Primer pair, gene probe, kit and application of primer pair, gene probe and kit in identification of pseudomonas aeruginosa

Abstract

The invention discloses a primer pair, a gene probe, a kit and application of the primer pair, the gene probe and the kit in identification of pseudomonas aeruginosa. Based on an RPA (recombinase polymerase amplification) rapid amplification principle, the primer pair, the gene probe and / or the kit are used for carrying out specific amplification on conserved sequence gene segments of the pseudomonas aeruginosa, so that accurate identification on the pseudomonas aeruginosa is realized. The invention provides a method which is more accurate and easier to operate for identifying the pseudomonas aeruginosa, so that more complex identification requirements of the pseudomonas aeruginosa in the aspects of production and scientific research in a plurality of fields such as biology, medicine and food are met.

Description

Technical Field

[0001] The present application belongs to the field of biomedical detection, and specifically relates to a primer pair, a gene probe and a kit and their application in identifying Pseudomonas aeruginosa. Background Art

[0002] Pseudomonas aeruginosae is a Gram-negative, aerobic, long rod-shaped bacterium belonging to the genus Pseudomonas. It is an opportunistic infection bacterium and one of the common pathogens causing chronic infections and nosocomial infections. It is also an opportunistic infection for plants. It generally affects the lungs and urinary tract, or causes burns, wounds and other blood infections, such as sepsis. Although it is very rare, Pseudomonas aeruginosae can also cause pneumonia. Incomplete cleaning of contact lenses can also cause corneal infection. Bacterial resistance monitoring in various countries over the years shows that this bacterium is one of the top pathogens causing nosocomial infections. In some areas, its detection rate can reach 10.78%, ranking first among non-fermenting bacteria. The results of bacterial resistance monitoring show that the resistance rate of Pseudomonas aeruginosa to imipenem and meropenem is gradually increasing. At present, the detection of drug-resistant bacteria in clinical practice is mainly carried out by detecting the results of drug sensitivity tests using paper diffusion methods, test strips and automated microbial systems, or by using PCR methods and NGS sequencing methods to detect pathogenic strains and drug-resistant genes. These conventional detection methods take a long time to operate, have high laboratory requirements, rely on expensive experimental equipment, and are complex to operate for strain isolation and processing, and there is a certain risk of infection, which makes it inconvenient to perform detection at the grassroots level or on-site. Therefore, the development of an efficient, accurate, sensitive, easy-to-operate, and readily monitorable method for detecting and analyzing Pseudomonas aeruginosa is of great significance for the scientific research and application of Pseudomonas aeruginosa.

[0003] RPA isothermal amplification technology, also known as recombinase polymerase amplification technology (RPA), is a new nucleic acid isothermal amplification technology developed by Piepenburg et al. in 2006 using protein recombination and repair involved in cell DNA synthesis. It is known as a nucleic acid detection technology that can replace PCR. RPA technology mainly relies on three enzymes: recombinase that can bind to single-stranded nucleic acids (oligonucleotide primers), single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase. The reaction process of RPA first involves the binding of the recombinase to the primer to form a protein-DNA complex, and then the protein-DNA complex searches for homologous sequences in double-stranded DNA. Once the primer finds the target homologous sequence, a chain exchange reaction occurs and DNA synthesis is initiated, completing exponential amplification of the target sequence on the template within minutes. The replaced DNA chain binds to SSB (single-stranded DNA binding protein) to keep the DNA in a single-stranded state and prevent it from being hydrolyzed by nucleases. The mixture of these three enzymes is also active at room temperature, with the optimal reaction temperature at around 39°C. It only requires a small amount of sample preparation and can amplify as few as 1-10 DNA copies within 10 minutes. It has the advantages of high sensitivity, selectivity, portability, rapidity, and the ability to perform multiple amplifications.

[0004] The main principle of colloidal gold test strips is to use cellulose acetate membrane as a carrier, utilize the capillary action of the microporous membrane, and slowly transfer the liquid dripped on one end of the membrane strip to the other end, and use the colloidal gold to show the color reaction through the antigen-antibody binding of the streaked line. The biggest feature of this method is that it is simple to operate, fast and rapid, sensitive and accurate, safe and simple, low cost, and does not require professional personnel and instruments and equipment. The negative and positive color development is obvious, and the test results can usually be observed by the naked eye within 3-5 minutes.

[0005] Currently, the traditional methods for identifying Pseudomonas aeruginosa include: imaging, histopathology, direct culture, serological methods, drug sensitivity testing, molecular methods and NGS sequencing methods.

[0006] The limitations of these traditional methods for P. aeruginosa identification are:

[0007] 1. These identification methods all require the cultivation of Pseudomonas aeruginosa strains, which takes a long time and is prone to contamination during the cultivation process.

[0008] 2. These identification methods can only identify a specific type of strain, and have serious lags in the diagnosis and treatment of diseases (after symptoms appear, the results cannot be known in time).

[0009] 3. Traditional bacterial molecular identification takes a long time (about 1-2 hours) to extract the bacterial genome, the steps are complicated, and improper operation during the extraction process can easily cause environmental pollution.

[0010] 4. Conventional strain identification is costly and time-consuming, and is inconvenient and costly for clinical patients.

[0011] Therefore, there is an urgent need for a method for identifying Pseudomonas aeruginosa that is rapid, sensitive, highly specific and easy to operate. Summary of the invention

[0012] In order to solve the technical problem that the prior art lacks a method for identifying Pseudomonas aeruginosa that is rapid, sensitive, highly specific and easy to operate, the present invention mainly innovates on the principle of combining RPA isothermal amplification technology with test strip detection, and provides a primer pair, a gene probe and a kit and their application in identifying Pseudomonas aeruginosa.

