Multi-modal presentation detection method based on simultaneous detection of multiple bacteria

Abstract

The invention discloses a multi-modal presentation detection method based on simultaneous detection of multiple bacteria, and belongs to the technical field of analysis and detection. The method comprises the following steps: preparing a bacterial target; the method comprises the following steps: preparing a bacterial target, then preparing a functional magnetic bead of a bacterial response type probe, adding the prepared functional magnetic bead of the bacterial response type probe into the bacterial target for incubation, identifying the target by the bacterial response type probe, cutting an RNA site to release an RCA amplification primer, adding an annular template, phi 29 DNA polymerase, dNTPs and T4 polynucleotide kinase for an RCA amplification reaction to obtain an RCA product, adding a molecular beacon, and carrying out fluorescence detection on the RCA product. And detection is carried out through photographing of a fluorescence spectrophotometer, an inverted fluorescence microscope or a smart phone. The detection method has good specificity on escherichia coli, coconut venom pseudomonas and staphylococcus aureus, has the advantages of portability, simplicity, high result reliability and accuracy, low cost and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and specifically relates to a multimodal presentation detection method based on simultaneous detection of multiple bacteria. Background Art

[0002] Food contamination is a major health problem that is prevalent worldwide. Among them, foodborne pathogens are the main cause of food safety problems and are widely present in contaminated food, drinking water and animal-derived products. Common pathogens such as Staphylococcus aureus, Escherichia coli and Pseudomonas cerebroventris each have significant pathogenic properties: Staphylococcus aureus can cause acute gastroenteritis, Escherichia coli may induce diarrheal enteritis, and Pseudomonas cerebroventris is closely related to severe systemic infections in immunosuppressed patients. When these pathogens coexist, their interactions may aggravate the severity of the infection and lead to serious consequences such as multiple organ failure. In addition, the presence of complex contamination significantly increases the risk of food safety. Therefore, the development of technologies for rapid, accurate and simultaneous detection of multiple pathogens is particularly important.

[0003] Although the existing detection methods have developed rapidly, most of them are optimized for the detection of single pathogens, which is difficult to meet the needs of rapid, sensitive and accurate detection of multiple bacteria coexisting in complex samples. For example, the traditional culture method is widely used due to its high sensitivity and specificity, but it is time-consuming and cumbersome to operate; molecular detection technology (such as PCR) has good accuracy, but it relies on expensive large-scale equipment and is difficult to distinguish between dead and live bacteria; immunoassays are convenient for on-site detection, but the sensitivity and specificity are significantly affected by the quality of antibodies, and there are certain limitations on the repeatability of the results. In addition, most of these detection technologies present the test results through a single mode, which makes it difficult to balance the detection efficiency and the accuracy of result verification. Therefore, it is urgent to develop a multimodal presentation method based on multiple bacterial detection, which provides sensitive, convenient and intuitive detection results by combining multiple signal detection technologies, such as fluorescence spectroscopy quantitative measurement, microscopic imaging and intelligent device-assisted detection. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a multimodal presentation detection method based on the simultaneous detection of multiple bacteria. The detection method is based on a bacterial response probe composed of different DNAzymes and RCA amplification primers that respond to different bacterial targets, cutting RNA sites to release RCA amplification primers, triggering three different RCA amplifications to produce different RCA products, and combining different molecular beacons to produce different fluorescence signals and different colors. By measuring different fluorescence signals, multiple bacteria can be quickly distinguished, thereby constructing a portable, simple, low-cost, and highly reliable and accurate method for simultaneous detection of multiple bacteria.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The present invention provides a detection method based on multimodal presentation of multiple bacteria simultaneous detection, comprising the following steps:

[0007] (1) Target preparation: After the bacteria are cultured in a culture medium, centrifuge at 5000-13000 rpm for 5-30 min. The supernatant is CEM. The precipitate is resuspended in a buffer solution and ultrasonically lysed. Centrifuge at 5000-13000 rpm for 5-30 min. The supernatant is CIM.

[0008] (2) Preparation of functionalized magnetic beads with bacteria-responsive probes: Take the magnetic beads washed with magnetic bead buffer, add the bacteria-responsive probe modified with biotin, shake and mix well, and incubate for 40 to 80 minutes;

[0009] (3) cleavage reaction: adding the functionalized magnetic beads of the bacteria-responsive probe prepared in step (2) to the bacterial target prepared in step (1) for incubation, wherein the bacteria-responsive probe recognizes the target, cleaves the RNA site and releases the RCA amplification primer;

[0010] (4) RCA amplification reaction: adding a circular template, phi 29 DNA polymerase, dNTPs, and T4 polynucleotide kinase to the solution obtained in step (3) to carry out RCA amplification reaction to obtain an RCA product;

[0011] (5) Signal detection: Add molecular beacons to the solution obtained in step (4) and detect using a fluorescence spectrometer, an inverted fluorescence microscope or a smartphone for photography.

