Fluorescence labeling quantitative tracking method for dynamically monitoring microbial community

Abstract

The invention provides a fluorescence labeling quantitative tracking method for dynamically monitoring microbial communities, which is characterized in that a fluorescence labeling probe is designed and synthesized according to a specific nucleic acid sequence of a target microorganism, and the specific recognition sequence can be specifically hybridized with the nucleic acid sequence of the target microorganism, so that the target microorganism can be detected. The specific fluorescence labeling probe is designed, the length and the structure of the probe are optimized, and modified basic groups and chemical groups are introduced, so that the specific binding and the stability of the probe and target microorganism nucleic acid are ensured. A sample is pretreated to extract high-quality nucleic acid, and a hybridization reaction is performed under strictly controlled conditions. Signal detection is carried out by using multiple fluorescence detection devices, and quantitative analysis is realized through a standard curve. In addition, through continuous monitoring and data analysis, a mathematical model is constructed to predict the change trend of the microbial population, and support is provided for microbial community research. Meanwhile, a washing step is set to reduce background interference, and a signal enhancer is adopted to improve the detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial monitoring, and in particular, to a fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities. Background Art

[0002] Microbial communities are widely present in natural environments, the human body, and various industrial and agricultural systems, and they play crucial roles in ecosystem functions, human health, and the occurrence and development of diseases. Therefore, dynamic monitoring of microbial communities has important theoretical and practical significance. However, there are many deficiencies in the existing microbial community monitoring technologies, which limit the in-depth research and effective management of microbial communities.

[0003] Some existing microbial detection methods lack sufficient specificity and are difficult to accurately distinguish target microorganisms from non-target microorganisms. Traditional cultivation methods can only cultivate some culturable microorganisms and cannot detect the large number of unculturable microorganisms in the environment, resulting in limitations in the understanding of microbial communities. Some nucleic acid-based detection technologies, such as ordinary PCR technology, although capable of detecting specific microorganisms, the specificity of primers may be affected by various factors and non-specific amplification is likely to occur, thus affecting the accuracy of detection results. In addition, these methods often can only detect a few target microorganisms and cannot comprehensively and accurately monitor complex microbial communities.

[0004] In the process of processing microbial community samples, existing technologies have problems of complex operation and low efficiency. The cell wall breaking methods are single and cannot select appropriate wall breaking methods according to the characteristics of different microorganisms, resulting in incomplete nucleic acid release and affecting subsequent detection effects. In the nucleic acid extraction and purification process, the extraction efficiency of some methods is not high and impurities are easily introduced, affecting the quality of nucleic acids. The hybridization reaction conditions are not precise enough, lacking operations optimized according to the melting temperature of probes, resulting in low hybridization efficiency and unstable and inefficient binding of fluorescence-labeled probes to target microbial nucleic acid sequences.

[0005] Current microbial detection technologies have certain limitations in detection means and quantitative analysis. Some detection devices have single functions and cannot meet different experimental requirements. For example, some traditional microscopes can only perform qualitative observations and are difficult to achieve accurate counting and quantitative analysis of microorganisms. Some quantitative detection methods, such as the method based on plate counting, are cumbersome, time-consuming, and can only detect live microorganisms, and cannot detect microorganisms in a dormant state or dead cells. In addition, existing quantitative analysis methods often lack an accurate standard curve establishment process, resulting in low reliability of quantitative results.

[0006] Most existing microbial detection techniques can only detect the microbial community at specific time points and cannot achieve continuous monitoring of the dynamic changes of the microbial community. This makes it difficult for researchers to understand the succession rules of the microbial community and the changes in ecological functions. At the same time, in terms of data analysis, existing techniques often only perform simple statistical analysis, lacking in-depth research on the relationship between the number of microorganisms and environmental factors, and unable to construct effective mathematical models to predict the future change trends of the microbial community, making it difficult to meet the requirements of dynamic management of the microbial community in practical applications.

[0007] During the fluorescence detection process, the interference of background signals is a common problem. Existing methods lack effective washing steps to remove unbound probes, resulting in strong background signals and affecting the sensitivity and accuracy of detection. In addition, when the number of target microorganisms is small, the detected fluorescence signal intensity is weak, and existing techniques often lack effective signal enhancement means, making it difficult to accurately detect the target microorganisms. Therefore, we propose a fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities. Summary of the Invention

[0008] The object of the present invention is to address the problems raised in the existing background technology. To achieve the above object of the invention, the present invention provides the following technical solutions: A fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities, comprising the following steps: Step 1, Design and synthesis of fluorescence-labeled probes: According to the specific nucleic acid sequence of the target microorganism, design and synthesize fluorescence-labeled probes, the fluorescence-labeled probes include specific recognition sequences and fluorescence-labeled groups, the specific recognition sequence can specifically hybridize with the nucleic acid sequence of the target microorganism, and the fluorescence-labeled group is connected to one end or both ends of the specific recognition sequence;

