Method and application of detecting pathogens based on SERS-based CRISPR / Cas system combined with microfluidic paper analysis device

Abstract

The present invention discloses a method for detecting pathogens by combining a SERS-based CRISPR / Cas system with a microfluidic paper analysis device and its application. The method only requires extracting the genome of the pathogen in a sample, and after RPA isothermal amplification, the CRISPR / Cas system can be used to react with a sandwich gold nanostar probe on a μPAD to achieve detection of the microbial pathogen Salmonella. The detection range is wide, the method is simple, fast and easy to operate.

Description

Technical Field

[0001] The present invention belongs to the field of microbial detection, and specifically relates to a method for detecting pathogens by combining a SERS-based CRISPR / Cas system with a microfluidic paper analysis device and an application thereof. Background Art

[0002] Pathogenic bacteria are currently recognized internationally as the main cause of food microbial contamination. According to the World Health Organization, there were 550 million cases of diarrhea in 2010, of which 63.7% were attributable to bacterial infection. Bacterial diarrhea deaths accounted for 81.4%. Some pathogenic bacteria, such as Salmonella, can cause foodborne diseases even at very low or very low levels. The European Commission (EC) has long enforced food microbiological standards, stipulating that Salmonella tests for foods such as minced meat, milk powder and cheese should be "negative". This requires that the detection sensitivity of Salmonella should be reduced to the single cell level. We also know that in some cases, the pathogenicity of bacteria is closely related to concentration. This can be confirmed by the EC regulation, which stipulates that for certain ready-to-eat foods, Listeria monocytogenes should be less than 100 CFU / g, which means the importance of quantitative detection. Therefore, whether from any perspective of prevention, routine monitoring, large-scale screening or emergency response to food safety incidents, it is imperative to develop rapid, highly sensitive, on-site, and cost-effective biosensing methods to determine whether contamination is present.

[0003] Traditional standard methods rely on microbial culture and biochemical testing, which is time-consuming and laborious. In recent years, a series of new methods have been established, including molecular amplification technologies such as PCR and qPCR, antigen-antibody-based immunoassays, gene chips, mass spectrometry, and various biosensors. However, they usually have disadvantages such as long time, expensive instruments, poorly trained personnel, low sensitivity, and long turnaround time, which make on-site testing impossible.

[0004] Microfluidic paper analytical devices (μPADs), in addition to their widespread use in paper lateral flow-based assays, have attracted widespread attention in PON detection because they provide a sample-based detection platform. They are miniaturized, disposable, and customizable in a range of analytical scenarios, including food safety, making them a promising tool, especially in resource-limited and point-of-care testing (POCT). Although μPADs are simple to manufacture, economical, and end-user friendly, they suffer from two major limitations: low detection sensitivity and limited quantitative detection capabilities.

[0005] The CRISPR / Cas system has been used not only for gene editing but also for molecular diagnostics. A notable example is CRISPR / Cas12a, which, as a member of the Class II V CRISPR system, specifically recognizes and cuts target DNA under the guidance of CRISPR RNA (crRNA). When the target DNA is recognized, the trans-cleavage activity of Cas12a is triggered, resulting in the arbitrary fragmentation of ssDNA. Using this anti-cleavage property as a biocatalytic signal generator and amplifier, CRISPR / Cas12a has been made into a new type of biosensing platform, such as DETECTR. In recent years, a number of CRISPR / Cas12a-based methods have been established to detect nucleic acids, single nucleotide polymorphisms, viruses, bacteria, enzymes, small molecules, ions, etc. with different signal readouts (colorimetry, fluorescence, electrochemistry, SERS, etc.). Typically, signal amplification technologies such as RPA are also applied in CRISPR / Cas12a-based detection to further improve sensitivity.

[0006] Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical method based on enhanced Raman scattering of molecules adsorbed on or near SERS-active surfaces, such as noble metal nanostructures. It has been widely used for sensitive and selective detection of chemical and biological molecules. SERS-based CRISPR / Cas detection methods are attractive for PON detection due to their high sensitivity and selectivity and have been used to identify SARS-CoV-2 from clinical samples or other viruses. Recently, CRISPR / Cas12a-based lateral flow assays have achieved single-base mutations in double-stranded DNA with SERS signal readout. However, these methods have never been applied on μPADs. Summary of the invention

[0007] In view of the above-mentioned deficiencies, the purpose of the present invention is to provide a method for detecting pathogens based on a SERS-based CRISPR / Cas system combined with a microfluidic paper analysis device, which provides a new idea for detecting pathogenic microorganisms in food.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for detecting pathogens using a SERS-based CRISPR / Cas system combined with a microfluidic paper analysis device, the method comprising the following steps:

[0010] 1) Extract the pathogen genome and perform RPA isothermal amplification to obtain the genome to be tested;

[0011] 2) adding the sandwich gold nanostar probe to the loading area of ​​the μPAD analysis device for drying;

[0012] 3) adding the genome to be tested in step 1) into the CRISPR / Cas system for reaction to obtain a CRISPR / Cas reaction system;

[0013] 4) adding the CRISPR / Cas reaction system in step 3) to the μPAD analysis device having the sandwich gold nanostar probe dried in step 2) to react, and forming a detection point in the detection area of ​​the μPAD analysis device;

[0014] 5) Using a handheld Raman instrument, perform Raman detection on the detection area of ​​the μPAD analysis device in step 4).