[0012] Furthermore, the bacteria described in step (1) are one or more of Escherichia coli, Pseudomonas cocovenenans and Staphylococcus aureus.

[0013] Furthermore, the culture condition in step (1) is shaking culture at 30-37° C. for 8-20 hours.

[0014] Furthermore, the biotin-modified bacteria-responsive probe described in step (2) is shown in SEQ ID NO. 4-6.

[0015] Furthermore, the cutting reaction time in step (3) is 30 to 120 minutes.

[0016] Furthermore, the nucleotide sequence of the circular template described in step (4) is shown in SEQ ID NO.1-3.

[0017] Furthermore, in step (4), the amount of phi 29 DNA polymerase and T4 polynucleotide kinase in the RCA amplification reaction system is 2 to 10 U; the amount of circular template is 5 to 20 μM, the RCA amplification time is 1 to 3 hours, and the amplification temperature is 25 to 35°C.

[0018] Furthermore, the molecular beacons described in step (5) include three molecular beacons, as shown in SEQ ID NOs. 7-9.

[0019] Furthermore, the amount of molecular beacon added during signal detection in step (5) is 10-70 pmol.

[0020] Furthermore, in step (5), a fluorescence spectrometer is used to collect fluorescence signals at 440 nm, 518 nm, and 667 nm respectively under the conditions of excitation wavelength of 350 nm, emission of 360-500 nm, excitation of 494 nm, emission of 500-600 nm, excitation of 643 nm, emission of 650-750 nm, wherein the slit range is 10 nm.

[0021] Furthermore, in step (5), an inverted fluorescence microscope is used, the light source controller is turned on and adjusted to 50% intensity, the filters are adjusted to correspond to blue light, green light and red light respectively, the exposure time, black, gamma value and white are adjusted, and finally the image is photographed and saved.

[0022] Furthermore, 30 μL of the samples detected by the inverted fluorescence microscope in step (5) were respectively transferred into eight rows of tubes, and photographed using a smart phone under a suitable excitation light source.

[0023] The detection method described in the present invention is only used to obtain information as an intermediate result, and is not used as a method to directly derive the diagnosis result of a disease or information on health status. This method is a non-disease diagnostic method.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The method of the present invention can detect multiple foodborne bacteria at the same time. Compared with the limitation of traditional culture methods that can usually only detect a single target, it significantly improves the detection efficiency and can be extended to the simultaneous detection of other pathogens, and has potential application value. It quantitatively measures the fluorescence signal through a fluorescence spectrometer, verifies the stability through microscope imaging, and realizes portable and naked-eye detection through smartphone photography, providing multi-level and diversified result expression methods. This multimodal strategy not only meets the needs of professional laboratory analysis, but is also suitable for rapid on-site detection. It uses smartphones combined with fluorescent probes to achieve preliminary detection, which provides the possibility for on-site detection and remote monitoring with limited resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1It is a schematic diagram of the working principle of the detection method of the present invention.

[0027] Figure 2 It is a feasibility analysis diagram of RCA amplification of three bacteria in Example 2 of the present invention, A to C correspond to the feasibility analysis diagrams of RCA amplification of Escherichia coli, Pseudomonas cocovenenans and Staphylococcus aureus, respectively, wherein L-ECT, L-BCT, and L-SAT are linear templates, C-ECT, C-BCT, and C-SAT are circular templates (linear templates for cyclization reaction), ECI-RCA is the RCA product amplified by the primers released by Escherichia coli in the amplification system of three different bacteria, BCI-RCA is the RCA product amplified by the primers released by Pseudomonas cocovenenans in the amplification system of three different bacteria, and SAI-RCA is the RCA product amplified by the primers released by Staphylococcus aureus in the amplification system of three different bacteria.

[0028] Figure 3 This is a graph of the optimization of the concentration of the circular template in Example 3 of the present invention, wherein A is a fluorescence spectrum graph of the optimization of three circular templates, and BD are the bar graphs of the optimization of the concentration of the circular templates of Staphylococcus aureus, Pseudomonas cocovenenans, and Escherichia coli, respectively.

