Method for detecting clostridium difficile through fluorescence of silver nano-cluster compiled and guided by G-rich sequence enhanced DNA promoted by entropy-driven isothermal amplification primer

Abstract

The invention relates to a method for detecting clostridium difficile through fluorescence of a G-rich sequence enhanced DNA (Deoxyribose Nucleic Acid) compiled silver nanocluster promoted by an entropy-driven isothermal amplification primer. The detection method comprises the following steps: 1, screening a proper primer c-Apt combined with the aptamer Apt; 2, preparing magnetic beads modified by double-stranded DNA formed by combining the aptamer Apt and the primer c-Apt into a premixed reaction solution; 3, mixing RNase H2 of a target object clostridium difficile with the premixed reaction solution, and carrying out incubation reaction; 4, carrying out identification and magnetic separation; 5, carrying out entropy driven amplification on the primer c-Apt; 6, enhancing the fluorescence of the DNA-compiled silver nano-cluster by a G-rich sequence; and 7, identifying a fluorescence signal, and judging positive / negative. According to the method, the entropy-driven isothermal amplification technology is utilized to amplify the primers and the G-rich sequence close to the silver nano-cluster enhanced fluorescence to doubly amplify fluorescence signals, so that the accuracy and the practicability of the method are improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for detecting Clostridium difficile by using an entropy-driven isothermal amplification primer to promote G-rich sequence-enhanced DNA-directed silver nanocluster fluorescence. Background Art

[0002] Clostridioides difficile is a Gram-positive, large rod-shaped bacillus. The use of antibiotics can lead to overgrowth of the bacterium. With the widespread use of broad-spectrum antibiotics, the incidence of Clostridioides difficile-associated diarrhea has continued to increase, and outbreaks have occurred in recent years. Its epidemic strains have undergone genetic mutations, increased the ability to produce toxins, and increased the mortality rate and recurrence rate of patients. At present, the technology for rapid and sensitive detection of Clostridioides difficile has yet to be developed. Therefore, the development of rapid, sensitive, and convenient detection methods for Clostridioides difficile is crucial for public health security and the stable development of social economy.

[0003] Entropy-driven isothermal amplification (EDC) is a new in vitro nucleic acid amplification technology that has low temperature requirements and does not require expensive and sophisticated temperature cycling equipment. It can be completed at a constant temperature. Using EDC technology to detect Clostridium difficile can help reduce the difficulty of detection operations, shorten detection time, and reduce detection costs. It does not require reliance on specific machines and can be interpreted by the naked eye.

[0004] Over the past few decades, many fluorescence detection methods such as organic fluorophores and quantum dots have been developed, but there are problems such as high modification cost and poor photostability. Noble metal nanoclusters are considered to be alternatives to organic fluorophores and quantum dots in the fields of chemical sensing, bioassays and bioimaging due to their fluorescence emission characteristics. In particular, silver nanoclusters (AgNCs) have attracted considerable attention. In order to improve the accuracy and reliability of Clostridium difficile detection, the present invention is committed to designing a method for enhancing the fluorescence of silver nanoclusters with G-rich sequences to enhance the signal amplification efficiency of the system. Summary of the invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a method for detecting Clostridium difficile by using entropy-driven isothermal amplification technology to amplify primers and promote G-rich sequences to enhance DNA-directed silver nanoclusters fluorescence. The detection method has the advantages of being rapid, sensitive and convenient.

[0006] The present invention specifically adopts the following technical solutions:

[0007] In a first aspect, the present application claims protection for an entropy driven isothermal amplification technology amplification primer, the sequence of the primer being SEQ ID NO:7.

[0008] Secondly, the present application requests protection for a method for preparing amplification primers using entropy-driven isothermal amplification technology, wherein a cDNA that complementarily binds to the aptamer Apt is designed at the junction of the neck loop where the aptamer Apt has strong binding ability to the RNase H2 of the target Clostridium difficile, and the length of the cDNA is increased by 2 bases from 14 bases to 24 bases, and a cDNA with a length of 24 bases is selected as the primer.

