CN110734990A - Detection reagents, kits and their applications - Google Patents

Abstract

The invention discloses a detection reagent, a kit and an application thereof. The detection reagent includes any one or a combination of any one or more of the following four primer pairs, the four primer pairs include: a first primer pair, including sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The first primer and the second primer shown; the second primer pair, including the third primer and the fourth primer whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively; the third primer pair, including the sequence as shown in The fifth primer and sixth primer shown in SEQ ID NO: 5 and SEQ ID NO: 6; the fourth primer pair, including the seventh primer and the sixth primer whose sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Eight primers. The detection method provided by the present invention is to amplify gene fragments of appropriate length with labeled specific primers, which can ensure high specificity of detection and improve the accuracy of detection results.

Description

Detection reagents, kits and their applications

technical field

The invention relates to a detection reagent and a kit, in particular to a detection reagent, a kit and application thereof for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae and applications thereof, belonging to molecular biology and The field of immunology technology.

Background technique

Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae and other pathogenic bacteria widely exist in nature, which can cause pollution to food and water bodies, thereby threatening the life safety of humans and animals.

In order to prevent the harm of such pathogenic bacteria, a variety of pathogenic bacteria detection technologies have been developed. For example, for Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae, the current conventional detection methods include sample processing, enrichment culture, isolation and culture, and staining observation. This detection method is the "gold standard for clinical detection". ”, but the workload is large, and the detection cycle is long and takes about a week.

Others such as physiological and biochemical identification are more accurate, but the disadvantage is that the operation is complicated and time-consuming, but it is also time-consuming. The chromogenic plate detection can effectively detect E. coli, but the disadvantage is that it is time-consuming, requires professional operation, and the reagents are expensive. Membrane filtration technology is a quantitative test and has many of the same advantages as test-tube fermentation technology. The most obvious advantage of membrane filtration technology over test-tube fermentation technology is that it is very convenient to detect larger volumes of water samples, which can increase the detection sensitivity and Reliability, the disadvantage is that the specificity is not high, and the results are easily affected by other bacteria in the water sample and cause misjudgment. Generally, it is used as a speculative experiment and needs to be further confirmed by other methods; the enzyme activity detection method (enzyme substrate method) is better than The traditional method saves time and labor (18-24h), and has high specificity, but the disadvantage is that the cost of reagents is high; the biggest problem of immunological methods is the cross-reactivity of commercial monoclonal antibodies or polyclonal antibodies with various intestinal bacteria. It is easy to cause false positives, and it is necessary to prepare monoclonal or polyclonal antibodies with higher specificity.

In this way, the methods based on molecular biology have unique advantages. Molecular biological identification can overcome the above shortcomings. Pathogens can be detected within a few hours, and the operation is simple and convenient. On the one hand, compared with the traditional methods of microbial isolation, culture and identification, PCR detection technology reduces the length of the detection cycle and saves the cost of detection; it is suitable for the clinical detection of Escherichia coli. Epidemiological investigations and investigations on the infection of individual cows and herds in dairy herds are an effective and good method for early prevention and control of Escherichia coli, accurate identification of pathogens, early and rapid clinical treatment, and reduction of economic losses. On the one hand, PCR detection technology has high specificity and sensitivity, but the disadvantage is that this method needs to combine electrophoresis tank, agarose gel and other instrument reagents, uses nucleic acid dyes and other toxic substances, and requires a gel imager for later analysis. Safety requirements are high and toxic.

However, the existing detection methods have disadvantages such as complicated operation, easy to be affected by the environment, easy to be infected, not suitable for early diagnosis, and have large errors in the detection results. Therefore, a rapid, accurate and convenient diagnostic method is urgently needed.

SUMMARY OF THE INVENTION

The main purpose of the present invention is to provide a more sensitive, fast, convenient, safe and cheap detection reagent, kit and application for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and application thereof. Overcome the deficiencies in the prior art.

In order to realize the foregoing invention purpose, the technical scheme adopted in the present invention includes:

An embodiment of the present invention provides a detection reagent, which includes any one or a combination of the following four primer pairs, wherein the four primer pairs include:

The first primer pair, including the first primer and the second primer whose sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;

The second primer pair includes the third primer and the fourth primer whose sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;

The third primer pair includes the fifth primer and the sixth primer whose sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively;

The fourth primer pair includes the seventh primer and the eighth primer whose sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

Further, the 5' end of at least one primer contained in at least one primer pair also has a nucleic acid label, and the nucleic acid label includes FITC, FAM, HEX, TET, TAMRA, ROX, VIC, NED, Alexa Flour, biological Any one of prime, digoxin, cy3, and cy5.

Further, the 5' ends of the two primers contained in each primer pair have the nucleic acid label.

Further, the detection reagent also includes DNA polymerase and conventional PCR components.

The embodiment of the present invention also provides a kit, which includes the detection reagent and a nucleic acid immunochromatography test strip, wherein the nucleic acid immunochromatography test strip includes a bottom plate, a sample pad, a gold label pad, a nitrocellulose Membrane and absorbent pad, the nitrocellulose membrane is provided with a detection strip (sprayed with digoxigenin antibody) and a quality control strip (sprayed with goat anti-mouse secondary antibody).

Further, a complex formed by a first antibody and colored particles is attached to the gold label pad, a second antibody is arranged on the detection band, and a third antibody is arranged on the quality control band; The first antibody is the antibody of the nucleic acid label, the second antibody is the antibody or ligand of the acid label; the third antibody is the antibody capable of binding to the antibody of the specified species.

