Burkholderia pseudomallei detection system and application thereof

Abstract

The invention provides a gene marker, crRNA, a composition and a molecular beacon probe for detecting burkholderia pseudomallei, and provides a method for detecting the burkholderia pseudomallei. The invention constructs a novel detection platform with good specificity, high sensitivity and short time consumption, and is suitable for rapid and efficient detection of Burkholderia pseudomallei.

Description

Technical Field

[0001] The present invention relates to the field of molecular biological diagnostic technology and microbial detection technology, and more particularly to a method for detecting Burkholderia pseudomallei using CRISPR-Cas12a. Background Art

[0002] Burkholderia pseudomallei (B. pseudomallei), also known as melioidosis, is a Gram-negative short bacillus that is widely found in rivers and muddy areas in tropical and subtropical regions. Infection with it can lead to a serious and fatal melioidosis. Strengthening the prevention, diagnosis and treatment of melioidosis has important social significance. With the increasing frequency of international trade, cooperation and exchanges, many imported cases have been reported, posing a great threat to global public health. Melioidosis caused by B. pseudomallei infection has various symptoms and is known as "a hundred diseases". The traditional diagnostic method of B. pseudomallei mainly includes isolation and culture of bacteria and immunological detection, but there are disadvantages such as time-consuming detection process, the need for specialized biosafety equipment, and well-trained laboratory personnel, so it is very easy to be missed or misdiagnosed. Melioidosis has an acute onset, high mortality rate, high recurrence rate, and lack of effective vaccines. Therefore, it is urgent to develop advanced diagnostic methods to quickly and accurately detect B. pseudomallei.

[0003] Molecular diagnostic technology based on CRISPR-Cas12a can identify pathogen-specific nucleic acid sequences and achieve specific diagnosis. At present, the B. pseudomallei detection technology based on CRISPR-Cas12a generally requires pre-amplification of the sample to be tested. This step increases the complexity of the detection process while improving the sensitivity. At the same time, multiple operation steps introduce the risk of sample contamination. On the other hand, the reaction system only contains a single type of crRNA, and detection is performed by targeting a single conserved gene. Although this detection method is relatively simple and direct, once the target site of the strain mutates, it is very easy to produce false negative results. In addition, the linear fluorescent probe has a high background response signal and low detection sensitivity. To solve the above problems, it is necessary to develop a new B. pseudomallei fast and efficient detection method. Summary of the invention

[0004] In order to solve the problems existing in the existing molecular diagnosis technology of B. pseudomallei, the present invention is based on the CRISPR-Cas12a system, and through strategies such as pan-genomics screening of crRNA, combining multiple crRNAs, and designing molecular beacon probes, a new detection platform with good specificity, high sensitivity, and short time consumption is constructed, which is suitable for the rapid and efficient detection of B. pseudomallei.

[0005] The present invention first provides a gene marker for detecting Burkholderia pseudomallei, whose nucleotide sequence is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0006] The present invention also provides a crRNA for detecting Burkholderia pseudomallei based on CRISPR-Cas12a, which is designed based on the above-mentioned gene markers and PAM sequences, and the PAM sequence site is TTTN.

[0007] In one embodiment according to the present invention, the nucleotide sequence of crRNA is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23.

[0008] The present invention also provides a composition for detecting Burkholderia pseudomallei, comprising Lbcas12a protein and crRNA;

[0009] The crRNA is selected from one or more of the crRNAs having nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23.

[0010] In one embodiment according to the present invention, the composition comprises a ssDNA or a molecular beacon probe, wherein the ssDNA or the molecular beacon probe comprises a FAM fluorescent group and a BHQ1 quenching group;

[0011] Preferably, the nucleotide sequence of the ssDNA fluorescent probe is SEQ ID NO.49;

[0012] The nucleotide sequence of the molecular beacon is selected from SEQ ID NO.50-61.

[0013] In one embodiment of the present invention, the Lbcas12a protein is derived from Lachnospiraceaebacterium ND2006.

