CN1699596A - Bacteria trace rapid simultaneous detection method - Google Patents

Abstract

The present invention relates to a fast synchronous inspection method of micro content bacteria, which comprises: 1) mould board preparation, 2) PCR augmentation, the augmentation guide object is the general guide object of bacteria ribosomal ribonucleic acid gene, 3) gel electrophoresis of denaturization gradient, i.e. gel electrophoresis of denaturization gradient for augmentation outgrowth to determine sorts. The augmentation of 16S rRNA gene segment of bacteria is realized through UPPCR technology, obtaining all corresponding bacteria gene segment sequence information, and then DGGE is employed to appraise the augmentation outgrowth of mixed bacteria, so as to confirm the inspected bacteria. The invention provides a fast synchronous inspection method of micro content bacteria by combining the technologies of UPPCY and DGGE, which achieves the advantages of micro content, fast and synchronous inspection for mixed bacteria, especially pathogenic bacteria.

Description

Bacteria trace rapid simultaneous detection method

technical field

The invention relates to a method for rapid and synchronous detection of microscopic bacteria, especially pathogenic bacteria.

Background technique

Pathogenic bacteria seriously endanger people's health and industrial and agricultural production. To control the disease caused by it, it should be diagnosed in time to take corresponding preventive measures. In recent years, PCR (polymerase chain reaction) technology has been applied to the detection of pathogenic bacteria. These detection techniques can be divided into two categories, namely PCR with specific primers and PCR with non-specific primers. The former has different primer sequences depending on the target bacteria, and different primer sequences may require different amplification conditions. If the bacteria to be tested are unknown, it is difficult to achieve the purpose of rapid and specific detection (Peng X X, Zhang J Y, Wang S Y, et al, Immuno-capture PCR for Detection of Aeromonas hydrophila. J. Microbiol. Methods. 2002; 49(3): 335-338): The latter is generally synthesized according to the conservation of the bacterial 16S rRNA (ribosomal ribonucleic acid) gene Universal primers, the amplified products often need to cooperate with RFLP (restriction fragment polymorphism analysis), SSCP (single-strand conformation polymorphism) or sequence analysis to be confirmed, if the amplified products come from more than two pathogenic bacteria, then It is difficult to get the result directly. Based on the above situation, we designed the use of antibodies to specifically identify the bacteria to be tested, and then used the universal primers of the bacterial 16S rRNA gene for PCR amplification (UPPCR), and established the universal primer immunocapture PCR technology, which can quickly and specifically detect pathogenic bacteria in the mixture The purpose (CN02101983.5, Xiamen University, general primer PCR method for immunocapture method of detecting bacteria). Because there are many types of pathogenic bacteria, some pathogenic bacteria also have more subtypes and serotypes. Using the antibody capture method UPPCR for detection requires more materials.

Contents of the invention

The purpose of the present invention is to provide a method for detecting multiple bacteria rapidly and synchronously by using UPPCR-DGGE (universal primer PCR amplification-denaturing gradient gel electrophoresis). The technical scheme is to first use 16S rRNA gene universal primers to amplify to obtain the gene fragments of all bacteria in the sample to be tested, and then perform DGGE analysis to determine the bacteria.

Concrete steps of the present invention are as follows:

1) Prepare the template:

2) PCR amplification (UPPCR): the amplification primer is a universal primer for bacterial ribosomal ribonucleic acid gene;

3) Denaturing gradient gel electrophoresis (DGGE): The amplified product was subjected to denaturing gradient gel electrophoresis, and compared with the standard control, the species of mixed bacteria was determined.

The template can be prepared by direct thermal denaturation method or thermal denaturation method after antibody capture. The steps of said direct thermal denaturation method are to take mixed bacterial culture, heat until lysed, and centrifuge to obtain supernatant. The step of the heat denaturation method after antibody capture is to take the mixed culture bacterial solution and add it coated with more than two kinds of specific antibodies, heat to lyse, and take the supernatant. Then UPPCR was performed.

The present invention uses UPPCR technology to amplify the 16S rRNA gene fragments of bacteria, thereby obtaining information on the corresponding gene fragment sequences of all bacteria in the culture medium, and then uses DGGE to identify the amplification products of these mixed bacteria to determine the detected bacteria. Therefore, a detection technique for rapid simultaneous identification of mixed bacteria using UPPCR and DGGE was established. The basic principle of the present invention is that the 16S rRNA gene of bacteria is highly conserved, and the universal primer can amplify almost all bacteria, so the information of all bacteria in the specimen to be tested can be obtained. According to the sensitivity of DGGE separation, the mixed bacterial products are separated one by one, and finally compared with the standard map, the bacteria can be identified, so as to achieve the purpose of trace, rapid and simultaneous detection of mixed bacteria, especially pathogenic bacteria.

