CN106636387B - Salmonella nucleic acid rapid detection kit, test paper and detection method - Google Patents

Abstract

The invention relates to a Salmonella nucleic acid rapid detection kit, test paper and detection method, and relates to the design of Salmonella-specific primers, the optimization of constant temperature amplification conditions for helicase, and the establishment of nucleic acid film chromatography detection test strips, which can realize nucleic acid detection Visual observation of the results. The entire reaction process requires only one constant temperature device, and the test result can be obtained in only ¹ hour from the pretreatment to the completion of the detection of a single sample. Sexual pathogens have no cross-reaction, and the detection test strip of the invention is easy to operate, has high sensitivity, strong specificity and high efficiency, and is especially suitable for on-site detection and clinical diagnosis in grassroots units.

Description

Salmonella nucleic acid rapid detection kit, test paper and detection method

technical field

The invention relates to a rapid detection method for nucleic acid isothermal amplification products, which utilizes nucleic acid thin film chromatography detection strips to detect target sequences, and is applied in the detection process of food-borne pathogenic bacteria, in particular to a salmonella helicase isothermal amplification And nucleic acid thin film chromatography test strips.

technical background

Analysis of past food safety incidents shows that food poisoning caused by bacterial contamination has a high frequency, a large degree of impact and a wide range of coverage. The incidence of human and animal infections caused by food-borne pathogens is still high. The common pathogens are mainly Salmonella, Escherichia coli O157, Staphylococcus aureus, Listeria and so on. Salmonella, as a pathogenic bacteria that can cause zoonotic diseases, exists widely in nature. There are more than 2,500 Salmonella serotypes that have been identified. Among them, the main serotypes that cause food-borne salmonellosis are Enteritis, Typhimurium and Salmonella choleraesuis. Three kinds of endotoxins released after the lysis of the bacteria invade the intestinal mucosa, nerves and blood vessels, causing headache, vomiting, diarrhea, fever and other poisoning symptoms. It can be seen that the food safety situation is still severe, and the effective monitoring of bacterial contamination has become the main solution to avoid food contamination incidents. Therefore, rapid, sensitive and specific detection methods are essential for clinical diagnosis and food safety. Conventional standard testing adopts the traditional culture method. The pathogenic bacteria to be tested need to be tested separately according to the specified testing steps. The testing period is long, the operation is complicated, and the workload is large. The testing personnel need to have professional experience, and the testing results are easily affected by human factors. Misjudgment occurred.

The detection method using the basic principles of immunology is mainly based on the specific immune binding reaction between its antigen and antibody. The corresponding specific antibody is obtained by stimulating the organism with the specific antigenic substance of the microorganism itself. Among them, the use of enzyme-linked immunosorbent assay kits and colloidal gold test strips has developed into a fairly mature technology in the detection of food-borne pathogens, involving a wide range of detection. Due to its simple operation, convenience and quickness, low price and accurate results, this type of product has gradually entered the grassroots testing and medical departments and has even been used in the self-testing of ordinary households, bringing many conveniences to people's daily diagnosis of diseases. However, there are still some deficiencies in the current research stage and practical application of immunization technology. The main reason is that the preparation procedure of monoclonal antibodies is cumbersome and consumes a lot of manpower and material resources. It is not ideal and directly affects the specificity and sensitivity of the detection results. In the actual detection process, false positive or false negative reports often occur, indicating that the stability of the method needs to be further improved.

In recent years, with the in-depth research and rapid development of molecular biology, the detection methods of bacteria and other pathogens in the actual test have been transferred from the traditional single biochemical experiments to molecular biology methods such as polymerase chain reaction (PCR) technology, gene chip , probe hybridization and other techniques. The polymerase chain reaction is a method of selectively amplifying DNA or RNA fragments in vitro by using specific primers to simulate the interior of cells. This DNA replication method can achieve exponential amplification of specific gene fragments in a very small amount of genome in a few hours. , significantly improving the detection sensitivity has become the most sensitive detection technology at the current research level, with the advantages of high accuracy and specificity, and has played a role in many fields such as microbial detection, diagnosis of infectious and genetic diseases, and detection of gene mutations. important role. However, PCR technology requires precise thermal cycling process, and it is limited to the reaction process and must rely on precise instruments and equipment, thus limiting its application in on-site detection and grassroots areas where facilities are lacking.