[0013] The present invention designs a pair of conservative primers and probes based on the conserved region lasB gene of Pseudomonas aeruginosa, and uses the RPA amplification method to establish a visualization nucleic acid test strip detection technology for Pseudomonas aeruginosa detection. The lasB gene is an important virulence factor and synthetic elastase of Pseudomonas aeruginosa, so the lasB conserved sequence gene is selected as the target gene for detection. The detection first extracts the genome of the sample, then performs RPA amplification on the extracted sample, and finally drips the RPA amplified sample onto the test strip, and indicates whether the strain exists in the sample by the development of the test strip.

[0014] In a first aspect, the present invention provides a primer pair for specific amplification of Pseudomonas aeruginosa, the primer pair comprising a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the reverse primer is shown in any one of SEQ ID NOs: 4-6;

[0015] Preferably, the nucleotide sequence of the forward primer is as shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO: 4; or,

[0016] The nucleotide sequence of the forward primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 5; or,

[0017] The nucleotide sequence of the forward primer is shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO:6;

[0018] And / or, the 5' end of the reverse primer is conjugated with a label, and the label is preferably biotin.

[0019] The second aspect of the present invention provides a gene probe coupled with digoxigenin labeling, wherein the nucleotide sequence of the gene probe is shown in SEQ ID NO:7.

[0020] The third aspect of the present invention provides a primer-probe combination, the primer-probe combination comprising the primer pair as described in the first aspect and / or the gene probe as described in the second aspect of the present invention;

[0021] Preferably, the primer-probe combination comprises a primer pair whose nucleotide sequence of the forward primer is shown in SEQ ID NO:1 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO:4, and a gene probe whose nucleotide sequence is shown in SEQ ID NO:7; or, the primer-probe combination comprises a primer pair whose nucleotide sequence of the forward primer is shown in SEQ ID NO:2 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO:5, and a gene probe whose nucleotide sequence is shown in SEQ ID NO:7; or, the primer-probe combination comprises a primer pair whose nucleotide sequence of the forward primer is shown in SEQ ID NO:3 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO:6, and a gene probe whose nucleotide sequence is shown in SEQ ID NO:7;

[0022] More preferably, the 5' end of the reverse primer is conjugated with a label, and the label is preferably biotin.

[0023] A fourth aspect of the present invention provides a test strip, the test strip comprising a sample pad, a conjugation pad, a nitrocellulose membrane and an absorption pad, the conjugation pad comprising microsphere solution 1 and microsphere solution 2; the nitrocellulose membrane comprising a detection line T line and a quality control line C line;

[0024] The microsphere solution 1 contains microspheres labeled with streptavidin; the microsphere solution 2 contains microspheres labeled with mouse anti-human antibody; the detection line T contains digoxigenin antibody, and the quality control line C contains goat anti-mouse antibody;

[0025] In some preferred embodiments, the test strip meets one or more of the following conditions:

[0026] (1) The particle size of the microspheres is 100-300 nm, for example, 200 nm;

[0027] (2) the molar concentration ratio of streptavidin in the microsphere solution 1 to the digoxigenin antibody in the detection line T is 1:1; and,

[0028] (3) The binding pad is a colloidal gold binding pad, and the amount of gold sprayed onto the microspheres is, for example, 6 μl / cm.

[0029] In the present invention, the detection line T has digoxin antibody, and the quality control line C has goat anti-mouse antibody; there are two kinds of microspheres on the colloidal gold binding pad: one is a microsphere labeled with streptavidin (SA), and the other is a microsphere labeled with mouse anti-human antibody. When the test strip is used for sample detection, since the amplified positive sample is labeled with biotin and digoxin, the sample is added to the glass fiber sample pad and flows to the colloidal gold binding pad. At this time, biotin will bind to the microspheres labeled with SA to generate a "microsphere-SA-biotin-positive fragment-digoxin" complex, which will reach the detection line T line along the flow direction. At this time, the complex will bind to the digoxin antibody and intercept the complex at the detection line T line, so the positive fragment will be colored on the detection line T line; if there is no positive fragment in the sample, the complex will not be formed, so the band will not appear on the detection line T line; another microsphere on the colloidal gold binding pad is a mouse anti-human microsphere, which will flow forward with the addition of the sample and will not react with the digoxin antibody on the detection line T line, but will continue to flow forward to the quality control line C line, react with the goat anti-mouse antibody on the quality control line C line, and the microsphere will be intercepted and colored at the quality control line C line.

[0030] The fifth aspect of the present invention provides a use of the primer pair described in the first aspect of the present invention, the gene probe described in the second aspect of the present invention, the primer-probe combination described in the third aspect of the present invention and / or the test strip described in the fourth aspect of the present invention in the preparation of a kit for identifying Pseudomonas aeruginosa.

[0031] The sixth aspect of the present invention provides a kit for identifying Pseudomonas aeruginosa, wherein the kit is selected from one or more of the primer pair described in the first aspect of the present invention, the gene probe described in the second aspect of the present invention, the primer-probe combination described in the third aspect of the present invention, and the test strip described in the fourth aspect of the present invention;

[0032] In some preferred embodiments, the kit further comprises a nucleic acid extraction reagent and / or a sample release buffer; the nucleic acid extraction reagent is an alkaline reagent, and the sample release buffer comprises a phosphate buffered saline solution.

[0033] In some more preferred embodiments, the nucleic acid extraction reagent is KOH and / or the sample release buffer is PBST+0.05% Triton.

[0034] In some embodiments, the method for preparing the test strip comprises the following steps:

[0035] i) preparing microsphere solution 1 and microsphere solution 2;

[0036] ii) The test strip is sprayed with gold film.

