A circular DNA template precursor for detecting Clostridium difficile infection, its preparation method and application

Abstract

The present invention provides a circular DNA template precursor for detecting Clostridium difficile, a preparation method thereof and an application thereof. The circular DNA template precursor includes a three-dimensional self-assembly reaction using a reaction system composed of H1 and H2 hairpin probes and a trigger strand Trigger. The three-dimensional self-assembly reaction includes Trigger triggering the H1 hairpin structure to form a T / H1 intermediate, the H1 hairpin structure being unfolded and binding to H2 to produce an H1 / H2 structure, and the DNA template of H2 being cyclized to obtain the circular DNA template precursor. When Clostridium difficile toxin is present, the trigger strand Trigger can be released again, and can continuously catalyze the hairpin structure of the H1 DNA hairpin probe without being consumed. By adopting the technical scheme of the present invention, signal cascade three-stage amplification is realized through three-dimensional self-assembly-amplification reaction and silver-enhanced gold nanoparticles, so as to realize highly sensitive and highly specific detection of Clostridium difficile infection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology. Specifically, it relates to a circular DNA template precursor for Clostridioides difficile detection, its preparation method and application. Background Art

[0002] Clostridioides difficile infection (CDI) is a hospital-acquired infectious disease caused by Clostridioides difficile. Its pathogenic bacteria widely exist in the hospital environment and have become the most common cause of diarrhea in inpatients. After Clostridioides difficile infection, the mild cases can cause diarrhea, while the severe cases can present as toxic enteritis, pseudomembranous enteritis, or even death. Due to the characteristics of high incidence, high recurrence rate, and high mortality of Clostridioides difficile infection, it is one of the most important hospital infections that lead to poor prognosis and death of patients. The main reasons for the difficulty in controlling Clostridioides difficile infection and the high number of new patients are the long time, low efficiency, and high misdiagnosis rate of early diagnosis and differential diagnosis of Clostridioides difficile infection, which restricts the timeliness and effectiveness of clinical treatment. Therefore, how to establish a new method for sensitive, simple, and intelligent detection of Clostridioides difficile infection at the bedside is still a technical bottleneck to be solved urgently.

[0003] Clostridioides difficile infection (CDI) is one of the most important hospital infections that lead to poor prognosis and death of patients. Toxin release is the key to the pathogenesis of Clostridioides difficile infection. Most toxigenic strains can produce two toxins, toxin A (TcdA) and toxin B (TcdB), both of which can trigger a host cell cascade reaction, resulting in severe intestinal damage. Currently, the conventional glutamate dehydrogenase (GDH) detection method cannot distinguish toxigenic and non-toxigenic Clostridioides difficile, resulting in low detection specificity and unable to meet the diagnosis of clinical toxigenic Clostridioides difficile. The cytotoxicity assay (CCTA) and the toxigenic culture (TC) are the gold standard methods for detecting free toxins and toxigenic Clostridioides difficile respectively, with high detection specificity. However, these methods require a lot of time, delaying the patient's treatment decision and infection control. The toxin enzyme immunoassay (EIAs) also has high specificity, but the sensitivity of toxin detection by this method is low and the misdiagnosis rate is high.

[0004] In recent years, the detection methods using nucleic acid amplification tests (NAATs) have developed rapidly, with advantages such as high sensitivity, strong specificity, short detection time, and simple operation. However, this method has high costs, requires specialized equipment and professional operators, and only detects the toxin genes of Clostridium difficile, not the toxins themselves. It is difficult to distinguish asymptomatic colonization of Clostridium difficile from Clostridium difficile infection, with poor clinical specificity and easy to cause false positives.

[0005] Based on the above analysis, Summary of the Invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a DNA hydrogel for detecting Clostridium difficile, its preparation method and application. By using intelligent nano self-assembled hydrogel materials, signal three-stage amplification is achieved through three-dimensional self-assembled amplification reaction and silver-enhanced gold nanoparticles, so as to realize highly sensitive and highly specific detection of Clostridium difficile.