Further, the third antibody is goat anti-mouse polyclonal antibody, and the colored particles are spherical red colloidal gold particles labeled with fluorescein isothiocyanate antibody.

In some specific embodiments, the sample pad, the gold standard pad, the nitrocellulose membrane and the absorbent pad are all placed on the bottom plate, the sample pad is located at one end of the bottom plate, and the absorbent pad is located at the other end of the bottom plate, so The gold label pad is located near one end of the sample pad and a part of it is placed at the bottom of the sample pad. The nitrocellulose membrane is arranged in the middle of the gold label pad and the absorbent pad and overlaps with the gold label pad and the absorbent pad. There are sprayed detection strips and quality control strips on the film, the detection strips are located on the side close to the gold standard pad, the quality control strips are located on the side close to the water-absorbing pad, and the water-absorbing filter paper pads and the water-absorbing pads are respectively arranged on the lateral layer The two ends of the matrix membrane are separated, the conjugate pad is arranged on the water-absorbing filter paper pad, and there is a certain interval between the gold standard pad and the water-absorbing pad.

In some more specific embodiments, the primary antibody (eg FITC antibody) and colloidal gold particles were incubated together, and the first antibody was coated on the surface of colloidal gold particles to form colored cross-links by electrostatic binding force, and the cross-links were dropped on the gold pad to dry for use. The antibody was immobilized on the nitrocellulose membrane in the shape of a line to form a detection band (line), and the third antibody was immobilized in the shape of a line on the nitrocellulose membrane to form a quality control band (line).

Preferably, the water-absorbing filter paper pad is glass fiber RB 65, preferably, the lateral chromatography matrix membrane is a nitrocellulose membrane Sartorius CN 140, preferably, the water-absorbing pad is CH 37, preferably, the The base plate is SM 31-40.

Further, the embodiment of the present invention also provides a developing solution (0.4% NaCl) for nucleic acid sequence detection, and the developing solution can effectively develop PCR amplification products, so that the antigen and antibody can be effectively combined.

The embodiments of the present invention also provide the application of the detection reagent or the kit in preparing a product for detecting pathogenic bacteria.

The embodiment of the present invention also provides a product applied to a pathogenic bacteria detection method, the product includes the kit according to any one of claims 5-7, and the detection method includes:

Using at least one primer pair among the four primer pairs, the nucleic acid extracted from the sample to be tested is amplified by PCR amplification reaction,

Add the amplified product dropwise to the nucleic acid immunochromatography test paper of the kit, and observe whether a colored band is formed on the detection band and the accusation band; if a visible colored band is formed on both the detection band and the quality control band If there is no colored band on the detection band, and there is a visible colored band on the quality control band, it is judged as negative; if there is no colored band on the quality control band, it is judged as invalid.

Further, the pathogenic bacteria include any one or two or more of Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae.

Compared with the prior art, the advantages of the present invention include:

1) The present invention improves the sensitivity of detecting food-borne pathogens by combining PCR amplification technology and antigen-antibody specific binding technology, and uses colloidal gold-labeled antibody technology and PCR product amplification dual strategy to achieve final signal amplification, thereby Realize the visual detection of Escherichia coli;

2) The method provided by the present invention has the advantages of rapidity, visualization, and ultra-sensitivity; it is of great significance for improving the detection sensitivity of food-borne pathogenic bacteria, reducing costs and realizing the convenience, and is relatively insufficient in the experimental environment. Popularization or rapid testing of E. coli would be of great help;

3) As a general technology platform for nucleic acid amplification product detection, the method provided by the invention and its nucleic acid immunochromatography test strip can be widely used in agriculture, animal husbandry and food industry, clinical detection of food-borne pathogens, customs inspection Inspection, environmental monitoring, infectious disease prevention and control and other fields. Common food-borne pathogens such as Staphylococcus aureus, Salmonella, Enterohemorrhagic Escherichia coli, Listeria monocytogenes, Shigella, and Vibrio parahaemolyticus are the nucleic acid immune layers of the present invention Analysis of the detection object of the test strip.

Description of drawings

1a is a schematic diagram of the detection principle of the detection kit for Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae in a typical implementation case of the present invention;

Figure 1b is a schematic diagram of the judgment results of the detection kits for Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae in a typical implementation case of the present invention;

Fig. 2a is the colloidal gold solution appearance figure of different particle diameters in the embodiment of the present invention 1;

Fig. 2b is the scanning diagram of ultraviolet spectrophotometer of colloidal gold solution of different particle sizes in the embodiment of the present invention 1;

Fig. 2c is the color rendering effect diagram of the colloidal gold solution test strips of different particle sizes in the embodiment of the present invention 1;

Fig. 3a is the specific detection nucleic acid electrophoresis analysis diagram of Escherichia coli in the embodiment 2 of the present invention;

Fig. 3b is the specific detection nucleic acid immunochromatographic test strip analysis diagram of Escherichia coli in the embodiment of the present invention 2;

Fig. 4a is the nucleic acid electrophoresis analysis diagram of sensitivity detection of Escherichia coli in Example 3 of the present invention;

Fig. 4b is the sensitivity detection nucleic acid immunochromatographic test strip analysis diagram of Escherichia coli in Example 3 of the present invention;

Fig. 5a is the colloidal gold solution appearance figure of different particle diameters in the embodiment of the present invention 4;

