Method for detecting sulfate reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe

Abstract

The invention discloses a method for detecting sulfate reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probes, and belongs to the technical field of biology and analytical chemistry. According to the invention, a conserved region of a conservative gene adenyloyl sulfate reductase gene APS of sulfate reducing bacteria is taken as a target molecule, crRNA with specificity is designed to be combined with Cas12a to form a complex, when the complex is specifically combined with the target molecule, a trans-cleavage effect of CRISPR / Cas can be started, and then a free non-specific ssDNA fluorescent probe in a system is cleaved, so that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result is accurate. A detection experiment proves that the fluorescence intensity and the content of the sulfate reducing bacteria are in a forward linear relationship, and the method does not need to carry out enlarged culture on the sulfate reducing bacteria, can be used for carrying out real-time detection on the to-be-detected substance and is simple and convenient to operate. The method has the advantages of mild reaction conditions, high selectivity, short detection period, low cost and great popularization value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biology and analytical chemistry, and specifically relates to a method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probes. Background Art

[0002] Sulfate-Reducing Bacteria (SRB) is a general term for corrosive microorganisms that use sulfate or other oxidized sulfides as electron donors and obtain growth and metabolic energy from them. H2S produced during the growth and metabolic activities of SRB will cause serious corrosion to steel components in marine environments and underground humid environments. Current detection and identification methods for corrosive microorganisms include colony culture methods, detection methods based on bacterial characteristic metabolic compounds, and cell-based detection methods. Among them, the colony culture method has high reliability, but its operation is complicated, time-consuming, and has low application value in marine environments; the detection method based on characteristic metabolites is simple to operate and has a wide range of applications, but it still has the problem of long time consumption; the cell-based detection method has a short detection cycle, but the stability and specificity need to be further improved. Therefore, it is an urgent need to research and develop a highly sensitive and highly specific corrosion microorganism detection technology.

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) is a natural "immune system" in microorganisms. When viruses invade bacteria, bacteria can capture foreign genetic material fragments and integrate them into their own genome CRISPR sequences, and then accurately cut viral DNA through Cas nucleases to resist viral invasion. CRISPR and CRISPR associated proteins (Cas) are called CRISPR / Cas systems. Cas12a is a type 2 V Cas protein. It has no cutting activity when not activated. When it binds to a specific crRNA, the conformation of Cas12a will change and it will bind to crRNA to form a Cas12a-crRNA binary complex, which can specifically recognize the target DNA and activate its endonuclease activity. When Cas12a sequentially cuts single-stranded or double-stranded DNA in cis, the active site will continue to be exposed, showing trans-cutting activity, and non-specifically cutting the surrounding ssDNA. In view of the characteristics of Cas12a, a variety of highly sensitive and specific nucleic acid detection platforms have been reported using CRISPR / Cas12a technology. However, to date, no research has been reported on methods for highly sensitive and specific detection of sulfate-reducing bacteria based on CRISPR / Cas12a technology. Summary of the invention

[0004] In order to overcome the technical defects of the existing traditional sulfate-reducing bacteria SRB detection, the object of the present invention is to provide a method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probes. The present invention uses the conservative gene adenylate sulfate reductase gene APS conservative region of sulfate-reducing bacteria as the target molecule, and designs a specific crRNA that can be combined with Cas12a to form a complex. When the complex specifically binds to the SRB target molecule, the trans-cutting effect of CRISPR / Cas can be activated, thereby causing the free non-specific ssDNA fluorescent probe in the system to be cut, thereby showing fluorescent characteristics. The detection method of the present invention has the advantages of high sensitivity, strong specificity, rapid and efficient, user-friendly and low price.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe, comprising the following steps:

[0007] (1) Using nucleic acid amplification technology, the specific target gene fragment adenylate sulfate reductase gene APS sequence in sulfate-reducing bacteria SRB was amplified;

[0008] (2) adding the PCR product amplified in step (1) to a reaction system containing CRISPR / Cas12a and ssDNA fluorescent probes for constant temperature incubation, wherein the reaction system includes crRNA specific for the target gene, Cas12a protein, and FAM-ssDNA-BHQ1 fluorescent probe for fluorescence detection; under the mediation of crRNA, guiding the Cas12a protein to specifically recognize the adenylate sulfate reductase gene APS and activate the Cas12a nuclease activity, thereby cutting the fluorescent probe modified with FAM and BHQ1 at both ends to form FAM-ssDNA and BHQ1-ssDNA;

[0009] (3) The green fluorescence intensity of the cut FAM-ssDNA is detected under blue light excitation. A standard curve is obtained based on the relationship between a series of concentrations of adenylate sulfate reductase gene APS standards and the green fluorescence intensity. The bacterial supernatant of the sample to be tested is added to the reaction system of step (2) for testing, thereby determining whether the sample to be tested contains sulfate-reducing bacteria and simultaneously determining the content of sulfate-reducing bacteria.