[0014] In one embodiment of the present invention, the nucleotide sequence of the crRNA is SEQ ID NO: 17, or is selected from one of the following combinations:

[0015] Combination 1: crRNAs having nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17 mixed in equal amounts;

[0016] Combination 2: crRNAs having nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 mixed in equal amounts;

[0017] Combination 3: crRNAs with nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19 mixed in equal amounts;

[0018] Combination 4: crRNAs with nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 mixed in equal amounts.

[0019] Another aspect of the present invention also provides the use of the above-mentioned gene marker, crRNA or composition in the preparation of a preparation or kit for detecting Burkholderia pseudomallei.

[0020] Another aspect of the present invention provides a method for detecting Burkholderia pseudomallei, comprising:

[0021] The above composition and 1×NEBuffer TM R2.1 buffer was premixed and incubated at 25°C for 10 minutes, followed by the addition of genomic DNA of the sample to be tested and incubation in ddH 2 The system was supplemented with 5% O to 20 μL, and the reaction was carried out at 37°C for 90 minutes. During the reaction, the fluorescence intensity was collected every 3 minutes.

[0022] In one embodiment of the present invention, the reaction is carried out in a q-PCR fluorescence quantitative analyzer or an enzyme-labeled instrument.

[0023] The beneficial effects of the above technical solution of the present invention are as follows:

[0024] After the present invention uses pan-genome to integrate 3245 B.pseudomallei genome information, 12 crRNAs with high conservatism and wide coverage are designed, which minimizes the probability of strain misdetection and missed detection caused by gene diversity; by combining the strategy of crRNA, different regions of the B.pseudomallei genome are simultaneously targeted, greatly improving the sensitivity of detection and ensuring the accuracy of the test results; by designing molecular beacons, molecular beacon probes are used to significantly reduce background fluorescence signals and improve fluorescence quenching efficiency. The detection method constructed by the present invention does not require nucleic acid pre-amplification reaction, avoids the drawbacks such as sample cross contamination, complex experimental operation, and long reaction time, and further broadens the detection ability of the method to B.pseudomallei. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following drawings are used to describe in detail the non-limiting embodiments to make their features, purposes and advantages more obvious:

[0026] Figure 1 Schematic diagram of the principle of CRISPR-Cas12a detection of B. pseudomallei;

[0027] Figure 2 Flowchart for designing crRNA based on pan-genomics;

[0028] Figure 3 Schematic diagram of the recombinant plasmid pMD19-T-Bp;

[0029] Figure 4 This is a graph showing the effect of a single crRNA on the detection efficiency of B. pseudomallei;

[0030] Figure 5 Effect of combined crRNA on detection efficiency of B. pseudomallei;

[0031] Figure 6 Schematic diagram of the molecular beacon probe and its effect on the detection efficiency of B. pseudomallei;

[0032] Figure 7 Evaluation of detection specificity of the optimized CRISPR-Cas12a system. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1 Design of specific crRNA based on pan-genome

[0035] The workflow diagram of pan-genome-based design of specific crRNA is as follows Figure 2 As shown. First, download the B. pseudomallei genomes collected as of April 2024 from the NCBI GenBank database (https: / / github.com / kblin / ncbi-genome-download), totaling 3245. Then, use Prokka to annotate each genome to better confirm the gene coding sequence and other genomic information features. Next, use Panaroo to perform pan-genome analysis on the integrated genomic information and identify 100% conserved genes in 3245 genomes. Finally, use the R package CaSilico to design 12 specific crRNAs based on 11 conserved gene core regions (nucleotide sequences are SEQ ID NO.1-11), as shown in SEQ ID NO.12-23, and then design an off-target crRNA (SEQ ID NO.24) as a negative control. The crRNA was synthesized and purified by Shenzhen BGI Co., Ltd.

[0036] Example 2 Preparation of target substrate dsDNA

[0037] In order to ensure the concentration accuracy and template consistency of the subsequent performance test experiments, the 12 specific crRNA targeting base sequences designed above were assembled to form a target sequence array. B. pseudomallei BPC006 was selected as the template for preparing the target substrate, and 12 primers (SEQ ID NO.25-48, synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.) were used to amplify the 12 crRNA targeting base sequences and their adjacent genomic regions to obtain 12 PCR products. Subsequently, overlap PCR and seamless cloning technology were used to assemble the 12 PCR products in the order of their original positions in the genome, and then the assembled fragments were connected to the pMD19-T vector (Takara, Japan) to generate the recombinant plasmid pMD19-T-Bp (such as Figure 3 The target sequence array was amplified and purified by PCR using primers A-gRNA01-F / A-gRNA12-R (SEQ ID NO. 25 and 48) with pMD19-T-Bp as a template to obtain the target substrate dsDNA. The concentration of dsDNA was determined using Nanodrop (Thermofisher, USA) to facilitate subsequent experiments.