[0007] To achieve the above object, the present invention provides the following technical solution, a preparation method of a circular-DNA template precursor for detecting Clostridium difficile, including performing a three-dimensional self-assembled amplification reaction using a reaction system composed of an H1 DNA hairpin probe, an H2 DNA hairpin probe, and a trigger strand Trigger; the three-dimensional self-assembled amplification reaction includes the released trigger strand Trigger triggering the H1 DNA hairpin probe, opening the hairpin structure of the H1 DNA hairpin probe and forming a T / H1 intermediate, and the unfolded hairpin structure of the H1 DNA hairpin probe binding to the H2 DNA hairpin probe to generate an H1 / H2 structure, enabling the DNA template of the H2 DNA hairpin probe to circularize to obtain a circular-DNA template precursor, and the trigger strand Trigger sequence is released again; the released trigger strand Trigger can continuously catalyze the hairpin structure of the H1 DNA hairpin probe and interact with it without being consumed; the trigger strand Trigger is designed and obtained based on complete complementary pairing with the aptamer of toxin A or toxin B released by Clostridium difficile.

[0008] The gene sequence of the trigger strand Trigger is designed with complete complementary pairing to the gene sequence of the aptamer of toxin A or toxin B. When the circular-DNA template precursor is applied to Clostridium difficile infection, when there is Clostridium difficile toxin in the system, the chain length of the gene sequence of Clostridium difficile toxin complementary to the aptamer gene sequence is longer than the chain length of the aptamer gene sequence complementary to the trigger trigger. Clostridium difficile toxin binds to the aptamer to release the trigger strand Trigger, and the trigger trigger is displaced to release the trigger strand trigger, thereby achieving continuous catalysis.

[0009] Preferably, the sequence of the aptamer of the toxin is as shown in SEQ ID NO.7.

[0010] Preferably, the sequence of the trigger chain Trigger is as shown in SEQ ID NO.1.

[0011] Preferably, the sequence of the H1 DNA hairpin probe is as shown in SEQ ID NO.2.

[0012] Preferably, the sequence of the H2 DNA hairpin probe is as shown in SEQ ID NO.3.

[0013] The above is a preferred embodiment, and the sequence of the trigger chain Trigger is designed according to the sequence of the aptamer of toxin B released by Clostridium difficile. Or designing the sequence of the trigger chain Trigger using toxin A can also achieve the same technical effect.

[0014] Preferably, the reaction system further includes phi29 buffer.

[0015] Preferably, the volume ratio of the addition of the H1 DNA hairpin probe, the H2 DNA hairpin probe and the trigger chain Trigger is 1:1:1.

[0016] Preferably, the annealing program of the cycle conditions of the three-dimensional self-assembly reaction includes: denaturation: 95 °C, 2 min; annealing: 65 °C, 2 min, 60 °C, 6 min, 60 °C - 20 °C, decreasing by 0.5 °C every 30 seconds, 80 cycles; 20 °C, 30 s; 4 °C, 10 min.

[0017] Based on the above technical solution, the present invention also provides a circular-DNA template precursor for detecting Clostridium difficile.

[0018] The above preparation method or the obtained circular DNA template precursor can be applied to the detection of Clostridium difficile infection.

[0019] By using the circular DNA template precursor as a raw material to synthesize a DNA hydrogel to determine whether the sample to be tested is infected with Clostridium difficile; if the DNA hydrogel is successfully synthesized, the sample to be tested is positive; if the DNA hydrogel cannot be synthesized, the sample to be tested is negative.

[0020] Alternatively, using the three-dimensional self-assembly reaction of the circular DNA template precursor, combined with the PCR amplification reaction, to synthesize the DNA hydrogel, and to judge the Clostridium difficile infection, the judgment method is the same.

[0021] As another one of the objects of the present invention, the present invention also provides a method for detecting Clostridium difficile infection, which includes using a circular DNA template precursor, as well as specific primers, gold nanoparticles and PCR reaction reagents to perform an amplification reaction, and judging positive or negative by whether the DNA hydrogel is synthesized.