Fig. 5b is the colloidal gold solution ultraviolet spectrophotometer scanning diagram of different particle diameters in the embodiment of the present invention 4;

Fig. 5c is the color rendering effect diagram of the colloidal gold solution test strips of different particle diameters in the embodiment of the present invention 4;

Fig. 6a is the specific detection nucleic acid electrophoresis analysis figure of Staphylococcus aureus in the embodiment of the present invention 5;

Fig. 6b is the specific detection nucleic acid immunochromatographic test strip analysis diagram of Staphylococcus aureus in the embodiment of the present invention 5;

Fig. 7a is the nucleic acid electrophoresis analysis diagram of sensitivity detection of Staphylococcus aureus in the embodiment of the present invention 6;

Figure 7b is an analysis diagram of a nucleic acid immunochromatographic test strip for the sensitivity detection of Staphylococcus aureus in Example 6 of the present invention;

Fig. 8a is the appearance diagram of the colloidal gold solution of different particle diameters in the embodiment of the present invention 7;

Fig. 8b is the ultraviolet spectrophotometer scanning diagram of the colloidal gold solution of different particle diameters in the embodiment of the present invention 7;

Figure 8c is a color rendering effect diagram of the colloidal gold solution test strips of different particle sizes in the embodiment of the present invention 7;

Fig. 9a is the specificity detection nucleic acid electrophoresis analysis figure of Streptococcus dysgalactiae in the embodiment of the present invention 8;

Fig. 9b is the specific detection nucleic acid immunochromatographic test strip analysis diagram of Streptococcus dysgalactiae in the embodiment of the present invention 8;

Fig. 10a is the nucleic acid electrophoresis analysis diagram of sensitivity detection of Streptococcus dysgalactiae in Example 9 of the present invention;

Figure 10b is an analysis diagram of a nucleic acid immunochromatography test strip for the sensitivity detection of Streptococcus dysgalactiae in Example 9 of the present invention;

Figure 11a is the appearance diagram of the colloidal gold solution of different particle sizes in Example 10 of the present invention;

Fig. 11b is the scanning diagram of ultraviolet spectrophotometer of colloidal gold solution with different particle sizes in Example 10 of the present invention;

Figure 11c is a color rendering effect diagram of colloidal gold solution test strips with different particle sizes in Example 10 of the present invention;

Figure 12a is a specific detection nucleic acid electrophoresis analysis diagram of Streptococcus agalactiae in Example 11 of the present invention;

Figure 12b is an analysis diagram of a nucleic acid immunochromatographic test strip for the specific detection of Streptococcus agalactiae in Example 11 of the present invention;

Fig. 13a is a nucleic acid electrophoresis analysis diagram of sensitivity detection of Streptococcus agalactiae in Example 12 of the present invention;

Figure 13b is an analysis diagram of nucleic acid immunochromatographic test strips for the sensitivity detection of Streptococcus agalactiae in Example 12 of the present invention.

Detailed ways

In view of the deficiencies in the prior art, the inventor of the present application was able to propose the technical solution of the present invention after long-term research and extensive practice. The technical solution, its implementation process and principle will be further explained as follows.

Explanation of technical terms used in the description of the present invention:

Colloidal gold: Gold sol, also known as colloidal gold, is a stable, uniform and monodispersed gold particle suspension in liquid formed after gold salt is reduced to gold.

Nucleic acid polymerases: Enzymes that synthesize long nucleic acid chains; are divided into two categories: DNA polymerases and RNA polymerases.

Primer: The starting material for DNA synthesis. Typically a pair of single-stranded oligonucleotides, after hybridization to a template, DNA synthesis begins at the 3' end.

Labeling: A method of conjugating a detectable signal molecule (eg, hapten, fluorescence, radioactivity, etc.) to a single-stranded oligonucleotide.

Hybridization: In particular, complementary DNA single strands form a double-stranded structure through base pairing.

Development: The PCR product after the amplification reaction starts to move towards the detection line and the quality control line from the bottom end of the water-absorbing pad of the test strip under the chromatographic action of the development buffer.

Nucleic acid: Generic term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Antigens and Haptens: Substances that are immunogenic. Usually macromolecular proteins or cellular components. But some small molecules are also immunogenic, called hapten. Haptens are often used to label probes.

Antibody: A protein molecule that can specifically bind to an antigen or hapten.

Complex: A specific combination of two or more molecules.

Primer-dimer: In the polymerase chain reaction (PCR) process, a dimer molecule formed by the annealing of primer pairs or a single primer to each other, the dimer composed of two different primers is a heterodimer, It may cause false positives due to the binding of the antibody with two labels, and the dimer formed by the annealing of a single primer is a homodimer, and only one label will not cause false positives, but too many Dimer formation reduces amplification efficiency.

Immunochromatographic test strips: Medical tools for rapid detection, also known as Chromogenic Film Chromatography.

The method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae provided in the embodiment of the present invention greatly simplifies the detection procedure after nucleic acid amplification and saves the detection cost; the interpretation of the results is simple, clear and intuitive (the detection principle and The results are shown in accompanying drawings 1a and ab); the method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus agalactiae provided in the embodiment of the present invention is used as a novel detection technology after nucleic acid amplification, Has the following advantages:

Wide application: as a general technology platform for nucleic acid amplification product detection, the method and test strip provided by the present invention can be widely used in agriculture, animal husbandry and food industry, clinical detection of food-borne pathogens, customs inspection and inspection, environmental surveillance, infectious disease prevention and control, etc. Common food-borne pathogens such as Staphylococcus aureus, Salmonella, Enterohemorrhagic Escherichia coli, Listeria monocytogenes, Shigella, and Vibrio parahaemolyticus are all nucleic acid amplifications of the present invention The object of the product detection test strip.