[0015] Furthermore, the preparation method of the sandwich gold nanostar probe comprises the following steps:

[0016] Preliminary preparation: All glassware used in the preparation process needs to be soaked in aqua regia (HNO3:HCl=3:1) for more than 30 minutes, and then rinsed with ultrapure water;

[0017] Preparation of nanostar seeds: Using the seed culture method, heat the HAuCl4 solution until it boils, then quickly add trisodium citrate dihydrate solution to the boiling solution, and continue stirring until the solution gradually changes from light yellow to wine red, thereby obtaining a nanostar seed solution;

[0018] Preparation of sandwich gold nanostars: ultrapure water, HAuCl4, and HCl were added to a centrifuge tube, and then the prepared nanostar seed solution was added. After stirring evenly, silver nitrate solution and ascorbic acid solution were quickly added. After oscillation for 30 seconds, the solution quickly changed from light red to dark green. Then, sodium dodecyl sulfate solution was added, stirred and mixed, centrifuged, the supernatant was discarded, and the solution was resuspended with ultrapure water to obtain a gold nanostar solution AuNSs. 4-MBA was added, and the solution was incubated at 37°C for 2 hours. CTAC, HAuCl4, and AA were added, stirred and mixed, centrifuged, the supernatant was discarded, and the solution was resuspended with ultrapure water to obtain a sandwich gold nanostar solution AuNS@4-MBA@Au.

[0019] Two single-stranded DNA powders modified with thiol SH- at one end, i.e., DNA1 and DNA2, were added with ddH2O to prepare a solution containing DNA1 and DNA2;

[0020] The solution containing DNA1 and DNA2 was added to the AuNS@4-MBA@Au solution, and the mixture was frozen at -20°C. After thawing, the mixture was centrifuged and the supernatant was discarded. The mixture was centrifuged and eluted three times with HEPES buffer and then resuspended to obtain the bound sandwich gold nanostar probe, namely, AuNS@4-MBA@Au@DNA probe.

[0021] Furthermore, the sequences of the two SH-DNAs are respectively as follows:

[0022] DNA1:SH-AAAAAAAAACCCAGGTTCTCT-3';

[0023] DNA2: 5'-TCACAGATGCGTAAAAAAAAA-SH.

[0024] Furthermore, the specific steps of the construction method of the μPAD analysis device are as follows:

[0025] First, use a wax printer to print wax on cellulose filter paper to make walls and wax valves. Then heat it on a hot plate at 120 degrees Celsius for 90 seconds to allow the melted wax to penetrate into the cellulose filter paper. The wax blocks the micropores to form a hydrophobic barrier. The wax valve divides the μPAD into a loading area and a detection area. A 1×2.2 cm adhesive strip is pasted on the back of the device as a back pad to retain the solution, and the μPAD analysis device is constructed.

[0026] Furthermore, the CRISPR / Cas system is selected from the CRISPR-Cas12a system.

[0027] Furthermore, the pathogen is Salmonella enterica serovar Typhimurium.

[0028] Furthermore, the method for detecting pathogens using a SERS-based CRISPR / Cas system combined with a microfluidic paper analysis device is used in the preparation of microbial detection reagents.

[0029] By adopting the above scheme, the present invention has the following advantages:

[0030] 1. The present invention is a method for detecting Salmonella enterica serovar Musciurus using a CRISPR / Cas system combined with μPAD (i.e., RPA-Cas12a-μPAD). The method only includes pre-treatment of the sample and SERS detection of the sandwich gold nanostar probe, and the concentration of the target DNA of the Salmonella genome can be detected. More importantly, the method only needs to extract the genome of Salmonella in the sample, and after RPA isothermal amplification, the detection of microbial pathogenic bacteria Salmonella can be realized on μPAD, which greatly broadens the application of CRISPR-Cas12a and SERS in the field of molecular detection.

[0031] 2. The present invention has established a method for detecting Salmonella by combining CRISPR-Cas12a system with SERS through a series of experiments, that is, the correlation coefficient R of the standard curve of the Salmonella genome detected in the EP tube without amplification and after PCR amplification 2 The values ​​were 0.97 and 0.98, respectively, with good linearity. At the same time, the correlation coefficient R 2The value of is 0.97. Therefore, the number of microbial copies detected by the standard curve in the present invention is more accurate.

[0032] 3. The RPA-Cas12a-μPAD detection method established by the present invention has a low detection limit, the minimum detection limit of samples without amplification is 10-100 CFU / mL, and can detect up to 10 8 CFU / mL, with a wide detection range and high sensitivity.

[0033] 4. The RPA-Cas12a-μPAD detection method established by this method has strong specificity. Taking the detection of Salmonella as an example, there is no obvious Raman signal enhancement for microorganisms such as Escherichia coli, yeast, and Bacillus rhizogenes, which proves that the present invention has good selectivity for Salmonella and is not affected by other microorganisms.