Description of drawings

Figure 1 is the direct method to obtain the UPPCR product spectrum of the pathogenic bacteria template and the DGGE analysis result of the product. In Figure 1, A. UPPCR results. M, Molecular weight standard; 1, Pseudomonas fluorescens; 2, Vibrio anguillarum; 3, Vibrio riverina; 4, Aeromonas hydrophila; Bacteria; 7, 6 kinds of bacteria mixed. B. DGGE results; 1, Pseudomonas fluorescens; 2, Vibrio anguillarum; 3, Vibrio riverina; 4, Aeromonas hydrophila; Bacteria, 7, 6 kinds of bacteria mixed.

Fig. 2 is the UPPCR product pattern and the DGGE analysis result of the pathogenic bacteria template obtained by the antibody method. In Figure 2, A. UPPCR results. M, molecular weight standard; 1, Shigella dysenteriae serotype 1; 2, Shigella flexneri serotype 1; 3, Shigella flexneri type 1a; 4, Shigella flexneri type 3a; . B. DGGE results. 1, Shigella Shigella serotype 1; 2, Shigella baumannii serotype 1; 3, Shigella flexneri type 1a; 4, Shigella flexneri type 3a; 5, a mixture of 4 strains.

Detailed ways

The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

Example 1

Use the direct thermal denaturation method to prepare the template, inoculate the bacteria in LB medium, and culture them on a shaker at 28°C for 12-18 hours as seed bacteria, and then inoculate them in the same medium as above at a ratio of 1:50 (bacteria:medium). Incubate on a shaking table at 28-37°C for 12-13 hours; take 0.5ml of the bacterial solution into a sterile 1.5ml centrifuge tube, centrifuge at 3,500 rpm for 20min, discard the supernatant; wash the precipitate with distilled water once, and add 0.8ml of sterile double-distilled water , mix well, boil in 100℃ water bath for 10min; cool the centrifuge tube in 0℃ ice bath after water bath, then centrifuge at 12,000rpm for 10min, absorb 15～30μl (15μl for 25μl PCR reaction system, 15μl for 50μl PCR reaction system 30 μl) supernatant was used as a PCR reaction template.

Pipette 2.5 μl 10× buffer, 0.5 μl dNTP (nucleotide mixture, each 10 mM), 0.7 μl MgCl ₂ (25 mM), 0.16 μl primer mixture (primer 1: 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGGCACAAGCGGTGGAGCATGTG, where at the 5' end GC clamp of 40 nucleotides was added to stabilize the resolution of DGGE; primer 2: 5′-GCCCGGGAACGTATTCACCG), 0.2μl Taq enzyme (5u·μl ^-1 ), and 15μl template in each PCR tube , and finally make up to a volume of 25 μl with sterile double-distilled water, mix the reagents thoroughly and centrifuge slightly, and collect them at the bottom of the tube; place the small centrifuge tube after adding the sample in a PCR amplification instrument, and the amplification program is 94 ℃ for 10min, then 94℃ for 1min, 68℃ for 1min, 72℃ for 3min, after that, the annealing temperature decreased by 0.5℃ for each cycle until 58℃, then repeated 20 PCR cycles with the annealing temperature at 58℃, and finally 72℃ for 10min; amplification Finally, 5 μl of the PCR product was taken, mixed with an appropriate amount of loading buffer (0.25% bromophenol blue, 40% sucrose aqueous solution), and spotted on 2% agarose gel containing 0.5 μg/mL ethidium bromide (EB). 80V electrophoresis for 30min, then observe the results.