At present, nucleic acid isothermal amplification technology has developed rapidly at home and abroad, and now widely used loop-mediated isothermal amplification, chain substitution amplification, rolling circle amplification, nickase nucleic acid isothermal amplification, and helicase-dependent isothermal amplification Wait. The reaction process only needs to be carried out at a constant temperature and can achieve specific amplification of DNA or RNA, and the sensitivity can reach the detection level of PCR technology.

Invention patents "Salmonella Enteritidis Constant Temperature Fluorescence Detection Primer Set, Kit and Detection Method" (Publication No. CN105219870A) and "Salmonella Enteritidis Strand Displacement Constant Amplification (SDA) Rapid Detection Kit and Detection Method" (Publication No. CN105219870A) No. CN102382884A) Description According to different amplification principles, the amplification of Salmonella DNA nucleic acid sequence is realized under constant temperature conditions, but this kind of method still needs to combine gel electrophoresis and imaging system to process and analyze the experimental data, and does not significantly simplify the inspection operation steps and inability to demonstrate its strengths in public health emergencies.

In the constant temperature amplification process, the upstream and downstream primers modified with specific markers are used to introduce two different types of nucleic acid markers into the amplification product, which can be captured by the specific binding antibody or its ligand. The detection principle is based on the approximate double Antibody sandwich method colloidal gold immunochromatographic analysis method, this method has been used in the detection of isothermal amplification products for various food-borne pathogenic bacteria and virus genes, and the nucleic acid detection of Salmonella, the publication number is CN102329790A Invention patent " Double-labeled nucleic acid isothermal amplification method and detection test strip" provides a method for rapidly detecting LAMP reaction products by using probes labeled with biotin and fluorescein isothiocyanate. However, since this amplification system requires the participation of multiple internal and external primers, it is difficult to avoid the formation of non-specific amplification products such as primer-dimers, so this type of amplification results is not suitable for the detection of test strips.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the deficiencies of the prior art, and to provide a rapid detection technology for detecting harmful food-borne pathogen-Salmonella by using helicase constant temperature amplification, especially a rapid and accurate detection technology. , Sensitive Salmonella nucleic acid rapid detection kit, test paper and detection method.

The technical scheme that the present invention realizes the object is as follows:

A Salmonella nucleic acid rapid detection kit, comprising forward primer invA-F: sequence 5'-ATTTCTATGTTCGTCATTCCATTACCTACC-3', reverse primer invA-R: sequence: 5'-ATGTAGAACGACCCCATAAACACCAA-3', using digoxigenin and biotin respectively Label the 5' ends of the upstream and downstream primers, gold-labeled antibodies, antibodies against the species of gold-labeled antibodies, and ligands of the chromogenic substance biotin.

Moreover, an amplification system is also included, and the amplification system includes 10×Annealing Buffer, dNTP, MgSO ₄ , NaCl, and Enzyme Mix.

Moreover, the gold-labeled antibodies are digoxigenin and biotin.

Also, streptavidin is the ligand substance of the chromogenic substance biotin.

Moreover, the gold-labeled antibody is a mouse anti-digoxigenin antibody, and the anti-gold-labeled antibody is a goat anti-mouse secondary antibody.

A Salmonella nucleic acid rapid detection test paper, comprising a sample pad, a gold label pad, a nitrocellulose membrane and an absorbent paper. One end of the sample pad overlaps with one end of the gold label pad, and the other end of the gold label pad overlaps with the end of the nitrocellulose membrane. The other end of the cellulose membrane is overlapped with the absorbent paper. A detection line and a quality control line are embedded in parallel between the nitrocellulose membranes. The detection line is on the side of the gold pad, and the quality control line is between the detection line and the end of the absorbent paper. ;

The gold-labeled pad is sprayed with a gold-labeled antibody, the detection line is coated with the ligand substance of the chromogenic substance biotin, and the quality control line is coated with an antibody of the anti-gold-labeled antibody species.