[0034] 5. The sandwich gold nanostar probe (AuNS@4-MBA@Au@DNA probe) designed by the present invention can accurately detect Salmonella. It only needs to simply design the primer sequence and can be used to detect other pathogenic microorganisms. The detection method is accurate, has a wide detection range, is simple, fast and easy to operate.

[0035] 6. Compared with other traditional methods, the design of the present invention has the advantages of simple method and high sensitivity. At the same time, compared with other detection methods (plate culture, qPCR), the design of the present invention greatly reduces the detection cost, thus facilitating large-scale rapid detection of samples.

[0036] 7. The principle of the method for detecting Salmonella by the RPA-Cas12a-μPAD system in the present invention is that the Cas12a-crRNA complex will only stimulate the accessory cutting activity of Cas12a when there is a specific target DNA that can complementarily bind to crRNA, that is, it can cut unrelated single-stranded DNA (ssDNA). If ssDNA is complementary to the SH-DNA connected to the sandwich gold nanostar probe and the Raman intensity is measured, the cutting effect can be converted into a Raman signal output. When Cas12a-crRNA detects the corresponding target DNA, it can cut the linker-ssDNA, so that it cannot complementarily pair with the SH-DNA1 / 2 bound to the sandwich gold nanostar probe, and the sandwich gold nanostar probe free in the supernatant has a strong Raman signal;

[0037] In simple terms, the strength of the Raman signal has a linear relationship with the concentration of the target DNA, that is, the unamplified genome of Salmonella, so the concentration of the Salmonella genome, that is, the copy number of the Salmonella genome (CFU / mL), can be converted into a corresponding Raman signal.

[0038] 8. Under the action of the μPAD analysis device, the separation of AuNS@4-MBA@Au@DNA probes in different aggregation states can be achieved by natural capillary-driven fluid flow through the fiber filter paper, without the need for external force or additional centrifugation to pull down the AuNS@4-MBA@Au@DNA probes, saving costs and speeding up the detection rate.

[0039] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a feasibility Raman spectrum diagram of the CRISPR-Cas12a system combined with SERS for detecting Salmonella of the present invention;

[0041] Figure 2 A feasibility Raman intensity bar graph of the CRISPR-Cas12a system combined with SERS for detecting Salmonella of the present invention;

[0042] Figure 3 This is a UV characterization diagram of the CRISPR-Cas12a system combined with SERS for detecting Salmonella of the present invention;

[0043] Figure 4 This is a DLS graph of the CRISPR-Cas12a system of the present invention combined with SERS to detect Salmonella;

[0044] Figure 5 Optimization diagram of linker ssDNA for detecting Salmonella using the CRISPR-Cas12a system combined with SERS of the present invention

[0045] Figure 6 This is a pH optimization diagram for detecting Salmonella using the CRISPR-Cas12a system combined with SERS of the present invention;

[0046] Figure 7 This is a diagram showing the optimization of the amount of Cas12a enzyme for detecting Salmonella using the CRISPR-Cas12a system of the present invention combined with SERS;

[0047] Figure 8 This is a diagram showing the optimization of the cleavage time for detecting Salmonella using the CRISPR-Cas12a system combined with SERS of the present invention;

[0048] Fig. 9This is a buffer optimization diagram for detecting Salmonella using the CRISPR-Cas12a system combined with SERS of the present invention;

[0049] Fig.10 The CRISPR-Cas12a system of the present invention combined with SERS to detect the Log of Salmonella PCR amplification CFU / mL Linear fit standard curve with Raman intensity;

[0050] Fig.11 This is a bar graph of the LOD of the CRISPR-Cas12a system combined with SERS to detect Salmonella after PCR amplification;

[0051] Fig.12 Log for the detection of Salmonella without amplification by the CRISPR-Cas12a system combined with SERS of the present invention CFU / mL Linear fit standard curve with Raman intensity;

[0052] Fig.13 This is a LOD bar graph of the CRISPR-Cas12a system combined with SERS to detect Salmonella without amplification;

[0053] Fig.14 This is a Raman intensity bar graph of the CRISPR-Cas12a system of the present invention for specific detection of different pathogenic bacteria and cells;

[0054] Fig.15 Raman spectra of the CRISPR-Cas12a system of the present invention for specific detection of different pathogenic bacteria and cells;

[0055] Fig.16 It is a Raman intensity heat map of the Cas12 of the present invention combined with the mutated crRNA and the target specifically recognized;

[0056] Fig.17 It is a diagram of the construction of μPAD of the present invention and the operation steps of Cas12a-μPAD;

[0057] Fig.18 A schematic diagram of SERS detection points in the detection area of ​​the μPAD analysis device of the present invention;

[0058] Fig.19 Raman map of the feasibility experiment of μPAD combined with Cas12a of the present invention;

[0059] Fig. 20 This is a bar graph of the feasibility experiment conducted by μPAD combined with Cas12a of the present invention;

[0060] Fig.21TEM characterization image of AuNS@4-MBA@Au@DNA1 and DNA2 probes attached to the μPAD surface of the present invention;

[0061] Fig. 22 The μPAD physical picture and Log of the RPA-Cas12a-μPAD of the present invention for detecting milk samples CFU / mL Linear fit standard curve with Raman intensity;

