DNAzyme capable of specifically recognizing acinetobacter baumannii as well as screening method and application of DNAzyme

Abstract

The invention provides DNAzyme capable of specifically recognizing acinetobacter baumannii as well as a screening method and application of the DNAzyme. The DNAzyme comprises a catalytic core sequence and a target recognition sequence, the nucleotide sequence of the catalytic core sequence is SEQ ID NO. 1; the nucleotide sequence of the target recognition sequence is SEQ ID NO. 2. A specific nucleic acid sequence of the acinetobacter baumannii is accurately locked through a target recognition sequence SEQ ID NO.2, so that high detection specificity is ensured, and cross reaction with other pathogenic bacteria is avoided; a catalytic core sequence SEQ ID NO.1 efficiently cuts a substrate chain after successful recognition, and a detectable signal change is generated, so that efficient and specific recognition and detection of the acinetobacter baumannii are realized, and a reliable basis is provided for clinical infection diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pathogenic bacteria detection and molecular biology, and in particular, to a DNAzyme capable of specifically recognizing Acinetobacter baumannii, and a screening method and application thereof. BACKGROUND

[0002] Acinetobacter baumannii (AB) is one of the most important pathogenic bacteria of nosocomial infection. More than 36% of ICU acquired pneumonia in Asia is related to Acinetobacter baumannii. Bloodstream infections caused by Acinetobacter baumannii account for more than 12% of all Acinetobacter baumannii hospital-acquired infections. AB infection increases the mortality rate of patients, the demand for ICU admission, the risk of secondary infection and readmission, the length of hospital stay and cost, and aggravates the medical burden. Clinically, the high-risk groups of AB infection need to be concerned.

[0003] Clinical microbiology laboratories often use traditional biochemical tests and automated bacterial identification systems (such as API20NE, Vitek 2, Phoenix and MieroSean WalkAway, etc.) to identify Acinetobacter. However, the members of Acb complex (mainly including A. baumannii, A. pittii, A. nosocomialis and Acinetobacter genogroup 13TU / 3) have very similar biochemical phenotypes and are difficult to distinguish. Therefore, it is usually necessary to use molecular biology methods to identify Acinetobacter to the species level. Pulsed field gel electrophoresis (PFGE) is the "gold standard" for bacterial molecular typing, and the most commonly used high-resolution technique is clamped homogeneous electric field electrophoresis (CHEF). However, it is time-consuming (2-4 days), requires high operation, and has no unified operation standard and standard interpretation. PCR is mainly based on detecting OXA-51 gene or 16S rRNA gene amplification analysis, however, the genes of Acb complex (Acb) are similar. The results of Nemec et al. show that the similarity of 16S rRNA genes between Acb strains is very high (97-98.1%), so caution is needed when using this method to identify A. baumannii, and hospital Acinetobacter and A. pittii interfere with the test. Metagenomic next-generation sequencing (mNGS) is a non-biased sequencing that can sequence and analyze all microbial nucleic acids detected in the sample. mNGS has the characteristics of broad spectrum and high sensitivity, and can achieve species-level identification and detect drug resistance genes in a relatively short time (usually 1-3 days), which can be applied to hospital infection outbreak prevention and control and drug resistance genome research, and has important significance for the anti-infection treatment of patients with multi-drug resistant bacteria infection and the development of hospital infection prevention and control measures. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can identify species within the Acb complex, and has a good guiding effect on clinical empirical medication. However, mNGS and MALDI-TOF MS require special equipment, are expensive, and require high technical requirements for operators, which are not suitable for primary hospitals and are not suitable for routine early screening of AB.

[0004] Therefore, there is an urgent need to provide a scheme for rapidly and simply detecting A. baumannii. SUMMARY

[0005] The main purpose of the present application is to provide a DNAzyme capable of specifically recognizing A. baumannii, and a screening method and application thereof, so as to at least solve the problems of high operation requirement and difficulty in early screening of A. baumannii in the prior art.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a DNAzyme capable of specifically recognizing Acinetobacter baumannii, comprising a catalytic core sequence and a target recognition sequence; the nucleotide sequence of the catalytic core sequence is SEQ ID NO. 1; and the nucleotide sequence of the target recognition sequence is SEQ ID NO. 2.