[0038] Example 3 CRISPR-Cas12a detection of B. pseudomallei reaction

[0039] Under the guidance of crRNA, Cas12a specifically recognizes the target substrate dsDNA, thereby activating the trans-cleavage function, cutting the single-stranded DNA probe, separating its fluorescent group from the quenching group and emitting fluorescence, and the fluorescence intensity is collected and analyzed by the instrument, such as Figure 1 shown.

[0040] First, prepare the CRISPR-Cas12a reaction system: 50nM LbCas12a protein, 1×NEBuffer TM r2.1, 500nM ssDNA probe and 100nM crRNA. These components were pre-incubated at 25 ° C for 10 minutes to ensure that the Cas12a-crRNA complex was formed. After the incubation, different concentrations of target substrate dsDNA were added to the system to start the detection reaction so as to evaluate the activity of Cas12a at different substrate concentration levels. Finally, an appropriate amount of enzyme-free sterile water was added to make the volume of the final reaction system 20 μL. The entire reaction system was placed in a qPCR tube and incubated at 37 ° C for 90 minutes. During the entire incubation process, the FAM channel on the ABIQ5 real-time fluorescence PCR system (ThermoFisher, USA) was used to collect fluorescence signals every 3 minutes.

[0041] The results showed that when the concentration of the target substrate dsDNA was controlled at 200 pM and other reaction conditions remained unchanged, only the type of crRNA was changed, and the performance differences of different crRNAs were characterized by different fluorescence intensities. Figure 4 As shown: gRNA0212, gRNA0169, gRNA0172, gRNA0270 and gRNA0257 are the top five crRNAs with the best performance.

[0042] Example 4 Combining multiple crRNAs to optimize the CRISPR-Cas12a detection system

[0043] In order to further optimize the detection performance of the CRISPR-Cas12a system, multiple crRNAs targeting the chromosomal genome were combined to increase the number of Cas12a-crRNA complexes, thereby improving the efficiency of trans-cleavage (e.g. Figure 5 a).

[0044] Therefore, in this example, the top five crRNAs with the best performance were combined to verify the effects of the four crRNA combinations on the detection efficiency of the CRISPR-Cas12a system:

[0045] Combination 1: gRNA0212+gRNA0169

[0046] Combination 2: gRNA0212+gRNA0169+gRNA0172

[0047] Combination 3: gRNA0212+gRNA0169+gRNA0172+gRNA0270

[0048] Combination 4: gRNA0212+gRNA0169+gRNA0172+gRNA0270+gRNA0257

[0049] Ensure that other reaction conditions are as described in Example 3, only the type of gRNA is changed and the crRNA concentration in each combination is equal and the total concentration is consistent with the reaction concentration of a single crRNA. Figure 5 b The results show that compared with the single crRNA with the best performance (gRNA0212), the four combinations of crRNA can enhance the fluorescence signal generated per unit time. Among them, combination 4 (5 crRNA combinations) has the greatest improvement in the detection efficiency of the CRISPR-Cas12a system.

[0050] Example 5 Design of molecular beacons to optimize CRISPR-Cas12a detection system

[0051] Designing a molecular beacon with a closer spatial distance between the fluorescent group and the quenching group can significantly reduce the background fluorescence signal of the probe while improving the fluorescence quenching efficiency, thereby improving the sensitivity of the CRISPR-Cas12a detection system ( Figure 6 a, 6c).