[0062] Fig.23 The μPAD physical picture and Log of the RPA-Cas12a-μPAD detecting chicken samples of the present invention CFU / mL Linear fit standard curve with Raman intensity;

[0063] Fig.24 This is a bar graph showing the LOD of milk samples detected by RPA-Cas12a-μPAD of the present invention;

[0064] Fig.25 This is a bar graph showing the LOD of chicken samples detected by RPA-Cas12a-μPAD of the present invention;

[0065] Fig.26 Two qPCR result graphs comparing the present invention with the RPA-Cas12a-μPAD method;

[0066] Fig. 27 The standard curve diagram of the SYBR Green method and the TapMan method of the present invention;

[0067] Fig.28 This is a comparison chart of the positive and negative multiples of the Salmonella genome determined by the TapMan method of the present invention;

[0068] Fig.29 It is a comparison chart of the positive and negative multiples of the SYBR Green method of the present invention;

[0069] Fig.30 This is a comparison chart of negative and positive Raman intensity multiples of the Salmonella genome in the juice determined by the present invention;

[0070] Fig.31 The ROC curve diagram formed by comparing the method of detecting Salmonella by combining the CRISPR-Cas12a system of the present invention with SERS and two methods of qPCR;

[0071] Fig.32 This is a repeatability diagram of the Raman signal after the negative and positive samples of the present invention are measured 10 times respectively;

[0072] Fig.33 This is a Raman signal reproduction diagram of the samples after the Raman signals of 10 different negative and positive samples are measured respectively. DETAILED DESCRIPTION

[0073] The present invention is described in detail below in conjunction with the accompanying drawings and examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment involved in the present invention are conventional reagents, methods and equipment in the technical field.

[0074] Example 1: Extraction of Salmonella genome

[0075] 1.1 Bacterial culture and colony forming unit (CFU) test

[0076] Salmonella was grown in Luria-Bertani broth. Liquid cultures were performed in flasks at 37°C using a shaker. For colony forming unit (CFU) assays, serial dilutions of the bacteria were prepared and 100 μL of the diluted samples were spread on Hektoen enteric (HE) agar plates at 37°C for 24 h. The number of colonies on each plate was recorded.

[0077] 2.1 Extraction of Salmonella genome

[0078] Take 300 μl of bacterial solution to a 1.5 ml centrifuge tube, centrifuge at 12000 r / min for 2 min, discard the supernatant, add extraction solution (25% Chelex-100, 0.5% NP40, prepared with TE, pH = 9.0) to the precipitate, incubate at 56°C for 30 min, place in a 100°C water bath for 10 min, centrifuge at 12000 r / min for 2 min, take the supernatant, and extract the DNA of Salmonella.

[0079] Example 2: Design and purification of crRNA

[0080] Perform RPA isothermal amplification according to the TwistAmp Basic Kit manual:

[0081] The synthesized customized Salmonella DNA template dry powder was centrifuged at 12000 rpm for 10 min, then dissolved and diluted to 5 μM using DEPC water, and then the prepared solution was polymerized in a PCR instrument to polymerize the DNA double strands. The annealing system is shown in Table 1, and the annealing program is shown in Table 2. An appropriate amount of the polymerized double-stranded DNA annealing product was taken for subsequent experiments, and the remaining part was stored in a -20°C refrigerator.

[0082] Table 1 Annealing system

[0083]

[0084]

[0085] The above substances were added into the EP tube in sequence, vortexed to mix, and centrifuged instantaneously.

[0086] Table 2 Annealing schedule

[0087]

[0088] After annealing, the HiScribe T7 fast and efficient RNA synthesis kit was used for the transcription step. The specific CrRNA transcription system is shown in Table 3.

[0089] Table 3 Transcription system

[0090]

[0091] The above substances were added to the EP tube in sequence, vortexed to mix, centrifuged instantaneously, and incubated for transcription at 37°C in a PCR instrument for 16 h.

[0092] Take out the transcription sample incubated overnight at 37°C in the PCR instrument, add 2 μL DNase I (2000U / mL) to the reaction system in the previous step, mix evenly with a pipette, centrifuge instantly to make all the solution pool at the bottom of the tube, place it on a thermal cycler and incubate the reaction at 37°C for 30 minutes to completely eliminate the DNA template in the system, and then purify RNA according to the instructions of the Monarch RNA purification kit. The purified RNA is purified using Eppendorf The RNA concentration was measured using a basic ultra-micro nucleic acid quantifier and finally stored in a -80°C refrigerator.

