Salmonella high-sensitivity sensing detection method based on fluorescent nano-diamond

Abstract

The invention provides a lateral chromatography test strip for salmonella detection and a detection method thereof, and belongs to the field of food safety diagnosis. The fluorescent nano-diamond is a nano-diamond containing a nitrogen vacancy color center, red fluorescence near 670 nm can be excited under exciting light of 540 nm, and meanwhile, the fluorescent nano-diamond can change internal quantum spinning and filter background fluorescence through microwave regulation and control, so that the detection limit is increased; the test strip disclosed by the invention can obtain first signal amplification through a Cas12 specific targeting activation mechanism in an actual sample with relatively low concentration, and then performs second amplification through a fluorescent signal of the fluorescent nano-diamond, so that rapid and high-sensitivity detection of salmonella is realized. According to the present invention, the signal output mode is changed by replacing the labeling material, the sensitivity of the test strip is improved, the Cas12 enzyme has accurate nucleic acid targeting and cutting functions, non-target nucleic acid can be cut, the detection signal is further amplified, the detection specificity and the detection sensitivity are improved, and the Cas12 enzyme has characteristics of high sensitivity and high sensitivity. And the fluorescent signal can be quickly and visually read, so that the fluorescent probe can be used for on-site detection, the timely and quick diagnosis requirement is met, and the fluorescent probe has important application potential and economic value in the field of food safety diagnosis.

Description

Technical Field

[0001] The present invention designs a simple, rapid, specific and sensitive microbial diagnosis technology, which belongs to the field of microbial detection technology Background Art

[0002] Nowadays, infectious diseases caused by pathogenic microorganisms are seriously threatening the life and health of people all over the world.

[0003] Pathogenic microorganism nucleic acid diagnostic technology has a wide range of applications, such as POCT, food safety, environmental testing and other fields. Foodborne diseases are the number one problem in food safety. They are mainly caused by pathogenic microorganisms and are highly contagious, such as Salmonella, Listeria, Staphylococcus aureus, etc. Every year, 80 to 94 million people in the world are infected with Salmonella, of which 59,000 to 155,000 people die. Therefore, it is necessary to establish effective detection technology to block the spread of pathogens in a timely manner.

[0004] Fluorescent nanodiamonds are diamonds with a particle size in the range of 1nm to 200nm. They have good biocompatibility, low cytotoxicity, and fluorescence without photobleaching. Fluorescent nanodiamonds (FNDs) containing nitrogen vacancy (NV) centers (defects with optical transitions within the band gap) have received considerable attention as a spin system used as qubits in quantum computing and communication, as well as quantum sensing. Such applications stem from the fact that the negative NV (NV-) center spin state can be optically initialized and read out, and manipulated using both DC and microwave magnetic fields. FNDs also have attractive fluorescence properties, including high quantum yield, no blinking, surface groups that can be used for biofunctionalization, and ease of large-scale production, such as by high-pressure and high-temperature diamond milling. Sensing applications of NV centers include magnetic field quantification, temperature sensing, and biolabeling, the latter including cell imaging, drug delivery, and contrast enhancement in magnetic resonance imaging. A key advantage of NV- centers is that, compared to neutral (NV 0 ) centers, their fluorescence can be selectively modulated by spin manipulation, thus enabling signal separation in imaging in high-background environments.

[0005] The CRISPR / Cas system is one of the emerging nucleic acid detection technologies in recent years. It is a new immune system produced by microorganisms to adapt to the external environment. It is suitable for most archaea and bacteria. It is mainly composed of two parts: the CRISPR locus and the Cas gene cluster. The CRISPR locus includes three parts: the leader sequence, the spacer sequence and the repeat sequence. The principle of CRISPR diagnosis is: when the target nucleic acid exists in the system to be tested, the Cas protein specifically recognizes the target sequence under the guidance of the guide RNA to form a complex, which is then stimulated to produce incidental cutting activity against single-stranded RNA, chopping up the fluorescent labeled probes in the system, emitting a fluorescent signal, completing the detection process, and judging whether the target nucleic acid exists by whether the detection system emits light. So far, Cas nucleases including Cas9, Cas12, and Cas13 have been widely used in gene editing, molecular diagnosis and other fields, but CRISPR-Cas9 has the disadvantage of not being able to detect multiple targets in the same sample. The cleavage activity of Cas13 on single-stranded RNA requires the target RNA to activate, which is costly and requires strict operation. The most common application is to use the non-specific ssDNA cleavage activity of Cas12 for biological diagnostic testing. CRISPR-Cas12 has precise nucleic acid targeting and cleavage functions, and can cleave non-target nucleic acids, further amplify the detection signal, and improve the detection sensitivity. The discovery of Ca12's trans-cleavage activity has made many portable diagnostic tools possible.