[0010] As a preferred embodiment of the present invention, the nucleic acid amplification technique described in step (1) is PCR, and the primer sequences used for PCR amplification are as follows:

[0011] The nucleotide sequence of SRB-PCR-F is shown in SEQ ID No. 1;

[0012] The nucleotide sequence of SRB-PCR-R is shown in SEQ ID No.2.

[0013] As a preferred embodiment of the present invention, the PCR amplification system in step (1) includes: 40-60 ng of SRB plasmid template, 9-11 μM of forward primer, 9-11 μM of reverse primer, 9-11 mM of dNTP mixture, buffer, 0.5-2 μL of DNA polymerase, and enzyme-free water supplemented to 50 μL.

[0014] As a preferred embodiment of the present invention, the PCR amplification program in step (1) is: pre-denaturation at 95°C for 10 min; 95°C for 15-30 s, 55°C for 30s-60s, 72°C for 30s-60s, for a total of 30 to 40 cycles; and finally extension at 72°C for 10 min.

[0015] As a preferred embodiment of the present invention, the incubation temperature in step (2) is 30-38°C, and the incubation time is 30-60 min.

[0016] As a preferred embodiment of the present invention, the nucleotide sequence of the specific crRNA described in step (2) is shown in SEQ ID No.4.

[0017] As a preferred embodiment of the present invention, the structure of the FAM-ssDNA-BHQ1 fluorescent probe in step (2) is as follows: 5'-FAM-NNNNN-BHQ1-3', where NNNNN represents any base sequence.

[0018] As a preferred embodiment of the present invention, the concentration of Cas12a described in step (2) is 100-300nM, the concentration ratio of Cas12a to crRNA is 1:0.5-1:5, and the concentration of FAM-ssDNA-BHQ1 fluorescent probe is 150-1500nM.

[0019] As a preferred embodiment of the present invention, in step (3), the wavelength of the blue light is 470-500 nm, and the wavelength of the green fluorescence is 520-540 nm.

[0020] As a preferred embodiment of the present invention, the concentration range of adenylate sulfate reductase gene APS in the standard in step (3) is 10 2 ~10 8 copies / μL.

[0021] As a preferred embodiment of the present invention, the sample to be tested in step (3) is pre-treated by thermal lysis to obtain a bacterial supernatant.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The fluorescence intensity of the detection method of the present invention is in a positive linear relationship with the bacterial content of sulfate-reducing bacteria, and has the advantages of high sensitivity, strong specificity, rapid and efficient, user-friendly and low price. The method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe of the present invention does not require the expansion and cultivation of sulfate-reducing bacteria, and can detect the substance to be detected in real time, and is easy to operate. The reaction conditions of the present invention are mild, the selectivity is high, the detection cycle is short, the cost is low, and it has great promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the gel electrophoresis of genomic DNA extracted from sulfate-reducing bacteria;

[0025] Figure 2 Amplification curve (A) and standard curve (B) of the gold standard RT-qPCR method for detecting the standard plasmid of sulfate-reducing bacteria;

[0026] Figure 3 It is the detection sensitivity of the standard plasmid of sulfate-reducing bacteria detected by the CRISPR / Cas12a and ssDNA fluorescent probe detection method of the present invention, wherein A is a fluorescence detection graph and B is a fitted line graph;

[0027] Figure 4 This is the specific verification result of the detection method of sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe of the present invention, wherein A is a fluorescence detection graph, and B is a fluorescence intensity comparison graph of the detection endpoint. DETAILED DESCRIPTION

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0029] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0030] Example 1