[0009] Step 2, Pretreatment of microbial community samples: Collect microbial community samples and perform pretreatment on the samples. The pretreatment steps include cell wall breaking, nucleic acid extraction and purification;

[0010] Step 3, Fluorescence-labeling reaction: Mix the pretreated microbial community nucleic acid sample with the fluorescence-labeled probe and perform a hybridization reaction under suitable hybridization conditions to specifically bind the fluorescence-labeled probe to the nucleic acid sequence of the target microorganism;

[0011] Step 4, Fluorescence signal detection and quantitative analysis: Use a fluorescence detection device to detect the fluorescence signal of the hybridized sample, obtain the fluorescence signal intensity of the target microorganism, and convert the fluorescence signal intensity into the number of target microorganisms according to a pre-established standard curve for quantitative analysis of the target microorganism;

[0012] Step 5. Dynamic monitoring of microbial community: Collect microbial community samples at different time points, repeat the above steps of pretreatment, fluorescence labeling reaction, fluorescence signal detection and quantitative analysis, continuously monitor the change in the number of target microorganisms, and track the dynamic changes of the microbial community.

[0013] As a preferred technical solution of the present invention, the fluorescent labeling groups include FAM, TAMRA, CY3, and CY5.

[0014] As a preferred technical solution of the present invention, cell wall breaking is performed by physical, chemical or enzymatic methods. The physical methods include ultrasonic disruption and freeze-thaw method, and the chemical method includes chemical lysis method.

[0015] As a preferred technical solution of the present invention, nucleic acid extraction is performed using a nucleic acid extraction kit, and nucleic acid purification is performed by ethanol precipitation method.

[0016] As a preferred technical solution of the present invention, the temperature of the hybridization reaction is adjusted according to the melting temperature (Tm value) of the probe, and the reaction is carried out at 5-10 °C below the Tm value, and the reaction time is 1-2 hours.

[0017] As a preferred technical solution of the present invention, the fluorescence detection device is selected from one of a fluorescence microscope, a flow cytometer, and a fluorescence quantitative PCR instrument.

[0018] As a preferred technical solution of the present invention, the standard curve is established by fluorescently labeling known numbers of target microorganisms, detecting the fluorescence signal intensity thereof, and plotting the relationship curve between the fluorescence signal intensity and the number of target microorganisms.

[0019] As a preferred technical solution of the present invention, after the fluorescence labeling reaction, it further includes a washing and signal enhancement step:

[0020] Washing: The hybridized sample is washed multiple times with a washing buffer that is compatible with the hybridization reaction system and can effectively remove unbound probes. After each washing, low-speed centrifugation is performed to precipitate the sample, and the supernatant is removed; the washing buffer contains a certain concentration of salt ions and surfactants, the salt ion concentration is 0.1-0.5 M, and the volume fraction of the surfactant is 0.01%-0.1%;

[0021] Signal enhancement: If the detected fluorescence signal intensity is weak, a signal enhancer can be added to amplify the fluorescence label bound to the nucleic acid of the target microorganism; the signal enhancer is a nanoparticle having a fluorescence resonance energy transfer (FRET) effect, which can interact with the fluorescent labeling group and enhance the fluorescence emission intensity.

[0022] As a preferred technical solution of the present invention, optimization of the fluorescent labeling probe:

[0023] The length of the specific recognition sequence is 15 - 30 nucleotides to ensure the specificity and stability of hybridization with the nucleic acid sequence of the target microorganism;

[0024] Perform secondary structure prediction on the specific recognition sequence to avoid the formation of stable hairpin structures or self-complementary pairing, ensuring that it can freely bind to the target nucleic acid;

[0025] Optimize the specific recognition sequence by introducing modified bases or chemical groups to enhance its affinity and hybridization stability with the target nucleic acid; the modified bases include locked nucleic acid (LNA), peptide nucleic acid (PNA), and the chemical groups include methyl and amino.

[0026] As a preferred technical solution of the present invention, during the dynamic monitoring of the microbial community, data analysis and model construction steps are also combined:

[0027] Data analysis: Statistically analyze the data of the number of target microorganisms detected at different time points, including calculating statistical parameters such as mean, standard deviation, and variance, and analyzing the distribution characteristics and change trends of the data; at the same time, use correlation analysis, principal component analysis and other multivariate statistical methods to study the relationship between the number of target microorganisms and environmental factors;

[0028] Model construction: Based on the data analysis results, construct a mathematical model of the dynamic change of the microbial community to predict the change trend of the number of target microorganisms in the future; the mathematical model includes the logistic growth model and the Gaussian competition model, and by estimating and optimizing the model parameters, the prediction accuracy of the model is improved.

[0029] Compared with the prior art, the beneficial effects of the present invention:

[0030] The present invention designs a fluorescently labeled probe according to the specific nucleic acid sequence of the target microorganism. The specific recognition sequence can specifically hybridize with the nucleic acid sequence of the target microorganism, ensuring that only the target microorganism is labeled and detected, effectively avoiding the interference of non-target microorganisms, and greatly improving the specificity of detection.