Simple operation: just drop the amplified nucleic acid sample directly on the nucleic acid reagent detection plate, no professional operation is required, not only the invention embodiment can be used, but also can be used in front-line prevention and control institutions and farms; fast: detection 5 Interpret the results in minutes; convenient: no electrophoresis or gel imaging system, and can be directly observed with the naked eye; sensitive: compared with agarose gel electrophoresis, the detection sensitivity is increased by nearly 10-100 times; high specificity: used in the detection process The specific labeled primers make the results more accurate; the cost is low: the detection cost is much lower than that of gel electrophoresis and ELISA detection.

In a typical implementation case of the present invention, a method for detecting Escherichia coli or Staphylococcus aureus or Streptococcus dysgalactiae or Streptococcus agalactiae using a nucleic acid immunochromatographic test strip method comprises the following steps:

1) Provide two unique amplification primers:

Wherein, the sequence (SEQ ID NO: 1) of the upstream primer Ecoil-FITC-F (that is, the aforementioned first primer) used in the detection of Escherichia coli is 5'-ATCAACCGAGATTCCCCCAGT-3', and the 5' end uses fluorescein isothiocyanate. (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 2) of the downstream primer Ecoil-DIG-R (that is, the aforementioned second primer) is 5'-TCACTATCGGTCAGTCAGGAG-3', and the 5' end is labeled with such as digoxigenin ( DIG) and other nucleic acid markers; under suitable amplification conditions, a DNA fragment with a length of 254 bp can be amplified; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;

The sequence (SEQ ID NO: 3) of the upstream primer nuc-FITC-F (that is, the aforementioned third primer) used in the detection of Staphylococcus aureus is 5'-CGATTGATGGTGATACGGTT-3', and the 5' end uses fluorescein isothiocyanate. (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 4) of the downstream primer nuc-DIG-R (ie, the fourth primer) is 5'-CTCTTTTTTCGCTTGTGCTT-3', and the 5' end uses digoxigenin (DIG), etc. Nucleic acid marker labeling, under suitable amplification conditions, can amplify a DNA fragment with a length of 356bp; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;

The sequence (SEQ ID NO: 5) of the upstream primer sdy-FITC-F (that is, the fifth primer) used in the detection of Streptococcus dysgalactiae is 5'-TAAAGGTGC AACTGCATCACTA-3', and the 5' end uses fluorescein isothiocyanate. (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 6) of the downstream primer sdy-DIG-R (ie, the sixth primer) is 5'-AGTCACATGGTGGATTTTCCA-3', and the 5' end uses digoxigenin (DIG), etc. Nucleic acid marker labeling, under suitable amplification conditions, can amplify a DNA fragment with a length of 282bp; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;

The sequence (SEQ ID NO: 7) of the upstream primer cfb-F (that is, the seventh primer) used in the detection of Streptococcus agalactiae is 5'-TAATAATCAAGCCCAGC-3', and the 5' end uses fluorescein isothiocyanate (FITC) and other nucleic acid markers; the sequence (SEQ ID NO: 8) of the downstream primer cfb-R (ie, the eighth primer) is 5'-CCTTTTGTTCTAATGCC-3', and the 5' end is marked with nucleic acid markers such as digoxigenin (DIG), Under suitable amplification conditions, a DNA fragment with a length of 388bp can be amplified; when there is no nucleic acid template to be detected, there is no specific nucleic acid fragment amplification product;

2). Cross-linking a universal antibody of a marker (such as the aforementioned nucleic acid marker, named as marker 1) (ie, the aforementioned first antibody such as anti-digoxigenin antibody) and colloidal gold particles, forming on the surface of the colloidal particles. coated antibody;

3) An antibody or ligand (ie, a second antibody, such as FITC antibody) of another marker (such as the aforementioned nucleic acid marker, named as marker 2) is immobilized in a line shape on the membrane on the test strip (such as A detection band (line) is formed on the nitrocellulose membrane);

4) When there is a specific amplification product to be detected, due to the action of the upstream and downstream primers, the amplification product simultaneously carries two of the above three labeling substances, forming Marker 1-Amplification Product-Marker 2 The complex, wherein marker 1 is the first nucleic acid marker, marker 2 is the second nucleic acid marker;

5) Combine the marker 1-amplification product-marker 2 formed in step 4) with the antibody of marker 1 coated on the surface of colloidal gold particles to form an anti-marker 1 antibody-marker 1-amplification product-marker 2 colored particle complexes;

6) The colored particle complex obtained in step 5) flows upward along the fiber through the capillary phenomenon in the solution to the line coated with the antibody or ligand of Marker 2. ) combined and deposited, stayed on the detection line, formed a colored line visible to the naked eye, and judged as positive;

7) When the specific amplification product does not exist, the above step 4)-step 6) does not occur, the marker 1-amplification product-marker 2 complex cannot be formed, and the marker deposited on the detection line cannot be formed. On the antibody (ligand) of 2, no visible band was formed, and it was judged as negative.