[0031] The actual sample of sulfate-reducing bacteria SRB (Dalian Zhiwei Microbiology Technology Co., Ltd.) was pretreated by thermal lysis. Under sterile conditions, 1 mL of the sample suspension was taken into a sterile centrifuge tube, centrifuged at 10,000 × g for 3 min, and the supernatant was discarded; resuspended with 300 μL of sterile deionized water, centrifuged at 10,000 × g for 3 min, and the supernatant was discarded; 200 μL of sterile deionized water was added and boiled at 100°C for 10 min; the thermal lysis solution was centrifuged at 10,000 × g for 3 min, and the supernatant was used as the substrate for the subsequent amplification reaction. Figure 1 The gel electrophoresis diagram of the genomic DNA extracted from sulfate-reducing bacteria. The target fragment was amplified by PCR amplification technology to obtain the detection standard. The primers used to amplify the specific gene fragment are as follows: SRB-PCR-F (SEQ ID No. 1) and SRB-PCR-R (SEQ ID No. 2).

[0032] Specifically, the nucleotide sequence of SEQ ID No.1:

[0033] 5′-CGCTGAAATGACCATGATGG-3′;

[0034] The nucleotide sequence of SEQ ID No.2:

[0035] 5′-CGCGCATTTCACGAAGC-3′.

[0036] The forward primer is 10 μM, the reverse primer is 10 μM, the dNTP mixture is 10 mM, the buffer, the DNA polymerase is 1 μL, and the enzyme-free water is added to 50 μL for target fragment amplification.

[0037] The PCR program was as follows: pre-denaturation at 95°C for 10 min; 95°C for 15-30 s, 55°C for 30 s-60 s, 72°C for 30 s-60 s, for a total of 30-40 cycles; and finally extension at 72°C for 10 min.

[0038] Specifically, the amplified nucleotide sequence of the detection standard (the conserved region of the adenylate sulfate reductase gene APS) is shown in SEQ ID No.3.

[0039] Construct a CRISPR / Cas12a reaction system, add 200nM Cas12a, 200nM crRNA (SEQ ID No. 4), 1000nM FAM-Reporter-BHQ1, 2μL amplification product, 2μL Cas12a buffer and make up to 20μL with RNase-free water, incubate at 37°C for 40min, and immediately detect the fluorescence intensity of 528nm under 485nm excitation light.

[0040] The nucleotide sequence of SEQ ID No.4:

[0041] 5'-UAAUUUCUAACUAAGUGUAGAUCGCUGAAAUGACCAUGAUGG-3'

[0042] The structure of FAM-Reporter-BHQ1 is as follows:

[0043] 5′-FAM-TTTTT-BHQ1-3′.

[0044] Example 2

[0045] Characterization of the sensitivity of the detection method of the present invention to the sulfate-reducing bacteria SRB standard

[0046] In order to investigate the detection sensitivity of the detection method of the present invention to the standard plasmid of sulfate-reducing bacteria SRB (adenylyl sulfate reductase gene APS conservative region inserted plasmid, the plasmid standard vector is pUC57-Brick, and the standard cloning site is EcoRV), the sensitivity of this method was compared with the gold standard real-time quantitative RT-qPCR detection method.

[0047] Specifically, different contents of 10 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 After the sample of the SRB standard plasmid of 100 copies / μL sulfate-reducing bacteria was PCR amplified by the primers (SEQ ID No.1, SEQ ID No.2) used for the specific gene fragment system of the present invention, the specific gene fragment system was detected by RT-qPCR and the method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe of the present invention.

[0048] The specific process of the method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe of the present invention is the same as that of Example 1.

[0049] Figure 2 The amplification curve and standard curve results of the gold standard RT-qPCR method for detecting sulfate-reducing bacteria are shown. Figure 3The sensitivity of the method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probes in detecting samples with different SRB contents was demonstrated. The method was tested to directly identify as low as 10 2 copies / μL sulfate-reducing bacteria SRB. In comparison, real-time quantitative RT-qPCR can only detect a minimum of 10 3 copies / μL.

[0050] Example 3

[0051] Characterization of the detection specificity of the detection method of the present invention for sulfate-reducing bacteria SRB

[0052] In order to investigate the specific selectivity of the detection method of the present invention for sulfate-reducing bacteria SRB, sulfate-reducing bacteria (SRB) isolates, Escherichia coli (Escherichia coli, E.coil), Bacillus subtilis (Bacillus subtilis), and Lactococcus lactis (L.lactis) were used as test samples, and the method in Example 1 was used for detection to evaluate the detection specificity of the present method.