[0093] Example 3: Preparation of Sandwich Gold Nanostar Probes

[0094] 1. Preparation of sandwich gold nanostars:

[0095] Preliminary preparation: All glassware used in the preparation process needs to be soaked in aqua regia (HNO3:HCl=3:1) for more than 30 minutes, and then rinsed with ultrapure water;

[0096] Preparation of nanostar seeds: Using the seed culture method, add 100 mL (1 mM) HAuCl4 solution to a 250 mL Erlenmeyer flask and heat until boiling. Then quickly add 3 mL (1% mass fraction) trisodium citrate dihydrate solution to the solution, stirring continuously, the solution gradually changes from light yellow to wine red, continue to add for 15 minutes, cool to room temperature, obtain nanostar seed solution, and store at 4 ° C for later use;

[0097] Preparation of sandwich gold nanostars: Add 20 mL of ultrapure water to a 50 mL centrifuge tube, then add HAuCl4 to a concentration of 0.25 mM, add 20 μL of 1 M HCl, and then add 200 μL of the prepared nanostar seed solution. After stirring evenly, quickly add 200 μL of silver nitrate solution (2 mM) and 100 μL of ascorbic acid solution (0.1 M). After oscillating for 30 seconds, the solution quickly changes from light red to dark green. Then add 200 μL of sodium dodecyl sulfate solution (2%) and stir for 5 minutes. After centrifugation at 1300 g for 15 minutes, discard the supernatant and resuspend with 20 mL of ultrapure water to obtain a gold nanostar solution (AuNSs) and place it in a 4 ° C refrigerator away from light for later use;

[0098] 40 μL of 1 mM 4-MBA was added to 4 mL of AuNSs solution and incubated at 37 °C for 2 h. Then 2 mL of CTAC (0.2 M), 60 μL of HAuCl4 (10 mM), and 120 μL of AA (0.2 M) were added and stirred for 20 min. After that, the mixture was centrifuged at 1000 g for 20 min, the supernatant was discarded, and the mixture was resuspended in ultrapure water to obtain a sandwich gold nanostar solution (AuNS@4-MBA@Au), which was stored in a refrigerator at 4 °C away from light.

[0099] 2. Preparation of sandwich gold nanostar probes by synthesis of sandwich gold nanostars and thiol DNA:

[0100] Two single-stranded DNAs with thiol (SH-) modified at one end, namely DNA1 and DNA2 dry powder, were added to ddH2O to prepare 100μM DNA1 and DNA2 solutions. The sequences of the two thiol DNAs are as follows:

[0101] DNA1: SH-AAAAAAAAACCCAGGTTCTCT-3'

[0102] DNA2: 5'-TCACAGATGCGTAAAAAAAAA-SH

[0103] Take 5uL of thiol DNA1 and DNA2 (100uM) respectively and add them to 200uL sandwich gold nanostar solution, shake and mix well, freeze and bind for 2h at -20℃. After thawing, centrifuge at 12000rpm for 30min at 4℃, discard the supernatant, repeat centrifugation and elute 3 times with buffer A (5mM HEPES, pH 7.6), and then resuspend with buffer B (10mM HEPES, 300mM NaCl, pH 7.6) to obtain the bound sandwich gold nanostar probe (AuNS@4-MBA@Au@DNA probe), which is placed at 4℃ for use.

[0104] Example 4: Construction of μPAD analysis device

[0105] Wax was first printed on Whatman grade 4 filter paper (i.e., cellulose filter paper) using a wax printer to make walls and valves, and then heated on a hot plate at 120 degrees Celsius for 90 seconds to allow the melted wax to penetrate the paper. The wax blocked the micropores to form a hydrophobic barrier. In addition, a 30% transparency wax valve was printed on the filter paper at the same time as the channel wall. The wax valve is a hydrophobic area that is used on the μPAD to separate the loading area and the detection area. The liquid solution is retained in the loading area by the wax valve. After adding 0.4μL of isopropanol, the wax dissolved and the solution flowed. In addition, a 1×2.2cm adhesive strip was attached to the back of the device as a back pad to retain the solution;

[0106] The construction and step-by-step operation of μPAD are as follows Fig.17 As shown, Fig.18 The detection using μPAD is shown and the position indicated by the arrow is the SERS test point.

[0107] Example 5: Feasibility experiment of CRISPR-Cas12a system combined with SERS to detect Salmonella

[0108] 1. Experimental methods:

[0109] Prepare 5 different samples, namely Positive, No Cas12a, No crRNA, No target and Only reporter, each consisting of 100μL system; Positive: 200nM Cas12a, 250nM crRNA and 1nM target DNA complementary to crRNA, 15nM linker DNA and HEPES buffer (5mM HEPES, 150mM NaCl, 10mM MgCl2, pH7.6), No Cas12a means that the Cas12a enzyme is removed from the Positive sample, and similarly, No crRNA and No target. Only reporter has only 15nM linker DNA; react at 37℃ for 10min, and then inactivate at 65℃ for 8min. 50 μL of each sample was taken and added to EP tubes containing 25 μL of AuNS@4-MBA@Au@DNA1 and DNA2, respectively. The tubes were incubated at 37°C for 10 min and then centrifuged at 5000 rpm / min for 3 min. The Raman signals of the supernatants of the five samples were measured respectively.

[0110] 2. Experimental results

[0111] like Figure 1 and Figure 2As shown, a strong Raman signal is generated when and only when Cas12a, crRNA, and target DNA complementary to crRNA are present at the same time, which verifies that Cas12a has the ability of accessory cleavage triggered by nucleic acid specific recognition, proves the feasibility of the method of the present invention, and has specific recognition ability.