[0037] In some embodiments, the preparation of the microsphere solution 1 and the microsphere solution 2 in i) specifically comprises the following steps:

[0038] a) Mix the two tubes of microspheres with a particle size of 100-300 nm and the activation buffer in a volume ratio of 1:7;

[0039] b) Add streptavidin to the first tube at a final concentration of 0.1 to 0.15 mg / mL; add mouse anti-human antibody to the second tube at a final concentration of 0.1 to 0.15 mg / mL;

[0040] c) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide at a final concentration of 1-10 mg / mL to the two tubes of solution in b) respectively, placing the mixture on a constant temperature shaker at a speed of 80-120 rpm and a temperature of 22-26° C. for reaction for 50-70 minutes;

[0041] d) After the reaction is completed, ultrasonicate, add blocking agents respectively, ultrasonicate, and continue to place in a constant temperature shaker at a speed of 80 to 120 rpm and a temperature of 22 to 26° C. for blocking, and the blocking time is 80 to 100 minutes;

[0042] e) After blocking, centrifuge for 20 minutes, discard the supernatant, resuspend the precipitates in the first tube and the second tube with PBS to obtain microsphere solution 1 and microsphere solution 2, and store them in a refrigerator at 2-8°C for later use.

[0043] In some preferred embodiments, the particle size of the microspheres is 200 nm; the activation buffer is a HEPES solution; the final concentration of streptavidin is 0.125 mg / mL; the final concentration of the mouse anti-human antibody is 0.125 mg / mL; the final concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1 mg / mL; the blocking agent is a CE510 solution; the rotation speed is 100 rpm, the temperature is 24°C, the reaction time is 60 mins; and the blocking time is 90 mins.

[0044] In some more preferred embodiments, the HEPES solution has a concentration of 5.6 g / L and a pH of 7.45.

[0045] In some embodiments, the test strip gold-sprayed film in ii) specifically comprises the following steps:

[0046] (1) diluting the microsphere solution 1 and the microsphere solution 2 prepared in step (i) by 15 to 25 times with a PBS solution;

[0047] (2) first spraying the diluted microsphere solution 1 of (1) onto the test strip at a concentration of 5 to 7 μl / cm, and drying; then spraying the diluted microsphere solution 2 of (1) onto the test strip at a concentration of 5 to 7 μl / cm, and drying;

[0048] (3) Using a film-spraying gold sprayer, digoxin antigen protein is sprayed on the nitrocellulose membrane as the detection line T line, and goat anti-mouse antibody is sprayed as the quality control line C line to obtain a test strip.

[0049] In some preferred embodiments, the dilution is 20-fold; and the concentration is 6 μl / cm.

[0050] The seventh aspect of the present invention provides a method for identifying Pseudomonas aeruginosa in a sample to be tested for non-diagnostic purposes, the method comprising using the primer pair described in the first aspect of the present invention, the gene probe described in the second aspect of the present invention, the primer-probe combination described in the third aspect of the present invention, the test strip described in the third aspect of the present invention and / or the kit described in the sixth aspect of the present invention.

[0051] In some preferred embodiments, the method comprises the following steps:

[0052] (I) performing RPA amplification on the DNA extracted from the sample to be tested using the primer pair described in the first aspect of the present invention, the gene probe described in the second aspect of the present invention, and / or the primer-probe combination described in the third aspect of the present invention to obtain a Pseudomonas aeruginosa gene-specific RPA amplification product; and,

[0053] (II) The RPA amplification product and the sample release buffer are mixed at a volume ratio of 1:5, and added to the test strip as described in the fourth aspect of the present invention.

[0054] In some more preferred embodiments, the method further comprises:

[0055] (III) determining whether Pseudomonas aeruginosa is present in the sample to be tested based on the information on the test strip obtained after implementing step (II);

[0056] The judgment conditions for judging the positive or negative of the sample are: when the test strip has a detection line T line and a quality control line C line, it means that the result is positive and Pseudomonas aeruginosa exists in the sample; when the test strip has only a quality control line C line but no detection line T line, it means that the result is negative and Pseudomonas aeruginosa does not exist in the sample; when the test strip has no quality control line C line, the result is invalid.

[0057] In some embodiments, the DNA sample is obtained by a nucleic acid extraction reagent (alkaline reagent, such as KOH or NaOH).

[0058] In some embodiments, the nucleic acid extraction reagent is 0.1 M KOH.

[0059] In the present invention, "non-diagnostic purpose" application scenarios include, for example, detecting Pseudomonas aeruginosa in the environment, detecting Pseudomonas aeruginosa on the surface of contact lenses, etc.

[0060] An eighth aspect of the present invention provides a system for Pseudomonas aeruginosa, the system comprising:

[0061] (1) an information collection module: used to collect information on the test strip obtained by using the method according to the seventh aspect of the present invention; the information may be a photograph of the test strip; and,

[0062] (2) Identification module: used to determine whether the sample to be tested contains Pseudomonas aeruginosa; the judgment criteria are:

[0063] a. If there is no quality control line C, the identification result is considered invalid; if there is a quality control line C, proceed to step b;

[0064] b. If there is a T line, it is judged as positive for Pseudomonas aeruginosa; if there is no T line, it is judged as negative for Pseudomonas aeruginosa.

[0065] (3) Data storage module: used to store information, including user information, detection time and / or judgment results, to facilitate subsequent recording and tracking of pathogen infection information.

[0066] A ninth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program implements the identification system as described in the eighth aspect of the present invention.

[0067] The tenth aspect of the present invention provides an electronic device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor implements the identification system as described in the eighth aspect of the present invention when executing the computer program.

[0068] The eleventh aspect of the present invention provides a primer pair as described in the first aspect of the present invention, the gene probe as described in the second aspect of the present invention, the primer-probe combination as described in the third aspect of the present invention, the test strip as described in the fourth aspect of the present invention, the kit as described in the sixth aspect of the present invention, the identification system as described in the eighth aspect of the present invention, the computer-readable storage medium as described in the ninth aspect of the present invention and / or the electronic device as described in the tenth aspect of the present invention for use in identifying whether a sample to be tested contains Pseudomonas aeruginosa.

[0069] In some embodiments, the application is for non-diagnostic purposes, such as detecting Pseudomonas aeruginosa in the environment, detecting Pseudomonas aeruginosa on the surface of contact lenses, etc.