[0022] Alternatively, after performing a three-dimensional self-assembly reaction by using a reaction system composed of an H1 DNA hairpin probe, an H2 DNA hairpin probe and a trigger strand Trigger to obtain a circular DNA template precursor, an amplification reaction is then carried out, and it is judged whether the sample to be tested is infected with Clostridium difficile by whether the DNA hydrogel can be obtained.

[0023] Preferably, the detection method includes:

[0024] Providing a circular DNA template precursor, and the preparation method is the same as the foregoing technical solution;

[0025] Preparing an amplification reaction system, including specific primers, gold nanoparticles, a circular DNA template precursor and PCR reaction reagents;

[0026] The specific primers include a first specific primer, a second specific primer and a third specific primer;

[0027] The sequences of the first specific primer, the second specific primer and the third specific primer respectively include those shown in SEQ ID NO.4-6;

[0028] After the circular DNA template precursor, the first specific primer, gold nanoparticles and PCR reaction reagents are incubated for the first time, the second specific primer and the third specific primer are added for reaction, and after the reaction ends, a second incubation is carried out to terminate the reaction. If the reaction product is the DNA hydrogel, it is judged as positive.

[0029] Preferably, the conditions for the first incubation include incubating at 30 °C for 2 hours.

[0030] Preferably, the conditions for the second incubation include incubating at 65 °C for 10 min.

[0031] Preferably, the volume ratio of the addition of the circular DNA template precursor, the first specific primer, the second specific primer and the third specific primer is 6∶2∶1∶1.

[0032] Preferably, the PCR reaction reagents include phi29 polymerase reaction buffer, BSA, dNTP, T4 ligase, phi29 polymerase and DEPC water (nuclease-free water).

[0033] With the above technical solution, the H1 DNA hairpin probe and the H2 DNA hairpin probe are three-dimensionally self-assembled through the release of the trigger chain Trigger to form a circular DNA template precursor, realizing the first amplification of the detection signal for Clostridium difficile infection; using the circular DNA template precursor and combining with specific primers and phi29 polymerase reagent to perform an amplification reaction to obtain a DNA hydrogel, realizing the second amplification of the detection signal for Clostridium difficile infection, which can significantly improve the sensitivity of detecting the toxin released by Clostridium difficile, and improve the specificity of detecting Clostridium difficile through the designed specific primers.

[0034] In order to further improve the detection sensitivity and avoid false negatives, based on the above solution, the present invention stains the DNA hydrogel by silver staining method, and uses the gold nanoparticles added during the preparation of the DNA hydrogel to complete the reduction of silver ions in the silver nanoparticle-enhanced staining agent, realizing the third signal amplification; the end of the first specific primer is modified with biotin, and the gold nanoparticles can specifically bind to biotin, thus providing a basis for the staining of the DNA hydrogel.

[0035] Specifically, the staining method includes: washing the DNA hydrogel with DEPC water; staining the DNA hydrogel with the silver nanoparticle-enhanced staining agent in a light-proof environment, reducing silver ions to metallic silver, and the DNA hydrogel shows black, completing the staining and realizing the third amplification of the signal; after the staining, if the obtained DNA hydrogel is black, it is judged as positive.

[0036] As one of another object of the present invention, the circular DNA template precursor provided by the above technical solution is used for the preparation of the DNA hydrogel, and the preparation method of the DNA hydrogel includes:

[0037] Performing a three-dimensional self-assembly amplification reaction with a reaction system composed of an H1 DNA hairpin probe, an H2 DNA hairpin probe and a trigger chain Trigger to obtain a circular DNA template precursor; the trigger chain Trigger is designed to be complementary and paired according to the aptamer of the toxin released by Clostridium difficile; the sequence of the trigger chain Trigger is shown in SEQ ID NO.1;

[0038] The sequence of the H1 DNA hairpin probe is shown in SEQ ID NO.2;

[0039] The sequence of the H2 DNA hairpin probe is shown in SEQ ID NO.3;

[0040] After the first incubation of the first specific primer, gold nanoparticles and PCR reaction reagents, the second specific primer and the third specific primer are added for reaction. After the reaction ends, a second incubation is carried out. After terminating the reaction, the DNA hydrogel is obtained.