[0007] The second aspect of the present application provides a screening method of DNAzyme, which is applied to the DNAzyme capable of specifically recognizing Acinetobacter baumannii, and comprises the following steps:

[0008] Step 1: design and synthesize an initial library containing a specific DNA sequence of 94 bases, each DNA sequence consisting of three parts: a fixed sequence at both ends, a random sequence of 40 bases in the middle, and a fixed RNA cleavage site rA, the initial random sequence library containing 10 13 -10 16 sequence different DNA molecules to ensure that the target DNAzyme is included;

[0009] Step 2: using Acinetobacter baumannii CEM as the target and non-Acinetobacter baumannii CEM strains as the control, the initial DNA library is fixed by magnetic beads, and then incubated with the control and the target CEM;

[0010] Step 3: separate the magnetic beads from the solution, collect the supernatant containing the cleaved nucleic acid fragments, and use the fixed sequence in step 1 as the amplification primer to amplify the nucleic acid fragments in the solution by PCR, thereby reconstructing the full-length DNA library containing the complete fixed sequence, random region and RNA cleavage site rA for the next round of screening;

[0011] Step 4: repeat steps 2 to 3 for a total of ten rounds, each round using Acinetobacter baumannii CEM nucleic acid as the target and non-Acinetobacter baumannii CEM nucleic acid as the control for differential screening, and obtaining the enriched target DNAzyme sequence.

[0012] Optionally, in step 2, the magnetic beads and the solution are separated by using a magnetic stand.

[0013] Optionally, in step 2, the non-Acinetobacter baumannii includes Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

[0014] The third aspect of the present application provides the application of the DNAzyme capable of specifically recognizing Acinetobacter baumannii in the clinical detection of Acinetobacter baumannii infection.

[0015] Optionally, the target recognition sequence of the DNAzyme has a fluorescent signal group at the 3' end, and the fluorescent signal group is FAM or RhB.

[0016] The fourth aspect of the present application provides application of the DNAzyme capable of specifically recognizing Acinetobacter baumannii in a fluorescent detection probe for detecting Acinetobacter baumannii.

[0017] The fifth aspect of the present application provides a kit capable of specifically recognizing and detecting Acinetobacter baumannii, wherein the kit comprises the DNAzyme.

[0018] The DNAzyme capable of specifically recognizing Acinetobacter baumannii, the screening method and the application thereof, comprise a catalytic core sequence and a target recognition sequence; the nucleotide sequence of the catalytic core sequence is SEQ ID NO. 1; and the nucleotide sequence of the target recognition sequence is SEQ ID NO. 2. The specific nucleic acid sequence of Acinetobacter baumannii is accurately locked by the target recognition sequence (SEQ ID NO. 2), so that the detection is highly specific, and cross-reactions with other pathogenic bacteria are avoided; after successful recognition, the catalytic core sequence (SEQ ID NO. 1) efficiently cuts the substrate chain to produce a detectable signal change, thereby realizing efficient and specific recognition and detection of Acinetobacter baumannii, and providing a reliable basis for clinical infection diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings accompanying the specification of this application are used to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0020] Figure 1 is a schematic diagram of the screening principle of the DNAzyme capable of specifically recognizing AB according to the embodiment of the present application;

[0021] Figure 2 is a screening result diagram according to the embodiment of the present application;

[0022] Figure 3 is a probe affinity identification diagram according to the embodiment of the present application;

[0023] Figure 4 is a bacterial detection principle diagram according to the embodiment 3 of the present application;

[0024] Figure 5 is a sample detection result diagram according to the embodiment of the present application. DETAILED DESCRIPTION

[0025] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0026] DNAzyme refers to a nucleic acid molecule fragment with special structure and catalytic function, which is screened from an artificially synthesized DNA / RNA library by in vitro screening, namely Systematic Evolution of Ligands by Exponential Enrichment (SELEX), and usually consists of a substrate strand and an enzyme strand. The two DNA strands are partially complementary and hybridized by base pairing to form a double-stranded system. The presence of the target activates the activity of the DNAzyme, which cuts a single RNA base buried in the substrate strand DNA, so that the double strand is self-decomposed and the target molecule is released for recognition by the next DNAzyme copy. The catalytic cutting DNAzyme can trigger multiple DNAzyme copies to start self-cleavage by using a single target molecule, and effectively guarantees the high sensitivity recognition of low-abundance candidate biomarkers through the catalytic signal amplification mechanism.