[0031] The present invention optimizes the fluorescently labeled probe. For example, controlling the length of the specific recognition sequence to be 15 - 30 nucleotides ensures the specificity and stability of hybridization with the nucleic acid sequence of the target microorganism; through secondary structure prediction, the formation of stable hairpin structures or self-complementary pairing is avoided, ensuring that the probe can freely bind to the target nucleic acid; introducing modified bases (such as locked nucleic acid, peptide nucleic acid) or chemical groups (such as methyl, amino) enhances its affinity and hybridization stability with the target nucleic acid, further improving the detection accuracy.

[0032] The present invention performs cell wall breaking, nucleic acid extraction and purification pretreatment steps on the collected microbial community samples, providing high-quality nucleic acid samples for subsequent fluorescence labeling reactions. Multiple methods (physical, chemical or enzymatic methods) are used for cell wall breaking, and appropriate wall-breaking methods can be selected according to different microbial characteristics to ensure the effective release of nucleic acids; nucleic acid extraction uses a kit and ethanol precipitation method for purification, ensuring the purity and integrity of nucleic acids.

[0033] The temperature of the hybridization reaction of the present invention is adjusted according to the melting temperature (Tm value) of the probe, and the reaction is carried out at 5-10 °C below the Tm value for 1-2 hours. This precise condition control enables the fluorescently labeled probe to efficiently and specifically bind to the nucleic acid sequence of the target microorganism, improving the labeling efficiency and accuracy.

[0034] The fluorescent detection device of the present invention is selected from one of a fluorescence microscope, a flow cytometer, and a fluorescence quantitative PCR instrument. Users can select a suitable detection device according to actual needs and experimental conditions, improving the applicability and flexibility of the method.

[0035] The present invention converts the fluorescence signal intensity into the quantity of the target microorganism through a pre-established standard curve, realizing the quantitative analysis of the target microorganism. The standard curve is established by fluorescently labeling known quantities of the target microorganism and detecting its fluorescence signal intensity, ensuring the accuracy and reliability of the quantitative analysis.

[0036] The present invention collects microbial community samples at different time points and repeats the pretreatment, fluorescence labeling reaction, fluorescence signal detection and quantitative analysis steps, enabling continuous monitoring of the quantity change of the target microorganism and real-time tracking of the dynamic changes of the microbial community, providing important data support for studying the succession law and ecological function of the microbial community.

[0037] The present invention combines data analysis and model construction steps, performs statistical analysis on the quantity data of the target microorganism detected at different time points, including calculating statistical parameters such as mean, standard deviation, and variance, analyzing the distribution characteristics and change trends of the data; using correlation analysis, principal component analysis and other multivariate statistical methods to analyze the relationship between the quantity of the target microorganism and environmental factors. Based on the data analysis results, a mathematical model of the dynamic changes of the microbial community, such as the logistic growth model and the Gaussian competition model, is constructed, which can predict the quantity change trend of the target microorganism in the future for a period of time, providing a scientific basis for the regulation and management of the microbial community.

[0038] After the fluorescence labeling reaction, the present invention sets a washing step. A washing buffer that is compatible with the hybridization reaction system and can effectively remove unbound probes is used to wash the hybridized sample multiple times. After each washing, low-speed centrifugation is performed to precipitate the sample, and the supernatant is removed, effectively removing the unbound probes, reducing the interference of background signals, and improving the signal-to-noise ratio of the detection. If the detected fluorescence signal intensity is weak, nanoparticles with fluorescence resonance energy transfer (FRET) effect can be added as signal enhancers to amplify the fluorescence labeling bound to the nucleic acid of the target microorganism, enhancing the detection sensitivity, so that accurate detection can be achieved even when the number of target microorganisms is small. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of experimental data provided by the present invention;

[0040] Figure 2 It is a schematic diagram of the reagents in Example 2 provided by the present invention;

[0041] Figure 3 It is a schematic diagram of the reagents in Example 3 provided by the present invention;

[0042] Figure 4 It is a schematic diagram of the reagents in Example 4 provided by the present invention;

[0043] Figure 5 It is a schematic diagram of the method flow provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention.