[0006] In this study, we established a method for rapid, specific, accurate and sensitive detection of Salmonella by combining fluorescent nanodiamonds with Cas12a test strips. Our method is characterized by rapid, high sensitivity and high specificity. FND also has attractive fluorescence properties, including high quantum yield, no blinking and photobleaching, so it can replace traditional nanogold as a labeling material, and the detection depends on the recognition of the extracted Salmonella nucleic acid by the Cas12a protein and the subsequent trans-cleavage signal; visualization, because the detection signal comes from the fluorescence generated by the cleavage probe FND, which can be clearly observed under a convenient fluorescence detection instrument. This system is suitable for early detection of pathogens in a wide range of field environments, and can effectively achieve detection to limit their spread. Summary of the invention

[0008] A method for detecting pathogens by a test strip combining fluorescent nanodiamond and Cas12a, characterized in that the steps of the method include: material preparation, sgRNA design and synthesis, bacterial culture and DNA extraction, modification and characterization of fluorescent nanodiamonds, design and synthesis of probes, establishment of Cas12a detection system, verification of the feasibility of fluorescent nanodiamonds, and assembly and debugging of test strips.

[0009] The diamond modification antibody described above uses Anti-6-FAM antibody. The particles are concentrated by centrifuging 100 μl FND–PG at 21,130 g for 7.5 minutes. The supernatant is then removed and the FND is resuspended in anhydrous N,N-dimethylformamide (DMF, 99.8%, Sigma-Aldrich). After resuspension in DMF, the colloidal solution is sonicated in an ultrasonic bath for 5 minutes. The washing and sonication steps are repeated 3 times to remove water. After the last centrifugation, the particles are resuspended in 100 μl 50 mg ml -1 DSC (≥95%, Sigma-Aldrich) in DMF and placed in a thermostatic shaker at 300 rpm for 3.5 h at room temperature. Excess reagent was removed by three cycles of centrifugation and resuspension in DMF (as described above). After the third centrifugation, the pellet was resuspended in 100 μl of deionized water and 13.7 μl of antibody (1 mg ml -1 ) was added to the activated FND. The mixture was placed in a shaker at 300 rpm overnight at room temperature for 15 hours. The remaining succinimidyl carbonate was quenched by adding 10 μl Tris-HCl pH 7.5 (1 M). After 30 minutes, unbound reagent was removed by three centrifugation cycles and resuspended in deionized water (100 μl) and stored in 100 μl PBS containing 0.1 wt% BSA.

[0010] The design and synthesis of the sgRNA described above is a primer set designed based on the conserved sequence region of the Salmonella-specific gene.

[0011] The nucleotide sequence shown in SEQ ID NO: 1 is: sgRNA: 5'-UAGCGCGACCAUAGUGAAGA-3'.

[0012] The nucleotide sequence shown in SEQ ID NO: 2 is: ssDNA: 5'-TAGCGCGACCATAGTGAAGA-3'.

[0013] The nucleotide sequence shown in SEQ ID NO:3 is: ssDNA-FQ:6-FAM-TTTTTTTTTTTT-BHQ.

[0014] The above-mentioned bacterial culture and DNA extraction were carried out by inoculating bacteria into LB medium (LB: 10 g / L tryptone, 5 g / L yeast powder, 5 g / L NaCl) and culturing at 37°C in a shaking incubator (180 rpm) for 12 h, and then extracting genomic DNA using a kit.