[0027] The present application is illustrated by the following examples:

[0028] In the present application, unless specified, the raw materials and equipment used can be purchased from the market or commonly used in the art.

[0029] The DNAzyme provided in the present application specifically recognizes Acinetobacter baumannii, and includes a catalytic core sequence and a target recognition sequence. The nucleotide sequence of the catalytic core sequence is SEQ ID NO. 1, and the nucleotide sequence of the target recognition sequence is SEQ ID NO. 2. The catalytic core sequence is a nucleotide sequence with catalytic activity in the DNAzyme, also known as the enzyme strand. The catalytic core sequence can catalyze the RNA cleavage reaction of the substrate strand, i.e. the target recognition sequence. When the DNAzyme binds to the target, the catalytic core sequence plays a role to cut the substrate strand, thereby producing a detectable signal change, so as to realize the specific recognition and detection of Acinetobacter baumannii. The target recognition sequence binds to the target molecule specific to Acinetobacter baumannii. Only when the target recognition sequence successfully binds to the target molecule, the catalytic core sequence can play a role to trigger the subsequent chemical reaction. Thus, the target recognition sequence SEQ ID NO. 2 accurately locks the specific nucleic acid sequence of Acinetobacter baumannii, ensures high specificity of detection, and avoids cross-reaction with other pathogenic bacteria; the catalytic core sequence SEQ ID NO. 1 efficiently cuts the substrate strand after successful recognition, produces a detectable signal change, and thus realizes efficient and specific recognition and detection of Acinetobacter baumannii, providing a reliable basis for clinical infection diagnosis.

[0030] The present application also provides a method for screening a DNAzyme specifically recognizing Acinetobacter baumannii, comprising the following steps:

[0031] Step 1, design and synthesize an initial library containing 94-base specific DNA sequences, each DNA sequence consisting of three parts: fixed sequences at both ends, a random sequence of 40 bases in the middle, and a fixed RNA cleavage site rA, the initial random sequence library contains 10 13 -10 16 sequence different DNA molecules to ensure that the target DNAzyme is included;

[0032] Step 2, using Acinetobacter baumannii CEM as the target, and non-Acinetobacter baumannii CEM strains as the control, the initial DNA library is incubated with the control and target CEM after being immobilized by magnetic beads;

[0033] Step 3, separate the magnetic beads from the solution and collect the supernatant containing the cleaved nucleic acid fragments. Use the fixed sequence in step 1 as the amplification primer to amplify the nucleic acid fragments in the solution by PCR, and reconstruct the full-length DNA library containing the complete fixed sequence, random region and RNA cleavage site rA for the next round of screening;

[0034] Step 4: Repeat steps 2 to 3 for a total of ten rounds, each round using Acinetobacter baumannii CEM nucleic acid as the target and non-Acinetobacter baumannii CEM nucleic acid as the control for differential screening to obtain enriched target DNAzyme sequences.