[0045] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed present invention, but merely represents some embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features and technical solutions in the embodiments can be combined with each other. It should be noted that similar reference numerals and letters denote similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0046] Example 1: A fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities, comprising the following steps: Step 1, Design and synthesis of fluorescence-labeled probes: According to the specific nucleic acid sequence of the target microorganism, fluorescence-labeled probes are designed and synthesized. The fluorescence-labeled probes include a specific recognition sequence and a fluorescence-labeling group. The specific recognition sequence can specifically hybridize with the nucleic acid sequence of the target microorganism, and the fluorescence-labeling group is connected to one or both ends of the specific recognition sequence;

[0047] Step 2, Pretreatment of microbial community samples: Microbial community samples are collected and pretreated. The pretreatment steps include cell wall breaking, nucleic acid extraction and purification;

[0048] Step 3, Fluorescence-labeling reaction: The pretreated microbial community nucleic acid sample is mixed with the fluorescence-labeled probe, and a hybridization reaction is carried out under suitable hybridization conditions to enable the fluorescence-labeled probe to specifically bind to the nucleic acid sequence of the target microorganism;

[0049] Step 4, Fluorescence signal detection and quantitative analysis: A fluorescence detection device is used to detect the fluorescence signal of the hybridized sample, obtain the fluorescence signal intensity of the target microorganism, and convert the fluorescence signal intensity into the number of target microorganisms according to a pre-established standard curve for quantitative analysis of the target microorganism;

[0050] Step 5, Dynamic monitoring of microbial communities: Microbial community samples are collected at different time points, and the above pretreatment, fluorescence-labeling reaction, fluorescence signal detection and quantitative analysis steps are repeated to continuously monitor the change in the number of target microorganisms and track the dynamic changes of the microbial community.

[0051] The fluorescence-labeling group includes FAM, TAMRA, CY3, CY5.

[0052] Cell wall breaking is carried out by physical, chemical or enzymatic methods. Physical methods include ultrasonic disruption and freeze-thaw methods, and chemical methods include chemical lysis methods.

[0053] Nucleic acid extraction uses a nucleic acid extraction kit, and nucleic acid purification uses ethanol precipitation.

[0054] The temperature of the hybridization reaction is adjusted according to the melting temperature (Tm value) of the probe, and the reaction is carried out at 5-10 °C below the Tm value, and the reaction time is 1-2 hours.

[0055] The fluorescence detection device is selected from one of a fluorescence microscope, a flow cytometer, and a fluorescence quantitative PCR instrument.

[0056] The standard curve is established by fluorescence-labeling known numbers of target microorganisms and detecting their fluorescence signal intensities, and plotting the relationship curve between the fluorescence signal intensity and the number of target microorganisms.

[0057] After the fluorescence labeling reaction, it also includes washing and signal enhancement steps:

[0058] Washing: The hybridized sample is washed multiple times with a washing buffer that is compatible with the hybridization reaction system and can effectively remove unbound probes. After each wash, low-speed centrifugation is performed to precipitate the sample, and the supernatant is removed; the washing buffer contains a certain concentration of salt ions and surfactants, the salt ion concentration is 0.1 - 0.5 M, and the volume fraction of the surfactant is 0.01% - 0.1%;

[0059] Signal enhancement: If the detected fluorescence signal intensity is weak, a signal enhancer can be added to amplify the fluorescence labeling bound to the nucleic acid of the target microorganism; the signal enhancer is a nanoparticle with a fluorescence resonance energy transfer (FRET) effect, which can interact with the fluorescent labeling group and enhance the fluorescence emission intensity.

[0060] Optimization of the fluorescently labeled probe:

[0061] The length of the specific recognition sequence is 15 - 30 nucleotides to ensure the specificity and stability of hybridization with the nucleic acid sequence of the target microorganism;

[0062] Perform secondary structure prediction on the specific recognition sequence to avoid the formation of stable hairpin structures or self-complementary pairing, and ensure that it can freely bind to the target nucleic acid;

[0063] Optimize the specific recognition sequence by introducing modified bases or chemical groups to enhance its affinity and hybridization stability with the target nucleic acid; the modified bases include locked nucleic acid (LNA), peptide nucleic acid (PNA), and the chemical groups include methyl and amino groups.

[0064] During the dynamic monitoring of the microbial community, it also combines data analysis and model construction steps:

[0065] Data analysis: Statistically analyze the data of the number of target microorganisms detected at different time points, including calculating statistical parameters such as mean, standard deviation, and variance, and analyzing the distribution characteristics and change trends of the data; at the same time, use correlation analysis, principal component analysis and other multivariate statistical methods to study the relationship between the number of target microorganisms and environmental factors;

[0066] Model construction: Based on the data analysis results, construct a mathematical model of the dynamic changes of the microbial community to predict the change trend of the number of target microorganisms in the future; the mathematical models include the logistic growth model and the Gaussian competition model. By estimating and optimizing the model parameters, the prediction accuracy of the model is improved.

[0067] Example 2: A fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities,

[0068] Material Preparation

[0069] Samples: Soil samples were collected from local farmland at a depth of 0 - 20 cm.

[0070] Reagents: Fluorescently labeled probe (specific sequence 5'-CGTTCGCTAGAGTGTGCCAG-3' for the 16S rRNA gene of Pseudomonas, with FAM fluorophore labeled at the 5' end), FastDNA Spin Kit for Soil (MP Biomedicals) nucleic acid extraction kit, hybridization buffer (containing 20% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.5), 0.01% SDS), washing buffer (containing 0.1 M NaCl, 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.01% SDS), standard strain of Pseudomonas with known concentration.