[0009] In a third aspect, the present application claims a method for detecting Clostridium difficile by using G-rich sequence-enhanced DNA-directed silver nanoclusters for fluorescence, comprising the following steps:

[0010] Step 1: Prepare primers as described above:

[0011] Step 2: The double-stranded DNA (dsDNA) formed by the combination of the biotin-modified aptamer Apt and the primer is fixed on the magnetic beads modified with streptavidin to prepare a premixed reaction solution;

[0012] Step 3: Mix the RNase H2 of Clostridium difficile with the premixed reaction solution and perform an incubation reaction;

[0013] Step 4: the aptamer Apt recognizes the RNase H2 of Clostridium difficile, so that the primer bound to the aptamer Apt is released;

[0014] Step 5: Design the DNA single strand X1 and DNA single strand X2 required for entropy-driven isothermal amplification, the primer and DNA single strand X2 are partially complementary to the DNA single strand X1, add the fuel chain F and the primer released in step 4 to the triplex of the primer, DNA single strand X2 and DNA single strand X1 pre-combined at a molar ratio of 1:1:1, the primer added later combines with the DNA single strand X1 and competes with the DNA single strand X2, and obtains a triplex structure formed by the combination of DNA single strand X1 and the primer at a molar ratio of 1:2, stimulates the combination of fuel chain F and DNA single strand X1, releases the primer 1 times more than the added amount, and the primer continues to perform a new round of entropy-driven isothermal amplification;

[0015] Step 6: Design a hairpin DNA sequence Y1 and a single-stranded DNA X3, wherein the loop of the hairpin DNA sequence Y1 is a C-rich sequence, synthesize silver nanoclusters (AgNCs) on the loop, add the primers amplified in step 5, and the primers open the hairpin DNA sequence Y1, promote the binding of the single-stranded DNA X3 with a G-rich sequence to the hairpin DNA sequence Y1, and enhance the fluorescence of the silver nanoclusters;

[0016] Step 7: Identify the fluorescent signal and determine positive / negative;

[0017] Wherein, the sequences of DNA single strand X1, DNA single strand X2, and fuel strand F in step (5) are: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, respectively;

[0018] In the step (6), the sequences of the hairpin DNA sequence Y1 and the DNA single strand X3 are: SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0019] Furthermore, in step (2), the magnetic beads and dsDNA are fixed by slowly shaking and mixing at 25° C. for 1 hour.

[0020] Furthermore, in step (3), the reaction between RNase H2 of Clostridium difficile and magnetic beads modified with dsDNA is carried out by incubating at 25° C. for 20 minutes.

[0021] Furthermore, in step (5), the concentration of the primers is 2 μM, the reaction time of the entropy-driven isothermal amplification reaction is 40 min, and the reaction temperature is 37° C.

[0022] Furthermore, in step (5), a small amount of the primers are amplified by entropy-driven isothermal amplification to obtain a large amount of the primers, thereby completing exponential amplification, reducing the detection limit and improving sensitivity.

[0023] Furthermore, in the step (6), the molar ratio of the hairpin DNA sequence Y1, AgNO3, and NaBH4 is hairpin DNA sequence Y1:AgNO3:NaBH4=1:16:8.

[0024] Furthermore, the concentration of AgNCs in step (6) is 3 μM.

[0025] Furthermore, the buffer used for the synthesis of AgNCs in step (6) is phosphate buffered saline (PBS) with a concentration of 300 mM and a pH of 7.0-7.4.

[0026] Furthermore, in step (6), the fluorescence of the silver clusters is enhanced 100 times.

[0027] Furthermore, in step (6), the excitation wavelength of the silver cluster fluorescence is 493 nm, and the emission wavelength is 582 nm.

[0028] The present invention adopts the above solution, which has the following advantages compared with the prior art:

[0029] (1) In the magnetic separation part, the primer (referred to as primer c-Apt, the same below) will be released only when the aptamer Apt recognizes the target RNase H2 of Clostridium difficile, which has high sensitivity and strong specificity.

[0030] (2) A specific amplification primer c-Apt was designed for the aptamer Apt, and the exponentially growing primer c-Apt was obtained by the entropy-driven isothermal amplification method, which shortened the detection time and saved production and detection costs. It has good detection sensitivity and strong specificity.

[0031] (3) This method solves the problems of high cost and complicated operation of fluorescent dye labeling.