[0052] First, six molecular beacon probes with a loop length of 5 to 20 bp were synthesized (S6L5, S6L8, S6L11, S6L14, S6L17 and S6L20, synthesized by Sangon Biotech (Shanghai) Co., Ltd.), whose sequences are shown in SEQ ID NOs. 50-55. The other reaction conditions were kept the same as those described in Example 3, and only the type of molecular beacon probe was changed. Figure 6 bThe results show that molecular beacon probes with different loop lengths can significantly enhance the fluorescence intensity ratio. Among them, S6L8 exhibits superior detection performance, and its signal-to-noise ratio is 42 times higher than that of linear ssDNA.

[0053] Based on the structure of S6L8, the detection performance of different stem lengths was further explored. Six molecular beacon probes with stem lengths of 3 to 9 bp were synthesized (S3L8, S4L8, S5L8, S7L8, S8L8 and S9L8, synthesized by Sangon Biotech (Shanghai) Co., Ltd.), and their sequences are shown in SEQ ID NO.56-61. Figure 6 dThe results show that the stem length is not absolutely positively or negatively correlated with the performance. According to the comprehensive optimization of stem length and loop length, S5L8 showed the best detection performance and had the highest fluorescence intensity ratio at each test time. After optimization, the CRISPR-Cas12a detection system has higher detection sensitivity and signal-to-noise ratio, which is suitable for use in detecting low-concentration samples.

[0054] Example 6 Specificity evaluation of CRISPR-Cas12a detection system

[0055] This example uses 21 B. pseudomallei genomes and 8 other common clinical pathogen genomes, all of which are extracted using the bacterial genomic DNA extraction kit DP302 from Tiangen Biochemical Technology (Beijing) Co., Ltd. Different genomic DNAs were used as substrate dsDNA to judge the specificity of the CRISPR-Cas12a detection system. Figure 7 The results showed that only 21 B. pseudomallei target genomes could induce strong fluorescence signals, while 8 non-target genomes failed to produce obvious fluorescence signals. This indicates that the CRISPR-Cas12a detection system for B. pseudomallei established and optimized in the present invention has good detection specificity and can effectively distinguish target and non-target biological samples.

[0056] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

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Claims

1. A gene marker for detecting Burkholderia pseudomallei, wherein the nucleotide sequence is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:

11.

2. Detection of crRNA of Burkholderia pseudomallei based on CRISPR-Cas12a, characterized in that: Designed based on the gene marker and PAM sequence as claimed in claim 1, the PAM sequence site is TTTN.

3. The crRNA as claimed in claim 2, wherein the nucleotide sequence is SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO:

23.

4. A composition for detecting Burkholderia pseudomallei, comprising Lbcas12a protein, crRNA, ssDNA or a molecular beacon probe; The crRNA is selected from one or more of the crRNAs having nucleotide sequences of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23; The ssDNA or molecular beacon probe contains a FAM fluorescent group and a BHQ1 quenching group; Preferably, the nucleotide sequence of the ssDNA fluorescent probe is SEQ ID NO.49; The nucleotide sequence of the molecular beacon probe is selected from SEQ ID NO.50-61.

5. The composition of claim 4, wherein the Lbcas12a protein is derived from Lachnospiraceae bacteriumND2006.

6. The composition according to claim 4, wherein the nucleotide sequence of the crRNA is preferably SEQ ID NO: 17, or is selected from one of the following combinations: Combination 1: crRNAs having nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 17 mixed in equal amounts; Combination 2: crRNAs having nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 mixed in equal amounts; Combination 3: crRNAs having nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 19 mixed in equal amounts; Combination 4: crRNAs with nucleotide sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 mixed in equal amounts.

7. Use of the gene marker according to claim 1, the crRNA according to claim 2 or 3, or the composition according to any one of claims 4 to 6 in the preparation of a preparation or kit for detecting Burkholderia pseudomallei.

8. A method for detecting Burkholderia pseudomallei, comprising: The composition according to any one of claims 4 to 6 and 1×NEBuffer TM r2.1 buffer was premixed and incubated at 25°C for 10 minutes, then the genomic DNA of the sample to be tested was added and the system was supplemented to 20 μL with ddH2O, and the reaction was carried out at 37°C for 90 minutes, and the fluorescence intensity was collected once every 3 minutes during the reaction.

9. The method according to claim 8, characterized in that The reaction is carried out in a q-PCR fluorescence quantitative analyzer or an enzyme-labeled instrument.

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