained through verification, wherein the total reaction system comprises 1 mu L of DNA template, the concentration of primer is 80nM, and MgSO4The concentration was 3.5mM, the amount of dNTP added was 4.5. mu.L, and finally 3.5. mu.L of Enzyme Mix was added, and the mixture was allowed to react at 64 ℃ for 60 min.
Example 2
Preparation of salmonella nucleic acid film chromatography detection test paper strip
1. Preparation of colloidal gold-labeled antibody
Firstly, 1mL of 10nm colloidal gold solution is put into a 1.5mL clean centrifugal tube, and a certain amount of K is dripped2CO3Adjusting the colloidal gold solution to the optimum pH value by the solution;
dripping 15 mu L of antibody with the concentration of 1 mu g/mu L into the colloidal gold solution, rapidly shaking for 5min, and standing the uniformly mixed solution for 1h at 4 ℃;
thirdly, 20 mu L of 20% BSA solution and 10 mu L of 20% PEG 20000 solution are dripped into the mixture, and the uniformly mixed solution is kept stand for 30min at the temperature of 4 ℃;
fourthly, centrifuging for 15min at the low temperature of 4 ℃ at 2000rpm, carefully transferring supernatant into a clean centrifugal tube, and discarding unmarked colloidal gold precipitate;
fifthly, centrifuging the supernatant for 30min at 10000rpm and 4 ℃ low temperature, removing the supernatant, retaining the marked colloidal gold particles for precipitation, redissolving the colloidal gold particles by 5 times by using a gold-labeled working solution, and storing the colloidal gold particles at 4 ℃.
2. Optimization of working conditions of salmonella nucleic acid thin film chromatography detection test strip
Selecting four different types of cellulose nitrate membranes AE 98, Milipore HF90s, Milipore HF135s and Milipore HF180s, and verifying the color development effect of the test paper strip;
secondly, the concentrations of the coated streptokinase avidin of the detection lines are respectively selected to be 0.5, 1.0, 1.5 and 2mg/mL, the goat anti-mouse secondary antibody coated by the quality control line is respectively selected to be diluted by 60, 80, 100 and 120 times, and the color development effect of the test paper strip is verified;
thirdly, the sample loading developing solution respectively adopts PBS buffer solution (pH 7.4), PBS buffer solution (pH 7.4) containing 0.1% BSA, PBS buffer solution (pH 7.4) containing 0.1% Tween 20 and Tris EDTA (TE, pH 8.0), and the test strip color developing effect is verified;
wherein a nitrocellulose membrane with the model of Milipore HF135s is selected, the detection line is coated with streptavidin with the concentration of 2mg/mL, the quality control line is coated with goat-anti-mouse secondary antibody diluted by 80 times, and the test strip has the best color development effect under the condition of adopting Tris EDTA loading buffer solution.
3. Salmonella nucleic acid film chromatography detection test strip assembly
Uniformly coating a colloidal gold-labeled antibody on a gold-labeled pad with the specification of 83mm multiplied by 3mm dropwise according to 30 muL/cm, and drying the gold-labeled pad at room temperature overnight in vacuum for later use.
And the streptavidin is coated by a detection line (T line), the concentration is 2mg/mL, the goat anti-mouse secondary antibody is coated by a quality control line (C line), the dilution is 100 times, C, T lines are drawn on a nitrocellulose membrane (NC membrane) according to 1 muL/cm by using a membrane drawing instrument, and the solution is dried overnight at 37 ℃ for later use.
And thirdly, drying the sample pad and the absorbent paper at 37 ℃ overnight, assembling the sample pad and the absorbent paper into a test strip together with the gold label pad, the NC film and the absorbent paper, cutting the test strip into the test strip with the specification of 60mm multiplied by 3.7mm by a slitter, fixing the test strip in a plastic shell, sealing the test strip at room temperature, and keeping the test strip in a dark and dry manner.
Example 3
Establishment of test paper strip detection method for salmonella nucleic acid thin film chromatography detection
The method comprises the steps of taking 1 mu L of salmonella DNA as a template to be added into a PCR tube containing a salmonella helicase constant-temperature amplification reaction system, taking an equal amount of sterile double distilled water as negative control, and reacting the reaction system at 68 ℃ for 60min to perform salmonella helicase constant-temperature amplification.