[0041] The sequences of the first specific primer, the second specific primer and the third specific primer respectively include those shown in SEQ ID NO.4-6.

[0042] Based on the above technical solution, when this detection method is applied to a kit,

[0043] The DNA hydrogel material prepared by the technical solution provided by the present invention is an intelligent nano self-assembled hydrogel material. Through three-dimensional self-assembly-amplification reaction and silver-enhanced gold nanoparticles, cascade three-stage amplification of signals is realized, so as to achieve highly sensitive and highly specific detection of Clostridium difficile.

[0044] When the above technical solution is applied to rapid detection, the present invention also provides a kit for detecting Clostridium difficile virus, which at least includes H1 DNA hairpin probe, H2 DNA hairpin probe, trigger chain Trigger and phi29 buffer; and the first specific primer, the second specific primer, the third specific primer, gold nanoparticles and PCR reaction reagents; it also includes other necessary components of the kit.

[0045] The beneficial effects achieved by the technical solution provided by the present invention compared with the prior art:

[0046] 1. By adopting the technical solution of the present invention, cascade three-stage amplification of the detection signal of Clostridium difficile is realized through three-dimensional self-assembly reaction, amplification reaction and silver-enhanced gold nanoparticles, so as to achieve highly sensitive and highly specific detection of Clostridium difficile.

[0047] 2. By adopting the technical solution of the present invention, the trigger chain Trigger triggers the H1 DNA hairpin probe, and the hairpin of the H1 DNA hairpin probe sequence is opened and self-assembled and combined with the H2 DNA hairpin probe sequence to generate the H1 / H2 structure, so that the DNA template of the H2 DNA hairpin probe is circularized to obtain a circular DNA template precursor, and the trigger chain Trigger can be released again to catalyze the self-assembly reaction cyclically without being consumed.

[0048] 3. The present invention uses PCR amplification technology to combine the circular DNA template precursor obtained by self-assembly with specific primers, and directly judges whether it is infected with Clostridium difficile through the reaction product. The detection result can be materialized, the judgment method is intuitive, and the sensitivity is high.

[0049] 4. The present invention realizes the cascaded three - stage amplification of signals through three - dimensional self - assembly - amplification reaction and silver - enhanced gold nanoparticles, thereby achieving the highly sensitive and highly specific detection of Clostridium difficile. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1a It is a schematic structural diagram of the H1 DNA hairpin probe provided in Example 1 of the present invention.

[0051] Figure 1b It is a schematic structural diagram of the H2 DNA hairpin probe provided in Example 1 of the present invention.

[0052] Figure 2 It is a schematic diagram of the electrophoresis results of the trigger chain Trigger, H1 DNA hairpin probe, H2 DNA hairpin probe and circular - DNA template precursor in Example 1 of the present invention.

[0053] Figure 3a It is a photo of the synthesized DNA hydrogel and amplification in Example 1 of the present invention.

[0054] Figure 3b It is an imaging diagram of nucleic acid staining of the synthesized DNA hydrogel in Example 1 of the present invention.

[0055] Figure 3c It is a physical photo of the synthesized DNA hydrogel in Example 1 of the present invention.

[0056] Figure 4 It is a photo of the DNA hydrogel synthesized in Comparative Example 1 of the present invention.

[0057] Figure 5 It is a comparative photo of the judgment of Clostridium difficile infection by synthesizing DNA hydrogels in Example 1 and Comparative Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] 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 are described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0059] The principle of primer and probe design in the present invention is to design the gene sequence of the complementary paired trigger strand Trigger according to the aptamer of toxin A or toxin B released by Clostridium difficile, and force the H1 DNA hairpin probe to open its hairpin structure and form a T / H1 intermediate with the trigger strand Trigger by releasing the trigger strand Trigger. The H1 hairpin structure that cannot base pair is unfolded and combined with the H2 hairpin structure to produce a more stable H1 / H2 structure, further triggering the cyclization of the DNA template of the H2 hairpin structure to obtain a circular DNA template precursor.

