A probe cluster, a kit and its application in detecting Clostridium difficile

Abstract

The present invention relates to the field of molecular biology detection technology, and specifically to a probe cluster, a kit and its application in detecting Clostridium difficile, wherein the kit includes a recombinase-mediated isothermal nucleic acid amplification system, amplification primers of the conservative sequence of the toxin B gene of Clostridium difficile, four sets of locked nucleic acid-modified G-quadruplex / heme DNA enzyme probes and competitive probes. In this method, nucleic acid amplification and its hybridization with the probe are carried out under constant temperature (42°C-45°C), and there is no need to purify the amplified product, and the steps are few and the speed is fast, and all detections can be completed within 40 minutes. The method has high sensitivity, good specificity, does not rely on complex instruments, and the results can be directly observed with the naked eye, which is suitable for the rapid detection of Clostridium difficile.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and infection immunology technology detection, and specifically to a primer and locked nucleic acid modified probe composition for detecting Clostridium difficile. The present invention also relates to a kit and method for detecting Clostridium difficile. Background Art

[0002] Clostridioides diffcile (CD) is a Gram-positive bacterium named for its difficulty in culturing. It is the main pathogen causing hospitalization for infectious diarrhea. Clostridioides diffcile infection (CDI) can cause varying degrees of diarrhea, fever, bloody stools, and even intestinal perforation and abdominal infection, and even lead to death of patients. CD is highly toxic, highly resistant to most antibiotics, with an increasing number of multidrug-resistant strains and a high recurrence rate after infection. It is a very difficult to treat infectious disease, and the continued increase in its morbidity and mortality poses a serious threat to public health. CDI progresses rapidly, and the development to the later stage will seriously affect the prognosis, so it is particularly important to diagnose it early and accurately.

[0003] At present, the main laboratory test for diagnosing CDI is to prove the presence of the bacteria or the toxins it secretes in stool samples. CD microbial culture is the "gold standard" for determining CDI, but CD culture is difficult, has poor sensitivity, is time-consuming, and expensive, and requires a special anaerobic culture system. Therefore, this method is not currently routinely used in clinical microbiology laboratories. Cytotoxicity tests are costly, slow, and highly professional, and have high technical requirements for equipment and testing personnel. In addition, although the enzyme-linked immunosorbent assay (ELISA) has high specificity, it has low sensitivity due to the easy degradation of related toxins and the existence of false negative results; the related sensors have good selectivity, stability, and accuracy, but require an electrochemical workstation, which is a problem for grassroots institutions with fewer equipment resources or inspectors who need to conduct on-site testing. These undoubtedly limit the application of this method.

[0004] Toxin B (TcdB) is the most critical in the pathogenic process of Clostridium difficile. TcdB alone can induce death in animal models. TcdB is a representative gene of CD. It is a good method to amplify and detect its conserved gene sequence. However, the traditional nucleic acid detection method is to first amplify a trace amount of target nucleic acid by PCR, and then perform solid phase hybridization when the target nucleic acid is amplified to a detectable level, which requires complicated operation steps, a long hybridization process and complex instruments. With the continuous development of molecular biology technology, the emergence of constant temperature nucleic acid amplification technology has solved the limitations of PCR technology in instruments. It has the advantages of high sensitivity, less time consumption, simplicity and portability. However, the current constant temperature nucleic acid amplification methods all have certain disadvantages, such as the complex products of LAMP (Loop-Mediated Isothermal Amplification) and it is difficult to further analyze the products; the cost of synthesizing rolling circle amplification padlock probes in RCA is high; SDA (Rolling Circle Amplification) requires modified dNTP as substrate, the target sequence preparation is complex, and the detection of amplified products still requires special equipment. Recombinase polymerase amplification (RPA) is a constant temperature amplification technology involving recombinase, single-stranded DNA binding protein (GP32) and strand displacement Bsu polymerase. It can react continuously at 23-45°C, and the reaction time is only 5-20 minutes, avoiding the need for thermal cycling equipment. It is very suitable for disease diagnosis and on-site testing. It is currently the fastest nucleic acid constant temperature amplification technology. However, the RPA amplification product is double-stranded DNA, which is not very ideal for the amplification of single-stranded oligonucleotide products required for nucleic acid hybridization (asymmetric amplification), and it increases the purification steps and costs.

[0005] The G-quadruplex / heme DNA enzyme system has been widely used in nucleic acid detection. It has the advantages of low cost, rapid hybridization, and results that can be observed with the naked eye. This enzyme has been successfully applied to the nucleic acid detection of tumor mutant genes, viral RNA, and bacterial DNA. Locked nucleic acid (LNA) is a bicyclic nucleotide derivative that has the same phosphate backbone as DNA / RNA in structure, which reduces the flexibility of the ribose structure, thus having better recognition ability and strong affinity for DNA / RNA, and the hybridization product formed is not easy to dissociate even at high temperatures. When LNA is used as a detection probe, the required length is shorter, the concentration is lower, and the water solubility is better, which can capture the target nucleic acid more accurately and stably. The organic combination and improvement of the above technologies are expected to solve the above problems.