[0070] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0071] The reagents and raw materials used in the present invention are commercially available.

[0072] The positive and progressive effects of the present invention are:

[0073] The present invention designs specific RPA amplification primers and gene probes for the conserved sequence gene fragments of Pseudomonas aeruginosa, and can perform RPA amplification identification on all types of Pseudomonas aeruginosa. Utilizing the specific amplification primers and gene probes of the present invention, the false positive phenomenon caused by the generation of primer dimers during the RPA amplification process can be effectively avoided, making the identification result more accurate. It has the advantages of being fast, sensitive, highly specific, easy to operate, and low in cost. The present invention proposes a method and system for identifying a strain of Pseudomonas aeruginosa, which can more conveniently, quickly, accurately, and economically identify the type of Pseudomonas aeruginosa, is conducive to the identification and research of Pseudomonas aeruginosa, and provides substantial reference suggestions for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The figure is the gel electrophoresis diagram of nucleic acid extraction from different strains using different alkaline reagents; M is Marker; NC is negative control; the order of samples is: 1, Staphylococcus aureus; 2, Pseudomonas aeruginosa; 3, Acinetobacter baumannii; 4, Klebsiella pneumoniae; 5, Rhizopus oryzae; 6, Mucor circinelloides.

[0075] Figure 2 This is the detection diagram after adding the probe into the RPA amplification system.

[0076] Figure 3 The electrophoresis diagram is the result of RPA amplification using different primers for Pseudomonas aeruginosa.

[0077] Figure 4 The figure is a gel electrophoresis diagram of RPA products labeled with different markers; wherein M is Marker; lanes 1-8 are: 1. RPA product labeled with digoxigenin; 2. RPA product labeled with digoxigenin combined with digoxigenin antibody-5; 3. RPA product labeled with digoxigenin combined with digoxigenin antibody-6; 4. RPA product labeled with digoxigenin combined with digoxigenin antibody-12; 5. RPA product labeled with digoxigenin combined with digoxigenin antibody-13; 6. RPA product labeled with digoxigenin combined with digoxigenin antibody-14; 7. RPA product labeled with FITC; 8. RPA product labeled with FITC combined with FITC antibody.

[0078] Figure 5 The figure is the electrophoresis diagram of RPA products labeled with biotin and digoxigenin at different concentrations; wherein lanes 1-8 are: 1. RPA product labeled with biotin and digoxigenin (labeled product); 2. Labeled product: SA=4:1; 3. Labeled product: SA=2:1; 4. Labeled product: SA=1:1; 5. Labeled product: SA=1:2; 6. Labeled product: DIG-13=4:1; 7. Labeled product: DIG-13=2:1; 8. Labeled product: DIG-13=1:1.

[0079] Figure 6 This is a graph showing the test results of comparing test strips using different sample buffers.

[0080] Figure 7 This is a graph showing the results of a strain sample-specific test strip test.

[0081] Figure 8 This is the result of the sensitivity test strip test of Pseudomonas aeruginosa samples; the sample order is 1:1.77*10 7 CFU / ml, 2:1.77*10 6 CFU / ml, 3:1.77*10 5 CFU / ml, 4:1.77*10 4 CFU / ml, 5:1.77*10 3 CFU / ml, 6:1.77*10 2 CFU / ml, 7: sterile water.

[0082] Fig. 9 Schematic diagram of the identification system for microbial species.

[0083] Fig.10 A schematic diagram of the structure of an electronic device. DETAILED DESCRIPTION

[0084] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0085] Example 1 Optimization of sample nucleic acid extraction method

[0086] First, the present invention provides a method for quickly extracting a genome DNA sample of Pseudomonas aeruginosa. The steps of extracting a Pseudomonas aeruginosa sample with a sample release agent are as follows: take 20 μl of sample, add 95 μl of 0.1M KOH, 0.5mM EDTA, and vortex to mix; place in a 90°C metal bath for incubation for 5 minutes; then let it stand at room temperature to restore the temperature to room temperature, and a flocculent precipitate is precipitated, placed in a centrifuge at 10,000 rpm for 2 minutes, take the supernatant and add the sample preservation solution (Tris-HCl buffer, pH 8.0, 20 μl) to obtain an extracted genome solution.

[0087] The sample crude extraction method was tested on fungi, Gram-positive bacteria and Gram-negative bacteria respectively. For the selection of alkaline reagents, 0.1M sodium hydroxide (NaOH) or 0.1M potassium hydroxide (KOH) was selected as the test reagent, and the remaining steps were consistent with the sample release agent treatment method for Pseudomonas aeruginosa.

[0088] The extracted DNA of Pseudomonas aeruginosa samples was verified by PCR amplification. The specific amplification primers and probe sequences of Pseudomonas aeruginosa are shown in Table 3; the primer pair for bacterial verification selected 16sDNA-F / R (as shown in Table 3), and the primer pair for fungal verification selected ITS1 / ITS4 primer pair (as shown in Table 3). The PCR amplification system and amplification procedure are as follows:

[0089] Table 1 PCR amplification system

[0090]

[0091] Table 2 PCR amplification program

[0092]

[0093] The amplification results were identified using 2% agarose gel. Figure 1 As shown in the figure, it can be seen that the selection of potassium hydroxide (KOH) as the alkaline hydrolysis reagent in the lysis reagent can play a significant role in the extraction, and the extracted sample can be normally subjected to the subsequent amplification reaction.

[0094] Example 2 Addition of probes to the system

[0095] For the traditional RPA amplification combined with test strip detection method, only a pair of labeled forward and reverse primers need to be designed, but primer dimers will be generated during the detection process, resulting in false positive test results in the test strip.

[0096] The present invention adds a labeled specific primer (i.e., probe PA-lasB-PR, SEQ ID NO: 7) to the RPA amplification, so that the primer dimer cannot be detected in the test strip. Figure 2As shown, after adding the probe, the detection line T of NC in the RPA amplification system did not appear, and without adding the probe, the primer dimers appearing in the RPA amplification system would bind to the detection line T and cause a false positive.