DGGE: 7L1×TAE (trishydroxymethylaminomethane acetic acid electrophoresis) buffer solution (trishydroxymethylaminomethane 48.4g, glacial acetic acid 11.42ml, 0.5M disodium edetate 20ml pH8.0, add distilled water to 200ml is 50X buffer solution, autoclaved, stored at room temperature) into the electrophoresis tank, put the temperature controller on the electrophoresis tank, connect the wires, turn on the switch, pump and heater on the temperature controller in turn; set the temperature The controller is set to a preset temperature of 56°C, and the temperature change rate is 200°C/h to preheat the electrophoretic fluid; a set of 16cm×16cm parallel gradient gel interlayers is assembled with clean glass plates and spacers, and the It is locked on the glue rack to keep it horizontal; use two matching syringes to connect with the hose respectively, mark "LO" and "HI" on the syringes, and use the "LO" syringe and "HI" syringe to inhale 0 % denaturing gel and 70% denaturing gel (add 320μl denaturing gradient indicator solution to 70% denaturing gel); set the volume of the gradient mixer (cam wheel) to 14.5mL, and connect it with a Y-shaped tube and a 16-gauge needle A pair of syringes are respectively fixed on both sides of the gradient mixer; turn the gear, pour the glue into the interlayer, insert a clean comb, and wait for coagulation; after coagulation, pull out the comb and remove the interlayer; the two interlayers are respectively fixed on In the electrophoresis tank, pour 350ml 1×TAE buffer solution into the upper tank, put the temperature controller in the correct direction, and then turn on the power switch, pump and heater to make the system reach the set 56°C; use 5μl PCR to amplify The sample is mixed with an equal amount of 2× gel loading staining solution (0.25% bromophenol blue, 40% sucrose aqueous solution), and the sample is added in the washed sample well (in order to improve the sensitivity, the concentration of the PCR product can be increased by concentration treatment) ); adjust the voltage to 20V for 20 minutes, then run 100°C electrophoresis for 10-12 hours, and turn on the temperature controller at the same time to keep the system at 56°C; In a staining dish containing 250ml 1×TAE buffer and 25μl 0mg/ml ethidium bromide, stain for 5-10 minutes; after staining, move the gel to a dish containing 250ml 1×TAE buffer, decolorize for 5-20 minutes ; Scan the stained gel in a multi-color gel imaging system and save the results.

Example 2

Similar to Example 1, the difference is that the template is prepared by thermal denaturation after antibody capture, and antibodies (such as type and group-specific antibodies) that can react with various bacteria are diluted with pH9.6, 0.01mol/L carbonic acid buffer Coat each well of a 96-well enzyme-linked reaction plate (1-10 μg/ml), 50 μl per well, and store in a refrigerator at 4°C overnight; then use pH 7.4, 0.01mol/L phosphate-Tween buffer (PBS- T) Wash the plate 3 times, each time for 3-5 minutes; add 50 μl of 10% calf serum to each well, seal at 36-38°C for 1 hour, and shake dry; add 20-40 μl of bacterial liquid culture solution to each well (add 20 μl , 50μl PCR reaction system plus 40μl), incubate at 36-38°C for 1-2h, wash the plate 5 times with PBS-T washing solution, 3-5min each time. Add 20-40 μl (consistent with the volume of the added bacterial solution) sterile double-distilled water to each well, heat in a boiling water bath for 8-10 minutes, and draw 15-30 μl (15 μl for 25 μl PCR reaction system, 30 μl for 50 μL PCR reaction system) lysate as PCR reaction template.

Example 3

Bacterial template preparation: using Pseudomonas fluorescens, Vibrio anguillarum, Vibrio fluvialis, Aeromonas hydrophila, Providencia rettii rettgeri), Aeromonas sobria (Aeromonas sobria), strains preserved by the applicant. The above-mentioned bacteria were inoculated separately and mixed in LB medium, a total of 7 tubes were cultured on a shaker at 28°C for 12-18 hours, and used as seed bacteria. Then inoculate the above-mentioned same culture medium at a ratio of 1:50 (bacteria:medium), culture on a shaking table at 28°C for 12-13 hours; take 0.5ml of the bacteria solution into a new sterile 1.5ml small centrifuge tube, and run at 3,500 rpm Centrifuge for 20 minutes, discard the supernatant; wash the precipitate with distilled water once, add 0.8ml of sterile double distilled water, mix well, boil in a water bath at 100°C for 10 minutes; put the small centrifuge tube after the water bath in an ice bath at 0°C to cool, Then centrifuge at 12,000 rpm for 10 min, and absorb 15 μL of the supernatant as a PCR reaction template.