[0060] The gene sequence of the trigger strand Trigger is designed to be completely complementary paired with the gene sequence of the aptamer of toxin A or toxin B. The circular DNA template precursor is applied to Clostridium difficile infection. When there is Clostridium difficile toxin in the system, the chain length of the gene sequence of Clostridium difficile toxin complementary to the aptamer gene sequence is longer than that of the aptamer gene sequence complementary to the trigger. The Clostridium difficile toxin binds to the aptamer to release the trigger strand Trigger, and the trigger strand trigger is displaced to release the trigger strand trigger, thus realizing continuous catalysis.

[0061] The Trigger sequence is released again, and the released Trigger continuously catalyzes the interaction of the hairpin structures without being consumed, thus realizing the further cycle of Trigger to amplify the fluorescence signal.

[0062] Furthermore, the above circular DNA template precursor is used to judge Clostridium difficile infection. The judgment method includes using the circular-DNA template precursor to synthesize a DNA hydrogel to judge whether the sample to be tested is infected with Clostridium difficile; if the DNA hydrogel is successfully synthesized, the sample to be tested is positive; if the DNA hydrogel cannot be synthesized, the sample to be tested is negative.

[0063] Unless otherwise specified, the reagents or reactants used in the present invention are all commercially available.

[0064] The technical solution of the present invention will be described below through specific examples.

[0065] Example 1

[0066] This example provides a method for detecting Clostridium difficile. The specific steps include:

[0067] Step 1, prepare the gene sequence of the trigger strand Trigger designed to be complementary paired with the aptamer of toxin B released by Clostridium difficile. The nucleotide sequence of the trigger strand Trigger is shown in SEQ ID NO.1; the sequence of the aptamer of toxin B (toxB aptamer) is shown in SEQ ID NO.7.

[0068] Step 2: Provide H1 DNA hairpin probe and H2 DNA hairpin probe that can be triggered by it, as well as biotinylated specific primer-1, specific primer-2, and specific primer-3. Among them, the nucleotide sequence of the H1 DNA hairpin probe is shown in SEQ ID NO.2.

[0069] The nucleotide sequence of the H2 DNA hairpin probe is shown in SEQ ID NO.3.

[0070] The nucleotide sequence of specific primer-1 is shown in SEQ ID NO.4.

[0071] The nucleotide sequence of specific primer-2 is shown in SEQ ID NO.5.

[0072] The nucleotide sequence of specific primer-3 is shown in SEQ ID NO.6.

[0073] Specifically, the sequences of SEQ ID NO.1-6 are shown in Table 1, where bio refers to biotin.

[0074] Table 1 Sequence Listing

[0075]

[0076] See Figure 1a and Figure 1b , which are the H1 and H2 hairpin secondary structures simulated by software for the present invention. The synthesized circular-DNA template precursor is shown by the dashed box in the figure.

[0077] Step 3: Three-dimensional self-assembly reaction. Prepare the reaction system of the circular-DNA template precursor. As shown in Table 2, mix the H1 DNA hairpin probe, H2 DNA hairpin probe, and Trigger component of the trigger strand in a PCR tube, and supplement with 10×phi29 buffer to 20 μL to obtain the reaction system.

[0078] In this example, a fecal specimen of Clostridium difficile is selected as the test sample. After centrifuging and filtering the test sample, the supernatant is mixed with the above reaction system. When Clostridium difficile toxin is present, the Clostridium difficile toxin binds to the aptamer to release the trigger strand Trigger, thereby completing the subsequent reaction.

[0079] As another implementation manner, the test sample can also be added to the reaction system of the amplification reaction.

[0080] After mixing the circular-DNA template precursor, PCR reaction reagents, and the sample to be tested prepared in this step, if there is Clostridium difficile toxin in the sample to be tested, that is, the sample to be tested is infected with Clostridium difficile, the aptamer binds to the template sequence of the toxin, thereby releasing the trigger strand Trigger, and the trigger strand Trigger continuously catalyzes the hairpin structure in a cycle, enabling the reaction to continue.