[0006] Early, rapid and accurate diagnosis is helpful to buy time for the treatment of CDI. Therefore, it is of great significance to develop a rapid, specific, sensitive, relatively low-cost and easy-to-operate method for detecting Clostridium difficile nucleic acid. Summary of the invention

[0007] The present invention aims to amplify the conserved region of the TcdB gene of Clostridium difficile by RPA, and assist with the use of four groups of split G-quadruplex / heme DNA enzyme probes modified with locked nucleic acid to perform visual isothermal detection of Clostridium difficile. The method includes a recombinase-mediated isothermal nucleic acid amplification system, Clostridium difficile toxin B gene amplification primers, locked nucleic acid-modified probes and a G-quadruplex / heme DNA enzyme system. The method is carried out under isothermal conditions (42°C-45°C), and all detection can be completed within 40 minutes. The method has high sensitivity, good specificity, does not rely on complex instruments, and the results can be directly observed with the naked eye, which is suitable for the rapid detection of Clostridium difficile.

[0008] The purpose of the present invention is to provide a probe cluster and a kit for detecting Clostridium difficile.

[0009] To achieve the purpose of the present invention, the technical solution adopted by the present invention is:

[0010] 1. Extract genomic DNA from feces;

[0011] 2. TcdB is a representative gene of CD. The conserved sequence of the gene was amplified by RPA at constant temperature (42°C-45°C) using upstream and downstream primers. The conserved sequence is shown in SEQ ID No.1, and its complementary paired sequence is shown in SEQ ID No.2; the upstream primer sequence is shown in SEQ ID No.3, and the downstream primer sequence is shown in SEQ ID No.4;

[0012] 3. Maintaining a constant temperature (42°C-45°C), directly adding detection probe cluster I to the amplified product, which includes locked nucleic acid modified probe group 1 and probe group 2, wherein the sequences of the detection probe groups 1A and 1B are shown in SEQ ID No.5 and SEQ ID No.6, respectively, and the sequences of the detection probe groups 2A and 2B are shown in SEQ ID No.7 and SEQ ID No.8, respectively, wherein the sites modified with locked nucleic acid are indicated in italics;

[0013] 4. Maintaining a constant temperature (42°C-45°C), directly adding detection probe cluster II to the product in 3, which includes probe group 3 and probe group 4 modified with locked nucleic acid, wherein the sequences of detection probe groups 3A and 3B are shown in SEQ ID No.9 and SEQ ID No.10, respectively, and the sequences of detection probe groups 4A and 4B are shown in SEQ ID No.11 and SEQ ID No.12, respectively, wherein the sites modified with locked nucleic acid are indicated in italics; at the same time, probe cluster III, i.e., competition probe group, includes 4 nucleic acid sequences to prevent the combination of probe group 1 with probe group 4, and probe group 2 with probe group 3, thereby reducing the background signal of the blank control group, wherein the 4 competition probe groups are competition probe 1A, competition probe 1B, competition probe 2A, and competition probe 2B, and the sequences are shown in SEQ ID No.13, SEQ ID No.14, SEQ ID No.15, and SEQ ID No.16, respectively;

[0014] 5. Place the product at room temperature for 5 minutes, add the substrate to develop color for 5 minutes, observe the results with the naked eye, take photos with a smartphone, and if conditions permit, use an enzyme reader to detect the absorbance value of the product;

[0015] 6. Determination of test results: When the color of the sample group is dark green, the test result is positive, indicating that the sample contains Clostridium difficile; when it is colorless, the test result is negative, indicating that the sample does not contain Clostridium difficile.

[0016] Compared with the prior art, the beneficial effects and advantages of the present invention are:

[0017] 1. The present invention uses a conserved sequence of the genomic DNA of Clostridium difficile as a template, adds a specific primer combination for detecting the target gene in Clostridium difficile, and uses the RPA technology with the fastest amplification speed. It only takes 15 minutes of amplification to meet the detection requirements of this method.

[0018] 2. Improved commercial RPA amplification buffer, the main components are: 50mM Tris-pH 8.0, 100mM KCl, 14mM MgCl2, 5% Carbowax 20M, 200μM dNTPs, etc. The amplification buffer does not contain dithiothreitol (DTT), but does not affect the RPA amplification efficiency, and can also avoid the inhibitory effect of DTT on the results of the G-quadruplex / heme DNA enzyme catalysis reaction, thereby avoiding further purification of the RPA product, reducing the processing steps, saving time, and increasing practicality and simplicity.

[0019] 3. Using four groups of split DNA enzyme probes modified with locked nucleic acid to directly detect double-stranded DNA amplified by RPA can improve sensitivity and specificity, and the total time for two hybridizations is only 10 minutes; and avoiding the step of amplifying single-stranded DNA using asymmetric RPA technology increases simplicity and saves time. The previous method for detecting salivary Helicobacter pylori based on recombinase amplification and peptide nucleic acid-assisted split DNAzyme probes (Yingying Wang et al. Sensors & Actuators: B. Chemical, 396 (2023), 134582) has many steps and takes a long time (100 minutes). High temperature inactivation (80°C) and other steps are required in the middle of the detection, which increases the complexity of the detection. And the result shows a lighter green color, which is not easy to distinguish with the naked eye.

[0020] 4. After the above nucleic acid amplification and nucleic acid hybridization are completed, the sample only needs to be placed at room temperature for 5 minutes and then the substrate is added and observed for 5 minutes to obtain the test result. In summary, the entire test and result determination can be completed within 40 minutes.

[0021] 4. Use competitive probe cluster III to block the binding of probe group 1 and probe group 4, and probe group 2 and probe group 3, thereby reducing the background signal of the blank control group, making a correct judgment on the negative sample results, and avoiding false positive problems.