Moreover, the gold label pad was sprayed with colloidal gold labeled with anti-digoxigenin antibody, the detection line was coated with streptavidin, the streptavidin concentration was 2 mg/mL, and the quality control line was coated with 80-fold diluted Goat anti-mouse secondary antibody.

Moreover, the sample pad is made of glass fiber, and the overlapping portion of each part is 2-10 mm, and a PVC back plate is installed at the lower part of the test paper.

Also, the rapid test strips used a sample developer with PBS buffer pH 7.4 supplemented with 0.1% BSA and 0.1% Tween 20.

A rapid detection method for Salmonella nucleic acid, comprising the following steps:

(1) The monoclonal antibody such as mouse anti-digoxigenin antibody is adsorbed on the colloidal gold particles of the color-developing substance as a gold-labeled antibody, and the combination forming a stable structure is fixed on the gold-labeled pad, which is a pad made of glass fiber;

(2) Streptavidin is used as the ligand of the marker biotin to coat the nitrocellulose membrane in a linear form to form a detection line;

(3) Antibodies of the anti-gold-labeled antibody species, such as goat anti-mouse antibody, are also coated on the quality control line in a linear form that binds too much gold-labeled antibody;

(4) Design specific isothermal amplification upstream primers 5' and downstream primers 5' to modify antigens or hapten substances such as digoxigenin and biotin, respectively. The amplification reaction obtains the amplification product of the two markers digoxigenin-target fragment-biotin at the same time;

⑸ The digoxigenin-target fragment-biotin produced in step ⑷ is first combined with the digoxigenin antibody coated with colloidal gold particles on the gold label pad (glass fiber) to form the digoxigenin antibody-digoxigenin-target fragment- Biotin color particle complex;

(6) The chromogenic particle complex obtained in step (5) moves upward along the cellulose membrane by capillary action in the loading buffer to the detection line and is captured by the receptor streptavidin to obtain digoxin antibody-digoxigenin-target fragment- The biotin-streptavidin complex settles on the detection line to form a visible color band, and the gold-labeled antibody coated on the colloidal gold particles not bound to the amplification product and the sheep on the quality control line The anti-mouse antibody binds and two colored bands appear, which is a positive result;

Or, when there is no Salmonella nucleic acid amplification product, the above-mentioned steps (3)-(6) do not occur, and the digoxigenin antibody coated with colloidal gold particles on the gold label pad (glass fiber) cannot be combined, and the colloidal gold particles of the color-developing substance cannot be combined with the digoxigenin antibody. The substances coated on the detection line combine to form a colored band, and the goat anti-mouse antibody on the quality control line combines with the gold-labeled antibody coated on the colloidal gold particles to form a colored band, which is a negative result.

Compared with other technologies, the present invention has the following advantages:

The present invention develops a more efficient molecular detection method for detecting Salmonella by using the helicase constant temperature amplification technology combined with nucleic acid film chromatography detection strips, and the design of primers and selection of the optimal amplified fragment position and length can effectively avoid The formation of non-specific amplification and optimization to obtain the best development buffer enables nucleic acid test strips to achieve the most ideal color development effect. The detection method will not pollute the laboratory and the surrounding environment and does not require expensive equipment or professional operating skills, so it can handle the detection of large-scale samples under imperfect experimental conditions. The specific points are reflected in the following aspects:

1. The nucleic acid film chromatography detection test strip for detecting Salmonella helicase isothermal amplification products of the present invention has strong specificity, high sensitivity, simple operation and strong stability;

2. The amplification process is carried out in a closed reaction system to effectively avoid the contamination of amplification products;

3. The reaction speed is fast, and it only takes about 1 hour to complete all the detection steps of the sample;

4. The detection process does not require complex and expensive equipment and professional and technical personnel to operate;