[0081] If the sample to be tested is not infected with Clostridium difficile, the subsequent reaction cannot continue.

[0082] Table 2 Reaction system of circular-DNA template precursor

[0083]

[0084] Place the PCR tube with a 20 μL reaction system into a PCR instrument (model: Genepro), and set the annealing program using the following cycling conditions: Denaturation: 95 °C, 2 min; Annealing: 65 °C, 2 min, 60 °C, 6 min, 60 °C - 20 °C, decreasing by 0.5 °C every 30 s, 80 cycles; 20 °C, 30 s; 4 °C, 10 min.

[0085] Based on the above annealing program, the reaction system undergoes a three-dimensional self-assembly reaction. The released trigger strand Trigger forces the H1 DNA hairpin probe to open its hairpin structure and form a T / H1 intermediate, while the H1 hairpin structure that cannot base-pair is unfolded and combines with H2 to produce a more stable H1 / H2 structure, resulting in the cyclization of the DNA template of H2 to obtain a circular-DNA template precursor, and the Trigger sequence is released again.

[0086] When the sample to be tested contains Clostridium difficile toxin, the trigger strand Trigger can be released, and the released Trigger continuously catalyzes the interaction of the hairpin structures without being consumed, thereby achieving the first amplification of the detection signal.

[0087] Perform gel electrophoresis testing on the prepared circular-DNA template precursor, and evaluate the three-dimensional self-assembly performance of the H1 DNA hairpin probe and the H2 DNA hairpin probe. Specifically, it includes preparing a 15% nucleic acid non-denaturing polyacrylamide gel electrophoresis test, referring to Figure 2, it is possible to analyze the interaction between DNA strands and DNA hairpins. In the figure, lanes 1-5 represent the following respectively: Lane 1: Trigger strand; Lane 2: H2 DNA hairpin probe; Lane 3: H1 DNA hairpin probe; Lane 4: H1 DNA hairpin probe + H2 DNA hairpin probe; Lane 5: Trigger strand + H1 DNA hairpin probe + H2 DNA hairpin probe. As can be seen from the figure, the Trigger strand can trigger the H1 hairpin structure, open the H1 hairpin structure and form a T / H1 intermediate. The H1 hairpin structure is unfolded and combines with the H2 hairpin structure to form an H1 / H2 structure, causing the DNA template of the H2 DNA hairpin probe to circularize.

[0088] Step 4: Use the above-mentioned circular-DNA template precursor to synthesize a DNA hydrogel to determine whether the test sample is infected with Clostridium difficile. If the DNA hydrogel is successfully synthesized, the test sample is positive; if the DNA hydrogel cannot be synthesized, the test sample is negative. The specific steps include:

[0089] 1. Incubate the circular DNA template precursor prepared in Step 3 above, specific primer-1, gold nanoparticles, and PCR reaction reagents for the first time. The incubation conditions include incubating at 30 °C for 2 hours; the PCR reaction reagents include phi29 polymerase reaction buffer, BSA, dNTP, T4 ligase, Phi29 polymerase, and DEPC water.

[0090] 2. Add 0.5 μL of 100 μM specific primer-2 and 0.5 μL of 100 μM specific primer-3 and continue the reaction for 10 hours. After the reaction is completed, incubate at 65 °C for 10 min to terminate the reaction. If the reaction product is a DNA hydrogel, it is judged as positive.

[0091] The second-stage signal amplification of the detection signal is achieved through the action of phi29 enzyme for amplification reaction. Whether a DNA hydrogel can be obtained is used as the basis for judging whether the test sample is infected with Clostridium difficile. The judgment method can be materialized, simple, and intuitive.

[0092] Among them, the sequences of specific primer-1, second specific primer-2, and third specific primer-3 are SEQ ID NO.3-6 respectively, see Table 1; a biotin group is modified at the end of specific primer-1.

[0093] The reaction system for the synthesis of the DNA hydrogel is shown in Table 3.