In the method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae provided by the present invention, the specificity of the amplified product is ensured by the specific primers screened, and the modifiers at both ends of the primers are compatible with those on the test strip. The antigen-antibody specific binding doubles to ensure the accuracy of the detection results; and the detection method of the present invention avoids the complex operation link of gel electrophoresis after nucleic acid amplification, and integrates the test strip detection technology to make the detection method more safe, simple, and convenient. Fast, easy and cheap.

The method for detecting Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae provided by the present invention is to amplify gene fragments of appropriate length with labeled specific primers, which can ensure high specificity of detection and improve detection The accuracy of the results; the intuitive characteristics of the immunocolloidal gold (test strip) are maintained, and it has the advantages of rapidity, convenience, maturity, and cheapness.

The technical solution, its implementation process and principle will be further explained below with reference to the accompanying drawings and specific embodiments.

Example 1

The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.

1 Materials and methods

1.1 Materials

Colloidal gold solutions with different particle sizes from 10 nm to 50 nm are prepared by the examples of the present invention.

1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

1) Get 1 ml of colloidal gold solutions of different particle sizes (as shown in accompanying drawing 2a);

2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min 3min to remove unbound protein, take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, take the supernatant; add 80ul of reconstituted solution, age at 4°C for 2h; after taking out, take 6ul and drop them on the gold pad , dried at 25°C; assemble the dried gold label pads onto the test strips;

3) Culture the preserved Escherichia coli to the logarithmic phase, take the bacterial solution for nucleic acid amplification, and carry out PCR amplification with the above-mentioned (1.3) PCR reaction system; carry out different gradient dilutions of the PCR amplification products; Observe the results on test strips prepared from colloidal gold solutions of different particle sizes.

2 results

The appearance results and UV spectrophotometer scanning results of colloidal gold solutions with different particle sizes are shown in Figures 2a and 2b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; different particle sizes And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 2c. The larger the particle size of gold particles, the easier aggregation and precipitation will occur. Therefore, the colloidal gold solution larger than 35 nm is easy to aggregate during the treatment process and cannot obtain a stable and dispersed solvent, which is not suitable for The test strip was prepared for use. In the color development results shown in Figure 2c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.

Example 2

Test for the specific identification of Escherichia coli in contamination with different foodborne pathogens.

1 Materials and methods

1.1 Materials

The various zoonotic strains involved in this example are shown in Table 1.

Table 1 Experimental strains

Table 1 Bacterial strains for detection

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

The different strains were recovered and cultured, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Vitis epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, add 60ul dropwise to the test strip, and use a smartphone or camera to photograph the color development result of the test strip after five minutes of color development;

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results of specific identification between different strains of E. coli are shown in Figure 3a, and the identification results of nucleic acid immunochromatography test strips for the specific identification of different strains of E. coli are shown in Figure 3b. The results show that: the PCR amplification method established in the present invention The post-increase detection method has good specificity for Escherichia coli, and there is no obvious positive reaction of the test strip between different strains. , which can facilitate the use of grassroots workers, and is more conducive to the detection of Escherichia coli.

Example 3

Test the sensitivity of nucleic acid immunochromatographic strips to detect Escherichia coli.

1 Materials and methods

1.1 Materials

Escherichia coli was isolated, identified and preserved by the Invention Example of the School of Veterinary Medicine of Nanjing Agricultural University.

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

The Escherichia coli were recovered and cultured, and the bacterial solution was diluted by gradient and counted on a plate. The number of colonies corresponding to 1-9 in Figures 4a and 4b were 2 × 10 ⁸ cfu/ml, 2 × 10 ⁷ cfu/ml, and 2 × 10 ⁶ respectively. cfu/ml, 2×10 ⁵ cfu/ml, 2×10 ⁴ cfu/ml, 2×10 ³ cfu/ml, 2×10 ² cfu/ml, 2×10 ¹ cfu/ml, 2cfu/ml; simultaneous extraction Bacterial DNA was used as a PCR template, and E. coli was amplified by PCR using the PCR reaction system (1.3) above, and its sensitivity was verified by gel electrophoresis and nucleic acid immunochromatography test strips, respectively.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results of Escherichia coli detection sensitivity identification are shown in Figure 4a, and the identification results of Escherichia coli detection sensitivity identification nucleic acid immunochromatography test strips are shown in Figure 4b. The paper strip detection method has high detection sensitivity, and the lowest detection limit is 2×10 ² cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.

The Ecoil-FITC-F sequence (ie the first primer sequence) in Example 1-Example 3 is ATCAACCGAGATTCCCCCAGT; the Ecoil-DIG-R sequence (ie the second primer sequence) is: TCACTATCGGTCAGTCAGGAG.

Example 4

The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.

1 Materials and methods

1.1 Materials

Colloidal gold solutions with different particle sizes ranging from 10 nm to 50 nm are prepared by the examples of the present invention.

1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

1) get 1ml each of colloidal gold solutions of different particle sizes (as shown in accompanying drawing 5a);

2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min for 3 min to remove unbound proteins , take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, remove the supernatant; add 80ul of reconstituted solution, place it at 4 degrees for aging for 2h; after taking out, take 6ul of each drop on the gold pad, and dry at 25 degrees; The pads are assembled to the test strips.

3) Resuscitate the preserved Staphylococcus aureus, cultivate it to logarithmic phase, take the bacterial solution to carry out nucleic acid amplification, and carry out PCR amplification with the above-mentioned (1.3) PCR reaction system; carry out different gradient dilutions of the PCR amplification products; respectively; Add dropwise to test strips prepared from colloidal gold solutions with different particle sizes and observe the results.