[0029] Figure 4 It is the detection specificity diagram of three kinds of bacteria in Example 4 of the present invention, wherein A is a fluorescence spectrum diagram specific to Escherichia coli, B is a fluorescence spectrum diagram specific to Pseudomonas cocovenenans, C is a fluorescence spectrum diagram specific to Staphylococcus aureus, D is an imaging diagram specific to Escherichia coli, E is an imaging diagram specific to Pseudomonas cocovenenans, and F is an imaging diagram specific to Staphylococcus aureus.

[0030] Figure 5 This is an imaging diagram of the simultaneous detection of multiple bacteria in Example 5 of the present invention. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the specific implementation methods of the present invention are further described in detail below in conjunction with embodiments.

[0032] Table 1: Nucleic acid sequences used in the present invention

[0033]

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1 Construction of a multimodal presentation method for multiple bacterial detection

[0037] Different bacteria were placed in the corresponding culture medium and cultured until OD 600 =1 (Escherichia coli EC, Pseudomonas cocovenenans BC), OD 600 =2 (Staphylococcus aureus SA), centrifuge at 8000-13000rpm for 10min, the supernatant is CEM, resuspend the bacterial pellet below with 300μL reaction buffer, sonicate the resuspended bacterial solution in an ultrasonic machine for 30s, and then cool on ice for 2min, repeat the above operation process 6 times, and then centrifuge at 4℃, 8000rpm for 10min, the supernatant after centrifugation is the bacterial intracellular mixture (CIM). The obtained targets (CIM of Escherichia coli, CEM of Pseudomonas cocovenenans, and a mixture of CEM and CIM of Staphylococcus aureus) are incubated with bacterial response probes (SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6) composed of biotin-modified DNAzyme and RCA amplification primers connected to magnetic beads, respectively. Specifically, the bacterial response probes are composed of enzyme chains that respond to Escherichia coli, Pseudomonas cocovenenans, and Staphylococcus aureus, and substrates with RNA cleavage sites corresponding to each other, and one end of the bacterial response probe is modified with a biotin group and can be connected to the magnetic beads. The target is recognized by the bacterial response probe, and the RNA site is cut to release the RCA amplification primer. After adding phi29 DNA polymerase, T4 polynucleotide kinase (PNK), a circular template (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3) and dNTPs for RCA amplification, an RCA product is obtained. The RCA product can be respectively combined with molecular beacons (SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9) complementarily binds to open the stem-loop structure of the molecular beacon, thereby generating a stronger fluorescent signal. The three bacteria to be detected correspond to different molecular beacons, which generate different signals to detect the three bacteria. The generated signals are quantitatively measured by a fluorescence spectrometer, and taking photos with an inverted fluorescence microscope can realize quantitative and quantitative analysis, while assisting in verifying the authenticity of the fluorescence spectrometer results. Taking photos with a smartphone realizes portable naked-eye detection ( Figure 1 ).

[0038] Example 2 Characterization of RCA products by agarose gel electrophoresis

[0039] Take 5 μL of magnetic beads, wash them three times with magnetic bead buffer, add 3 μL (15 μM) of Escherichia coli, Pseudomonas cocovenenans and Staphylococcus aureus response probes (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6), make up to 25 μL with magnetic bead buffer, vortex oscillator at 1500 rpm for 35 min, place on a magnetic stand for 1 min after oscillation, remove the supernatant, and resuspend in 60 μL magnetic bead buffer to wash unbound enzyme chains and magnetic beads that have lost their magnetism. Repeat the washing process three times. Next, the magnetic beads were connected to the Escherichia coli, Pseudomonas cocovenenans and Staphylococcus aureus response probes composed of biotin-modified DNAzyme and RCA amplification primers for cleavage reaction. In 25 μL of 1× reaction buffer containing 200 nM biotin-modified DNAzyme and bacterial response probes composed of RCA amplification primers (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6), 25 μL of three bacterial targets (CIM of Escherichia coli, CEM of Pseudomonas cocovenenans, and a mixture of CEM and CIM of Staphylococcus aureus) were added respectively, and incubated at room temperature, wherein Escherichia coli was incubated for 60 min, Pseudomonas cocovenenans was incubated for 120 min, and Staphylococcus aureus was incubated for 30 min, and supernatants with amplification primers were obtained respectively. 10 μL 10×RB, 50 μL reaction supernatant, 5U phi29 DNA polymerase, 5U T4 polynucleotide kinase (PNK), 10pmol of prepared circular template (SEQID NO.1, SEQ ID NO.2, SEQ ID NO.3) and 10μL dNTPs (10mM) were mixed together and incubated at 30℃ for 2h. During the incubation, 0.36g agarose was weighed, added to 60ml 1×TAE buffer and mixed, heated in a microwave oven for 2min until the powder dissolved, added 1.2μL SYBR Safe nucleic acid dye and shaken, quickly poured into the gel plate, and waited for 2h to obtain a solidified 0.6% agarose gel. Place the agarose gel in a horizontal electrophoresis tank and add 1×TAE buffer until it covers the gel surface. 5μL sample was fully mixed with 1μL 6×loading buffer and then loaded on the agarose gel. Set the voltage to 80V and the time to 50min. After the run, the multispectral imager was used to image and analyze the gel samples of the two gels. The multispectral imager was used to image and analyze the gel samples of 0.6% agarose gel. The second and third channels of the three agarose gel imaging confirmed the synthesis of the circular template. The last three channels showed that only the bacteria corresponding to the bacterial response probe could be cut to trigger the RCA reaction, confirming that the three bacteria to be tested can all undergo RCA amplification to produce products ( Figure 2 ).