[0094] Table 3 Reaction system for the synthesis of DNA hydrogel

[0095] Component Dosage 10×phi29 polymerase reaction buffer 1.8 μL BSA (5 mg / mL) 0.5 μL dNTP (20 mM) 2 μL Bio-specific primer-1 (100 μM) 1 μL DEPC water 8.2 μL Circular-DNA template precursor 3 μL Phi29 polymerase (10 units / μL) 2.5 μL T4 ligase (400 units / μL) 1 μL Gold nanoparticles (80 μg / mL) 0.2 μL Total 20 μL

[0096] Step 5, staining: Wash the DNA hydrogel prepared in Step 4 above with Tris-HCL buffer (10 mM Tris, 5 mM MgCl2, 300 mM NaCl); stain the DNA hydrogel with a silver nanoparticle-enhanced stain for 1 minute in a light-protected environment to reduce silver ions to metallic silver, making the DNA hydrogel appear black to complete the staining; after staining, the black DNA hydrogel is obtained, which is judged as positive.

[0097] The silver nanoparticle-enhanced stain comprises 15 μL of Reagent A (S5020 from SIGMA) and 15 μL of Reagent B (S5145 from SIGMA).

[0098] In this example, gold nanoparticles are added during the synthesis of the DNA hydrogel, which can bind to the biotin group modified at the end of the specific primer-1. In the staining step, the gold nanoparticles play a liquefying role, reducing silver nanoparticles to silver atoms and forming a "silver shell" around the gold particles. Due to the liquefying effect of the gold particles, more silver ions are reduced, the "silver shell" increases, and finally the antigen position is amplified, thereby realizing the third amplification of the signal of the present invention and improving the detection sensitivity for detecting Clostridium difficile virus.

[0099] See Figure 3a - Figure 3c , wherein, Figure 3a The figures in are the DNA hydrogel products obtained by adding the trigger strand trigger in this example and their enlarged views respectively. Figure 3b is the imaging diagram of the DNA hydrogel obtained by nucleic acid staining; Figure 3c is the physical photograph of the obtained DNA hydrogel. It can be judged that the sample to be tested is positive through the DNA hydrogel obtained from the reaction product.

[0100] Comparative Example 1

[0101] This comparative example is basically the same as the steps of Example 1, except that the trigger strand trigger is not added and the other steps are the same.

[0102] See Figure 4 , which is the reaction result obtained in this comparative example, and no hydrogel product is obtained.

[0103] See Figure 5 , where A in the figure is the reaction product obtained after staining in Comparative Example 1, no DNA hydrogel is synthesized in the figure, and B is the black DNA hydrogel obtained after staining in Example 1.

[0104] It can be seen by comparison that the trigger strand trigger can catalyze the formation of a circular DNA template precursor, thereby providing a detection basis for the synthesis of DNA hydrogels in judging whether a sample is infected with Clostridium difficile.

[0105] The above are only the preferred embodiments of the present invention, and thus do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any changes, modifications, substitutions, integrations, and parameter changes made to these embodiments within the spirit and principle of the present invention, by means of conventional substitutions or capable of achieving the same functions without departing from the principle and spirit of the present invention, shall fall within the protection scope of the present invention.