[0032] (4) Due to its simple operation, good sensitivity, short detection time, and low detection cost, this method provides an efficient platform for detecting RNase H2 of the target Clostridium difficile by promoting the amplification of G-rich sequences through DNA-encoded AgNCs using the amplification primer c-Apt. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 Schematic diagram of the method for detecting Clostridium difficile by enhancing the fluorescence of DNA-directed silver nanoclusters for G-rich sequences;

[0035] Figure 2 is an electrophoresis diagram of the binding of cDNA of different lengths to the aptamer Apt, wherein: Figure 2A The molar ratio of aptamer Apt and primer c-Apt added is aptamer Apt: primer c-Apt = 1:2. Figure 2B The molar ratio of aptamer Apt and primer c-Apt added was aptamer Apt: primer c-Apt = 2:1;

[0036] Figure 3 The release of the primer c-Apt after magnetic selection is shown in the figure below:

[0037] Figure 4 shows the optimization of the reaction conditions of the method. Figure 4A It is the optimization of the concentration of primer c-Apt in triplex. Figure 4B is the optimization of the concentration of silver nanoclusters. Figure 4C It is the optimization of amplification reaction time. Figure 4D It is the optimization of amplification reaction temperature;

[0038] Figure 5 The standard curve of RNase H2 of the target Clostridium difficile at different concentrations;

[0039] Figure 6 is the selectivity graph of the method. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of 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.

[0041] Example 1 Sequence Design

[0042] The sequences for detecting Clostridium difficile of the present invention are shown in Table 1:

[0043] Table 1 Sequences related to Clostridium difficile detection

[0044]

[0045] Example 2 A method for detecting Clostridium difficile by using entropy-driven isothermal amplification technology to amplify primers and promote G-rich sequences to enhance DNA-directed silver nanoclusters fluorescence, see Attachment Figure 1 , including the following steps:

[0046] (1) Screening out a suitable primer c-Apt that binds to the aptamer Apt: A single-stranded DNA (cDNA) that is complementary to the aptamer Apt is designed at the junction of the neck loop where the aptamer Apt has a strong binding ability to the nuclease RNase H2 of Clostridium difficile. The length of the cDNA increases by 2 bases from 14 bases to 24 bases. The specific sequence is shown in Table 1 (from c-Apt-14 to primer c-Apt).

[0047] (2) 1 μM of aptamer Apt was reacted with 0.5 μM of complementary single-stranded DNA (cDNA) with different base numbers at 95°C for 5 min and then at 4°C for 20 min to allow double-stranded binding. Under the same experimental conditions, 0.5 μM of aptamer Apt was combined with 1 μM of cDNA with different base numbers.

[0048] (3) Then prepare 12% polypropylene gel and run the sample at 105V for 60min. The experimental results are as follows: Figure 2A and Figure 2B The results showed that the aptamer Apt could bind to cDNAs of different lengths. Considering the design of subsequent experiments, a cDNA with a length of 24 bases was selected as the primer c-Apt.

[0049] The aptamer Apt bound to the magnetic beads recognizes the target ribonuclease H2 (RNase H2) of Clostridium difficile and releases the primer c-Apt, including the following steps:

[0050] (1) The double-stranded DNA (dsDNA) formed by the combination of biotin-modified aptamer Apt and primer c-Apt is fixed on the magnetic beads modified with streptavidin to prepare a premixed reaction solution: 10 μM biotin-modified aptamer Apt and 20 μM primer c-Apt are reacted at 95°C for 5 min, and then reacted at 25°C for 30 min, and the two chains are combined to form dsDNA. 10 μL of the reaction solution is added to 90 μL of the magnetic bead solution modified with streptavidin, and the mixture is slowly shaken at 25°C for 1 h by flipping the inverted tube, and the magnetic beads are collected with a magnetic separation rack, and the supernatant is discarded. Wash with 50 mM HEPES buffer 5 times, and add 90 μL HEPES buffer to prepare a premixed reaction solution.

[0051] The HEPES buffer contained 150 mM NaCl, 6 mM KCl, 2.5 mM MgCl2, 2.5 mM CaCl2, 0.01% v / v Tween-20, pH=7.4.