[0040] Example 3 Optimization of circular template concentration

[0041] 10 μL 10×RB, 50 μL reaction supernatant (the supernatant with amplification primers in Example 2), 5U phi29 DNA polymerase, 5U T4 polynucleotide kinase (PNK), 10 μL dNTPs (10 mM) and 2 μL circular templates of different concentrations (the concentrations of the Staphylococcus aureus circular templates are 2.5, 5, 10, 15, and 20 μM, respectively; the concentrations of the Pseudomonas cerebrovenans circular templates are 2.5, 5, 7.5, 10, and 12.5 μM, respectively; the concentrations of the Escherichia coli circular templates are 2.5, 5, 7.5, 10, and 15 μM, respectively) are mixed together to form a reaction system. After incubation at 30° C. for 1-2 h and inactivation of phi29 DNA polymerase at 65° C. for 10 min, the fluorescence values ​​corresponding to different circular template concentrations can be quantitatively measured using a fluorescence spectrometer. The optimal concentration of the circular template for detecting Escherichia coli was 10 μM, the optimal concentration of the circular template for detecting Pseudomonas cocovenenans was 7.5 μM, and the optimal concentration of the circular template for detecting Staphylococcus aureus was 15 μM. Figure 3 ).

[0042] Example 4 Characterization of the specificity of simultaneous detection of multiple bacteria

[0043] In order to verify that this method is selective for the respective bacterial species when detecting Escherichia coli, Staphylococcus aureus and Pseudomonas cocovenenans, we selected Pseudomonas aeruginosa, Actinobacillus actinomycetemcomitans and Bacillus cereus for verification experiments. In 25 μL of 1× reaction buffer containing 200 nM biotin-modified DNAzyme and bacterial response probes (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6) composed of RCA amplification primers, 25 μL of targets prepared from the above-mentioned various bacteria (reference example 1 for target preparation) were added and incubated at room temperature, wherein Escherichia coli was incubated for 60 min, Pseudomonas cocovenenans was incubated for 120 min, and Staphylococcus aureus was incubated for 30 min. The supernatant with amplification primers was obtained and added to the RCA amplification system for reaction (the specific reaction parameters are the optimal conditions screened in Example 3). After the reaction, the fluorescence intensity of each tube was measured, and an inverted fluorescence microscope and a smartphone were used to take photos. The results are as follows: Figure 4 As shown, the fluorescence values ​​generated when using this method to detect Escherichia coli, Staphylococcus aureus and Pseudomonas cocovenenans respectively are much higher than the fluorescence values ​​generated when detecting other bacterial species, and the generated fluorescence color is also much deeper than that of other detected bacterial species. The method of the present invention has good detection specificity when detecting Escherichia coli, Staphylococcus aureus and Pseudomonas cocovenenans respectively.