[0097] Example 3 Optimization of primer pairs for RPA amplification of Pseudomonas aeruginosa

[0098] (1) According to the arranged sample sequence, the corresponding samples are marked on the RPA reaction 96-well plate (i.e., reaction plate, whose naming rule is "experiment date-sample number-site information") or 200 μl reaction tube of the biotechnology. Pseudomonas aeruginosae (ATCC15692) bacterial liquid samples are transferred from the sample tube in sequence using a pipette. Nucleic acid is extracted from the Pseudomonas aeruginosae liquid sample according to the optimized extraction method of Example 1. The sample should be added to the bottom of the reaction well; if the sample is not added to the bottom of the reaction well during the sample addition process, the sample should be centrifuged briefly after the sample addition is completed, and the extracted Pseudomonas aeruginosae genomic DNA product is obtained after the reaction is completed.

[0099] (2) RPA amplification was performed using the primer probe pairs in Table 3, PA-lasB-F1 / R1 / PR, PA-lasB-F2 / R2 / PR, and PA-lasB-F3 / R3 / PR, respectively. The primer sequences are shown in Table 3.

[0100] Table 3 RPA amplification primer pair sequences

[0101]

[0102] Note: 1. "F", "R" and "PR" in the primer names represent the upstream, downstream and probe sequences of the primers respectively.

[0103] 2. DIG-dT is the DIG labeling group, THF is the tetrahydrofuran molecule, C3-spacer is the blocking group at the 3' end, and the 5' end of the reverse primer (PA-lasB-R1, PA-lasB-R2 and PA-lasB-R3) is labeled with Biotin.

[0104] (3) Perform RPA amplification on the sample using the PA-lasB-F3 / R3 / PR primer probe, and directly perform RPA amplification on the DNA sample extracted from Pseudomonas aeruginosa to obtain the Pseudomonas aeruginosa gene-specific RPA amplification product. Configure the RPA reaction system shown in Table 4:

[0105] Table 4 RPA reaction system

[0106]

[0107]

[0108] After configuring the system as above, vortex mix, add 2.5 μl of the activator in the DNA constant temperature rapid amplification kit, vortex mix, and incubate in a 39°C constant temperature bath for 10 minutes.

[0109] (4) The obtained RPA amplification product was identified by 2% agarose gel electrophoresis. Figure 3 As shown in the electrophoresis diagram, it can be seen that the primers designed by the present invention can obtain relatively single and bright bands, indicating that sufficient DNA amount can be obtained by using the primers designed by the present invention for RPA amplification.

[0110] In addition, the electrophoresis band of lane 3 is brighter than that of lanes 1 and 2. The higher the brightness of the primers, the better the specificity of the primer amplification, the higher the amplification efficiency, and the higher the concentration of the amplified product; therefore, the primer probe pair (SEQ ID NO: 3, 6 and 7) is more obvious for the identification result. For low-concentration samples, primers with poor amplification efficiency may not amplify the target band, while primers with high amplification efficiency can amplify the target band, that is, the primer pair provided by the present invention can improve the sensitivity of detection.

[0111] Example 4 Preparation of digoxin antibody and FITC antibody

[0112] The following homemade antibodies are for cost considerations, and it is not necessary to use the homemade antibodies of the present invention. Those skilled in the art can prepare antibodies according to conventional methods, such as by the following subclone screening, hybridoma cells are inoculated into mice to collect ascites, and the method of purifying ascites is prepared and screened to obtain the target antibody with good specificity. Alternatively, conventional commercially available digoxin antibodies and FITC antibodies can also be selected. As long as the antibody used can produce a polymer binding with the RPA amplification product with the corresponding antigen marker, the technical effect of the present invention can be achieved.

[0113] The monoclonal antibody used in the example was developed on Balb / c mice using digoxin-BSA complex (Shanghai Maiye Biotechnology Co., Ltd., catalog number: Digoxin-BSA 230510#) as an immunogen. The first intradermal immunization was performed using digoxin-BSA complex (100 μg, as an immunogen) in 100 μl emulsion prepared with Freund's complete adjuvant, and 100 μg of immunogen in 100 μl emulsion prepared with Freund's incomplete adjuvant was used for enhanced immunization 25 days later. 50 μg of immunogen was used for intradermal immunization twice, with an interval of 4 weeks, and then 50 μg of immunogen was used in 100 μl Hanks balanced salt solution (China Bishi Biotechnology Co., Ltd.) for intravenous and intraperitoneal prefusion promotion (the IgG1 antibody strain in the example was obtained by fusion at the first immunization). Hybridoma cell lines secreting digoxin-specific antibodies were produced using standard fusion technology. After extensive screening, several anti-digoxin antibody clones with good specificity were selected for the development of rapid detection and screening of Pseudomonas aeruginosa.

[0114] Antibody subtype testing: A commercially available antibody subtype identification kit (ELISA) was used to identify the subtypes of several digoxin-specific antibodies that were screened. The identification results are shown in Table 5:

[0115] Table 5 Anti-digoxin antibody subtype identification results

[0116]

[0117] The monoclonal antibody used in the example was developed on Balb / c mice using FITC-OVA complex (Shanghai Maiye Biotechnology Co., Ltd., catalog number: FITC-OVA 240206#) as an immunogen. The first intradermal immunization was performed with FITC-OVA complex (100 μg, as an immunogen) in 100 μl emulsion prepared with Freund's complete adjuvant, and 25 days later, 100 μg of immunogen in 100 μl emulsion prepared with Freund's incomplete adjuvant was used for enhanced immunization. Then, 50 μg of immunogen was used for intradermal immunization twice, with an interval of 4 weeks, and then 50 μg of immunogen was used for intravenous and intraperitoneal prefusion promotion in 100 μl Hanks balanced salt solution (China Bishi Biotechnology Co., Ltd.) (the IgG1 antibody strain in the example was obtained by fusion at the first immunization). Hybridoma cell lines secreting digoxin-specific antibodies were produced using standard fusion technology. After extensive screening, anti-FITC antibody clones with good specificity were selected for the development of rapid detection of Pseudomonas aeruginosa.