UPPCR: Take a sterile new tip and add 2.5 μl 10× buffer, 0.5 μl dNTP (10 mM each), 0.7 μl MgCl ₂ (25 mM), 0.16 μl primer mixture (primer 1: 5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGGCACAAGCGGTGGAGCATGTG+primer 2: 5′-GCCCGGGAACGTATTCACCG), 0.2μl Taq enzyme (5u·μl ^-1 ), and 15μl template, and finally make up to a final volume of 25μl with sterile double-distilled water. Tube bottom; place the small centrifuge tube after adding the sample in the PCR amplification instrument, set the program as 94°C for 10 minutes, then 94°C for 1 minute, 68°C for 1 minute, and 72°C for 3 minutes. Until 58°C, repeat 20 PCR cycles with the annealing temperature at 58°C, and finally 72°C for 10 minutes; remove the centrifuge tube after the amplification is completed. Take 5 μl of the PCR product, mix it with an appropriate amount of loading buffer, spot the sample on 2% agarose gel containing 0.5 μg/mL ethidium bromide (EB), electrophoresis at 80 V for 30 min, and then observe the results.

DGGE: Carry out according to the method of embodiment 1, point 1 sample for each tube.

Example 4

Bacterial template preparation: Shigella dysenteriae serotype 1 (S.dysenteriae serotypel), baumannii serotype 1 (S.boydii serotype 1), flexneri serotype 1a (S.flexneri serotype 1a), Fu Shigella dysenteriae type 3a (S.flexneri serotype 3a), are all strains preserved by the applicant. The above four kinds of bacteria were inoculated separately and mixed in LB medium, a total of 5 tubes, cultured on a shaker at 37°C for 12-18 hours, as seed bacteria. Then inoculate it on the above-mentioned same culture medium at a ratio of 1:50 (bacteria liquid: culture medium), culture on a shaking table at 37°C for 12-13 hours overnight: take 20 μl of the bacteria liquid and add the antibody coated with Shigella (this antibody is effective against all Shigella). Incubate at 36-38°C for 1 hour in an enzyme-labeled plate that can produce specific binding, and wash the plate with PBS-T washing solution for 5 times, each time for 3-5 minutes. Add 20 μl of sterile double-distilled water to each well, heat in a boiling water bath for 8-10 minutes, and draw 15 μl of the lysate as a PCR reaction template.

UPPCR: same as embodiment 3.

DGGE: Same as Example 3.

Claims (7)