[0022] 5. The present invention constructs a visual constant temperature detection kit for Clostridium difficile in feces based on recombinase polymerase amplification and locked nucleic acid modified four groups of split DNA enzyme probes, which greatly simplifies the operation process of nucleic acid detection. Both nucleic acid amplification and probe hybridization are carried out at a constant temperature (42°C-45°C), without the need for expensive special equipment and recovery steps, and all tests can be completed within 40 minutes. The test results can be identified by the naked eye, and pictures can be taken and saved with a smartphone. This method has the advantages of being fast, simple, cheap, intuitive, low equipment dependence, high sensitivity and specificity, and is suitable for grassroots institutions and on-site testing where equipment is scarce. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below in combination with the drawings and specific embodiments.

[0024] Figure 1The schematic diagram and operation steps of four groups of split DNA enzyme probes based on recombinase polymerase amplification technology and locked nucleic acid modification and their application in the detection of Clostridium difficile.

[0025] Figure 2 This is a schematic diagram of the reaction when there is no target molecule DNA to be detected in the sample. The competitive probe group combines with probe group 1 and probe group 2 to avoid false positive results.

[0026] Figure 3 The use of locked nucleic acid modified split DNA enzyme probes to detect the artificially synthesized double-stranded DNA of the conserved gene of Clostridium difficile. Among them, (A) is the detection result using one set of probes; (B) is the detection result using two sets of probes; (C) is the detection result using four sets of probes; (D) is a statistical chart of the absorbance values ​​of the results of (A), (B), and (C) using an ELISA reader; (E) is the detection limit analysis using one set of probes; (F) is the detection limit analysis using two sets of probes; (G) is the detection limit analysis using four sets of probes.

[0027] Figure 4 The effect of the RPA reaction system on the color development reaction. Among them, (A) is the effect of DTT on the RPA amplification reaction; (B) is the effect of the RPA buffer without DTT on the color development reaction.

[0028] Figure 5 The parameters of the visualized constant temperature analysis platform for four groups of split DNA enzyme probes modified by recombinase polymerase amplification technology and locked nucleic acid are optimized. Among them, (A) is the color development result of probe group 1 and probe group 2, probe group 3 and probe group 4, and competitive probe group concentration optimization; (B) is the statistical graph of the color development result of probe group 1 and probe group 2, probe group 3 and probe group 4, and competitive probe concentration optimization after using an enzyme reader to detect the absorbance value; (C) is the color development graph of the effect of the number of LNA on each detection probe on the detection result; (D) is the statistical graph of the effect of the number of LNA on each detection probe on the detection result after using an enzyme reader to detect the absorbance value; (E) is the effect of the detection probe hybridization time on the detection result; (F) is the effect of the RPA amplification time on the detection result; (G) is the effect of the RPA amplification and hybridization temperature on the detection result.

[0029] Figure 6 The sensitivity and specificity detection of the visualized isothermal analysis platform based on recombinase polymerase amplification technology and locked nucleic acid modified four groups of split DNA enzyme probes and the detection of clinical samples of Clostridium difficile. Among them, (A) is the sensitivity detection; (B) is the specificity detection; (C) is the detection of clinical samples of Clostridium difficile. DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to specific embodiments.

[0031] LNA, or Locked Nucleic Acid, is a nucleic acid analog with special properties, in which the ribose part is connected to the 3' carbon atom through an additional 2'-5' phosphodiester bond to form an additional closed ring structure. This structure makes LNA have higher thermal stability and higher affinity, and is more stable than traditional DNA or RNA probes when binding to complementary DNA or RNA chains.

[0032] Example 1

[0033] Detection of Clostridium difficile based on recombinase polymerase amplification technology and four groups of split DNA enzyme probes modified with locked nucleic acid.

[0034] Reports have been published to explain the principle of the split G-quadruplex / heme DNA enzyme system (Zhuo Chen et al. Sensors & Actuators: B. Chemical, 321 (2020), 128559). That is, when each set of probes (including two single-stranded probes A and B) hybridizes with the target single-stranded DNA (ssDNA), the free G-quadruplex sequences on each probe can approach each other, so they can still form a G-quadruplex structure and bind to heme, thereby forming a G-quadruplex / heme DNA enzyme and catalyzing the substrate (ABTS 2- (2,2'-Azinobis(3-ethylbenzothiazine-6-sulfonic acid) diammonium salt)) forms a green free radical ABTS visible to the naked eye. - When there is no target ssDNA in the system, the free G-quadruplex sequences on the probes cannot approach each other, so DNA enzymes cannot be formed and color cannot be developed.

[0035] Two sets of probes can increase the DNAzyme concentration in the system compared to one set of probes, so that lower concentrations of target nucleic acids can be detected, which can improve sensitivity. In the present invention, four sets of probes are added to the system twice to further improve detection sensitivity. After extracting Clostridium difficile DNA from a stool sample, double-stranded target DNA (dsDNA) is first amplified using RPA. Then, probe group 1 and probe group 2 shown in Table 1 are added to the system, wherein probe group 1 includes locked nucleic acid-modified detection probe 1A and locked nucleic acid-modified detection probe 1B, and probe group 2 includes locked nucleic acid-modified detection probe 2A and locked nucleic acid-modified detection probe 2B. Under the action of recombinase and single-stranded DNA binding protein in the RPA system, probe group 1 and probe group 2 can hybridize with the invariant dsDNA to form two G-quadruplex structures; then, probe group 3 and probe group 4 shown in Table 1 are added, wherein probe group 3 includes locked nucleic acid-modified detection probe 3A and locked nucleic acid-modified detection probe 3B, and probe group 4 includes locked nucleic acid-modified detection probe 4A and locked nucleic acid-modified detection probe 4B. Probe group 3 and probe group 4 can hybridize with the invariant dsDNA to form another two G-quadruplex structures, that is, one target DNA can form four G-quadruplex structures and bind to heme to initiate a color development reaction. All probes can complementarily pair with two different target ssDNA segments. The target ssDNAs corresponding to probe group 1 and probe group 3 are continuously shifted regions on the same DNA chain, as are probe group 2 and probe group 4. Moreover, probe group 1 and probe group 4, and probe group 2 and probe group 3 are complementary pairs ( Figure 1 ). If probe group 3 and probe group 4 are not added, not only the detection limit will be affected, but also the detection time will be prolonged.