[0118] Antibody subtype testing: The commercially available antibody subtype identification kit (ELISA) was used to identify the subtype FITC-6 of the screened anti-FITC specific antibodies. The identification results are shown in Table 6:

[0119] Table 6 Identification results of anti-FITC antibody subtype FITC-6

[0120]

[0121] Example 5 Screening of markers and antibodies thereof

[0122] The reverse primers (PA-lasB-R3) labeled with digoxigenin (DIG) at the 5' end and fluorescein isothiocyanate (FITC) at the 5' end were selected respectively for performance comparison and identification. The amplification method is consistent with the PCR amplification system amplification detection method for detecting Pseudomonas aeruginosa in Table 1 described in Example 1. The amplified PCR products labeled with digoxigenin were incubated with digoxigenin antibodies (digoxigenin antibody-5, digoxigenin antibody-6, digoxigenin antibody 12, digoxigenin antibody-13 or digoxigenin antibody-14) at room temperature for 3 minutes; the amplified PCR products labeled with FITC were incubated with FITC antibodies at room temperature for 3 minutes (wherein the digoxigenin antibody and FITC antibody were prepared by Example 4). Finally, the incubated reactants were subjected to agarose gel electrophoresis to identify the binding efficiency of the antibody and the labeled PCR product. The identification results are shown in the figure. Figure 4 shown.

[0123] from Figure 4 It can be seen that the PCR products in lanes 4 and 5 can produce multimer binding with digoxigenin antibody, and the multimer band produced in lane 5 is brighter, which indicates that digoxigenin antibody-13 can produce multimer binding with PCR products and its efficiency is higher than that of other lanes. Therefore, digoxigenin was selected as the primer labeling and digoxigenin antibody-13 was selected as the subsequent labeling antibody.

[0124] Example 6 Selection of biotin and digoxigenin labeling concentrations

[0125] The markers biotin and digoxin on the PCR product need to be combined with streptavidin and digoxin antibody on the test strip later, and now concentration ratio screening is required. The remaining steps are consistent with the detection method for amplification of Pseudomonas aeruginosa described in Example 1. The amplified PCR products labeled with biotin and digoxin are gradient screened with streptavidin (SA) and digoxin antibody-13 (DIG-13) according to the molar concentration ratio, and incubated at room temperature for 3 minutes and then agarose gel electrophoresis is performed to identify the binding efficiency. The results are as follows: Figure 5 shown.

[0126] Depend on Figure 5It can be seen that in lanes 4 and 5, the PCR product can produce significant multimer binding with SA; in lane 8, the PCR product can produce significant multimer binding with DIG-13, indicating that the subsequent polymerization amplification of the signal value can be performed through the multimer. Therefore, the molar concentration of PCR product: SA: DIG-13 = 1:1:1 was selected for the test strip detection.

[0127] Example 7 Sample Release Buffer Optimization

[0128] Direct testing of the RPA-amplified sample with a test strip will result in the test strip not being able to develop (because the amplified sample is viscous, which makes it impossible to perform capillary action and develop on the test strip). Therefore, the sample release buffer was optimized. The sample concentration was selected as 1.77*10 5 The samples of Pseudomonas aeruginosa with CFU / ml were extracted and amplified by RPA. Water, PBS and PBST+0.05% Triton solution were selected for comparative detection and test. The results are as follows: Figure 6 shown.

[0129] From the above comparative tests of different sample release buffers, it can be seen that PBST+0.05% Triton can fully release the RPA amplified sample to obtain a more sensitive detection.

[0130] Example 8 Preparation of Test Strips

[0131] 1. Preparation of labeled microspheres

[0132] Take two tubes of 200 μl of 200 nm blue microspheres (Jiaxing Maijin Biotechnology Co., Ltd.), add 1400 μl of activation buffer (HEPES solution 5.6 g / L pH 7.45), and mix by pipetting; add 200 μl of microsphere 1 to the first tube 1mg / ml streptavidin (SA) solution, mouse anti-human antibody (0.2mg, Merck, catalog number: I6760) was added to the second tube of microspheres 2; EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) with a final concentration of 1mg / mL was added to the tube wall, and the mixture was quickly shaken up and down to be evenly mixed, and the tube was placed in a constant temperature reaction for 60mins (24℃, 100rmp); after the reaction, each tube was ultrasonicated for 5 times, 100μl of blocking agent (CE510 solution) was added, and then ultrasonicated for 5 times, and the tube was placed in a constant temperature shaker for blocking for 90mins (100rmp, 24℃); after the blocking was completed, centrifuged for 20min (13000rpm, 4℃), the supernatant was discarded, and the precipitate was resuspended with 2ml PBS storage buffer; it was stored in a 2-8℃ refrigerator for later use.

[0133] 2. Gold coating on test strips

[0134] The two tubes of labeled microsphere solutions prepared above were diluted 20 times with PBS solution, and then the microsphere 1 was sprayed with gold at a concentration of 6 μl / cm, and then dried in a dryer; then the microsphere 2 was sprayed with gold and dried to obtain a colloidal gold binding pad. Digoxin antibody (DIG-13) was sprayed on the nitrocellulose membrane using a film spraying instrument as the test line (T line) and goat anti-mouse antibody (Sigma, catalog number: A9309-1mL) as the quality control line (C line).

[0135] Take 15μl of RPA amplification product and add it to 75μl sample release buffer, vortex mix, and then add it to the treated test strip. Wait for about 3 minutes to judge whether it is positive or negative. When both the test line (T line) and the quality control line (C line) of the test strip have bands, it means that the result is positive and Pseudomonas aeruginosa exists; if only the quality control line has a band and there is no band at the test line, it means that the result is negative and Pseudomonas aeruginosa does not exist; if no band appears on the quality control line, the result is invalid.