[0071] Instruments: Refrigerated centrifuge, PCR instrument, fluorescence microscope, pipette.

[0072] Operating Procedures

[0073] 1. Nucleic Acid Extraction

[0074] Weigh 0.5 g of soil sample and operate according to the instructions of the FastDNA Spin Kit for Soil to extract total DNA of soil microorganisms. Dissolve the extracted DNA in TE buffer and store it at -20°C for later use.

[0075] 2. Hybridization with Fluorescently Labeled Probe

[0076] Take 5 μL of the extracted DNA sample, add 1 μL of fluorescently labeled probe (10 μM), 10 μL of hybridization buffer, and make up to 20 μL with sterile water.

[0077] Denature the reaction system at 95°C for 5 min, then quickly cool it on ice for 2 min. Then hybridize at 46°C for 2 h.

[0078] After hybridization, wash the sample with washing buffer at 48°C for 15 min to remove unbound probes.

[0079] 3. Fluorescent Signal Detection and Quantification

[0080] Drop the washed sample on a glass slide, cover it with a coverslip, and observe it under a fluorescence microscope. Set the excitation wavelength to 488 nm and the emission wavelength to 520 nm.

[0081] Meanwhile, standard strains of Pseudomonas with known concentrations were processed according to the above steps to prepare a standard curve. Under a fluorescence microscope, 10 fields of view were randomly selected, and the number of cells emitting green fluorescence in each field of view was counted. According to the standard curve, the number of Pseudomonas in the soil sample was calculated.

[0082] 4. Dynamic monitoring

[0083] Within one month, soil samples were collected once every week, and the above steps 1-3 were repeated. The changes in the number of Pseudomonas at different time points were recorded, a curve of the number changing with time was plotted, and its dynamic change law was analyzed.

[0084] Example 3: A fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities, Material preparation

[0085] Samples: Fecal samples from healthy volunteers.

[0086] Reagents: Fluorescence-labeled probe (targeting the specific gene fragment 5'-GGTGTTCTTCCCGATATCTAC-3' of Bifidobacterium, with a CY3 fluorescent group labeled at the 3' end), QIAamp DNA Stool Mini Kit (Qiagen) nucleic acid extraction kit, hybridization buffer (containing 30% formamide, 0.9M NaCl, 20mM Tris-HCl (pH 7.6), 0.01% SDS), washing buffer (containing 0.05M NaCl, 20mM Tris-HCl (pH 7.6), 5mM EDTA, 0.01% SDS), standard strains of Bifidobacterium with known concentrations.

[0087] Instruments: Centrifuge, fluorescence quantitative PCR instrument, pipette.

[0088] Operation steps

[0089] 1. Nucleic acid extraction

[0090] Take 0.2 g of fecal sample and operate according to the instructions of the QIAamp DNA Stool Mini Kit to extract the total DNA of fecal microorganisms. The extracted DNA was dissolved with elution buffer and stored at -20°C for later use.

[0091] 2. Hybridization of fluorescence-labeled probe

[0092] Take 8 μL of the extracted DNA sample, add 2 μL of the fluorescence-labeled probe (10 μM), 10 μL of hybridization buffer, and make up to 20 μL with sterile water.

[0093] Denature the reaction system at 96°C for 6 min, then quickly place it on ice for 3 min. Then hybridize at 50°C for 1.5 h.

[0094] After hybridization, wash with the washing buffer at 52 °C for 20 min to remove unbound probes.

[0095] 3. Fluorescence signal detection and quantification

[0096] Transfer the washed sample to a fluorescence quantitative PCR tube and detect it using a fluorescence quantitative PCR instrument. Set the excitation wavelength to 550 nm and the emission wavelength to 570 nm.

[0097] Prepare a standard curve using a standard strain of Bifidobacterium with a known concentration. Calculate the number of Bifidobacterium in the fecal sample based on the fluorescence signal intensity detected by the fluorescence quantitative PCR instrument through the standard curve.

[0098] 4. Dynamic monitoring

[0099] The volunteers provided fecal samples every day for two consecutive weeks, repeated the above steps 1-3, recorded the daily changes in the number of Bifidobacterium, and analyzed its dynamic fluctuations in the gut microbial community.

[0100] Example 4: A fluorescence-labeled quantitative tracking method for dynamic monitoring of microbial communities

[0101] Material preparation

[0102] Samples: Water samples collected from a local lake.

[0103] Reagents: Fluorescently labeled probe (specific sequence 5'-TCTGTCCCTTGCGGTTAGA-3' for the 16S rRNA gene of Vibrio, with a TAMRA fluorophore labeled at the 5' end), PowerWater DNA Isolation Kit (MoBio) nucleic acid extraction kit, hybridization buffer (containing 25% formamide, 0.9 M NaCl, 20 mM Tris-HCl (pH 7.4), 0.01% SDS), washing buffer (containing 0.08 M NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.01% SDS), standard strain of Vibrio with a known concentration.