[0035] Specifically, the screening principle is as shown in Figure 1 , first design and synthesize a random nucleic acid library, the middle of the sequence contains a random 40-base sequence, and each sequence contains an RNA bond (rA represents ribo-adenine) as a cleavage site. The initial random sequence library contains 10 13 -10 16 sequence different DNA molecules to ensure that the target DNAzyme is included; then using Acinetobacter baumannii CEM as the target and non-Acinetobacter baumannii strain CEM as the control, the library is incubated with CEM, rA is cut off, and then the nucleic acid fragments are released into the solution; then use the magnetic stand to separate, and then perform PCR amplification on the solution to reconstruct the full-length library, and obtain the amplified sequences for the next round of selection. Among them, the magnetic stand can quickly separate the magnetic beads from the solution, improve the efficiency and reduce the risk of contamination, and perform ten rounds of screening to obtain the sequence, which is removed and expanded to achieve enrichment of the target sequence. Among them, Acinetobacter baumannii CEM nucleic acid as the target positively selects specific binding DNAzyme, and non-Acinetobacter baumannii CEM nucleic acid as the control negatively excludes non-specific binding DNAzyme. Non-Acinetobacter baumannii includes Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, by limiting the range of non-target bacterial species, improving the specificity and targeting accuracy of DNAzyme screening.

[0036] The specific operation of screening is as follows:

[0037] I. Preparation of CEM:

[0038] Positive screening bacteria CEM: the target Acinetobacter baumannii strain is revived in 20 mL LB culture and incubated at 25°C with 180 rpm shaking until the OD600 (optical density at 600 nm) is about 1, the bacterial culture is loaded into a 1.5 mL EP tube and centrifuged at 11000g for 5 minutes at room temperature. The supernatant is transferred to a new 1.5 mL sterile EP tube and stored at -20°C as CEM of Acinetobacter baumannii.

[0039] Negative screening bacteria CEM: prepared in the same way as the positive screening bacteria CEM described above, and the negative screening bacteria include: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus.

[0040] Reaction buffer composition: Hepes (50mM HEPES, 150mM NaCl, and 15mM MgCL2, pH7.4).

[0041] II. Screening:

[0042] 1. First round of screening:

[0043] 1.1 Take 1 nmol of library (lib7-N40-rA-Biotin) and dissolve in 100 μL of reaction buffer.

[0044] 1.2 Take 800 μl of SA magnetic beads and wash the magnetic beads with 200 μL of reaction buffer for 5 times.

[0045] 1.3 Add the library to the magnetic beads and react at room temperature for 30 min. Use a magnet to fish the magnetic beads and remove the supernatant.

[0046] 1.4 Add 200 μL of reaction buffer to the magnetic beads and perform rinsing.

[0047] 1.5 Take 50 μL of negative screening bacteria CEM and add it to the SA magnetic beads from the previous step, incubate on a shaker for 60 min, use a magnet to fish the magnetic beads, and elute the supernatant into an EP tube. Wash the magnetic beads once with 150 μL of reaction buffer and add it to the Elution EP tube. Mark it as Elution-.

[0048] 1.6 Take 50 μL of positive screening bacteria CEM and add it to the SA magnetic beads from the previous step, incubate on a shaker for 60 min, use a magnet to fish the magnetic beads, and elute the supernatant into an EP tube. Wash the magnetic beads once with 150 μL of reaction buffer and add it to the Elution EP tube. Mark it as Elution+.

[0049] 1.7 Take eight-tube, add 30 μL PCR mix to each hole, take 1 μL elution- and elution+ each, dilute 10 times with reaction buffer, and add 2 μL each to the mix, centrifuge and mix. Fluorescent quantitative PCR detection.

[0050] 1.8 Add 2 mL elution- to the remaining elution+, and divide the sample into PCR tubes, 90 μL each, and perform PCR 25 cycles. The procedure is: 95°C 2 min; 95°C 1 min, 60°C 1 min, 72°C 1 min, 25 cycles.

[0051] 1.9 Concentrate the PCR product with n-butanol, and recover the PCR product (dsDNA) with an oligonucleotide purification kit.

[0052] 2. Second round of screening:

[0053] 2.1 Dilute the library to 1 μM*100 μL with Hepes. Take 100 μL SA magnetic beads, wash the magnetic beads with 200 μL Hepes 5 times, and finally temporarily store the magnetic beads in buffer.

[0054] 2.2 Add the first round of PCR product to the magnetic beads, and shake at room temperature for 30 min. Magnet fish the magnetic beads, and remove the supernatant.