[0136] Example 9 Sample-specific test strip detection

[0137] Specific detection was performed on different strain samples, and the samples were: Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas putida, Escherichia coli, Rhizopus oryzae, NC, and the above different strain types are common strains. The remaining steps are consistent with the detection method for Pseudomonas aeruginosa described in Example 3. RPA amplification verification was performed on the extracted DNA of different samples (the primer probe pairs used were SEQ ID NO: 3, 6 and 7), and the results are as follows Figure 7 shown.

[0138] Depend on Figure 7 It can be seen that the primer pair of the present invention can specifically detect Pseudomonas aeruginosa samples and is exclusive and specific to Pseudomonas aeruginosa samples.

[0139] Example 10 Sample sensitivity test strip detection

[0140] The Pseudomonas aeruginosa strains were diluted with sterile saline and different concentrations (1:1.5*10 7 CFU / ml, 2:1.5*10 6 CFU / ml, 3:1.5*10 5 CFU / ml, 4:1.5*10 4 CFU / ml, 5:1.5*10 3 CFU / ml, 6:1.5*10 2CFU / ml) samples were tested for sensitivity. The remaining steps were consistent with the method for detecting Pseudomonas aeruginosa described in Example 3. The extracted Pseudomonas aeruginosa sample DNA was subjected to RPA amplification verification (the primer probe pair used was SEQ ID NO: 3, 6 and 7), and the results were as follows: Figure 8 shown.

[0141] Depend on Figure 8 It can be seen that the primer pair provided by the present invention has a sample concentration of 1.5*10 3 CFU / ml, it can still be significantly detected. This proves that the primer probe pair of the present invention has a high sensitivity. And from the trend of the band depth, it can be seen that the lower the sample concentration, the lighter the positive band, so the concentration of the strain in the sample can be seen from the depth of the positive band.

[0142] Example 11 Microbial Species Identification System

[0143] System 61 for identification of microbial species: information collection module 51, identification module 52 and data storage module 53 (see Fig. 9 ).

[0144] The information collection module 51 is used to collect the test strip results and identify the uploaded result pictures;

[0145] The identification module 52 is used to determine whether the sample to be identified is Pseudomonas aeruginosa, and automatically upload and save the data. The judgment criteria for the identification are:

[0146] a. If there is no quality control line C, the identification result is considered invalid; if there is a quality control line C, proceed to step b;

[0147] b. If there is a T line, the result is positive for Pseudomonas aeruginosa; if there is no T line, the result is negative for Pseudomonas aeruginosa;

[0148] The data storage module 53 is used to store information, including user information, detection time and / or judgment results, to facilitate the subsequent recording and tracking of pathogen infection information.

[0149] Embodiment 12 Electronic device

[0150] This embodiment provides an electronic device, which can be expressed in the form of a computing device (for example, a server device), including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the microbial species identification system of Embodiment 11 of the present invention can be implemented when the processor executes the computer program.

[0151] Fig.10 The hardware structure diagram of this embodiment is shown, and the electronic device 9 specifically includes:

[0152] At least one processor 91, at least one memory 92, and a bus 93 for connecting different system components (including the processor 91 and the memory 92), wherein:

[0153] The bus 93 includes a data bus, an address bus, and a control bus.

[0154] The memory 92 includes a volatile memory, such as a random access memory (RAM) 921 and / or a cache memory 922 , and may further include a read-only memory (ROM) 923 .

[0155] The memory 92 also includes a program tool 925 having a set (at least one) of program modules 924, such program modules 924 including but not limited to: an operating system, one or more application programs, other program modules and program data, each of which or some combination may include the implementation of a network environment.

[0156] The processor 91 executes various functional applications and data processing by running the computer programs stored in the memory 92, such as processing the sequence information in the sample and the sequence information in the database.

[0157] The electronic device 9 can further communicate with one or more external devices 94 (e.g., keyboards, pointing devices, etc.). Such communication can be performed through an input / output (I / O) interface 95. In addition, the electronic device 9 can also communicate with one or more networks (e.g., local area networks (LANs), wide area networks (WANs), and / or public networks, such as the Internet) through a network adapter 96. The network adapter 96 communicates with other modules of the electronic device 9 through a bus 93. It should be understood that, although not shown in the figure, other hardware and / or software modules can be used in conjunction with the electronic device 9, including but not limited to: microcode, device drivers, redundant processors, external disk drive arrays, RAID (disk array) systems, tape drives, and data backup storage systems, etc.

[0158] It should be noted that although several units / modules or sub-units / modules of the electronic device are mentioned in the above detailed description, this division is merely exemplary and not mandatory. In fact, according to the embodiments of the present application, the features and functions of two or more units / modules described above can be embodied in one unit / module. Conversely, the features and functions of one unit / module described above can be further divided into multiple units / modules to be embodied.

[0159] Embodiment 13 Computer readable storage medium

[0160] An embodiment of the present invention provides a computer-readable storage medium having a computer program stored thereon, and when the program is executed by a processor, the steps of the microbial species identification system of embodiment 11 of the present invention are implemented.

[0161] The readable storage medium may include but is not limited to: a portable disk, a hard disk, a random access memory, a read-only memory, an erasable programmable read-only memory, an optical storage device, a magnetic storage device or any suitable combination of the above.

[0162] In a possible implementation manner, the present invention may also be implemented in the form of a program product, which includes program codes. When the program product is run on a terminal device, the program codes are used to enable the terminal device to execute the steps of implementing the above-mentioned identification method.

[0163] The program code for executing the present invention may be written in any combination of one or more programming languages, and may be executed entirely on a user device, partially on a user device, as an independent software package, partially on a user device and partially on a remote device, or entirely on a remote device.