5. It is suitable for the preliminary judgment of on-site rapid detection of food pathogenic bacteria.

Description of drawings

Figure 1 shows Salmonella primer specificity verification. M in the figure: DNA molecular weight marker DL2000; 1: blank control; 2: Salmonella enteritidis; 3: Salmonella paratyphi A; 4: Salmonella enterica subsp. enterica; 5: Salmonella choleraesuis; 6: Salmonella enterica enterica subsp. Type; 7: Escherichia coli O157:H7; 8: Escherichia coli (not O157:H7); 9: Shigella sonnei; 10: Shigella flexneri; 11: Enterobacter aerogenes; 12: Mono Listeria monocytogenes; 13: Staphylococcus aureus; 14: Campylobacter jejuni.

Figure 2 shows the structure of the nucleic acid thin film chromatography detection test strip. In the figure 1: sample pad; 2: nitrocellulose membrane; 3: back plate; 4: gold standard pad (glass fiber); 5: detection line; 6: quality control line; 7: absorbent paper.

Figure 3 shows the specificity verification of Salmonella nucleic acid thin film chromatography test strips. In the figure 1: Salmonella enteritidis; 2: Salmonella paratyphi A; 3: Salmonella enterica subsp. enterica; 4: Salmonella choleraesuis; 5: Salmonella enterica subsp. typhi serotype; 6: Escherichia coli O157:H7; 7: Escherichia coli (non-O157:H7); 8: Shigella sonnei; 9: Shigella flexneri; 10: Enterobacter aerogenes; 11: Listeria monocytogenes; 12: Staphylococcus aureus; 13: Campylobacter jejuni.

specific implementation

In order to understand the present invention, the present invention is further described below in conjunction with the examples: the following examples are illustrative, not restrictive, and the protection scope of the present invention cannot be limited by the following examples.

The invention combines the helicase constant temperature amplification technology and the nucleic acid thin film chromatography detection technology to establish a method for rapid detection of Salmonella nucleic acid. The invention aims to utilize the nucleic acid thin film chromatography to detect the isothermal amplification product of the helicase detected by the test strip, so as to realize the rapid detection of nucleic acid. Design specific primers for Salmonella labeled with biotin and digoxigenin respectively. The target nucleic acid is amplified by helicase at constant temperature to form an amplification product modified with two labels. The amplification product can be coated with a specific recognition Ligand and antibody nucleic acid thin film chromatography detection strips were detected. The nucleic acid molecule labeled with antigen or hapten biological small molecule can be recognized by its specific antibody or ligand to realize the detection of target nucleic acid. Among the commonly used nucleic acid markers, biotin can specifically bind to its ligand, streptavidin, while digoxin is easy to obtain its specific antibody. Therefore, the above two substances were selected as primer labels.

1. The present invention provides two specific amplification primers for Salmonella, select Salmonella specific invA gene to design forward primer invA-F: sequence 5'-ATTTCTATGTTCGTCATTCCATTACCTACC-3', reverse primer invA-R: sequence: 5'- ATGTAGAACGACCCCATAAACACCAA-3', digoxigenin and biotin label the 5' ends of the upstream and downstream primers, respectively. When the target nucleic acid exists, a DNA fragment with a size of 101 bp can be obtained by constant temperature amplification with helicase; if there is no target nucleic acid , no amplification product is formed, as shown in Figure 1.

2. The present invention provides a helicase isothermal amplification method for Salmonella: the helicase isothermal amplification reaction system for Salmonella includes forward and reverse primers (75nM), 10×Annealing Buffer (7μL), dNTP (3.5μL), MgSO ₄ (2 μL), NaCl (4 μL), Enzyme Mix (3.5 μL), supplemented to 50 μL with sterile ultrapure water. The extracted 1 μL Salmonella DNA was used as a template for positive control, and an equal amount of sterile ultrapure water was added as a negative control to a PCR tube containing a constant-temperature amplification reaction solution of Salmonella helicase, and the above reaction system was placed at 68 °C for reaction. The constant temperature amplification of Salmonella was carried out for 90 min.