And selecting PBS (pH 7.4) of 0.1% BSA and 0.1% Tween 20 as a sample loading developing solution, diluting 10 microliter of amplification product by the sample loading developing solution by 10 times, and then dropwise adding the amplification product onto a colloidal gold test strip for detection, wherein the test needs to be repeated for three times, and the detection result is observed after 5 minutes. The detection line and the quality control line of the positive result of the amplification product of the salmonella are both red; the blank control is negative and only the quality control line is red.
Example 4
Test strip specificity experiment for salmonella nucleic acid thin film chromatography detection
Other common food-borne pathogenic bacteria including Escherichia coli O157H 7, Shigella, Enterobacter aerogenes, Listeria monocytogenes, Staphylococcus aureus and Campylobacter jejuni are detected according to the method described in example 3, and the results of other experimental strains are negative except five strains of Salmonella by taking the same amount of sterile ultrapure water as negative control, and the experimental results are shown in FIG. 3, which indicates that the detection technology has high specificity.
Example 5
Salmonella nucleic acid film detection test strip pure culture sensitivity experiment
The method comprises the steps of detecting salmonella through pure bacteria liquid sensitivity, inoculating the salmonella into 10mL of LB liquid culture medium, and culturing overnight at 37 ℃;
1mL of sterile physiological saline is taken for counting bacteria, 10 times of gradient dilution is carried out on the pure bacteria liquid by using 0.9 percent of sterile physiological saline, and 1mL of sterile physiological saline is taken for extracting DNA and is used as an experimental template;
thirdly, testing according to a constant-temperature amplification reaction system, diluting 10 mu L of amplification product to 100 mu L with a sample loading developing solution, dropwise adding the diluted product to a test strip sample pad, repeating the test for three times, observing a detection result after 5min, and determining the sensitivity of the method in detecting pure bacterial liquid.
The salmonella pure culture solution is diluted to 4.5 multiplied by 10 in a concentration gradient way1CFU/mL, after helicase constant temperature amplification reaction, the test strip dripped on the test strip can show positive results, which shows that the sensitivity of the salmonella nucleic acid thin film chromatography test strip is 4.5 multiplied by 101CFU/mL。
Example 6
Salmonella nucleic acid film test strip actual sample sensitivity experiment
1. Solid state sample sensitivity test
Weighing 25g of solid sample under a sterile condition, placing the solid sample in a sterile homogenizing bag, and homogenizing for 3 min.
Secondly, injecting the sample into 225mL of SC culture solution, and culturing the sample until the concentration is 108Diluting CFU/mL Salmonella with 10 times gradient of 0.9% sterile physiological saline, adding 1mL bacterial liquid, and adding into the enriched bacterial liquid to obtain final concentration of 101CFU/mL，
And culturing the three fruits at 37 ℃ and 200 r/min. During the period, culture samples are collected every 2h,
fourthly, setting two parallel experiment groups for each part, extracting DNA by using a heat treatment method, taking a sample without bacteria as a blank control, carrying out an experiment according to a constant-temperature amplification reaction system,
fifthly, diluting 10 mu L of amplification product to 100 mu L with the sample loading developing solution, dropwise adding the diluted product to the test strip sample pad, repeating the test for three times, observing the detection result after 5min, and determining the sensitivity of the method for detecting the solid sample.
2. Liquid sample sensitivity test
Measuring 25mL of liquid or semisolid sample under a sterile condition, and injecting the sample into 225mL of SC culture solution;
the culture is carried out until the concentration is 108Diluting CFU/mL Salmonella with 10 times gradient of 0.9% sterile physiological saline, adding 1mL bacterial liquid, and adding into the enriched bacterial liquid to obtain final concentration of 101CFU/mL；
And culturing the three fruits at 37 ℃ and 200 r/min. Collecting culture samples every 2h during the period;
fourthly, setting two parallel experiment groups for each part, extracting DNA through a heat treatment method, taking a sample without bacteria as a blank control, and carrying out an experiment according to a constant-temperature amplification reaction system;
and fifthly, diluting 10 mu L of the amplification product to 100 mu L with the optimized sample loading developing solution, dropwise adding the diluted product to the test strip sample pad, repeating the test for three times, observing the detection result after 5min, and determining the sensitivity of the method for detecting the liquid sample.
Adding with a concentration of 101The actual sample of the CFU/mL salmonella can be detected after 2h of enrichment, and meanwhile, the sample without bacteria addition is not detected, and the result shows that the method can be applied to the rapid detection of the actual sample.