[0052] (2) Aptamer Apt recognizes RNase H2 of target Clostridium difficile, and primer c-Apt is released: 10 μL of target solution is added to the reaction solution described in (1), reacted at 25°C for 20 min, and the supernatant is collected by magnetic separation. Aptamer Apt recognizes RNase H2 of target Clostridium difficile. Since aptamer Apt has a stronger binding ability with RNase H2 of target Clostridium difficile, primer c-Apt bound to aptamer Apt is released. The results are shown in FIG. Figure 3 shown.

[0053] Entropy-driven isothermal amplification using primer c-Apt

[0054] Before adding primers, DNA single strand X1, DNA single strand X2, and primer c-Apt first form a triplex structure in the TAMg buffer system, 2 μM DNA single strand X1 and DNA single strand X2 react at 95°C for 5 min, then react at 4°C for 20 min, and then continue to add 2 μM primer c-Apt, incubate at 37°C for 30 min to form a triplex structure, then add the supernatant obtained above to the system, the primer c-Apt released in the supernatant serves as the initiator chain, and continue to add 2 μM fuel chain F, and the system is incubated at 37°C for 40 min for entropy-driven isothermal amplification reaction, and the primer c-Apt in the system in single-stranded form is exponentially amplified.

[0055] The TAMg buffer contained 45 mM Tris-Base, 12.5 mM MgCl2, pH=8.

[0056] G-rich sequences enhance the fluorescence of DNA-directed silver nanoclusters

[0057] 20 μM DNA single strand Y1 containing C-rich sequence was heated and annealed in PBS buffer solution to form a hairpin structure, then 1.6 mM AgNO3 was added, shaken vigorously for 2 min, incubated at 25°C in the dark for 20 min, and then 0.8 mM NaBH4 was added, and reduced at 25°C in the dark for 1 min to obtain silver nanoclusters that can emit weak fluorescence. 20 μM DNA single strand X3 was added to the solution for use.

[0058] The PBS buffer contains 225 mM NaH2PO4·2H2O, 75 mM Na2HPO4, pH: 7.0-7.4.

[0059] The hairpin DNA sequence Y1 of the synthetic silver cluster is added to the above system, and the primer c-Apt that has completed amplification is mixed with 3μM hairpin DNA sequence Y1. The primer c-Apt opens the hairpin DNA sequence Y1, promotes the binding of the DNA single strand X3 containing the G-rich sequence to the hairpin DNA sequence Y1, and the G-rich sequence is close to the silver nanocluster to enhance fluorescence. Further, under ultraviolet light irradiation, weak or no fluorescence is negative, and strong fluorescence is positive.

[0060] The excitation wavelength for reading the silver nanocluster fluorescence is 493 nm, and the emission wavelength is 582 nm.

[0061] Optimization of reaction conditions

[0062] In order to obtain the best reaction effect, the concentration of primer c-Apt in the triplex of the proposed method ( Figure 4A ), silver nanocluster concentration ( Figure 4B ), amplification reaction time ( Figure 4C ) and the amplification reaction temperature ( Figure 4D According to the optimization results, the concentration of primer c-Apt was 2 μM, the concentration of silver cluster was 3 μM, the reaction time was 40 min, and the reaction temperature was 37°C.

[0063] Detection performance verification of the method

[0064] In order to verify the detection performance and sensitivity of the proposed method, RNase H2 of the target Clostridium difficile at different concentrations was detected after the conditions were optimized. The results are shown in the figure. Figure 5When the concentration of the target DNA is lower than 100 nM, there is no signal output. When the concentration of the target DNA is in the range of 100 nM to 800 nM, the signal shows a good linear relationship. According to the 3σ method, the detection limit is calculated to be as low as 7 pM.

[0065] Selectivity verification of the method In order to verify the selectivity of the constructed method for Clostridium difficile, genomic DNA extracted from Salmonella, Shigella, Escherichia coli, Staphylococcus aureus and Clostridium difficile was tested, and a control group was added. The test results are shown in Figure 2. Figure 6 As shown, the results showed that there was a strong signal output only when RNase H2 of Clostridium difficile was present, proving that the constructed method had good selectivity.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An entropy driven isothermal amplification primer, characterized in that: The sequence of the primer is SEQ ID NO:

7.