3. The detection method of the Salmonella nucleic acid film chromatography detection test strip provided by the present invention comprises the following steps (the principle is shown in Figure 2):

(1) The monoclonal antibody such as mouse anti-digoxigenin antibody is used as the gold-labeled antibody to be adsorbed on the colloidal gold particles of the color-developing substance, and the conjugate to form a stable structure is fixed on the gold-labeled pad (that is, a glass fiber pad).

(2) Streptavidin is used as the ligand of the marker biotin and is coated on the nitrocellulose membrane in a linear form to form a detection line.

(3) Select the anti-gold-labeled antibody species, such as goat anti-mouse antibody, which is also coated on the quality control line in a linear form that binds too much gold-labeled antibody.

(4) Design specific isothermal amplification upstream primers 5' and downstream primers 5' to modify antigens or hapten substances such as digoxigenin and biotin, respectively. The amplification reaction obtained the amplification product of the two markers digoxigenin-target fragment-biotin at the same time.

⑸ The digoxigenin-target fragment-biotin produced in step ⑷ is first combined with the digoxigenin antibody coated with colloidal gold particles on the gold label pad (glass fiber) to form the digoxigenin antibody-digoxigenin-target fragment- Biotin chromogenic particle complex.

(6) The chromogenic particle complex obtained in step (5) moves upward along the cellulose membrane by capillary action in the loading buffer to the detection line and is captured by the receptor streptavidin to obtain digoxin antibody-digoxigenin-target fragment- The biotin-streptavidin complex settles on the detection line to form a visible color band, and the gold-labeled antibody coated on the colloidal gold particles not bound to the amplification product and the sheep on the quality control line The anti-mouse antibody binds and two colored bands appear, which is a positive result;

Or, when there is no Salmonella nucleic acid amplification product, the above-mentioned steps (3)-(6) do not occur, and the digoxigenin antibody coated with colloidal gold particles on the gold label pad (glass fiber) cannot be combined, and the colloidal gold particles of the color-developing substance cannot be combined with the digoxigenin antibody. The substances coated on the detection line combine to form a colored band, and the goat anti-mouse antibody on the quality control line combines with the gold-labeled antibody coated on the colloidal gold particles to form a colored band, which is a negative result.

4. The present invention also provides the structure of Salmonella nucleic acid film chromatography detection test strip as shown in Figure 2: it includes a sample pad, a binding pad, a nitrocellulose membrane (NC) and an absorbent paper on a PVC backboard in sequence. The overlapping part should be kept at least 2mm. The nitrocellulose membrane is equipped with a detection line (containing the ligand substance labeled with biotin) and a quality control line (including streptavidin). All materials should be dried at 37 °C overnight. .

5. The present invention optimizes the Salmonella nucleic acid film chromatography detection test strip: first, the reaction conditions of the constant temperature amplification of Salmonella helicase are optimized respectively, including primer concentration, MgSO ₄ concentration, dNTP concentration and reaction temperature, so as to obtain the optimal The optimal reaction conditions allow the amplification reaction to achieve optimal results. At the same time, the design of the colloidal gold detection test strip was optimized, including the type of nitrocellulose membrane, the coating concentration of the detection line and the quality control line, and the sample loading solution, so that the test strip showed the best color rendering effect.

The experimental methods in the following examples are conventional methods 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 isothermal amplification system for Salmonella helicase

Orthogonal experiments were carried out on Salmonella with primer concentration, MgSO ₄ , dNTP and reaction temperature as variables. The primer concentrations were 75 nm, 80 nm and 85 nm, respectively; the MgSO ₄ concentrations were 3 mM, 3.5 mM and 4 mM, and the dNTP addition amount was 2.5 μL, 3.5 μL and 4.5 μL; the reaction temperatures were 63°C, 64°C and 65°C, respectively. The amplification product was amplified by agarose gel electrophoresis and then used a gel imager to observe the amplification effect. It was verified that the total reaction system of Salmonella amplification was 50 μL, including 1 μL of DNA template, the primer concentration was 80 nM, and the MgSO ₄ concentration was 3.5mM, the amount of dNTP added was 4.5μL, and finally 3.5μL of Enzyme Mix was added, and the reaction was placed at 64°C for 60min.