[0036] Table 1

[0037]

[0038] When there is no target molecule DNA to be detected in the sample, probe cluster III binds to probe cluster I, that is, competition probe 1A shown in Table 1 binds to detection probe 1A of probe group 1, competition probe 1B binds to detection probe 1B of probe group 1, competition probe 2A binds to detection probe 2A of probe group 2, and competition probe 2B binds to detection probe 2B of probe group 2, thereby blocking the binding of probe group 1 to probe group 4 and probe group 2 to probe group 3, thereby avoiding the generation of false positive results ( Figure 2 ).

[0039] To simplify the operation process, prepare the corresponding buffers A, B, C1, C2, D, etc. before the test. The specific operation steps for the detection of Clostridium difficile using four groups of split DNA enzyme probes based on recombinase polymerase amplification technology and locked nucleic acid modification are as follows:

[0040] 1. Extract genomic DNA from feces using buffer A.

[0041] Take 1000 μL of buffer A (including the following concentrations of components: 50 mM Tris-pH 8.0, 5% BSA, 0.5% SDS), add 200 μL of stool samples collected from patients with Clostridium difficile infection, and mix thoroughly by vortex oscillator. Centrifuge the mixed solution at 10 000 × g for 10 min, take the supernatant and heat it at 95 ° C for 10 minutes in a micro-metal bath thermostat (LEOPARD, China) to inactivate the microorganisms in the stool sample, cool it to room temperature and set it aside as a template for the next nucleic acid amplification.

[0042] 2. Use buffer B to mix with template DNA and use RPA to amplify the conserved sequence of the representative gene TcdB of Clostridium difficile.

[0043] Take 8 μL of buffer B (comprising the following components: 50 mM Tris-pH 8.0, 100 mM KCl, 14 mM MgCl2, 5% Carbowax 20M, 200 μM dNTPs, 3 mM ATP, 50 mM creatine phosphate, 100 ng / μl creatine kinase, 30 ng / μL Bsu, 900 ng / μL GP32, 120 ng / μL uxsX and 30 ng / μL uvsY, and the final concentrations of 300 nM of upstream and downstream primers for amplifying the conservative region of Clostridium difficile toxin B, as shown in SEQ ID No. 3 and SEQ ID No. 4), and take 2 μL of the DNA template extracted in step 1 above and add it to buffer B to mix well, the total system is 10 μL, and the blank control group is added with 2 μL ultrapure water and 8 μL buffer B. Close the lid of the reaction tube and amplify at 42°C in a micro metal bath thermostat for 15 minutes.

[0044] 3. Add the amplified product to buffer C1 for nucleic acid hybridization.

[0045] After opening the reaction tube, add 45 μL of buffer C1 (including the following components in concentration: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100 (polyoxyethylene nonylphenol ether), 150 nM probe group 1, probe group 2, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro metal bath thermostat for 5 minutes.

[0046] 4. Add buffer C2 to the hybridization product for further nucleic acid hybridization.

[0047] After opening the lid of the reaction tube, add 40 μL of buffer C2 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 250 nM probe group 3, probe group 4, 200 nM competitive probe group), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro metal bath thermostat for 5 minutes.

[0048] 5. Use buffer D to observe the test results.

[0049] The hybridization product in the reaction tube was cooled at room temperature for 5 minutes. During the 5-minute waiting period, fresh buffer D (including the following concentrations of components) was prepared: 6 mM ABTS 2- , 2mM H2O2; ABTS 2- and H2O2 were dissolved in ultrapure water in advance to a final concentration of 150mM and 200mM, respectively). Then, 5μL of buffer D was added to the cooled hybridization product, and the color was developed at room temperature for 5 minutes. The results were observed with the naked eye and photographed with a smartphone. If conditions permit, the product can be detected by an ELISA reader at a wavelength of 414nm (OD 414 ).

[0050] 6. Determination of test results.

[0051] When the sample group is dark green, the test result is positive, indicating that the sample contains Clostridium difficile; when it is colorless, the test result is negative, indicating that the sample does not contain Clostridium difficile.

[0052] Example 2

[0053] The use of four groups of split DNA enzyme probes modified with locked nucleic acid to detect artificially synthesized double-stranded DNA of the conserved gene of Clostridium difficile can improve the detection sensitivity.

[0054] In order to verify the effectiveness of the detection probe, the detection system was first tested using an in vitro synthesized double-stranded DNA template, and a conserved nucleic acid sequence of the TcdB gene of Clostridium difficile was used as the test target. The DNA template sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, and the locked nucleic acid-modified detection probes 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, competitive probes 1A, 1B, 2A, and 2B are shown in SEQ ID No. 5 to SEQ ID No. 16 in Table 1, respectively.