[0112] Example 6: Characterization experiment of CRISPR-Cas12a system combined with SERS detection of Salmonella

[0113] 1. Experimental methods

[0114] UV characterization: 100 μL of each AuNSs, AuNSs@4-MBA, and AuNSs@4-MBA@Au solution were added to an opaque 96-well plate, and the full UV spectrum was scanned using an ELISA reader with a scanning range of 400-800 nm and a scanning interval of 2 nm.

[0115] DLS characterization: 1 mL of each sample of AuNPs, AuNSs@4-MBA, AuNSs@4-MBA@Au and After linking (after linkerssDNA was complementary to AuNSs@4-MBA@Au@DNA1 and DNA2) was taken, and the water content and particle size of the samples were measured by DLS.

[0116] 2. Experimental results

[0117] like Figure 3 As shown, after 4-MBA binds to AuNSs, the plasmon resonance on the AuNSs surface is slightly red-shifted, and then blue-shifts to 586 nm after being coated by the gold nanoshell;

[0118] like Figure 4 As shown, the average hydrated particle sizes of AuNPs, AuNSs, AuNS@4-MBA@Au and cross-linked AuNS@4-MBA@Au@DNA were 18.4±0.94, 58.1±4.37, 295.8±13.8 and 3000.5±247.4 nm, respectively.

[0119] Example 7: Optimization experiment of CRISPR-Cas12a system combined with SERS for detection of Salmonella

[0120] 1. Experimental methods

[0121] Optimize the concentration of ssDNA: Add two thiol DNA AuNS@4-MBA@Au@DNA probes to 50μL solution containing different concentrations of ssDNA, and the concentrations of ssDNA are 0, 1, 2, 5, 10, 15, 20, 30nM. The reaction is kept at 37°C for 10min, and after centrifugation at 5000rpm for 1min, the supernatant is collected and analyzed by Raman spectroscopy;

[0122] Optimization of Cas12a trans-cleavage duration: 200nM Cas12a, 250nM crRNA, 15nM ssDNA and 1nM target double-stranded DNA (dsDNA) were incubated in HEPES buffer (5mM HEPES, 150mM NaCl, 10mM MgCl2, pH 7.6) at 37°C for different durations of 0, 3, 5, 10, 20, 30, 40min, and then inactivated at 65°C for 8 minutes. 50μL of the reactant was mixed with 25μL of two AuNS@4-MBA@Au@DNA probes and incubated at 37°C for 5min, then centrifuged at 5000rpm for 1min, and the supernatant was collected and analyzed using a portable Raman spectrometer equipped with a 785nm laser beam;

[0123] Cas12a concentration, pH value, and buffer system optimization: Tris-buffered saline (TBS): 50mM Tris-HCl, 0.2MNaCl, pH=7.5; phosphate-buffered saline (PBS): 100mM NaH2PO4, 100mM Na2HPO4, pH=7.0; Tris buffer: 50mM Tris-HCl, 100mM NaCl, 1mM DTT, pH=7.0; HEPES buffer: 10mM HEPES, 300mMNaCl. The above reaction was carried out, the supernatant was collected and analyzed by Raman spectroscopy.

[0124] 2. Experimental results

[0125] like Figure 5-Figure 9 As shown,

[0126] To optimize the linker ssDNA concentration, Figure 5 It can be seen that as the concentration of linker ssDNA gradually increases, the SERS probes in the solution are gradually pulled down and aggregated, and the Raman intensity in the supernatant gradually decreases. When the concentration is 15nM, the Raman intensity reaches a plateau and tends to be stable. Therefore, the concentration of linker ssDNA is selected to be 15nM;

[0127] For the optimization of Cas12a cutting time, the Raman signal gradually increased from 0 to 10 min, and after 10 min, the Raman signal tended to be stable, so the Cas12a cutting time was set to 10 min;

[0128] For the optimization of pH, since the change of pH has little effect on the strength of Raman signal in the range of pH: 5-9, and the buffer pH of Cas12a reaction is 7.6, pH 8 is selected as the pH value after system optimization; for the optimization of Cas12a concentration, when the concentration of Cas12a is less than 200nM, the Raman signal gradually increases, and after being greater than 200nM, the intensity of the Raman signal tends to be stable, so 200nM is selected as the enzyme concentration of Cas12a; for the optimization of Cas12a reaction buffer, it can be clearly seen that in the reaction buffers of different concentrations, the strength of the Raman signal, among which the signal of HEPES Buffer is the highest, so HEPES Buffer is used as the reaction buffer of Cas12a.

[0129] Example 8: Standard curve, sensitivity, specificity and selectivity of CRISPR-Cas12a system combined with SERS for Salmonella detection

[0130] 1. Experimental methods

[0131] 1.1 Standard curve:

[0132] In the SERS-Cas12a system, 200nM LbCas12a, 250nM crRNA, 15nM linker DNA and different concentrations (10 0 —10 8 ) of Salmonella genomic target DNA was reacted in HEPES buffer (5mM, pH 7.6, NaCl 150 mM, 10mM MgCl2) at 37℃ for 10min, and then inactivated at 65℃ for 2min. 50μL of the reaction solution was added to two 25μL AuNS@4-MBA@Au@DNA probes at 37℃ for 5min. The mixture was centrifuged at 5000 rpm / min for 3min. The supernatant was collected for Raman spectroscopy analysis.