2 results

The apparent results of colloidal gold solutions with different particle sizes and the scanning results of UV spectrophotometers are shown in Figures 5a and 5b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 5c. The larger the particle size of gold particles, the more likely to cause aggregation and precipitation. Therefore, the colloidal gold solution larger than 35nm is easy to aggregate during the treatment process, and a stable dispersion solvent cannot be obtained, which is not suitable for Test strips were prepared for use. In the color development results shown in Figure 5c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.

Example 5

Test for specific identification of Staphylococcus aureus in different foodborne pathogenic contamination.

1 Materials and methods

1.1 Materials

The various zoonotic strains involved in the embodiments of the present invention are shown in Table 2.

Table 2 Experimental strains

Table 2 Bacterial strains for detection

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

The different strains were recovered and cultured, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecalis, and Staphylococcus epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results for the specific identification of different strains of Staphylococcus aureus are shown in Figure 6a, and the identification results of nucleic acid immunochromatography test strips for the specific identification of different strains of Staphylococcus aureus are shown in Figure 6b. The results show that: the present invention The established PCR-amplified detection method has good specificity for Staphylococcus aureus, and there is no obvious positive test strip reaction between different strains, and it has a good degree of identification. It can be observed with the naked eye under the circumstances, which is convenient for grass-roots workers to use, and is more helpful for the detection of Staphylococcus aureus.

Example 6

Test the sensitivity of nucleic acid immunochromatographic strips to detect Staphylococcus aureus.

1 Materials and methods

1.1 Materials

Various zoonotic strains involved in the embodiments of the present invention are shown in Table 3.

Table 3 Experimental strains

Table 3 Bacterial strains for detection

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

Staphylococcus aureus was revived and cultured, and the bacterial solution was serially diluted and counted on a plate. The number of colonies corresponding to 1-9 in Figures 6a and 6b were 4×10 ⁸ cfu/ml, 4×10 ⁷ cfu/ml, 4× 10 ⁶ cfu/ml, 4 x 10 ⁵ cfu/ml, 4 x 10 ⁴ cfu/ml, 4 x 10 ³ cfu/ml, 4 x 10 ² cfu/ml, 4 x 10 ¹ cfu/ml, 4 cfu/ml; At the same time, bacterial DNA was extracted as a PCR template, and the above-mentioned (1.3) PCR reaction system was used to carry out PCR amplification of Staphylococcus aureus, and then use gel electrophoresis and nucleic acid immunochromatography test strips to verify its sensitivity.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results of the detection sensitivity identification of Staphylococcus aureus are shown in Figure 7a, and the identification results of the nucleic acid immunochromatographic test strips for the detection sensitivity identification of Staphylococcus aureus are shown in Figure 7b. The results show: it can be seen that the detection sensitivity of Staphylococcus aureus established by the present invention The nucleic acid immunochromatography test strip detection method has high detection sensitivity and can reach the lowest detection limit of 4×10 ² cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.

The nuc-FITC-F sequence (ie, the third primer sequence) in Example 4-Example 6 is: CGATTGATGGTGATACGGTT; the nuc-DIG-R sequence (ie, the fourth primer sequence) is: CTCTTTTTTCGCTTGTGCTT.

Example 7

The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.

1 Materials and methods

1.1 Materials

Colloidal gold solutions with different particle sizes ranging from 10 nm to 50 nm are prepared by the examples of the present invention.

1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

1) Take 1 ml of colloidal gold solutions of different particle sizes (as shown in accompanying drawing 8a);

2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min for 3 min to remove unbound proteins , take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, remove the supernatant; add 80ul of reconstituted solution, place it at 4 degrees for aging for 2h; after taking out, take 6ul of each drop on the gold pad, and dry at 25 degrees; The pads are assembled to the test strips.

3) Resuscitate the preserved Streptococcus dysgalactiae, cultivate it to logarithmic phase, take the bacterial solution to carry out nucleic acid amplification, and use the above (1.3) PCR reaction system to carry out PCR amplification; carry out different gradient dilutions of the PCR amplification products; respectively; Add dropwise to test strips prepared from colloidal gold solutions with different particle sizes and observe the results.

2 results

The apparent results of colloidal gold solutions with different particle sizes and the scanning results of UV spectrophotometers are shown in Figures 8a and 8b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 8c. The larger the particle size of gold particles, the more likely to cause aggregation and precipitation. Therefore, colloidal gold solutions larger than 35 nm are easily aggregated during the treatment process and cannot obtain stable dispersion. The solvent is not suitable for The test strip was prepared for use. In the color development results shown in Figure 8c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.

Example 8

Testing the specific identification of Streptococcus dysgalactiae in different foodborne pathogenic contamination.

1 Materials and methods

1.1 Materials

Various zoonotic strains involved in the embodiments of the present invention are shown in Table 4.

Table 4 Experimental strains

Table 4 Bacterial strains for detection

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

The different strains were recovered and cultivated, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Streptococcus dysgalactiae, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Enterococcus faecalis, and Staphylococcus epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results of specific identification between different strains of Streptococcus dysgalactiae are shown in Figure 9a, and the identification results of nucleic acid immunochromatographic test strips for the specific identification of different strains of Streptococcus dysgalactiae are shown in Figure 9b. The results show that: the present invention The established PCR amplification detection method has good specificity for Streptococcus dysgalactiae, and there is no obvious positive test strip reaction between different strains, and it has a good degree of identification. It can be observed with the naked eye under circumstances, which is convenient for grass-roots workers to use, and is more helpful for the detection of Streptococcus dysgalactiae.