Example 2

Preparation of test strips for detection of Salmonella nucleic acid by thin film chromatography

1. Preparation of colloidal gold-labeled antibodies

(1) Take 1mL of 10nm colloidal gold solution and place it in a 1.5mL clean centrifuge tube, add a certain amount of K ₂ CO ₃ solution dropwise to adjust the colloidal gold solution to the optimum pH;

(2) Take 15 μL of antibody with a concentration of 1 μg/μL and drop it into the colloidal gold solution, shake it rapidly for 5 minutes, and let the mixed solution stand at 4°C for 1 hour;

(3) Add 20 μL of 20% BSA solution and 10 μL of 20% PEG 20000 solution dropwise, and let the mixed solution stand at 4°C for 30 minutes;

(4) Centrifuge at 2000rpm and 4°C for 15min, carefully transfer the supernatant to a clean centrifuge tube, and discard the unlabeled colloidal gold precipitate;

⑸ Centrifuge the supernatant for 30 min at 10,000 rpm and 4°C at low temperature, discard the supernatant, retain the labeled colloidal gold particles, reconstitute 5 times with gold-labeled working solution, and store at 4°C.

2. Optimization of working conditions for Salmonella nucleic acid thin film chromatography test strips

(1) Select four different types of nitrocellulose membranes AE 98, Milipore HF90s, Milipore HF135s, and Milipore HF180s to verify the color rendering effect of the test strip;

(2) The concentration of streptavidin coated on the detection line is 0.5, 1.0, 1.5, and 2 mg/mL, and the goat anti-mouse secondary antibody coated on the quality control line is diluted 60, 80, 100, and 120 times, respectively, and the test strip is verified. color rendering effect;

(3) PBS buffer (pH 7.4), PBS buffer containing 0.1% BSA (pH 7.4), 0.1% Tween 20 in PBS buffer (pH 7.4) and Tris EDTA (TE, pH 8.0) were used as loading developing solutions, respectively. Verify the color rendering effect of the test strip;

Milipore HF 135s nitrocellulose membrane was selected, the detection line was coated with streptavidin concentration of 2 mg/mL, the quality control line was coated with goat anti-mouse secondary antibody diluted 80 times, and Tris EDTA loading buffer was used Under the conditions of the test strip, the color rendering effect is the best.

3. Salmonella nucleic acid thin film chromatography test strip assembly

(1) Apply 30μL/cm of colloidal gold-labeled antibody dropwise to a gold-labeled pad with a size of 83mm×3mm, and dry it in a vacuum overnight at room temperature for use.

(2) The detection line (T line) is coated with streptavidin at a concentration of 2 mg/mL, and the quality control line (C line) is coated with goat anti-mouse secondary antibody, diluted 100 times, and the nitrocellulose membrane ( C and T lines were drawn on the NC film) according to 1 μL/cm, and dried at 37°C overnight for use.

(3) The sample pad and absorbent paper were also dried at 37°C overnight, assembled with the gold standard pad, NC film, and absorbent paper to form test strips, which were cut into test strips with a size of 60mm × 3.7mm using a strip cutter, and fixed on the test strip. In a plastic case, sealed at room temperature, protected from light, and stored in a dry place.

Example 3

Establishment of a test strip detection method for Salmonella nucleic acid film chromatography

(1) Add 1 μL of Salmonella DNA as a template to a PCR tube containing a constant temperature amplification reaction system of Salmonella helicase, and at the same time use an equal amount of sterile double-distilled water as a negative control. Helicase isothermal amplification.

(2) Select PBS buffer (pH 7.4) of 0.1% BSA and 0.1% Tween 20 as the sample loading developing solution, take 10 μL of the amplified product diluted 10 times with the loading developing solution and add it dropwise to the colloidal gold test strip for detection. The test needs to be repeated three times, and the test results are observed after 5 minutes. The amplification product of Salmonella is positive, and both the detection line and the quality control line are red; the blank control is negative, only the quality control line is red.