[0055] (1) Two synthetic complementary DNA template sequences were added to a prepared solution (50 mM Tris-pH 8.0, 100 mM KCl) at a final concentration of 10 μM, denatured at 95°C and immediately incubated at 50°C for 30 minutes to obtain complementary DNA double strands. The concentrations were adjusted to 0, 500, 1000, 2000, 3000, 4000, 5000, 6000, 8000, and 10000 nM, respectively;

[0056] (2) Take 10 μL of each double-stranded DNA after adjusting the concentration in (1) above, add 85 μL of buffer (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 150 nM probe group 1, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an enzyme reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure reproducibility. This is the experimental procedure using one set of probes (probe set 1);

[0057] (3) Take 10 μL of each double-stranded DNA after the concentration is adjusted in (1) above, add 85 μL of buffer C1 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 150 nM probe group 1, probe group 2, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an enzyme reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure repeatability. This is the experimental procedure using two sets of probes (probe set 1, probe set 2);

[0058] (4) Take 10 μL of each double-stranded DNA after adjusting the concentration in (1) above, add 45 μL of buffer C1 (including the following components at the following concentrations: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 150 nM probe group 1, probe group 2, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Open the lid and add 40 μL of buffer C2 (including the following components at the following concentrations: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 250 nM probe group 3, probe group 4, 200 nM competitive probe group), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for the components and final concentrations). Then, 5 μL of buffer D was added to the cooled hybridization product, and the color was developed at room temperature for 5 minutes. The results were observed with the naked eye, photos were taken with a smartphone, and the absorbance value (OD) of the product at a wavelength of 414 nm was detected using an ELISA reader. 414 ). All reactions were performed three times to ensure repeatability. This is the experimental procedure using four sets of probes (probe set 1, probe set 2, probe set 3, probe set 4);

[0059] The results showed that when only one, two, or four probe sets were used, the DNA samples all showed an obvious green change, but the corresponding optimal target ssDNA amounts were no less than 800 nM, 400 nM, and 200 nM, respectively. Figure 3 Since light absorption is linearly related to the concentration of target ssDNA ( Figure 3 EG), following the 3σ standard (Zhuo Chen et al. Sensors & Actuators: B. Chemical, 321 (2020), 128559), the detection limits (LODs) of probe groups 1, 2, and 4 were 2.44 nM, 1.14 nM, and 0.41 nM, respectively.

[0060] The above results show that the use of four probe sets can detect lower concentrations of target nucleic acids, which increases its detection sensitivity and application value. This method can be used for clear and efficient detection of Clostridium difficile target DNA. Therefore, the following experiments all use four probe sets.

[0061] Example 3

[0062] Effect of RPA reaction system on color development reaction.

[0063] This section attempts to determine whether the main components of the RPA amplification solution have an inhibitory effect on the activity of G-quadruplex / heme DNA enzyme. If there is no inhibitory effect, it is possible to avoid further purification of the RPA product and reduce the number of processing steps before nucleic acid analysis, thereby improving the practicality of G-quadruplex / heme DNA enzyme for nucleic acid hybridization. It has been previously discovered that several common components in nucleic acid amplification reaction systems have an effect on the activity of G-quadruplex / heme DNA enzyme, such as low concentrations of Mg 2+ (<6mM), DMSO (<6%) and additional potassium ions (<20mM) had no significant effect on the catalytic activity of DNA enzymes. However, dithiothreitol (DTT) is an inhibitor of G-quadruplex / heme DNA enzymes, so it is best not to add DTT to the nucleic acid amplification reaction system (ZhuoChen et al. Sensors & Actuators: B. Chemical, 321 (2020), 128559).

[0064] (1) Analyze the effect of RPA buffer B without DTT on RPA.

[0065] Take 2 μL of the Clostridium difficile nucleic acid DNA extracted in Example 1, add 8 μL of RPA buffer B without DTT (see Example 1 for each component and final concentration) and mix well. Close the lid of the reaction tube and amplify at 42°C in a micro-metal bath thermostat for 15 minutes. Then use a DNA cleaning recovery kit to purify the amplified product, then take a sample for agarose gel electrophoresis and take a picture;

[0066] (2) Analyze the effect of different concentrations of RPA buffer B without DTT on the color development reaction.

[0067] Take 70 μL of the artificially synthesized double-stranded DNA in Example 2 (concentration of 10000 nM), prepare 6 groups, add 10 μL of double-stranded DNA to each tube, then add RPA buffer B with volume concentrations of 0, 5%, 10%, 15%, 20%, and 30% (i.e., the volume is 0-30 μL respectively), and the part less than 30 μL is filled up to 30 μL with ultrapure water. Then add 25 μL of buffer C1 (including the following concentrations of each component: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 150 nM probe group 1, probe group 2, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubation at 42°C in a micro metal bath thermostat for 5 minutes. After opening the lid, add 30 μL of buffer C2 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 250 nM probe group 3, probe group 4, 200 nM competitive probe group), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an enzyme reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure reproducibility.

[0068] The results showed that RPA buffer B without DTT did not affect its nucleic acid amplification ( Figure 4 A), and low concentration of buffer B (no more than 15% by volume) had no significant inhibitory effect on the color development reaction ( Figure 4 In the actual detection, the volume ratio of buffer B is 10 μL / 100 μL, which is only 10%. Therefore, buffer B in this ratio does not affect the color development result.

[0069] The above results indicate that the detection probe can be directly added to the RPA amplification product without the need to recover and purify the RPA amplified nucleic acid product, which can increase the simplicity of the operation and save time.