[0055] 2.3 Add 500 μL melting buffer (200 mM NaOH, 500 mM NaCl), and shake at room temperature for 5 min. Magnet fish the magnetic beads, and remove the supernatant. Repeat 2 times.

[0056] 2.4 Add 200 μL reaction buffer to the magnetic beads, and perform rinsing, a total of 6 times.

[0057] 2.5 Take 50 μL anti-screening bacteria CEM and add to the SA magnetic beads from the previous step, shake at room temperature for 60 min, magnet fish the magnetic beads, and take the supernatant Elution to an EP tube. Wash the magnetic beads with 150 μL reaction buffer once, and add to the Elution EP tube. Mark as Elution-.

[0058] 2.6 Take 50 μL positive screening bacteria lysate and add to the SA magnetic beads from the previous step, shake at room temperature for 60 min, magnet fish the magnetic beads, and take the supernatant Elution to an EP tube. Wash the magnetic beads with 150 μL reaction buffer once, and add to the Elution EP tube. Mark as Elution+.

[0059] 2.7 Take eight-tube, add 30 μL mix to each tube, take 1 μL elution- and elution+ respectively, dilute 10 times with reaction buffer, and then add 2 μL to the mix, centrifuge and mix. Fluorescent quantitative PCR detection.

[0060] 2.8 Add 2 mL PCR mix to the rest of the Elution+, and then distribute to PCR tubes, 90 μL per tube, PCR 25 cycles. The procedure is: 95℃ 2 min; 95℃ 1 min, 60℃ 1 min, 72℃ 1 min, 25 cycles.

[0061] 2.9 Concentrate the PCR product with n-butanol, and recover the PCR product (dsDNA) with an oligonucleotide purification kit.

[0062] 3. The third to eighth rounds of screening, each round of screening is the same as above.

[0063] 4. The retention rate of each round of screening in the screening experiment results is shown in Table 1, with the increase of the screening rounds, the number of positive screening products gradually increases, and the eighth round has a significant increase compared with the first and third rounds, which can be subjected to high-throughput sequencing. Figure 2 Figure 2 Table 1: Screening results of each round of screeningFrom left to right, the bands correspond to the strains in order: control (buffer), E. coli (standard strain), K. pneumoniae (standard strain), S. aureus (standard strain), A. baumannii (standard strain), A. baumannii (clinical strain 1), E. coli (clinical strain 1), A. baumannii (clinical strain 2), A. baumannii (clinical strain 3).

[0064] 5. Sequencing:

[0065] 5.1 The obtained raw sequencing results are extracted by analysis software according to the tag primer, and a total of 1048575 sequences are obtained, of which there are 1476590 sequence types.

[0066] 5.2 The obtained sequences are analyzed, and one sequence to be sequenced (denoted as: ab-B21) is selected for analysis.

[0067] The sequence of ab-B21 is shown in SEQ ID NO. 3, and SEQ ID NO. 3 is:

[0068] 5 ′ -CCCACTAGAGCGGGGTGTCCAGCGGTCGGTGTCAGACCCCACGTCGGTTGCCGTCCAGCGATCTGTGTCC-3 ′ .

[0069] The above ab-B21 is used as the enzyme chain, and the corresponding Fs-AB is designed as the substrate chain. The fluorescence signal chain of AB is determined, and the end of F (6FAM) is modified. The two ends of ab-B21 and Fs-AB are partially complementary to each other. The sequence of Fs-AB is shown in SEQ ID NO. 4, and SEQ ID NO. 4 is:

[0070] 5 ′ -GGACACAGATCGCTGGACACTrAGGCTCTAGGGGGG-FAM-3 ′ .

[0071] The DNAzyme capable of specifically recognizing Acinetobacter baumannii is obtained, the nucleotide sequence of the catalytic core sequence (enzyme chain) is shown in SEQ ID NO. 1:

[0072] 5 ′ -CCCACTAGAGCGGGGTGTCCAGCGGTCGGTGTCAGACCCCACGTCGGTTGCCGTCCAGCGATCTGTGTCC-3 ′ ;

[0073] The nucleotide sequence of the target recognition sequence (substrate chain) is shown in SEQ ID NO. 2:

[0074] 5 ′ -GGACACAGATCGCTGGACACTrAGGCTCTAGGGGGG-3 ′ .