Claims (2)
1. A kit for rapidly detecting salmonella nucleic acid is characterized in that: comprises a forward primer invA-F sequence 5'-ATTTCTATGTTCGTCATTCCATTACCTACC-3', a reverse primer invA-R sequence: 5'-ATGTAGAACGACCCCATAAACACCAA-3', labeling the 5 ' ends of the forward primer and the reverse primer, the gold-labeled antibody, the antibody against the gold-labeled antibody species, and the ligand substance of the color development substance biotin with digoxin and biotin, respectively;
also comprises a helicase isothermal amplification system, wherein the amplification system comprises 10 x Annealing Buffer, dNTP and MgSO4、NaCl、Enzyme Mix；
The ligand substance of the color development substance biotin is streptavidin;
the gold-labeled antibody is a mouse anti-digoxin antibody, and the antibody of the anti-gold-labeled antibody species is a goat anti-mouse secondary antibody;
the kit also comprises salmonella nucleic acid rapid detection test paper which comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and absorbent paper, wherein one end of the sample pad is overlapped with one end of the gold-labeled pad, the other end of the gold-labeled pad is overlapped with the end of the nitrocellulose membrane, the other end of the nitrocellulose membrane is overlapped with the absorbent paper, detection lines and quality control lines are embedded in the nitrocellulose membrane at intervals in parallel, the detection lines are arranged on one side of the gold-labeled pad, and the quality control lines are arranged between the detection lines and the end of the absorbent paper;
the gold-labeled pad is sprayed with a gold-labeled antibody, the detection line is coated with a ligand substance of a chromogenic substance biotin, and the quality control line is coated with an antibody of an anti-gold-labeled antibody species;
the gold label pad is sprayed with colloidal gold of a mouse anti-digoxin antibody, a detection line is coated with streptavidin, the concentration of the streptavidin is 2mg/mL, and a quality control line is coated with a goat anti-mouse secondary antibody;
the sample pad is made of glass fiber, the overlapped part of each part is 2-10mm, and a PVC back plate is arranged at the lower part of the detection test paper;
the quick test strip uses a PBS buffer solution added with 0.1% BSA and 0.1% Tween 20, and pH7.4.
2. A method for rapid non-diagnostic detection of salmonella nucleic acid according to the kit of claim 1, wherein: which comprises the following steps:
the method comprises the steps of adsorbing a mouse anti-digoxin antibody serving as a gold-labeled antibody on colloidal gold particles of a chromogenic substance to form a stable-structure combination fixed on a gold-labeled pad;
streptavidin is used as a ligand substance of the marker biotin and is coated on the nitrocellulose membrane in a linear form to form a detection line;
thirdly, coating the goat anti-mouse antibody on the quality control line in a linear form to combine with excessive gold-labeled antibody;
the 5 'end of the forward primer and the 5' end of the reverse primer are respectively modified by digoxin and biotin, and when a salmonella nucleic acid amplification product to be detected exists, the amplification reaction of the nucleic acid is carried out under a constant temperature condition to obtain an amplification product which is simultaneously connected with two markers of digoxin-target fragment-biotin;
fifthly, combining the digoxin-target fragment-biotin generated in the step four with a digoxin antibody coated by colloidal gold particles on a gold label pad to form a digoxin antibody-digoxin-target fragment-biotin color developing particle complex;
sixthly, moving the color developing particle compound obtained in the step I upwards along the cellulose membrane in a sample buffer solution through capillary action to a detection line to be captured by a receptor streptavidin to obtain a digoxin antibody-digoxin-target fragment-biotin-streptavidin complex, settling on the detection line to form a macroscopic color developing strip, combining a gold-labeled antibody coated on colloidal gold particles which are not combined with an amplification product with a goat anti-mouse antibody on a quality control line, and obtaining two color developing strips as a positive result;
or, when no salmonella nucleic acid amplification product exists, the step sixteenth to sixteenth cannot be carried out, the obtained product cannot be combined with the digoxin antibody coated by the colloidal gold particles on the gold-labeled pad, the colored substance colloidal gold particles cannot be combined with the substance coated on the detection line to form a colored strip, and the negative result is obtained when the goat anti-mouse antibody on the quality control line is combined with the gold-labeled antibody coated on the colloidal gold particles to form a colored strip.
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