[0015] The Cas12a proteins described above were all purchased from NEB.

[0017] The establishment of the above-mentioned Cas12a feasibility detection system, the total reaction system is 20 μL, and the components include:

[0018] The detection system described above uses a fluorescence spectrometer to detect fluorescence signals at a wavelength of 540 nm.

[0019] The essential features and improvements of the present invention are: 1) Strong specificity All positive samples tested were detected, and none of the negative controls tested had positive results, which was consistent with the results of the gold standard qPCR. 2) High sensitivity 3) Wide range of applications 4) Fast detection speed 5) The equipment is simple and can be observed directly with the naked eye or in real time BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of the first detection method of the present invention; Figure 2 A schematic diagram of the feasibility verification of the first detection method of the present invention; Figure 3 Schematic diagram of the second detection method of the present invention; Figure 4 A schematic diagram of the feasibility verification of the second detection method of the present invention; DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with the embodiments.

[0026] The present invention conducts a Salmonella detection feasibility verification experiment, and the method specifically comprises the following steps:

[0027] Salmonella typhi was inoculated into LB medium and cultured overnight. DNA was extracted using a kit and DNA was used as the target. The extracted DNA was added to the Cas12a detection system. The system components included: 1× reaction buffer, 1μM Cas12a, 1μM sgRNA, 0.5μM ssDNA-FQ, 10× reaction buffer, ddH 2 O to 20 μL. After 10 minutes of cleavage reaction, take 5-10ul of the cleavage product and add it to the test strip. After 5 minutes, observe the fluorescence at 540nm using a convenient fluorescence detection instrument.

[0028] The present invention conducts a Salmonella detection feasibility verification experiment, and the method specifically comprises the following steps:

[0029] Salmonella typhi was inoculated into LB medium and cultured overnight. DNA was extracted using a kit and DNA was used as the target. The extracted DNA was added to the Cas12a detection system. The system components included: 1× reaction buffer, 1μM Cas12a, 1μM sgRNA, 0.5μM ssDNA-FQ, 10× reaction buffer, ddH 2 O to 20 μL. After 10 min of cleavage reaction, the cleavage product was adsorbed and fixed on a magnet, and the supernatant was taken and the fluorescence was observed at 540 nm under a fluorescence spectrometer.

Claims

1. A method for detecting Salmonella using a fluorescent nanodiamond test strip, characterized in that: The method comprises a test strip preparation step, a detection method and a pathogenic microorganism detection step.

2. Based on claim 1: It is characterized in that The test strip was prepared by spraying the conjugate pad with 120 nm diameter nanodiamonds, the surface of the nanodiamonds was modified with 6-FAM antibody, and the C line and T line were labeled with streptavidin and anti-rabbit antibody, respectively.

3. Based on claim 1: The fluorescence of the test strip is detected by adjusting the fluorescence signal through a square wave 4 Hz amplitude modulated microwave field, with excitation at a wavelength of 540 nm and emission at a wavelength of 670 nm.

4. Based on claim 3: The feasibility of the test strip detection is tested by magnetic bead adsorption, and the diamond modified with the antibody is connected to the ssDNA with FAM group and magnetic beads as the trans-cleavage probe FQ of Cas12.

5. Based on claim 4: Pathogenic microbial nucleic acid is extracted as a target, Cas12 recognizes the extracted target dsDNA under the mediation of specific sgRNA and performs cis and trans cleavage reactions, and the cleavage products are fixed and adsorbed with a magnet to measure the fluorescence of the supernatant.

6. Based on claim 5: dripping the cleavage product obtained in step 5 onto the sample pad of a test strip for incubation, and detecting the fluorescent signal of the fluorescent nanodiamond on the test strip by chromatography.

7. Based on the test strip detection technology combined with fluorescent nanodiamond and Cas12 according to claim 5, the specific target is the Salmonella genome SEQ NO: 1, and the probe FQ sequence is SEQ NO:

3.

8. Based on the test strip detection technology combined with fluorescent nanodiamond and Cas12 according to claim 5, the specific sgRNA needle Salmonella, the sgRNA sequence of Salmonella SEQ NO: 2.

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