Example 4

Salmonella nucleic acid thin film chromatography test strip specificity test

Other common foodborne pathogens including Escherichia coli O157:H7, Shigella, Enterobacter aerogenes, Listeria monocytogenes, Staphylococcus aureus, and Campylobacter jejuni were detected as described in Example 3, and Taking the same amount of sterile ultrapure water as a negative control, except for five Salmonella strains that were positive, all other experimental strains were negative. The experimental results are shown in Figure 3, indicating that the detection technology has high specificity.

Example 5

Sensitivity test of pure culture of Salmonella nucleic acid film detection test strip

(1) Sensitivity detection of Salmonella in pure bacterial liquid, inoculate it in 10mL LB liquid medium, and cultivate overnight at 37°C;

(2) Take 1 mL of each for bacterial count, carry out 10-fold gradient dilution of the pure bacterial solution with 0.9% sterile normal saline, and take 1 mL of each for DNA extraction as an experimental template;

(3) Carry out the test according to the constant temperature amplification reaction system, take 10 μL of the amplification product and dilute it to 100 μL with the sample developing solution, add it dropwise to the sample pad of the test strip, and repeat the test three times. Bacterial sensitivity.

The concentration gradient of the pure culture solution of Salmonella was diluted to 4.5×10 ¹ CFU/mL. After the helicase isothermal amplification reaction, the positive results were all shown on the test strip, indicating that the Salmonella nucleic acid film chromatography test strip The sensitivity was 4.5×10 ¹ CFU/mL.

Example 6

Sensitivity test of actual samples of Salmonella nucleic acid film test strips

1. Solid-state sample sensitivity experiment

(1) Weigh 25g of solid samples under sterile conditions and place them in a sterile homogenizing bag, and homogenize for 3 min.

(2) Inject the sample into 225 mL of SC culture medium, and after the Salmonella cultured to a concentration of 10 ⁸ CFU/mL, was diluted 10 times with 0.9% sterile normal saline, and then 1 mL of bacterial solution was added and placed in the sample enrichment solution to make the bacteria. Its final concentration is 10 ¹ CFU/mL,

(3) Incubate at 37°C and 200r/min. During the period, culture samples were collected every 2 h.

(4) Two parallel experimental groups were set to extract DNA by heat treatment, and the samples without bacteria were used as blank control, and the experiment was carried out according to the constant temperature amplification reaction system.

⑸ Take 10 μL of amplification product and dilute it to 100 μL with the sample developing solution, add it dropwise to the sample pad of the test strip, repeat the test three times, observe the detection results after 5 minutes, and determine the sensitivity of the method for detecting solid samples.

2. Liquid sample sensitivity test

(1) Measure 25mL liquid or semi-solid sample under sterile conditions, and inject the sample into 225mL SC culture medium;

(2) After the Salmonella cultured to a concentration of 10 ⁸ CFU/mL was diluted 10 times with 0.9% sterile saline, 1 mL of bacterial solution was added and placed in the sample enrichment solution to make the final concentration 10 ¹ CFU/mL. ;

(3) Incubate at 37°C and 200r/min. During the period, culture samples were collected every 2 h;

(4) Two parallel experimental groups were set for each part to extract DNA by heat treatment, and the samples without bacteria were used as blank control, and the experiment was carried out according to the constant temperature amplification reaction system;

⑸ Take 10 μL of amplification product and dilute it to 100 μL with the optimized sample developing solution, add it dropwise to the sample pad of the test strip, repeat the test three times, observe the detection results after 5 minutes, and determine the sensitivity of the method for detecting liquid samples.

The actual samples added with Salmonella at a concentration of 10 ¹ CFU/mL could be detected after 2 hours of enrichment, while the samples without bacteria were not detected. The results show that the method can be applied to the rapid detection of actual samples.