[0070] Example 4

[0071] Parameter optimization of a visualized isothermal analysis platform based on recombinase polymerase amplification technology and four groups of split DNA enzyme probes modified with locked nucleic acid.

[0072] This section attempts to optimize the parameters of the visualized isothermal analysis platform based on recombinase polymerase amplification technology and locked nucleic acid modified four groups of split DNA enzyme probes. The main optimized parameters are the concentration of probe cluster I (including probe group 1 and probe group 2), the concentration of probe cluster II (including probe group 3 and probe group 4), the concentration of probe cluster III (including four competitive probes), the number of LNA on each detection probe, RPA amplification time, detection probe cluster hybridization time, RPA amplification and hybridization temperature, etc.

[0073] (1) First, add the Clostridium difficile nucleic acid DNA extracted in Example 1 to RPA buffer B without DTT (the components and final concentrations are shown in Example 1) and mix well. Close the lid of the reaction tube and amplify at 42°C in a micro-metal bath thermostat for 15 minutes. After large-scale amplification, set aside;

[0074] (2) Concentration optimization of probe cluster I (including probe set 1 and probe set 2).

[0075] Prepare 8 groups, add 10 μL of the amplified product in (1) above to each tube, then add 10 μL of probe group 1 and probe group 2 with final concentrations of 0, 50, 100, 150, 200, 250, 300, 400 nM, respectively. The volume of the group less than 10 μL is made up to 10 μL with ultrapure water. Then add 35 μL of buffer C1 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 500 nM hemin) without probe group 1 and probe group 2, mix well, close the lid of the reaction tube, and incubate at 42°C in a micro metal bath thermostat for 5 minutes. After opening the lid, add 40 μL of buffer C2 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 250 nM probe group 3, probe group 4, 200 nM competitive probe group), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an enzyme reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure reproducibility.

[0076] (3) Concentration optimization of probe cluster II (including probe set 3 and probe set 4).

[0077] Prepare 8 groups, add 10 μL of the amplified product in (1) above to each tube. Then add 45 μL of buffer C1 (including the following concentrations of each component: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 150 nM probe group 1, probe group 2, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubation at 42°C in a micro metal bath thermostat for 5 minutes. After opening the lid, add 10 μL of probe group 3 and probe group 4 with final concentrations of 0, 50, 100, 150, 200, 250, 300, 400 nM respectively. The volume of the group less than 10 μL is filled up to 10 μL with ultrapure water. Then add 30 μL of buffer C2 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 200 nM competitive probe group) without probe group 3 and probe group 4, mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an enzyme reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure reproducibility.

[0078] (4) Concentration optimization of probe cluster III (including four competitive probes).

[0079] Prepare 8 groups, add 10 μL of the amplified product in (1) above to each tube, then add 45 μL of buffer C1 (including the following components at the following concentrations: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 0.0003% Triton X-100, 150 nM probe group 1, probe group 2, 500 nM hemin), mix well, close the lid of the reaction tube, and continue incubation at 42°C in a micro-metal bath thermostat for 5 minutes. After opening the lid, add 10 μL of the competitive probe groups with final concentrations of 0, 50, 100, 150, 200, 250, 300, and 400 nM, respectively. The volume of the group less than 10 μL is made up to 10 μL with ultrapure water. Then add 30 μL of buffer C2 (including the following concentrations of components: 50 mM Tris-pH 8.0, 150 mM NH4Cl, 250 nM probe group 3, probe group 4) without the competing probe group, mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an ELISA reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure reproducibility.

[0080] (5) Optimization of the number of LNAs on each detection probe.

[0081] Prepare 5 groups, add 10 μL of the amplification product in (1) above to each tube, then add 45 μL of buffer C1 (see Example 1 for each component and final concentration), close the lid of the reaction tube after mixing, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. The number of LNAs on probe group 1 and probe group 2 are 0, 2, 4, 6, and 8, respectively. Then add 40 μL of buffer C2 (see Example 1 for each component and final concentration), close the lid of the reaction tube after mixing, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. The number of LNAs on probe group 3 and probe group 4 are 0, 2, 4, 6, and 8, respectively, and correspond to the number of LNAs on probe group 1 and probe group 2. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then, 5 μL of buffer D was added to the cooled hybridization product, and the color was developed at room temperature for 5 minutes. The results were observed with the naked eye, photos were taken with a smartphone, and the absorbance value (OD) of the product at a wavelength of 414 nm was detected using an ELISA reader. 414 ). All reactions were performed three times to ensure reproducibility.

[0082] (5) Optimization of detection probe hybridization time.

[0083] Prepare 6 groups, add 10 μL of the amplified product in (1) above to each tube, then add 45 μL of buffer C1 (each component and final concentration are shown in Example 1), mix well, close the lid of the reaction tube, and continue to incubate at 42°C in a micro-metal bath thermostat for 1, 2, 5, 10, 15, and 20 minutes. After each group of incubation is completed, the product is immediately placed on ice. After the 20th minute group is finished incubating, all groups are opened and 40 μL of buffer C2 (each component and final concentration are shown in Example 1) are added, and incubated at 42°C in a micro-metal bath thermostat for 1, 2, 5, 10, 15, and 20 minutes. After each group of incubation is completed, the product is immediately placed on ice. After the 20th minute group is finished incubating, place it at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (each component and final concentration are shown in Example 1). Then, 5 μL of buffer D was added to the cooled hybridization product, and the color was developed at room temperature for 5 minutes. The results were observed with the naked eye, photos were taken with a smartphone, and the absorbance value (OD) of the product at a wavelength of 414 nm was detected using an ELISA reader. 414 ). All reactions were performed three times to ensure reproducibility.