2. A method for preparing amplification primers using entropy-driven isothermal amplification technology, characterized in that: A cDNA complementary to the aptamer Apt is designed at the neck loop junction where the aptamer Apt has strong binding ability with the target Clostridium difficile RNase H2. The length of the cDNA increases by 2 bases from 14 bases to 24 bases, and a cDNA with a length of 24 bases is selected as the primer.

3. A method for detecting Clostridium difficile by using G-rich sequence-enhanced DNA-directed silver nanoclusters fluorescence, characterized in that: The following steps are involved: Step 1: Prepare the primer as claimed in claim 1: Step 2: The double-stranded DNA (dsDNA) formed by the combination of the biotin-modified aptamer Apt and the primer is fixed on the magnetic beads modified with streptavidin to prepare a premixed reaction solution; Step 3: Mix the RNase H2 of Clostridium difficile with the premixed reaction solution and perform an incubation reaction; Step 4: the aptamer Apt recognizes the RNase H2 of Clostridium difficile, so that the primer bound to the aptamer Apt is released; Step 5: Design the DNA single strand X1 and DNA single strand X2 required for entropy-driven isothermal amplification, the primer and DNA single strand X2 are partially complementary to the DNA single strand X1, add the fuel chain F and the primer released in step 4 to the triplex of the primer, DNA single strand X2 and DNA single strand X1 pre-combined at a molar ratio of 1:1:1, the primer added later combines with the DNA single strand X1 and competes with the DNA single strand X2, and obtains a triplex structure formed by the combination of DNA single strand X1 and the primer at a molar ratio of 1:2, stimulates the combination of fuel chain F and DNA single strand X1, releases the primer 1 times more than the added amount, and the primer continues to perform a new round of entropy-driven isothermal amplification; Step 6: Design a hairpin DNA sequence Y1 and a single-stranded DNA X3, wherein the loop of the hairpin DNA sequence Y1 is a C-rich sequence, synthesize silver nanoclusters (AgNCs) on the loop, add the primers amplified in step 5, and the primers open the hairpin DNA sequence Y1, promote the binding of the single-stranded DNA X3 with a G-rich sequence to the hairpin DNA sequence Y1, and enhance the fluorescence of the silver nanoclusters; Step 7: Identify the fluorescent signal and determine positive / negative; Wherein, the sequences of DNA single strand X1, DNA single strand X2, and fuel strand F in step (5) are: SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively; In the step (6), the sequences of the hairpin DNA sequence Y1 and the DNA single strand X3 are: SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

4. The method according to claim 3, characterized in that In step (2), the magnetic beads and dsDNA are fixed by slowly shaking and mixing at 25°C for 1 hour.

5. The method according to claim 3, characterized in that: In step (3), the reaction between RNase H2 of Clostridium difficile and magnetic beads modified with dsDNA is carried out by incubating at 25° C. for 20 minutes.

6. The method according to claim 3, characterized in that: The concentration of the primers in step (5) is 2 μM, the reaction time of the entropy-driven isothermal amplification reaction is 40 min, and the reaction temperature is 37° C.

7. The method according to claim 3, characterized in that In step (5), a small amount of the primers are amplified by entropy-driven isothermal amplification to obtain a large amount of the primers, thereby completing exponential amplification, reducing the detection limit and improving sensitivity.

8. The method according to claim 3, characterized in that In the step (6), the molar ratio of the hairpin DNA sequence Y1, AgNO3, and NaBH4 is hairpin DNA sequence Y1:AgNO3:NaBH4=1:16:

8.

9. The method according to claim 3, characterized in that: The concentration of AgNCs in step (6) is 3 μM.

10. The method according to claim 3, characterized in that: The buffer used for the synthesis of AgNCs in step (6) is phosphate buffered saline (PBS) with a concentration of 300 mM and a pH of 7.0-7.

4.

11. The method according to claim 3, characterized in that In the step (6), the fluorescence of the silver clusters is enhanced 100 times.

12. The method according to claim 3, characterized in that In the step (6), the excitation wavelength of the silver cluster fluorescence is 493 nm, and the emission wavelength is 582 nm.

Images