Example 9

To test the sensitivity of nucleic acid immunochromatographic strips to detect Streptococcus dysgalactiae.

1 Materials and methods

1.1 Materials

Streptococcus dysgalactiae was isolated, identified and preserved by the Invention Example of the School of Veterinary Medicine of Nanjing Agricultural University.

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

Streptococcus dysgalactiae was revived and cultured, and the bacterial solution was serially diluted and counted on a plate. The number of colonies corresponding to 1-9 in Figures 9a and 9b were 1.6×10 ⁸ cfu/ml, 1.6×10 ⁷ cfu/ml, 1.6× 10 ⁶ cfu/ml, 1.6×10 ⁵ cfu/ml, 1.6×10 ⁴ cfu/ml, 1.6×10 ³ cfu/ml, 1.6×10 ² cfu/ml, 1.6×10 ¹ cfu/ml, 2cfu/ml; At the same time, bacterial DNA was extracted as a PCR template, and the above-mentioned (1.3) PCR reaction system was used for PCR amplification of Streptococcus dysgalactiae, and then gel electrophoresis and nucleic acid immunochromatography test strips were used to verify its sensitivity.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1×TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis are photographed and saved.

2 results

The electrophoresis results of the detection sensitivity identification of Streptococcus dysgalactiae are shown in Figure 10a, and the identification results of the nucleic acid immunochromatography test strips of the detection sensitivity of Streptococcus dysgalactiae are shown in Figure 10b. The nucleic acid immunochromatography test strip detection method has high detection sensitivity and can reach the lowest detection limit of 1.6×10 ³ cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.

In Example 7-Example 9, the sdy-FITC-F sequence (ie, the fifth primer sequence) is: TAAAGGTGCAACTGCATACTA; the sdy-DIG-R sequence (ie, the sixth primer sequence) is: AGTCACATGGTGGATTTTCCA.

Example 10

The effect of different colloidal gold particle size on the color rendering effect of the test strip was tested.

1 Materials and methods

1.1 Materials

Colloidal gold solutions with different particle sizes ranging from 10 nm to 50 nm are prepared by the examples of the present invention.

1.2 The appearance changes of gold nanoparticles with different particle sizes caused by different citric acid addition amounts are shown in the following table:

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

1) Take 1 ml of colloidal gold solutions of different particle sizes (as shown in Figure 11a);

2) Add 10 ul of 1M potassium carbonate solution to 1 ml of colloidal gold solution to adjust the pH of the solution, and mix well; add 4 ul of FITC antibody and incubate at four degrees for 1 h; then add blocking solution and incubate at room temperature for 30 min; centrifuge at 7000 r/min for 3 min to remove unbound proteins , take the supernatant and centrifuge again, centrifuge at 9400r/min for 20min, remove the supernatant; add 80ul of reconstituted solution, place it at 4 degrees for aging for 2h; after taking out, take 6ul of each drop on the gold pad, and dry at 25 degrees; The pads are assembled to the test strips.

3) Resuscitate the preserved Streptococcus agalactiae, cultivate it to logarithmic phase, take the bacterial solution to carry out nucleic acid amplification, and carry out PCR amplification with the above-mentioned (1.3) PCR reaction system; carry out different gradient dilutions of the PCR amplification products; Add dropwise to test strips prepared from colloidal gold solutions of different particle sizes, and observe the results.

2 results

The apparent results of colloidal gold solutions with different particle sizes and the scanning results of UV spectrophotometers are shown in Figures 11a and 11b. The colloidal gold solutions prepared in the embodiment of the present invention have stable properties and uniform particle size distribution to meet the experimental requirements; And the results of gradient dilution nucleic acid immunochromatography test strips are shown in Figure 11c. The larger the particle size of gold particles, the more likely to cause aggregation and precipitation. Therefore, colloidal gold solutions larger than 35 nm tend to aggregate during the treatment process and cannot obtain a stable dispersion solvent, which is not suitable for Test strips were prepared for use. In the color development results shown in Figure 11c, the test strip corresponding to 10 nm was judged to have the best color rendering effect from the degree of release from the gold pad, background clarity, color intensity, and color uniformity.

Example 11

Test for the specific identification of Streptococcus agalactiae in different foodborne pathogenic contamination.

1 Materials and methods

1.1 Materials

The various zoonotic strains involved in the embodiments of the present invention are shown in Table 5.

Table 5 Experimental strains

Table 5 Bacterial strains for detection

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

The different strains were recovered and cultured, the number of colonies was counted using a plate, bacterial DNA was extracted, and the above (1.3) PCR reaction system was used to detect Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Enterococcus faecalis, Vitis epidermidis. Cocci, yeast, Salmonella, Bacillus cereus, Listeria monocytogenes and pure water were amplified by PCR, and the specificity among different strains was verified by gel electrophoresis and nucleic acid immunochromatography strips respectively.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1 × TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results for the specific identification of different strains of Streptococcus agalactiae are shown in Figure 12a, and the identification results of nucleic acid immunochromatography test strips for the specific identification of different strains of Streptococcus agalactiae are shown in Figure 12b. The results show that: the present invention The established PCR amplification detection method has good specificity for Streptococcus agalactiae, and there is no obvious positive reaction of the test strip between different strains, and it has a good degree of identification. It can be observed with the naked eye under the circumstances, which is convenient for grass-roots workers to use, and is more helpful for the detection of Streptococcus agalactiae.