Claims

1. A primer pair for specific amplification of Pseudomonas aeruginosa, the primer pair comprising a forward primer and a reverse primer, characterized in that: The nucleotide sequence of the forward primer is shown in any one of SEQ ID NOs: 1-3, and the nucleotide sequence of the reverse primer is shown in any one of SEQ ID NOs: 4-6; Preferably, the nucleotide sequence of the forward primer is as shown in SEQ ID NO:1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:4; or, the nucleotide sequence of the forward primer is as shown in SEQ ID NO:2, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:5; or, the nucleotide sequence of the forward primer is as shown in SEQ ID NO:3, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:6; and / or, the 5' end of the reverse primer is conjugated with a marker, and the marker is preferably biotin.

2. A gene probe coupled with digoxigenin labeling, characterized in that: The nucleotide sequence of the gene probe is shown in SEQ ID NO:

7.

3. A primer-probe combination, characterized in that: The primer-probe combination comprises the primer pair according to claim 1 and / or the gene probe according to claim 2; Preferably, the primer-probe combination comprises a primer pair whose nucleotide sequence of the forward primer is shown in SEQ ID NO:1 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO:4, and a gene probe whose nucleotide sequence is shown in SEQ ID NO:7; or, the primer-probe combination comprises a primer pair whose nucleotide sequence of the forward primer is shown in SEQ ID NO:2 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO:5, and a gene probe whose nucleotide sequence is shown in SEQ ID NO:7; or, the primer-probe combination comprises a primer pair whose nucleotide sequence of the forward primer is shown in SEQ ID NO:3 and whose nucleotide sequence of the reverse primer is shown in SEQ ID NO:6, and a gene probe whose nucleotide sequence is shown in SEQ ID NO:7; More preferably, the 5' end of the reverse primer is conjugated with a label, and the label is preferably biotin.

4. A test strip comprising a sample pad, a conjugate pad, a nitrocellulose membrane and an absorption pad, characterized in that: The conjugate pad comprises microsphere solution 1 and microsphere solution 2; the nitrocellulose membrane comprises a detection line T line and a quality control line C line; The microsphere solution 1 contains microspheres labeled with streptavidin; the microsphere solution 2 contains microspheres labeled with mouse anti-human antibody; the detection line T contains digoxigenin antibody, and the quality control line C contains goat anti-mouse antibody; Preferably, the test strip meets one or more of the following conditions: (1) The particle size of the microspheres is 100-300 nm, for example, 200 nm; (2) the molar concentration ratio of streptavidin in the microsphere solution 1 to the digoxigenin antibody in the detection line T is 1:1; and, (3) The binding pad is a colloidal gold binding pad, and the amount of gold sprayed onto the microspheres is, for example, 6 μl / cm.

5. Use of the primer pair according to claim 1, the gene probe according to claim 2, the primer-probe combination according to claim 3 and / or the test strip according to claim 4 in preparing a kit for identifying Pseudomonas aeruginosa.

6. A kit for identifying Pseudomonas aeruginosa, characterized in that: The kit comprises one or more selected from the group consisting of the primer pair as claimed in claim 1, the gene probe as claimed in claim 2, the primer-probe combination as claimed in claim 3, and the test strip as claimed in claim 4; Preferably, the kit further comprises a nucleic acid extraction reagent and / or a sample release buffer; the nucleic acid extraction reagent is an alkaline reagent, and the sample release buffer comprises a phosphate buffered saline solution; More preferably, the nucleic acid extraction reagent is KOH and / or the sample release buffer is PBST+0.05% Triton.

7. A method for identifying Pseudomonas aeruginosa in a sample for non-diagnostic purposes, characterized in that: The method comprises using the primer pair as claimed in claim 1, the gene probe as claimed in claim 2, the primer-probe combination as claimed in claim 3, the test strip as claimed in claim 4 and / or the kit as claimed in claim 6; Preferably, the method comprises the following steps: (I) performing RPA amplification on the DNA extracted from the sample to be tested using the primer pair according to claim 1, the gene probe according to claim 2, and / or the primer-probe combination according to claim 3 to obtain an RPA amplification product; and, (II) mixing the RPA amplification product and the sample release buffer in a volume ratio of 1:5, and adding the mixture to the test strip as claimed in claim 4; More preferably, the method further comprises: (III) determining whether Pseudomonas aeruginosa is present in the sample to be tested based on the information on the test strip obtained after implementing step (II); The judgment conditions are: when the test strip has a detection line T line and a quality control line C line, it means that the result is positive and Pseudomonas aeruginosa exists in the sample to be tested; when the test strip has only a quality control line C line but no detection line T line, it means that the result is negative and Pseudomonas aeruginosa does not exist in the sample to be tested; when the test strip has no quality control line C line, the result is invalid.

8. A system for identifying Pseudomonas aeruginosa, characterized in that: The identification system comprises: (1) an information collection module: used to collect information on the test strip obtained by using the method according to claim 7; and, (2) Identification module: used to determine whether the sample to be tested contains Pseudomonas aeruginosa; the judgment criteria are: a. If there is no quality control line C, the identification result is considered invalid; if there is a quality control line C, proceed to step b; b. If there is a T line, it is judged as positive for Pseudomonas aeruginosa; if there is no T line, it is judged as negative for Pseudomonas aeruginosa; (3) Data storage module: used to store information, including user information, detection time and / or judgment results.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that: The computer program implements the authentication system according to claim 8.

10. An electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the computer program, the authentication system according to claim 8 is implemented.

11. Use of the primer pair according to claim 1, the gene probe according to claim 2, the primer-probe combination according to claim 3, the test strip according to claim 4, the kit according to claim 6, the identification system according to claim 8, the computer-readable storage medium according to claim 9 and / or the electronic device according to claim 10 in identifying whether a sample contains Pseudomonas aeruginosa; Preferably, the use is for non-diagnostic purposes.

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