[0053] The results are as follows Figure 4 As shown in the figure, the sulfate-reducing bacteria SRB isolate was positive under 485nm blue excitation light by fluorescence detection, and the fluorescence detection of Escherichia coli (E.coil), Bacillus subtilis (Bacillussubtilis), and Lactococcus lactis (L.lactis) by specific system was negative, indicating that the CRISPR / Cas12a system detection method has good accuracy and specificity.

[0054] The above-mentioned specific embodiments only describe the preferred embodiments of the present invention and do not limit the protection scope of the present invention. Without departing from the concept and spirit of the present invention, various changes / modifications / substitutions / combinations / simplifications made by ordinary technicians in this field to the technical solution of the present invention according to the text description / drawings provided by the present invention should all be equivalent replacement methods and should all fall within the protection scope of the present invention.

Claims

1. A method for detecting sulfate-reducing bacteria based on CRISPR / Cas12a and ssDNA fluorescent probe, characterized in that: The steps include: (1) Using nucleic acid amplification technology, the specific target gene fragment adenylate sulfate reductase gene APS sequence in sulfate-reducing bacteria SRB was amplified; (2) adding the PCR product amplified in step (1) to a reaction system containing CRISPR / Cas12a and ssDNA fluorescent probes for constant temperature incubation, wherein the reaction system includes crRNA specific for the target gene, Cas12a protein, and FAM-ssDNA-BHQ1 fluorescent probe for fluorescence detection; under the mediation of crRNA, guiding the Cas12a protein to specifically recognize the adenylate sulfate reductase gene APS and activate the Cas12a nuclease activity, thereby cutting the fluorescent probe modified with FAM and BHQ1 at both ends to form FAM-ssDNA and BHQ1-ssDNA; (3) The green fluorescence intensity of the cut FAM-ssDNA is detected under blue light excitation. A standard curve is obtained based on the relationship between a series of concentrations of adenylate sulfate reductase gene APS standards and the green fluorescence intensity. The bacterial supernatant of the sample to be tested is added to the reaction system of step (2) for testing, thereby determining whether the sample to be tested contains sulfate-reducing bacteria and simultaneously determining the content of sulfate-reducing bacteria.

2. The method according to claim 1, characterized in that The nucleic acid amplification technique described in step (1) is PCR, and the primer sequences used for PCR amplification are as follows: The nucleotide sequence of SRB-PCR-F is shown in SEQ ID No. 1; The nucleotide sequence of SRB-PCR-R is shown in SEQ ID No.

2.

3. The method according to claim 2, characterized in that The PCR amplification system in step (1) includes: 40-60 ng of SRB plasmid template, 9-11 μM of forward primer, 9-11 μM of reverse primer, 9-11 mM of dNTP mixture, buffer, 0.5-2 μL of DNA polymerase, and enzyme-free water supplemented to 50 μL; the nucleotide sequence after amplification is shown in SEQ ID No.

3.

4. The method according to claim 2, characterized in that The PCR amplification program in step (1) is: pre-denaturation at 95°C for 10 min; 95°C for 15-30 s, 55°C for 30s-60s, 72°C for 30s-60s, for a total of 30-40 cycles; and finally extension at 72°C for 10 min.

5. The method according to claim 1, characterized in that The incubation temperature in step (2) is 30-38°C, and the incubation time is 30-60 minutes.

6. The method according to claim 1, characterized in that The nucleotide sequence of the specific crRNA described in step (2) is shown in SEQ ID No. 4; the structure of the FAM-ssDNA-BHQ1 fluorescent probe is as follows: 5'-FAM-NNNNN-BHQ1-3', NNNNN represents any base sequence.

7. The method according to claim 1, characterized in that The concentration of Cas12a described in step (2) is 100-300nM, the concentration ratio of Cas12a to crRNA is 1:0.5-1:5, and the concentration of FAM-ssDNA-BHQ1 fluorescent probe is 150-1500nM.

8. The method according to claim 1, characterized in that In step (3), the wavelength of the blue light is 470-500 nm, and the wavelength of the green fluorescence is 520-540 nm.

9. The method according to claim 1, characterized in that The concentration range of adenylate sulfate reductase gene APS in the standard in step (3) is 10 2 ~10 8 copies / μL.

10. The method according to claim 1, characterized in that The sample to be tested in step (3) is pre-treated by thermal lysis to obtain a bacterial supernatant.

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