Example 12

To test the sensitivity of nucleic acid immunochromatographic strips for the detection of Streptococcus agalactiae.

1 Materials and methods

1.1 Materials

Streptococcus agalactiae was isolated, identified and preserved by the Invention Example of the School of Veterinary Medicine of Nanjing Agricultural University.

1.2 Primer Design

1.3 PCR amplification system:

Reaction conditions:

1.4 How to operate

Resuscitate and cultivate Streptococcus agalactiae, take the bacterial solution for gradient dilution and count it on a plate, and extract bacterial DNA as a PCR template. The above (1.3) PCR reaction system is used to carry out PCR amplification of Streptococcus agalactiae, and then use gel electrophoresis and gel electrophoresis respectively. Nucleic acid immunochromatography test strips verify its sensitivity.

1) Take 4 μL of PCR product, mix it with 196ul developing solution, and add 60ul dropwise to the test strip. After five minutes of color development, use a smartphone or camera to take the color development result of the test strip.

2) Take another 5 μL of PCR product and conduct electrophoresis in 1.5% agarose gel electrophoresis containing ethidium bromide. The electrophoresis conditions are: 1 × TAE buffer, voltage 125V, electrophoresis time 25min, and use a nucleic acid gel imager to observe The gel results after running the electrophoresis were photographed and saved.

2 results

The electrophoresis results of the detection sensitivity identification of Streptococcus agalactiae are shown in Figure 13a, and the identification results of the nucleic acid immunochromatographic test strips of the detection sensitivity of Streptococcus agalactiae are shown in Figure 13b. The nucleic acid immunochromatography test strip detection method has high detection sensitivity, and the lowest detection limit is 2 × 102cfu/mL, which is 10 times higher than the detection sensitivity of nucleic acid gel electrophoresis.

The cfb-FITC-F sequence (ie, the seventh primer sequence) in Examples 10-12 is: TAATAATCAAGCCCAGC; the cfb-DIG-R sequence (ie, the eighth primer sequence) is: CCTTTTGTTCTAATGCC.

It should be understood that the above-mentioned embodiments are only intended to illustrate the technical concept and characteristics of the present invention, and the purpose thereof is to enable those who are familiar with the art to understand the content of the present invention and implement it accordingly, and cannot limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included within the protection scope of the present invention.

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Claims (10)

1, detection reagents, characterized by comprising a combination of any or more of the following four primer pairs, said four primer pairs comprising:

the th primer pair comprises a th primer and a second primer with sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2 respectively;

a second primer pair comprising primers having the sequences shown in SEQ ID NO: 3. SEQ ID NO: 4, a third primer and a fourth primer;

a third primer pair comprising primers with sequences as shown in SEQ ID NO: 5. SEQ ID NO: 6, a fifth primer and a sixth primer;

a fourth primer pair comprising primers with sequences as shown in SEQ ID NO: 7. SEQ ID NO: 8, and a seventh primer and an eighth primer shown in the figure.

2. The detection reagent according to claim 1, wherein the 5' -end of at least primers of at least primer pairs further comprises nucleic acid markers, each of which comprises of FITC, FAM, HEX, TET, TAMRA, ROX, VIC, NED, Alexa Flourr, biotin, digoxin, cy3 and cy 5.

3. The detection reagent according to claim 2, wherein the nucleic acid label is provided at the 5' -end of each of the two primers contained in the primer pair.

4. The detection reagent of in any of claims 1-3, further comprising a PCR routine component.

The kit of , comprising the detection reagent of of claims 1-4 and a nucleic acid immunochromatographic strip, wherein the nucleic acid immunochromatographic strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad, and the nitrocellulose membrane is provided with a detection band and a quality control band.

6. The kit according to claim 5, wherein a complex formed by th antibody and colored particles is attached to the gold label pad, a second antibody is arranged on the detection band, and a third antibody is arranged on the quality control band, wherein the th antibody is an antibody of the nucleic acid marker, the second antibody is an antibody or a ligand of the acid marker, and the third antibody is an antibody capable of binding with an antibody derived from a specified species.

7. The kit of claim 6, wherein: the third antibody is goat anti-mouse polyclonal antibody, and the colored particles are spherical red colloidal gold particles marked by fluorescein isothiocyanate antibodies.

8. Use of the detection reagent according to in any of claims 1-4 or the kit according to in any of claims 5-7 for the preparation of a product for detecting a pathogenic bacterium.

A product for use in a method of detecting a pathogenic bacteria, wherein the product comprises a kit as claimed in any of claims 5 to 7 at , and wherein the method of detection comprises:

amplifying the nucleic acid extracted from the sample to be tested by a PCR amplification reaction using at least primer pairs among the four primer pairs,

dripping the amplified product onto the nucleic acid immunochromatographic test paper of the kit, and observing whether a colored band is formed on the detection band and the finger control band; if colored bands visible to naked eyes are formed on the detection band and the quality control band, the detection band and the quality control band are judged to be positive; if no colored band is formed on the detection band and a macroscopic colored band is formed on the quality control band, judging the detection band to be negative; and if no colored band is formed on the quality control band, judging that the band is invalid.

10. The product of claim 9, wherein the pathogenic bacteria comprise or more of Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae and Streptococcus agalactiae.

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