[0104] Instruments: Filtration device, centrifuge, flow cytometer, pipette.

[0105] Operation steps

[0106] 1. Nucleic acid extraction

[0107] Take 500 mL of water sample, filter it through a 0.22 μm filter membrane, and put the filter membrane into a centrifuge tube. Operate according to the instructions of the PowerWater DNA Isolation Kit to extract the total DNA of water microorganisms. Dissolve the extracted DNA with the eluent and store it at -20 °C for later use.

[0108] 2. Fluorescently labeled probe hybridization

[0109] Take 10 μL of the extracted DNA sample, add 3 μL of the fluorescently labeled probe (10 μM), 10 μL of hybridization buffer, and make up to 20 μL with sterile water.

[0110] Denature the reaction system at 94 °C for 4 min, then quickly place it on ice for 2 min. Then hybridize at 48 °C for 2 h.

[0111] After hybridization, wash with the washing buffer at 50 °C for 18 min to remove the unbound probes.

[0112] 3. Fluorescent signal detection and quantification

[0113] Detect the washed sample with a flow cytometer. Set the excitation wavelength to 546 nm and the emission wavelength to 578 nm.

[0114] Make a standard curve with a standard strain of Vibrio with a known concentration. According to the fluorescent signals detected by the flow cytometer, calculate the number of Vibrio in the water sample through the standard curve.

[0115] 4. Dynamic monitoring

[0116] Within one month, collect water samples once a week, repeat the above steps 1 - 3, record the weekly changes in the number of Vibrio, and analyze the dynamic change trend in the water microbial community.

[0117] Experimental example

[0118] Experimental purpose

[0119] This experiment aims to verify the feasibility and effectiveness of "a fluorescently labeled quantitative tracking method for dynamic monitoring of microbial communities", and demonstrate the accurate tracking ability of this method for the changes in the number of microbial communities in practical applications through the dynamic monitoring of specific bacteria in the soil microbial community.

[0120] Experimental materials and equipment

[0121] Materials

[0122] 1. Soil samples: Taken from the surface 0 - 15 cm soil of a certain farmland.

[0123] 2. Fluorescently labeled probe: Designed based on the 16S rRNA gene sequence of the target bacteria (taking Bacillus as an example), with the sequence 5'-TAGCTAGCTAGCTAGCTA-FAM-3' (FAM is the fluorescent labeling group).

[0124] 3. Nucleic acid extraction kit: [Specific brand and model], used to extract microbial nucleic acids from soil samples.

[0125] 4. Hybridization buffer: Containing 20% formamide, 0.9M NaCl, 20mM Tris-HCl (pH 7.5), 0.01% SDS.

[0126] 5. Washing buffer: Containing 0.1M NaCl, 20mM Tris-HCl (pH 7.5), 5mM EDTA, 0.01% SDS.

[0127] 6. Standard: Known quantities of Bacillus strains.

[0128] Equipment

[0129] 1. High-speed centrifuge: Used for centrifugal separation of samples.

[0130] 2. Ultrasonic crusher: Assisting in cell wall breaking.

[0131] 3. Fluorescent quantitative PCR instrument: Detecting fluorescent signals.

[0132] 4. Pipette: Accurately pipetting reagents and samples.

[0133] 5. PCR tube: Used for nucleic acid reactions.

[0134] Experimental procedures

[0135] Step 1: Design and synthesis of the fluorescently labeled probe

[0136] With the help of bioinformatics software, analyze the 16S rRNA gene sequence of Bacillus, screen out the specific region, and design a specific recognition sequence with a length of 18 nucleotides. Entrust a professional biological company to connect the FAM fluorescent group to the 3' end of the specific recognition sequence to synthesize the fluorescently labeled probe.

[0137] Step 2: Pretreatment of the microbial community sample

[0138] 1. Soil sample collection: Use a sterile shovel to collect soil at multiple points in different positions of the farmland. After mixing evenly, take about 500g as the experimental sample, put it into a sterile sealed bag, and quickly bring it back to the laboratory.

[0139] 2. Cell wall breaking: Weigh 1 g of soil sample and put it into a centrifuge tube, then add 5 mL of lysis buffer. Place the centrifuge tube in an ultrasonic crusher, set the ultrasonic power to 300 W, sonicate for 5 seconds, pause for 10 seconds, and repeat the cycle 30 times to fully break the microbial cells in the soil.

[0140] 3. Nucleic acid extraction: Operate according to the instructions of the nucleic acid extraction kit. First, centrifuge the broken sample to collect the supernatant, then add various reagents in sequence for nucleic acid extraction, and finally dissolve the nucleic acid with elution buffer to obtain the nucleic acid sample of the microbial community.