[0075] The sequence of the initial screening library Lib7-N40-rA is SEQ ID NO. 5:

[0076] 5 ′ -Biotin-GGACACAGATCGCTGGACACTrAGGCTCTAGGGGGGACTAGAGNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN CCGTCCAGCGATCTGTGTCC-3 ′ .

[0077] Example 2: Verification of the cleavage activity of the DNAzyme system ab-B21 capable of specifically recognizing Acinetobacter baumannii.

[0078] 201, sample preparation: the above ab-B21 is used as the enzyme chain, and the corresponding Fs-AB substrate chain is designed. The sequence of Fs-AB is: 5 ′-GGACACAGATCGCTGGACACTrAGGCTCTAGGGGGG-FAM-3 ′ Add 80 μL of reaction buffer and 10 μL of 100 μM DNAzyme substrate chain Fs-AB to an EP tube, then add 10 μL of 100 μM enzyme chain ab-B21; perform renaturation.

[0079] 202. The CEM bacterial lysate, from the highest concentration, was serially diluted 2-fold to 9 concentrations + 1 concentration 0 (10 μl) and added to the PCR 8-cell array. The renaturation product from the previous step was added 10 μl to each of the 8-cell arrays. After incubating on a shaker at room temperature in the dark for 2 hours, loading buffer was added at a 1:1 ratio, and the mixture was incubated at 95°C for 10 minutes before gel electrophoresis.

[0080] 203. Add the highest concentration of the reverse screening bacteria CEM to the PCR tube. Take 10 μL of the renaturation product from the previous step and add it to the reverse screening bacteria CEM. Incubate on a shaker at room temperature in the dark for 2 hours. Then add loading buffer at a 1:1 ratio and incubate at 95°C for 10 minutes before preparing for gel electrophoresis.

[0081] 204. Add samples sequentially according to the marker concentration (from lowest to highest) and reverse sieving; run the gel (100V, 40min), take pictures on a gel imaging system, plot the gel bands using ImageJ based on grayscale values, and calculate their affinity. The results are as follows: Figure 3 As shown.

[0082] 205. From Figure 3 It can be seen that ab-B21 has a significant effect. ab-B21 can shear CEM bacteria in the positive screening, and the shearing effect is obvious as the concentration of CEM in the positive screening increases.

[0083] Example 3: Synthesizing a probe for detecting Acinetobacter baumannii for bacterial detection.

[0084] Detection principle as follows Figure 4 As shown, the ab-B21 sequence was designed and synthesized as shown in SEQ ID NO.3, SEQ ID NO.3:

[0085] 5'-CCCACTAGAGCGGGGTGTCCAGCGGTCGGTGTCAGACCCCACGTCGGTTGCCGTCCAGCGATCTGTGTCC-3'.

[0086] The substrate chain sequence is shown in SEQ ID NO.4, which is:

[0087] 5'-GGACACAGATCGCTGGACACTrAGGCTCTAGGGGGG-FAM-3'.

[0088] The sequence of ab-B21 can be paired with the sequence of the substrate strand at both ends to form a DNAzyme-based nucleic acid detection system. This detection system is actually equivalent to a functional sensor, which can simulate the core function of a biological sensor, that is, specific recognition of the target and output of a detectable signal, thereby achieving accurate detection of Acinetobacter baumannii. With Acinetobacter baumannii CEM as the target and other strains CEM as the negative detection material, when the probe is incubated with CEM, DNAzyme (composed of ab-B21 and the substrate strand) can specifically recognize the target sequence and activate its catalytic activity, cutting rA (ribosyladenosine) in the substrate strand, and then diffusing the cleaved nucleic acid fragments into the solution. By running the gel to separate the cleaved and uncleaved bands, and detecting the fluorescence intensity change of different bands, it can be judged whether the target bacteria exist. The sample detection results are shown in Figure 5 The DNAzyme nucleic acid detection system exhibits similar functional characteristics of a biological sensor by accurately recognizing the target sequence, activating the catalytic reaction and outputting the fluorescence signal. It only has a strong fluorescence response when the target bacteria exist, while the non-target pathogenic bacteria and the blank sample produce weak fluorescence signals. This indicates that the DNAzyme nucleic acid detection system has a high specific recognition ability for Acinetobacter baumannii.