Claims (2)

1. a Salmonella nucleic acid rapid detection kit, is characterized in that: comprise forward primer invA-F: sequence 5'-ATTTCTATGTTCGTCATTCCATTACCTACC-3', reverse primer invA-R: sequence: 5'-ATGTAGAACGACCCCATAAACACCAA-3', use ground Goxigenin and biotin are respectively labeled with the 5' end of the forward primer and the reverse primer, the gold-labeled antibody, the antibody of the anti-gold-labeled antibody species, and the ligand substance of the chromogenic substance biotin; It also includes a helicase isothermal amplification system, and the amplification system includes 10×Annealing Buffer, dNTP, MgSO ₄ , NaCl, and Enzyme Mix; The ligand substance of the chromogenic substance biotin is streptavidin; The gold-labeled antibody is a mouse anti-digoxigenin antibody, and the antibody of the anti-gold-labeled antibody species is a goat anti-mouse secondary antibody; The kit also includes Salmonella nucleic acid rapid detection test paper, including sample pad, gold label pad, nitrocellulose membrane and absorbent paper, one end of the sample pad is overlapped with one end of the gold label pad, and the other end of the gold label pad is overlapped with the nitrocellulose membrane. The ends are overlapped, the other end of the nitrocellulose membrane is overlapped with the absorbent paper, and the detection line and the quality control line are embedded in parallel between the nitrocellulose membrane intervals. between the ends of the paper; The gold-labeled pad is sprayed with gold-labeled antibody, the ligand substance of the chromogenic substance biotin is coated on the detection line, and the antibody of the anti-gold-labeled antibody species is coated on the quality control line; The gold label pad is sprayed with mouse anti-digoxigenin antibody colloidal gold, the detection line is coated with streptavidin, the streptavidin concentration is 2 mg/mL, and the quality control line is coated with goat anti-mouse secondary antibody; The sample pad is made of glass fiber, the overlapping part of each part is 2-10mm, and a PVC backboard is installed at the lower part of the test paper; The rapid detection test paper used the sample developing solution of PBS buffer supplemented with 0.1% BSA and 0.1% Tween 20, pH 7.4. 2. the rapid detection method of the non-diagnostic purpose of test kit Salmonella nucleic acid according to claim 1, is characterized in that: it comprises the steps: (1) The mouse anti-digoxigenin antibody is adsorbed on the colloidal gold particles of the color-developing substance as a gold-labeled antibody, and the conjugate forming a stable structure is fixed on the gold-labeled pad; (2) Streptavidin is used as the ligand of the marker biotin to coat the nitrocellulose membrane in a linear form to form a detection line; (3) Coat the goat anti-mouse antibody on the quality control line in a linear form to bind too much gold-labeled antibody; (4) The 5' end of the forward primer and the 5' end of the reverse primer are modified with digoxigenin and biotin respectively. When there is a Salmonella nucleic acid amplification product to be tested, the nucleic acid amplification reaction is carried out under constant temperature conditions to obtain the simultaneous connection of the two markers. Digoxin-target fragment-biotin amplification product; ⑸ Combine the digoxigenin-target fragment-biotin produced in step ⑷ with the digoxigenin antibody coated with colloidal gold particles on the gold pad to form the digoxigenin antibody-digoxigenin-target fragment-biotin chromogenic particle Complex; (6) The chromogenic particle complex obtained in step (5) moves upward along the cellulose membrane by capillary action in the loading buffer to the detection line and is captured by the receptor streptavidin to obtain digoxin antibody-digoxigenin-target fragment- The biotin-streptavidin complex settles on the detection line to form a visible color band, and the gold-labeled antibody coated on the colloidal gold particles not bound to the amplification product and the sheep on the quality control line The anti-mouse antibody binds and two colored bands appear, which is a positive result; Or, when there is no Salmonella nucleic acid amplification product, the above steps ⑸-⑹ do not occur, and cannot be combined with the digoxigenin antibody coated with colloidal gold particles on the gold label pad, and the colloidal gold particles of the color-developing substance cannot be coated on the detection line. The substance combined to form a colored band, the goat anti-mouse antibody on the quality control line combined with the gold-labeled antibody coated on the colloidal gold particles, and a colored band appeared, which was a negative result.

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