[0044] Example 5 Simultaneous detection of multiple bacteria in the same system

[0045] In order to prove that this method can complete the simultaneous detection of Escherichia coli, Pseudomonas cocovenenans and Staphylococcus aureus in the same system, we selected three cases of using one bacterium, two bacteria and three bacteria alone for verification. In 25 μL of 1× reaction buffer containing 800nM biotin-modified DNAzyme and bacterial response probes (SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6) composed of RCA amplification primers, 25 μL of targets prepared from the above-mentioned various bacteria (reference example 1 for target preparation) were added, and incubated at room temperature, wherein Escherichia coli was incubated for 60 minutes, Pseudomonas cocovenenans was incubated for 120 minutes, and Staphylococcus aureus was incubated for 30 minutes to obtain a supernatant with amplification primers. The supernatant of each bacterium was evenly divided into four parts, wherein a portion of the supernatant obtained by incubation with Escherichia coli was added to the first tube, a portion of the supernatant obtained by incubation with Pseudomonas cocovenenans was added to the second tube, and a portion of the supernatant obtained by incubation with Staphylococcus aureus was added to the third tube. The supernatant obtained by incubating cocci was added to the fourth tube, the supernatant obtained by incubating Escherichia coli and Pseudomonas cerebrovenantis was added to the fifth tube, the supernatant obtained by incubating Escherichia coli and Staphylococcus aureus was added to the sixth tube, the supernatant obtained by incubating Pseudomonas cerebrovenantis and Staphylococcus aureus was added to the seventh tube, and the supernatant obtained by incubating Escherichia coli, Pseudomonas cerebrovenantis and Staphylococcus aureus was added to the seven tubes. The RCA amplification system was added to these seven tubes for reaction (the specific reaction parameters were the optimal conditions screened in Example 3), and photos were taken with a smartphone after the reaction was completed. The results are shown in Figure 5 As shown, fluorescent color is produced regardless of detecting one bacterium or multiple bacteria, indicating that this method can be used to detect multiple bacteria simultaneously.

Claims

1. A detection method based on multimodal presentation of multiple bacteria simultaneous detection, characterized in that: The steps include: (1) Target preparation: After the bacteria are cultured in a culture medium, centrifuge at 5000-13000 rpm for 5-30 min. The supernatant is CEM. The precipitate is resuspended in a buffer solution and ultrasonically lysed. Centrifuge at 5000-13000 rpm for 5-30 min. The supernatant is CIM. (2) Preparation of functionalized magnetic beads with bacteria-responsive probes: Take the magnetic beads washed with magnetic bead buffer, add the bacteria-responsive probe modified with biotin, shake and mix well, and incubate for 40 to 80 minutes; (3) cleavage reaction: adding the functionalized magnetic beads of the bacteria-responsive probe prepared in step (2) to the bacterial target prepared in step (1) for incubation, wherein the bacteria-responsive probe recognizes the target, cleaves the RNA site and releases the RCA amplification primer; (4) RCA amplification reaction: adding a circular template, phi 29 DNA polymerase, dNTPs, and T4 polynucleotide kinase to the solution obtained in step (3) to carry out RCA amplification reaction to obtain an RCA product; (5) Signal detection: Add molecular beacons to the solution obtained in step (4) and detect using a fluorescence spectrometer, an inverted fluorescence microscope or a smartphone for photography.

2. The detection method according to claim 1, characterized in that: The bacteria described in step (1) are one or more of Escherichia coli, Pseudomonas cocovenenans and Staphylococcus aureus; and the culture conditions are shaking culture at 30-37° C. for 8-20 hours.

3. The detection method according to claim 1, characterized in that: The biotin-modified bacteria-responsive probe described in step (2) is shown in SEQ ID NO. 4-6.

4. The detection method according to claim 1, characterized in that: The cutting reaction time in step (3) is 30 to 120 minutes.

5. The detection method according to claim 1, characterized in that: The nucleotide sequence of the circular template described in step (4) is shown in SEQ ID NO.1-3.

6. The detection method according to claim 1, characterized in that: In step (4), the dosage of phi29 DNA polymerase and T4 polynucleotide kinase in the RCA amplification reaction system is 2 to 10 U; the dosage of circular template is 5 to 20 μM, the RCA amplification time is 1 to 3 hours, and the amplification temperature is 25 to 35°C.

7. The detection method according to claim 1, characterized in that: The molecular beacons described in step (5) include three molecular beacons, as shown in SEQ ID NOs. 7-9.

8. The detection method according to claim 1, characterized in that: The amount of molecular beacon added during signal detection in step (5) is 10-70 pmol.

9. The detection method according to claim 1, characterized in that: In step (5), a fluorescence spectrometer is used to collect fluorescence signals at 440 nm, 518 nm, and 667 nm respectively under the conditions of excitation wavelength of 350 nm, emission of 360-500 nm, excitation of 494 nm, emission of 500-600 nm, excitation of 643 nm, and emission of 650-750 nm, wherein the slit range is 10 nm; Use an inverted fluorescence microscope, turn on the light source controller and adjust it to 50% intensity, adjust the filters to correspond to blue light, green light and red light respectively, adjust the exposure time, black, gamma value, white, and finally take pictures and save; 30 μL of the samples detected by inverted fluorescence microscope were transferred into eight rows of tubes respectively, and photos were taken with a smartphone under a suitable excitation light source.

Images