[0084] (6) Optimization of RPA amplification time.

[0085] The Clostridium difficile nucleic acid DNA extracted in Example 1 was added to RPA buffer B without DTT (each component and final concentration are shown in Example 1) and mixed. Six groups were prepared, and the total volume of each tube in each group was 10 μL. Close the lid of the reaction tube, and amplify at 42°C in a micro-metal bath thermostat for 5, 10, 15, 20, 40, and 60 minutes. After each group of amplification is completed, the product is immediately placed on ice. After the amplification of the 60th minute group is completed, all groups are added with 45 μL of buffer C1 (each component and final concentration are shown in Example 1), mixed and closed, and incubated at 42°C in a micro-metal bath thermostat for 5 minutes. After opening the lid, add 40 μL of buffer C2 (each component and final concentration are shown in Example 1), mix and close the lid of the reaction tube, and continue to incubate at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (each component and final concentration are shown in Example 1). Then, 5 μL of buffer D was added to the cooled hybridization product, and the color was developed at room temperature for 5 minutes. The results were observed with the naked eye, photos were taken with a smartphone, and the absorbance value (OD) of the product at a wavelength of 414 nm was detected using an ELISA reader. 414 ). All reactions were performed three times to ensure reproducibility.

[0086] (7) Optimization of RPA amplification and hybridization temperature.

[0087] The Clostridium difficile nucleic acid DNA extracted in Example 1 was added to RPA buffer B without DTT (see Example 1 for each component and final concentration) and mixed to prepare 7 groups, with a total volume of 10 μL per tube in each group. Close the lid of the reaction tube and amplify at 25, 30, 37, 40, 42, 45, and 50°C in a micro-metal bath thermostat for 15 minutes. Add 45 μL of buffer C1 (see Example 1 for each component and final concentration) to all groups, mix well, close the lid of the reaction tube, and incubate at 25, 30, 37, 40, 42, 45, and 50°C in a micro-metal bath thermostat for 5 minutes. After opening the lid, add 40 μL of buffer C2 (see Example 1 for each component and final concentration), mix well, close the lid of the reaction tube, and continue incubating at 25, 30, 37, 40, 42, 45, and 50°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color development is carried out at room temperature for 5 minutes. The results are observed with the naked eye, photos are taken with a smartphone, and the absorbance value (OD) of the product at a wavelength of 414 nm is detected using an ELISA reader. 414 ). All reactions were performed three times to ensure reproducibility.

[0088] The results showed that the probe concentration had a great influence on the signal-to-noise ratio of the results. The optimal concentration of probe cluster I was 150 nM, the optimal concentration of probe cluster II was 250 nM, and the optimal concentration of probe cluster III was 200 nM. Figure 5 If the concentration is too low, the color will be lighter, and if the concentration is too high, the background signal will increase. In addition, the number of LNAs required on each probe was also observed, and the results showed that the number of LNAs on each probe should not be less than 4 ( Figure 5 There was no difference in the incubation time of the detection probe at 20, 15, 10, and 5 minutes, but the color development efficiency decreased at 1 and 2 minutes ( Figure 5 Middle E). When RPA amplified for 60, 40, 20, and 15 minutes, the results of the four probes were the same, but when amplified for only 10 and 5 minutes, the color development efficiency was low ( Figure 5 For RPA amplification and hybridization temperature, 42-45°C is optimal ( Figure 5 Middle G).

[0089] According to the results of the above parameter optimization, after the present invention uses RPA to amplify the Clostridium difficile DNA template for 15 minutes, there is no need to denature and purify the product. The two added probes are incubated for only 5 minutes each, and the entire process is carried out at only 42-45°C. Then the color development reaction can be started and the results can be observed after 5 minutes.

[0090] Example 5

[0091] Sensitivity and specificity of the visualized isothermal analysis platform based on recombinase polymerase amplification technology and four groups of split DNA enzyme probes modified with locked nucleic acid and detection of CD clinical samples.

[0092] (1) Sensitivity detection

[0093] The sensitivity analysis of the RPA-LNA-DNAzyme visual isothermal analysis platform developed in the present invention is evaluated by the colony-forming units (CFU) of bacteria. CFU refers to the total number of bacterial colonies per unit volume. After calculating the CFU of bacteria, they are lysed and then diluted 10 times continuously. The retention concentrations are 0, 10, and 10, respectively. 0 , 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 / mL dilution, and 2 μL was taken respectively and used as template and subjected to RPA step to amplify the target region (159 nt) into double-stranded copies, and only 2 μL ultrapure water was added to the negative control group.

[0094] Add the above-mentioned 10-fold continuous dilutions of Clostridium difficile template DNA to RPA buffer B (each component and final concentration are shown in Example 1) and mix well. Prepare 8 groups with a total volume of 10 μL per tube in each group. Close the lid of the reaction tube and amplify at 42°C in a micro-metal bath thermostat for 15 minutes. Add 45 μL of buffer C1 (each component and final concentration are shown in Example 1) to all groups, mix well, close the lid of the reaction tube, and incubate at 42°C in a micro-metal bath thermostat for 5 minutes. After opening the lid, add 40 μL of buffer C2 (each component and final concentration are shown in Example 1), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (each component and final concentration are shown in Example 1). Then, 5 μL of buffer D was added to the cooled hybridization product, and the color was developed at room temperature for 5 minutes. The results were observed with the naked eye, photos were taken with a smartphone, and the absorbance value (OD) of the product at a wavelength of 414 nm was detected using an ELISA reader. 414 ). All reactions were performed three times to ensure reproducibility.