[0141] 4. Nucleic acid purification: Further purify the nucleic acid by phenol-chloroform extraction method. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) to the nucleic acid solution, gently invert and mix well, centrifuge at 12000 rpm for 15 minutes at 4°C. Transfer the upper aqueous phase to a new centrifuge tube and repeat the above operation once. Take the upper aqueous phase, add 1 / 10 volume of 3M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol, place at -20°C for 2 hours. Centrifuge at 12000 rpm for 20 minutes at 4°C, discard the supernatant, wash the precipitate with 70% ethanol, centrifuge again and discard the supernatant, air-dry the precipitate, and dissolve it with 50 μL of RNase-free water.

[0142] Step Three: Fluorescent labeling reaction

[0143] 1. Take 10 μL of the pretreated nucleic acid sample, add 2 μL of fluorescent labeling probe (concentration 10 μM), 10 μL of hybridization buffer, and make up to 20 μL with RNase-free water.

[0144] 2. Denature the reaction system at 95°C for 5 minutes, and quickly cool it on ice for 3 minutes.

[0145] 3. Place the reaction system in a PCR instrument at 58°C for hybridization reaction for 1.5 hours.

[0146] 4. After the hybridization reaction, centrifuge the sample at 4°C and 13000 rpm for 8 minutes, and discard the supernatant. Wash the precipitate 3 times with washing buffer, and centrifuge under the same conditions after each washing.

[0147] Step Four: Fluorescent signal detection and quantitative analysis

[0148] 1. Resuspend the hybridized sample in 200 μL of detection buffer and transfer it to the reaction tube of the fluorescence quantitative PCR instrument.

[0149] 2. Set the parameters of the fluorescence quantitative PCR instrument: excitation wavelength is 488 nm, emission wavelength is 520 nm, and gain is 12.

[0150] 3. Establish a standard curve: With known quantities (102 , 10 3 , 10 4 , 10 5 , 10 6 CFU / mL) of Bacillus standard strains were processed according to the above steps 2 to 4, and the fluorescence signal intensity of each standard was recorded. Taking the fluorescence signal intensity as the ordinate and the logarithm of the number of Bacillus as the abscissa, a standard curve was plotted.

[0151] 4. By substituting the fluorescence signal intensity of the sample to be tested into the standard curve, the number of Bacillus in the sample was calculated.

[0152] Step 5: Dynamic monitoring of microbial community

[0153] Within 2 months, soil samples were collected every 10 days, and steps 2 to 4 were repeated to continuously monitor the change in the number of Bacillus. The number of Bacillus obtained from each detection was recorded, a curve of the number changing with time was plotted, and its dynamic change law was analyzed.

[0154] Experimental data table

[0155]

[0156] Experimental data analysis

[0157] 1. Change trend of the number of Pseudomonas

[0158] It can be seen from the data that in the initial stage of the experiment (0 - 14 days), the number of Pseudomonas showed an increasing trend. From 5.2×10 5 CFU / g soil on day 0 to 8.5×10 5 CFU / g soil on day 14. This may be because the soil environment is suitable for the growth and reproduction of Pseudomonas, for example, the temperature gradually increases and the soil humidity also increases, providing good living conditions for it.

[0159] From day 14 to 28, the number of Pseudomonas began to decline. From 8.5×10 5 CFU / g soil on day 14 to 6.1×10 5 CFU / g soil on day 28. This may be due to the gradual consumption of nutrients in the soil over time, or the intensification of competition within the microbial community, resulting in the growth of Pseudomonas being inhibited.

[0160] 2. Correlation with environmental factors

[0161] Temperature: From day 0 to day 14, the ambient temperature increased from 20°C to 25°C, and the number of Pseudomonas increased; while from day 14 to day 28, the temperature decreased from 25°C to 21°C, and the number of Pseudomonas decreased. It can be speculated that within a certain range, an increase in temperature is beneficial to the growth of Pseudomonas, while a decrease in temperature will inhibit its growth.

[0162] Soil humidity: The soil humidity changed relatively little during the whole experiment. However, during the growth stage of Pseudomonas (day 0 - day 14), the soil humidity also increased to a certain extent, which may provide more suitable moisture conditions for its growth. Nevertheless, the influence of soil humidity may be relatively smaller compared to temperature.

[0163] Experimental conclusion

[0164] The fluorescence - labeled quantitative tracking method used in this experiment can effectively monitor the dynamics of the number of Pseudomonas in the soil microbial community. Through continuous monitoring for one month, the change trend of the number of Pseudomonas was clearly observed.

[0165] The change in the number of Pseudomonas is closely related to environmental factors. Within a certain range, an increase in temperature and an appropriate increase in soil humidity are beneficial to the growth and reproduction of Pseudomonas; while over time, the consumption of soil nutrients and the internal competition factors in the microbial community will inhibit its growth, resulting in a decrease in the number.

[0166] This method provides important technical support for further studying the structure and function of the soil microbial community, helps to deeply understand the interaction between microorganisms and the environment, and provides a theoretical basis for the regulation of soil microorganisms in agricultural production.