[0089] The construction and detection steps of the fluorescence detection probe based on DNAzyme are as follows:

[0090] 301. Sample preparation: Add reaction buffer and DNAzyme substrate strand Fs-AB to the EP tube, then add enzyme chain ab-B21, and centrifuge and mix well.

[0091] 302. Detection probe: The enzyme chain ab-B21 can be paired with the substrate strand Fs-AB at both ends to form a fluorescence detection probe.

[0092] 303. Sample detection: Add the clinical bacterial sample to the EP tube, and add 10 μl of the fluorescence detection probe obtained in the previous step to the EP tube. After incubation at room temperature in the dark for 20 min, add Loading buffer according to the same volume ratio of 1:1, and run the gel after incubation at 95℃ for 5 min.

[0093] The application also provides the application of the DNAzyme specifically recognizing Acinetobacter baumannii in clinical detection of Acinetobacter baumannii infection. The 3' end of the substrate strand sequence of the DNAzyme has a labeled fluorescent signal group, and the fluorescent signal group is FAM (which can be replaced by RhB). In the clinical sample detection scene, the clinical sample (such as sputum, blood, alveolar lavage fluid, etc.) of the suspected infected patient is collected, and the supernatant after the enrichment culture is mixed with the DNAzyme detection system of the application for incubation. The DNAzyme in the system can specifically recognize the marker of Acinetobacter baumannii. If Acinetobacter baumannii exists in the sample, the DNAzyme will bind to the target sequence and trigger the catalytic reaction, cut the substrate strand and release the detectable nucleic acid fragment. The reaction products are separated by gel electrophoresis, and the fluorescence intensity difference between the cleavage band and the uncut band is observed: if Acinetobacter baumannii exists in the sample, the cleavage reaction will release a large amount of nucleic acid fragments in the solution, and the fluorescence signal will be significantly enhanced; on the contrary, if there is no Acinetobacter baumannii in the sample, the DNAzyme cannot trigger effective cleavage, and the fluorescence signal is weak. Finally, whether the sample is infected with Acinetobacter baumannii is determined according to the fluorescence signal intensity, which provides a basis for clinical diagnosis. FAM and RhB are commonly used fluorescent groups with different excitation and emission wavelengths. The most suitable dye can be selected according to the specific experimental platform (such as the imaging system or the light source configuration of the fluorescence detection system) to achieve high sensitivity and high specificity detection effect.

[0094] The application also provides a kit for specifically recognizing and detecting Acinetobacter baumannii, which comprises the DNAzyme described in the application. The core functional component of the kit is an Acinetobacter baumannii-specific recognition and detection module based on DNAzyme, which uses a DNAzyme molecule to achieve precise targeting of the nucleic acid marker of Acinetobacter baumannii. The DNAzyme molecule is designed to specifically bind to Acinetobacter baumannii. When and only when Acinetobacter baumannii exists in the sample, the DNAzyme will activate its activity, accurately cut the substrate strand and release a detectable signal. The DNAzyme contained in the kit can effectively distinguish Acinetobacter baumannii from other common pathogenic bacteria (such as Pseudomonas aeruginosa and Staphylococcus aureus), avoiding false positive results caused by cross-reaction. At the same time, the kit provides an optimized reaction buffer system and operation process, which ensures that the DNAzyme has the maximum catalytic efficiency under the most suitable conditions, realizes the standardization of the whole process from clinical sample nucleic acid extraction to result interpretation, and significantly improves the speed and accuracy of clinical diagnosis of Acinetobacter baumannii infection.

[0095] The optimized reaction buffer system and operation process provided by the kit specifically include:

[0096] Optimized reaction buffer system: The optimized reaction buffer system provided with the kit is 1x reaction buffer (final concentration: 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 1 mM DTT, 0.01% Triton X-100), which is directly used without dilution.