[0095] The results showed that when the concentration of Clostridium difficile was greater than or equal to 100 CFU / mL ( Figure 6 In Figure 2A), the presence of Clostridium difficile was well detected, and no signal was obtained in the negative control, indicating that the system has good sensitivity.

[0096] (2) Specific detection

[0097] Several common intestinal bacteria were selected to evaluate the analytical specificity of the method. Common intestinal bacteria, such as Clostridium difficile standard strain (CD43255), Salmonella typhi (S.typhi C5), Escherichia coli (E.coli DH5α), Clostridium perfringens (C.perfringens), Streptococcus faecalis (S.faecalis), Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S.aureus), etc., were cultured, centrifuged and lysed at 100°C, and the supernatant was taken as the total DNA of the above bacteria, respectively, and added to RPA buffer B (each component and final concentration are shown in Example 1) as templates and mixed, and 8 groups were prepared, with a total volume of 10 μL per tube in each group. Close the lid of the reaction tube and amplify at 42°C in a micro-metal bath thermostat for 15 minutes. Add 45 μL of buffer C1 (each component and final concentration are shown in Example 1) to all groups, mix well, close the lid of the reaction tube, and incubate at 42°C in a micro-metal bath thermostat for 5 minutes. After opening the lid, add 40 μL of buffer C2 (see Example 1 for each component and final concentration), mix well, close the lid of the reaction tube, and continue incubating at 42°C in a micro-metal bath thermostat for 5 minutes. Cool at room temperature for 5 minutes. During the 5 minutes of waiting, prepare fresh buffer D (see Example 1 for each component and final concentration). Then take 5 μL of buffer D and add it to the cooled hybridization product. Color develop at room temperature for 5 minutes. Observe the results with the naked eye, take photos with a smartphone, and use an ELISA reader to detect the absorbance value (OD) of the product at a wavelength of 414 nm. 414 ). All reactions were performed three times to ensure reproducibility.

[0098] The results showed that the method could detect Clostridium difficile strains, while other bacterial strains were negative ( Figure 6 (B), indicating that the system has good specificity.

[0099] (3) Detection of Clostridium difficile clinical samples.

[0100] Feces of patients clinically diagnosed with Clostridium difficile infection were obtained, and genomic DNA was extracted from the feces using buffer A according to the method in Example 1, and subsequent RPA amplification and downstream detection were performed.

[0101] The results showed that the 6 stool samples infected with Clostridium difficile and the standard strain of Clostridium difficile (CD43255) were all bright green, and no signal was obtained in the negative control ( Figure 6 These results indicate that this method can detect Clostridium difficile in stool samples and has application value.

Claims

1. A probe cluster, characterized in that: Used to detect double-stranded DNA of target genes, the target genes include the conserved gene of Clostridium difficile toxin B, and the probe cluster includes probe group 1, probe group 2, probe group 3 and probe group 4; The probe group 1, probe group 2, probe group 3 or probe group 4 each comprises two single-stranded probes that can complementarily pair with two adjacent nucleic acid sequences on the single-stranded DNA of the target gene, and the 3' end and 5' end of each single-stranded probe respectively contain at least two locked nucleic acid modified bases; Probe group 1 and probe group 3 can hybridize with the continuously shifted regions on the same single-stranded DNA of the target gene, respectively; probe group 2 and probe group 4 can hybridize with the continuously shifted regions on another complementary single-stranded DNA of the target gene, respectively; and the two single-stranded probes of probe group 1 and probe group 4, and probe group 2 and probe group 3 are complementary pairs; The probe cluster further includes: competitive probe group I and competitive probe group II; Competitive probe group I is used to prevent the complementary pairing of two single-stranded probes of probe group 1 with two single-stranded probes of probe group 4, and competitive probe group II is used to prevent the complementary pairing of two single-stranded probes of probe group 2 with two single-stranded probes of probe group 3; The nucleotide sequences of the two single-stranded probes included in probe group 1 are shown as SEQ ID No.5 and SEQ ID No.6, respectively; the nucleotide sequences of the two single-stranded probes included in probe group 2 are shown as SEQ ID No.7 and SEQ ID No.8, respectively; the nucleotide sequences of the two single-stranded probes included in probe group 3 are shown as SEQ ID No.9 and SEQ ID No.10, respectively; the nucleotide sequences of the two single-stranded probes included in probe group 4 are shown as SEQ ID No.11 and SEQ ID No.12, respectively; Competitive probe I includes competitive probe 1A and competitive probe 1B, whose sequences are shown in SEQ ID No.13 and SEQ ID No.14 respectively; competitive probe II includes competitive probe 2A and competitive probe 2B, whose sequences are shown in SEQ ID No.15 and SEQ ID No.16 respectively.

2. A kit containing the probe cluster according to claim 1, characterized in that: The kit also contains heme, ABTS 2− .

3. The kit according to claim 2, characterized in that The kit also contains upstream and downstream primers for amplifying double-stranded DNA of target gene, recombinase, single-stranded DNA binding protein of target gene, and strand displacement Bsu polymerase.

4. Use of the probe cluster according to claim 1 in preparing a Clostridium difficile detection kit.

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