[0133] The correlation between SERS signal and Salmonella concentration was established, and the corresponding linear fitting was performed. Since the extracted Salmonella genomes were divided into amplified and unamplified, the standard curves were as follows: Fig.10 , Fig.12 shown.

[0134] 1.2 Sensitivity:

[0135] After establishing the standard curve of Raman signal and Salmonella concentration, the sensitivity of the amplified and non-amplified methods was calculated according to the LOD. Fig.11 , Fig.13 shown.

[0136] 1.3 Specificity:

[0137] Specificity of the SERS-Cas12a system: Genomic DNA was extracted from 8 different interfering organisms with the same concentration as Salmonella, including Agrobacterium tumefaciens, yeast, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Hela cells, HT29 cells, and MGC803 cells. PCR amplification was performed using Salmonella-specific primers, and then SERS-Cas12a detection was performed. Specificity is as follows Fig.14 , 15 As shown;

[0138] The specificity of SERS-Cas12a in crRNA design: three crRNAs with only one or two mutant nucleotides compared to the correct crRNA were synthesized, corresponding to four different target dsDNAs. Fig.16 As shown:

[0139] 1.4 Selectivity:

[0140] Genomic DNA was extracted from eight different interfering organisms at the same concentration as Salmonella and amplified by PCR using Salmonella-specific primers, followed by SERS-Cas12a detection.

[0141] 2. Experimental results

[0142] Standard curve: Figure 10-12 As shown in the figure, for the standard curves of the amplified and non-amplified methods, the linear correlation coefficient R 2 are 0.98 and 0.97, respectively, indicating that the intensity of the Raman signal is linearly dependent on the concentration of Salmonella;

[0143] Sensitivity: According to Fig.11 , 13 As shown in the LOD in the figure, the LOD after amplification can reach 1 CFU / mL, which is equivalent to 0.1 aM DNA; while the LOD without amplification can reach 10 1 CFU / mL, equivalent to 1aM DNA;

[0144] Specificity: such as Figure 14-16As shown, by detecting genomic DNA extracted from eight different interfering organisms with the same concentration as Salmonella, by measuring the Raman signal, it can be found that only when Salmonella is detected, the trans-cleavage activity of Cas12a is activated, so that the Raman signal is enhanced, which shows the selective and specific recognition ability of the SERS-Cas12a system for Salmonella. In addition, SERS-Cas12a can also analyze single nucleotide polymorphisms (SNPs) because it strictly activates trans-cleavage activity by recognizing target DNA through Cas12a-crRNA. Therefore, crRNA strictly recognizes its homologous target dsDNA and produces a strong Raman signal. In contrast, other base-mismatched crRNAs and non-homologous target dsDNA do not activate the trans-cleavage activity of CRISPR-Cas12a, so the Raman signal is very low. The results show that SERS-Cas12a has high specificity in identifying single nucleotide changes;

[0145] Selectivity: e.g. Figure 15-16 As shown, only when Salmonella was present, the Raman signal intensity increased significantly, proving that this method has good selectivity for Salmonella in the presence of different interfering organisms.

[0146] Example 9: Feasibility experiment of RPA-Cas12a-μPAD system for detecting Salmonella

[0147] 1. Experimental methods

[0148] 25 μL of AuNS@4-MBA@Au@DNA probe was premixed and dropped into the μPAD loading area. After 10 min, each μPAD was dried at 37 °C for 5 min, and then the linker ssDNA was added to 100 μL Cas12a trans-cleavage system (50 μL RPA amplification concentration was 10 0 -10 8 The target DNA was added to trigger Cas12a trans-cleavage within 10 min, and then inactivated at 65 °C for 5 min). After dissolving the wax valve with 0.4 μL isopropanol, the reaction mixture was infiltrated along the bridge liquid for 1 min. Raman spectroscopy was performed on the detection area of ​​each μPAD at the SERS test point. Raman spectra were recorded using near-infrared laser emission light with a wavelength of 785 nm under the conditions of a laser power of 150 mW and an integration time of 10 s.

[0149] Different concentrations of Salmonella culture were added to everyday food samples that were negative for Salmonella, such as milk and meat, and the genome of Salmonella was extracted and detected using the RPA-Cas12a-μPAD system.

[0150] 2. Experimental results

[0151] like Fig.19 , 20 As shown, the RPA-Cas12a-μPAD system only produces a strong SERS signal for positive samples;

[0152] like Fig.21 As shown, using scanning electron microscopy (SEM) technology, it can be proved that the test point is impregnated with SERS nanoparticles. This shows that the uncross-linked AuNS@4-MBA@Au@DNA probe can diffuse farther than the cross-linked nanoprobe and eventually reach the trapped test point;

[0153] like Figure 22-23 As shown, the SERS signal of the designed μPAD increases with the increase of Salmonella concentration, and a linear relationship can be obtained, indicating the specific reactivity of the designed μPAD to Salmonella;

[0154] like Figure 24-25 As shown, the LOD values ​​of milk and meat samples were both 1 CFU / mL;