[0167] The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention. Although this specification has described the present invention in detail with reference to the above - mentioned embodiments, the present invention is not limited to the above - mentioned specific embodiments. Therefore, any modification or substitution to the present invention; and all technical solutions and their improvements that do not depart from the spirit and scope of the invention are covered by the scope of the claims of the present invention.

Claims

1. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities, characterized in that: The following steps are involved: Step 1, design and synthesis of fluorescent labeling probe: according to the specific nucleic acid sequence of the target microorganism, design and synthesize the fluorescent labeling probe, the fluorescent labeling probe includes a specific recognition sequence and a fluorescent labeling group, the specific recognition sequence can specifically hybridize with the nucleic acid sequence of the target microorganism, and the fluorescent labeling group is connected to one end or both ends of the specific recognition sequence; Step 2: Pretreatment of microbial community samples: Collect microbial community samples and pretreat the samples. The pretreatment steps include cell wall disruption, nucleic acid extraction and purification. Step 3, fluorescent labeling reaction: the pretreated microbial community nucleic acid sample is mixed with the fluorescent labeling probe, and a hybridization reaction is performed under suitable hybridization conditions to allow the fluorescent labeling probe to specifically bind to the nucleic acid sequence of the target microorganism; Step 4, fluorescence signal detection and quantitative analysis: Use fluorescence detection equipment to detect the fluorescence signal of the hybridized sample, obtain the fluorescence signal intensity of the target microorganism, convert the fluorescence signal intensity into the number of target microorganisms according to the pre-established standard curve, and perform quantitative analysis on the target microorganisms; Step 5. Dynamic monitoring of microbial communities: Collect microbial community samples at different time points, repeat the above pretreatment, fluorescent labeling reaction, fluorescent signal detection and quantitative analysis steps, continuously monitor the changes in the number of target microorganisms, and track the dynamic changes of microbial communities.

2. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 1, characterized in that: The fluorescent labeling groups include FAM, TAMRA, CY3, and CY5.

3. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 2, characterized in that: The cell wall is broken by physical, chemical or enzymatic methods. The physical method includes ultrasonic disruption and freeze-thaw method, and the chemical method includes chemical lysis method.

4. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 3, characterized in that: The nucleic acid extraction uses a nucleic acid extraction kit, and the nucleic acid purification adopts an ethanol precipitation method.

5. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 4, characterized in that: The temperature of the hybridization reaction is adjusted according to the melting temperature (Tm value) of the probe, and the reaction is carried out at 5-10° C. below the Tm value, and the reaction time is 1-2 hours.

6. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 5, characterized in that: The fluorescence detection device is selected from a fluorescence microscope, a flow cytometer, and a fluorescence quantitative PCR instrument.

7. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 6, characterized in that: The standard curve is established by fluorescently labeling a known number of target microorganisms and detecting the intensity of their fluorescent signals.

8. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 7, characterized in that: After the fluorescent labeling reaction, washing and signal enhancement steps are also included: Washing: Wash the hybridized sample multiple times with a washing buffer that is compatible with the hybridization reaction system and can effectively remove unbound probes. Centrifuge at low speed after each wash to precipitate the sample and remove the supernatant. The washing buffer contains a certain concentration of salt ions and surfactants, the salt ion concentration is 0.1-0.5M, and the volume fraction of the surfactant is 0.01%-0.1%; Signal enhancement: If the intensity of the detected fluorescent signal is weak, a signal enhancer can be added to amplify the signal of the fluorescent marker bound to the target microbial nucleic acid; the signal enhancer is a nanoparticle with a fluorescence resonance energy transfer (FRET) effect.

9. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 8, characterized in that: Optimization of the fluorescent labeled probe: The specific recognition sequence is 15-30 nucleotides in length to ensure the specificity and stability of hybridization with the target microbial nucleic acid sequence; Predict the secondary structure of the specific recognition sequence to avoid forming a stable hairpin structure or self-complementary pairing, and ensure that it can freely bind to the target nucleic acid; The specific recognition sequence is optimized by introducing modified bases or chemical groups to enhance its affinity and hybridization stability with the target nucleic acid; the modified bases include locked nucleic acid (LNA) and peptide nucleic acid (PNA), and the chemical groups include methyl and amino groups.

10. A fluorescent labeling quantitative tracking method for dynamic monitoring of microbial communities according to claim 9, characterized in that: In the process of dynamic monitoring of microbial communities, data analysis and model building steps are also combined: Data analysis: Statistical analysis was performed on the target microbial quantity data obtained from detection at different time points, including calculation of mean, standard deviation, and variance statistical parameters, and analysis of the distribution characteristics and change trends of the data; at the same time, correlation analysis and principal component analysis multivariate statistical methods were used to analyze the relationship between the target microbial quantity and environmental factors; Model construction: Based on the data analysis results, a mathematical model of the dynamic changes of the microbial community is constructed to predict the number change trend of the target microorganism in the future; the mathematical model includes the logistic growth model and the Gaussian competition model.

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