[0097] Standard operation procedure: Use the matched reagent to process the clinical sample (sputum / blood, etc.); add 10 μL of 1x reaction buffer, 2 μL of DNAzyme (final concentration 1 μM), 2.4 μL of substrate strand (final concentration 1.2 μM) and 5 μL of sample nucleic acid into the PCR tube in turn, mix and centrifuge briefly; place the reaction tube in a 37°C constant temperature incubator for 45 minutes, and the DNAzyme specifically recognizes the Acinetobacter baumannii nucleic acid target and cuts the substrate strand to release the fluorescently labeled fragment; take 10 μL of the reaction product and add it to the gel, and perform electrophoresis at 100V for 20 minutes, and observe under the imaging instrument; positive samples appear specific fluorescent bands (FAM labeled), and negative samples have no bands or only substrate bands. The whole standardized operation ensures that the detection sensitivity is ≥10 3 CFU / mL, and has no cross-reaction with other pathogenic bacteria.

[0098] The optimized buffer system and operation procedure together ensure that the DNAzyme can still maintain high specificity and sensitivity in the complex clinical sample environment, while simplifying the experimental operation steps, reducing the risk of human error, and enabling non-professional laboratories to obtain stable and reliable detection results.

[0099] The above is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A DNAzyme capable of specifically recognizing Acinetobacter baumannii, characterized in that, The catalytic core sequence and the target recognition sequence are included. The nucleotide sequence of the catalytic core sequence is SEQ ID NO.

1. The nucleotide sequence of the target recognition sequence is SEQ ID NO.

2.

2. A method of screening for a DNAzyme, comprising the steps of, The DNAzyme capable of specifically recognizing Acinetobacter baumannii according to claim 1 comprises the following steps: Step 1, design and synthesize an initial library of DNA sequences of 94 bases, each DNA sequence consisting of three parts: a fixed sequence at both ends, a random sequence of 40 bases in the middle and a fixed RNA cleavage site rA, the initial random sequence library contains 10 13 -10 16 different DNA molecules to ensure that the target DNAzyme is included; Step 2: Using Acinetobacter baumannii CEM as the target and non-Acinetobacter baumannii CEM strains as the control, the initial DNA library is incubated with the control and the target CEM after being immobilized by magnetic beads; Step 3: The magnetic beads and the solution are separated, and the supernatant containing the cut and released nucleic acid fragments is collected. The nucleic acid fragments in the solution are amplified by PCR using the fixed sequence in step 1 as the amplification primer, and the full-length DNA library containing the complete fixed sequence, random region and RNA cleavage site rA is reconstructed for the next round of screening; Step 4: Repeat steps 2 to 3 for a total of ten rounds, each round using Acinetobacter baumannii CEM nucleic acid as the target and non-Acinetobacter baumannii CEM nucleic acid as the control for differential screening to obtain the enriched target DNAzyme sequence.

3. The DNAzyme screening method according to claim 2, characterized in that, In step 2, the magnetic beads and the solution are separated by using a magnetic stand.

4. The DNAzyme screening method according to claim 2, characterized in that, In step 2, the non-Acinetobacter baumannii includes Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

5. The DNAzyme capable of specifically recognizing Acinetobacter baumannii according to claim 1 is applied to the clinical detection of Acinetobacter baumannii infection.

6. The use of the DNAzyme capable of specifically recognizing Acinetobacter baumannii according to claim 1 in the clinical detection of Acinetobacter baumannii infection, characterized in that, The 3' end of the target recognition sequence of the DNAzyme has a fluorescent signal group, and the fluorescent signal group is FAM or RhB.

7. The DNAzyme capable of specifically recognizing Acinetobacter baumannii according to claim 1 is applied to the fluorescent detection probe for detecting Acinetobacter baumannii.

8. A kit capable of specifically recognizing and detecting Acinetobacter baumannii, characterized by: The kit comprises the DNAzyme according to claim 1.

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