Claims

1. A detection method for Clostridium difficile, characterized in that, It includes an amplification reaction with DNA extracted from human feces as a template, a reaction system composed of H1 DNA hairpin probe, H2 DNA hairpin probe and trigger strand Trigger, as well as specific primers, gold nanoparticles and PCR reaction reagents, and positive or negative is judged by whether the DNA hydrogel is synthesized; If the DNA hydrogel is successfully synthesized, the test sample is positive; if the DNA hydrogel cannot be synthesized, the test sample is negative; The detection method is for non-diagnostic purposes; A three-dimensional self-assembly reaction is carried out on the reaction system composed of H1 DNA hairpin probe, H2 DNA hairpin probe and trigger strand Trigger; The three-dimensional self-assembly reaction includes the trigger strand Trigger triggering the H1 DNA hairpin probe, opening the hairpin structure of the H1 DNA hairpin probe sequence and forming a T / H1 intermediate. The hairpin structure of the H1 DNA hairpin probe is unfolded and combined with the H2 DNA hairpin probe sequence to generate an H1 / H2 structure, enabling the DNA template loop of the H2 DNA hairpin probe to circularize, obtaining a circular DNA template precursor; the trigger strand Trigger sequence can be released again; the released trigger strand Trigger can continuously catalyze the hairpin structure of the H1 DNA hairpin probe and interact with it without being consumed; The trigger strand Trigger sequence is designed to be completely complementary to the aptamer of the toxin released by Clostridium difficile; The sequence of the trigger strand Trigger is shown as SEQ ID NO.1; The sequence of the H1 DNA hairpin probe is shown as SEQ ID NO.2; The sequence of the H2 DNA hairpin probe is shown as SEQ ID NO.3; The sequence of the aptamer of the toxin is shown as SEQ ID NO.7; The volume ratio of the addition of the H1 DNA hairpin probe, the H2 DNA hairpin probe and the trigger strand Trigger is 1:1:1; The specific steps include: Configure an amplification reaction system, including specific primers, gold nanoparticles and circular DNA template precursor, as well as PCR reaction reagents; The specific primers include a first specific primer, a second specific primer and a third specific primer; The sequences of the first specific primer, the second specific primer and the third specific primer are shown as SEQ ID NO.4 - 6 respectively; After the circular DNA template precursor, the first specific primer, gold nanoparticles and PCR reaction reagents are first incubated, the second specific primer and the third specific primer are added for amplification reaction, and after the reaction ends, a second incubation is carried out to terminate the reaction. If the reaction product is the DNA hydrogel, it is judged as positive; The conditions for the first incubation include incubation at 30°C for 2 hours; The conditions for the second incubation include incubation at 65°C for 10 min; The volume ratio of the addition of the circular DNA template precursor, the first specific primer, the second specific primer and the third specific primer is 6:2:1:1; The PCR reaction reagent includes phi29 polymerase reaction buffer, BSA, dNTP, T4 ligase, phi29 polymerase, and DEPC water.

2. The detection method of Clostridium difficile according to claim 1, wherein The annealing program in the cycle conditions of the three-dimensional self-assembly reaction includes: denaturation: 95 °C, 2 min; annealing: 65 °C, 2 min, 60 °C, 6 min, 60 °C - 20 °C, cooling rate 0.5 °C / 30 s, 80 cycles; 20 °C, 30 s; 4 °C, 10 min.

3. The detection method of Clostridium difficile according to any one of claims 1-2, characterized in that, It also includes staining; washing the DNA hydrogel with DEPC water; staining the DNA hydrogel with a silver nanoparticle-enhanced stain in a light-protected environment to reduce silver ions to metallic silver, and the DNA hydrogel appears black, completing the staining and achieving the third amplification of the signal; after the staining, the black DNA hydrogel is obtained.

4. A kit for detecting Clostridium difficile virus, at least including a reaction system composed of H1 DNA hairpin probe, H2 DNA hairpin probe, trigger strand Trigger, as well as specific primers, gold nanoparticles, and PCR reaction reagent; The sequence of the trigger strand Trigger is designed to be fully complementary and paired according to the aptamer of the toxin released by Clostridium difficile. The sequence of the trigger strand Trigger is shown as SEQ ID NO.

1. The sequence of the H1 DNA hairpin probe is shown as SEQ ID NO.

2. The sequence of the H2 DNA hairpin probe is shown as SEQ ID NO.

3. The sequence of the aptamer of the toxin is shown as SEQ ID NO.

7. The volume ratio of the addition of the H1 DNA hairpin probe, the H2 DNA hairpin probe, and the trigger strand Trigger is 1:1:

1. The specific primers include a first specific primer, a second specific primer, and a third specific primer. The sequences of the first specific primer, the second specific primer, and the third specific primer are shown as SEQ ID NOs. 4 - 6 respectively. The PCR reaction reagent includes phi29 polymerase reaction buffer, BSA, dNTP, T4 ligase, phi29 polymerase, and DEPC water.

Images