[0155] like Fig.26 A is the TapMan method, and B is the SYBR Green method. First, two qPCRs were performed based on SYBR Green and TaqMan. The standard curves of the two methods are shown in Fig. 27 , then, as Figure 28-30 As shown, a double-blind experiment was designed to distinguish contaminated samples from sterilized samples using RPA-Cas12a-μPAD and qPCR methods, and the receiver operating characteristic (ROC) curve was used accordingly. Fig.31 As shown, the area under the ROC curve (AUC) of AF-Cas12-iT was 1.0, the AUC of SYBR GreenqPCR was 0.884, and the AUC of TaqMan qPCR was 0.981;

[0156] like Fig.32 and 33 As shown, the repeatability and reproducibility of the RPA-Cas12a-μPAD system, the relative standard deviation (RSD) value was obtained, and all calculated RSD values ​​of repeatability and reproducibility were less than 6%, indicating acceptable repeatability and reproducibility. Repeatability and reproducibility are key issues in the development of the RPA-Cas12a-μPAD system. It is generally recommended that an RSD value of less than 10% is a prerequisite for verifying the reliability of the developed RPA-Cas12a-μPAD system.

[0157] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting pathogens by using a SERS-based CRISPR / Cas system combined with a microfluidic paper analysis device, which is not intended for the diagnosis and treatment of diseases, characterized in that: The method comprises the following steps: 1) Extract the pathogen genome and perform RPA isothermal amplification to obtain the genome to be tested; 2) adding the sandwich gold nanostar probe to the loading area of ​​the μPAD analysis device for drying; 3) adding the genome to be tested in step 1) into the CRISPR / Cas system for reaction to obtain a CRISPR / Cas reaction system; 4) adding the CRISPR / Cas reaction system in step 3) to the μPAD analysis device having the sandwich gold nanostar probe dried in step 2) to react, and forming a detection point in the detection area of ​​the μPAD analysis device; 5) using a handheld Raman instrument to perform Raman detection on the detection area of ​​the μPAD analysis device in step 4); The preparation method of the sandwich gold nanostar probe comprises the following steps: Preliminary preparation: All glassware used in the preparation process needs to be soaked in aqua regia (HNO3:HCl=3:1) for more than 30 minutes, and then rinsed with ultrapure water; Preparation of nanostar seeds: Using the seed culture method, heat the HAuCl4 solution until it boils, then quickly add trisodium citrate dihydrate solution to the boiling solution, and continue stirring until the solution gradually changes from light yellow to wine red, thereby obtaining a nanostar seed solution; Preparation of sandwich gold nanostars: ultrapure water, HAuCl4, and HCl were added to a centrifuge tube, and then the prepared nanostar seed solution was added. After stirring evenly, silver nitrate solution and ascorbic acid solution were quickly added. After oscillation for 30 seconds, the solution quickly changed from light red to dark green. Then, sodium dodecyl sulfate solution was added, stirred and mixed, centrifuged, the supernatant was discarded, and the solution was resuspended with ultrapure water to obtain a gold nanostar solution AuNSs. 4-MBA was added, and the solution was incubated at 37°C for 2 hours. CTAC, HAuCl4, and AA were added, stirred and mixed, centrifuged, the supernatant was discarded, and the solution was resuspended with ultrapure water to obtain a sandwich gold nanostar solution AuNS@4-MBA@Au. Two single-stranded DNA powders modified with thiol SH- at one end, i.e., DNA1 and DNA2, were added with ddH2O to prepare a solution containing DNA1 and DNA2; The solution containing DNA1 and DNA2 was added to the AuNS@4-MBA@Au solution, and then frozen at -20°C for mixing. After thawing, the solution was centrifuged and the supernatant was discarded. The solution was centrifuged and eluted three times with HEPES buffer and then resuspended to obtain the bound sandwich gold nanostar probe, namely, AuNS@4-MBA@Au@DNA probe. The sequences of the two SH-DNAs are as follows: DNA1:SH-AAAAAAAAACCCAGGTTCTCT-3'; DNA2: 5'-TCACAGATGCGTAAAAAAAAA-SH; The specific steps of the construction method of the μPAD analysis device are as follows: First, wax was printed on cellulose filter paper using a wax printer to make walls and wax valves. Then, the wax was heated on a hot plate at 120 degrees Celsius for 90 seconds to allow the melted wax to penetrate into the cellulose filter paper. The wax blocked the micropores to form a hydrophobic barrier. The wax valve divided the μPAD into a loading area and a detection area. A 1×2.2 cm adhesive strip was pasted on the back of the device as a back pad to retain the solution. Thus, the μPAD analysis device was constructed. The CRISPR / Cas system is selected from the CRISPR-Cas12a system; The pathogen is Salmonella enterica serovar Typhimurium; The correlation coefficient R between the standard curve of the Salmonella genome detected in the EP tube without amplification and after PCR amplification 2 The value of 0.98, with good linearity, and the correlation coefficient R of the standard curve detected by the RPA-Cas12a-μPAD method 2 The value of is 0.97; The method has a low detection limit, with a minimum detection limit of 10-100 CFU / mL for samples without amplification, and can detect up to 10 8 CFU / mL.

2. Use of the method according to claim 1 in preparing microbial detection reagents.

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