1. A rapid and synchronous detection method for bacterial traces, characterized in that the steps are: 1) prepare template; 2) PCR amplification: the amplification primer is a universal primer for bacterial ribosomal ribonucleic acid gene; 3) Denaturing gradient gel electrophoresis: take the amplified product and carry out denaturing gradient gel electrophoresis, and compare it with the standard control to determine the species of the mixed bacteria. 2. The method for rapid simultaneous detection of bacterial traces as claimed in claim 1, characterized in that said preparation template adopts direct thermal denaturation method or thermal denaturation method after antibody capture, and the step of said direct thermal denaturation method is to take mixed bacteria The culture is heated until lysed, centrifuged to take the supernatant; the step of the heat denaturation method after antibody capture is to take the mixed culture bacterial solution and add it coated with more than 2 kinds of specific antibodies, heat until lysed, and take the supernatant solution, followed by UPPCR. 3. The method for rapid simultaneous detection of bacterial traces according to claim 1, characterized in that said amplification primers are 5'-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGGCACAAGCGGTGGAGCATGTG and 5'-GCCCGGGAACGTATTCACCG primers. 4. The method for quick and simultaneous detection of bacterial traces as claimed in claim 2, characterized in that said direct thermal denaturation method is to inoculate the bacteria in LB culture medium and culture them on a shaker at 28°C for 12-18 hours as seed bacteria, and then use 1:50 (bacteria:medium) ratio, inoculate in the above same culture medium at 28°C or 37°C for 12-13 hours on a shaker; take 0.5ml of the bacterial solution into a sterile 1.5ml centrifuge tube, centrifuge at 3,500 rpm for 20min , discard the supernatant; wash the precipitate with distilled water once, add 0.8ml of sterile double distilled water, mix well, boil in a water bath at 100°C for 10min; cool the centrifuge tube after the water bath in an ice bath at 0°C, and then centrifuge at 12,000rpm After 10 minutes, 15-30 μl of the supernatant was taken as a template for the PCR reaction. 5. The rapid and simultaneous detection method of micro-bacteria as claimed in claim 2, characterized in that the antibody capable of reacting with various bacteria is diluted with pH 9.6, 0.01mol/L carbonic acid buffer in the thermal denaturation method after antibody capture Coat 1-10 μg/ml in each well of a 96-well enzyme-linked reaction plate, 50 μl per well, and store in a refrigerator at 4°C overnight; then wash the plate 3 times with pH 7.4, 0.01 mol/L phosphate-Tween buffer, 3-5 minutes each time; add 50 μl of 10% calf serum to each well, block at 36-38°C for 1 hour, and shake dry; add 20-40 μl of bacterial liquid culture solution to each well, incubate at 36-38°C for 1 hour, wash with PBS-T Wash the plate 5 times with the washing solution, each time for 3-5 minutes, add 20-40 μl sterile double-distilled water to each well, heat in a boiling water bath for 8-10 minutes, and absorb 15-30 μl lysate as a PCR reaction template. 6. The rapid and simultaneous detection method of bacterial traces as claimed in claim 1, characterized in that said PCR amplification is sequentially aspirating and adding 2.5 μl of 10× buffer, 0.5 μl of dNTP, 0.7 μl of MgCl ₂ 25 mM, and 0.16 μl of primers Mixed solution, primer 1: 5'-CGCCCGCCGCGCGCGGCGGG CGGGGCGGGGGCACGGGGGGGCACAAGCGGTGGAGCATGTG, in which a GC clamp of 40 nucleotides was added to the 5' end to stabilize the resolution of DGGE; primer 2: 5'-GCCCGGGAACGTATTCACCG, 0.2 μl Taq enzyme, and 15 μl template in each PCR tube, and finally make up to 25 μl with sterile double-distilled water, mix each reagent thoroughly and centrifuge slightly, and collect at the bottom of the tube; place the small centrifuge tube after adding the sample in the PCR amplification In the multiplier, the amplification program is 94°C for 10 minutes, then 94°C for 1 minute, 68°C for 1 minute, and 72°C for 3 minutes. After that, the annealing temperature decreases by 0.5°C in each cycle until it reaches 58°C, and then repeats 20 PCRs with the annealing temperature at 58°C. Cycle, last 10min at 72°C; after amplification, take 5μl of the PCR product, mix it with loading buffer, spot on 2% agarose gel containing 0.5μg/mL ethidium bromide, electrophoresis at 80V for 30min, and then observe the results . 7. The rapid and simultaneous detection method of bacterial traces as claimed in claim 1, characterized in that said DGGE is to pour 7L1×tris hydroxymethylaminomethane acetic acid electrophoresis buffer into the electrophoresis tank, and place the temperature controller on the electrophoresis tank. On the tank, connect the wires, turn on the switch, pump and heater on the temperature controller in turn; set the temperature controller to a predetermined temperature of 56°C, and the temperature change rate is 200°C/h to pre-prepare the electrophoretic solution. Heat; assemble a set of 16cm×16cm parallel gradient gel interlayer with a clean glass plate and spacers, and lock it on the gel filling frame to keep it horizontal; use two matching syringes to connect with the hose respectively. The syringes are marked "LO" and "HI", and the "LO" syringe and the "HI" syringe are used to inhale 0% denatured gel and 70% denatured gel respectively; set the volume of the gradient mixer to 14.5mL, and connect the Y-shaped tube A pair of syringes with 16-gauge needles are respectively fixed on both sides of the gradient mixer; turn the gear, pour the glue into the interlayer, insert a clean comb, and wait for coagulation; after coagulation, pull out the comb and remove the interlayer; The interlayers are respectively fixed in the electrophoresis tank, pour 350ml 1×TAE buffer solution into the upper tank, put the temperature controller in the correct direction, and then turn on the power switch, pump and heater to make the system reach the set 56°C; Mix 5 μl of PCR amplified sample with an equal amount of 2× gel staining solution, and add the sample into the cleaned sample hole; adjust the voltage to 20V for 20 minutes, and then perform electrophoresis at 100 for 10 to 12 hours. At the same time, turn on the temperature controller to make the system Maintain at 56°C; after electrophoresis is completed, turn off the power and heating system, remove the interlayer, and take out the gel block; put the gel into a staining plate containing 250ml 1×TAE buffer and 25μl 0mg/ml ethidium bromide, and stain for 5 ~10min; after staining, move the gel to a plate containing 250ml 1×TAE buffer, decolorize for 5-20min; scan the stained gel in a multi-color gel imaging system, and save the results.

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