CN116555485A - Method, application and device for detecting pathogenic microorganisms by surface enhanced Raman scattering biological nano sensor - Google Patents
Method, application and device for detecting pathogenic microorganisms by surface enhanced Raman scattering biological nano sensor Download PDFInfo
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Abstract
The invention discloses a method for detecting pathogenic microorganisms by a surface enhanced Raman scattering biological nano sensor based on CRISPR-Cas12a drive, which comprises the following steps: extracting pathogenic microorganism RNA, and then reversely transcribing the pathogenic microorganism RNA into cDNA by reverse transcription; specific crrnas designed in the CRISPR-Cas12a system Target recognition targets; the AuNPs@4-MBA@DNA nano probe and a CRISPR-Cas12a cutting reaction completed system are uniformly mixed in a system, placed, centrifuged and detected. The method can detect the sample by only simple pretreatment, CRISPR-Cas12a system reaction and hand-held microscopic Raman instant detection for 3 steps, can detect different microorganisms by only changing nucleic acid sequences, and can be used as a universal nucleic acid detection platform under various conditions.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to a method for detecting pathogenic microorganisms based on a Surface Enhanced Raman Scattering (SERS) biological nano sensor driven by CRISPR-Cas12a, application and a novel sleeve device.
Background
Nucleic acid detection is known as a gold standard for many infectious disease diagnoses due to its high sensitivity, specificity and lack of window-time limitations. Unlike current reverse transcription polymerase chain reaction (RT-qPCR), the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated (CRISPR-Cas) system is known as a next generation diagnostic technique, potentially completely altering the SARS-CoV-2 nucleic acid detection tool. CRISPR-Cas12a is a member of a class 2V CRISPR system. Upon recognition and binding to a specific target sequence (double-stranded DNA or single-stranded DNA), the Cas12a effector exhibits strong trans-cleavage activity (also known as collateral activity), resulting in non-specific cleavage of surrounding nucleic acids.
By exploiting this trans-cleavage activity, various CRISPR/Cas-based diagnostic (CRISPR-Dx) strategies have been developed for a range of applications. The CRISPR-Dx has the advantages of rapidness, simplicity, high sensitivity, specificity and the like, and can be combined with different output signals such as fluorescent signals, colorimetric signals and the like to be applied to SARS-CoV-2 detection. However, most of these CRISPR-based detection methods rely on pre-amplification of the target sequence. While preamplification typically uses multiple enzymes, custom primers, or expensive instrumentation; in addition, pre-amplification may lead to amplification bias, increase the complexity of detection, or cause nonlinear distortion when analyzing copies of the nucleic acid precursor. Therefore, the high-sensitivity and selective infectious disease detection without target pre-amplification has advantages and deserves scientific research.
Surface Enhanced Raman Scattering (SERS) is an enhancement phenomenon on some roughened noble metal surfaces developed based on raman spectroscopy. SERS has the advantages of ultra-high sensitivity, unique fingerprint information, and portable operation, and has been developed as an advanced detection tool. The work opens up a new way for CRISPR-based diagnosis, provides a new platform for SARS-CoV-2 detection and variants thereof, and is expected to be transferred to practical application.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provides a biological nano sensor based on CRISPR-Cas12a drive, which can detect pathogenic microorganisms on site by a one-tube method by using a novel sleeve device.
The technical scheme adopted for solving the technical problems is as follows:
a method for detecting pathogenic microorganisms by using a novel sleeve device and a tube method based on a CRISPR-Cas12a driven surface enhanced Raman scattering biological nano sensor comprises the following steps:
(1) Extracting pathogenic microorganism RNA, and then carrying out reverse transcription on the inner tube cover of the novel sleeve device to reversely transcribe the pathogenic microorganism RNA into cDNA, wherein the cDNA is used as a Target for subsequent experiments;
(2) Specific crrnas designed in the CRISPR-Cas12a system Target-recognize Target, when present, triggering the nucleic acid-specific recognition-induced accessory cleavage ability of Cas12a-crRNA complex, i.e., cleavage of unrelated single-stranded DNA, wherein the reactive accessory cleavage in the triggered CRISPR-Cas12a system is performed in the novel cannula device inner tube;
(3) The system formed by the cutting reaction of the AuNPs@4-MBA@DNA nano probe and the CRISPR-Cas12a is uniformly mixed in a system of an outer tube of the novel sleeve device, the sleeve device is placed at 37 ℃ for 10min, the sleeve device is centrifuged at 3000rpm for 1min, the relative centrifugal force is 850g or the sleeve device is filtered by a 1.2 mu m polyether sulfone membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a micro Raman spectrometer.
Further, the raman spectrometer in the step (3) is a handheld micro-raman spectrometer.
Further, the method comprises the following steps:
(1) Firstly, extracting viral RNA from pseudoviruses of novel coronaviruses by using a viral extraction kit, and then carrying out reverse transcription on an inner tube cover of a novel sleeve device to carry out reverse transcription on the viral RNA into cDNA, wherein the cDNA is used as a Target for subsequent experiments;
(2) Specific crrnas designed in the CRISPR-Cas12a system Target-recognize Target, when present, triggering the nucleic acid-specific recognition-induced accessory cleavage ability of Cas12a-crRNA complex, i.e., cleavage of unrelated single-stranded DNA, wherein the reactive accessory cleavage in the triggered CRISPR-Cas12a system is performed in the novel cannula device inner tube;
(3) The system formed by the cutting reaction of the AuNPs@4-MBA@DNA nano probe and the CRISPR-Cas12a is uniformly mixed in a system of an outer tube of the novel sleeve device, the sleeve device is placed at 37 ℃ for 10min, the sleeve device is centrifuged at 3000rpm for 1min, the relative centrifugal force is 850g or the sleeve device is filtered by a 1.2 mu m polyether sulfone membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a handheld micro-Raman spectrometer.
Further, the virus is a novel coronavirus SARS-CoV-2.
Further, the specific steps are as follows:
(1) Design and Synthesis of crRNA
Designing a crRNA sequence specific to SARS-CoV-2N gene sequence and carrying out gene synthesis, wherein the crRNA synthesis sequence is SEQ ID NO.1:
the crRNA template was centrifuged at 12000rpm for 5min, then dissolved and diluted to 4. Mu.M using DEPC water; then polymerizing the prepared solution with a T7 master in a PCR instrument to form a DNA double-strand, and performing a transcription step after annealing is finished; taking out a transcribed sample incubated for 16 hours at 37 ℃ in a PCR instrument, supplementing 50 mu L of the transcribed sample with RNase-free water, then adding 2 mu L of DNase I with the concentration of 2000U/mL into the sample, gently shaking and uniformly mixing, instantly centrifuging to ensure that the solution is completely collected at the bottom of a tube, placing the tube in a metal bath for incubation reaction at 37 ℃ for 30 minutes so as to thoroughly eliminate redundant DNA templates in a reaction system, and then carrying out RNA purification; after purification, determining the concentration of RNA for subsequent experiments, and storing the rest in a refrigerator at-80 ℃;
(1) Preparation of SERS nano-probe-AuNPs@4-MBA@DNA
The sodium citrate reduction method is adopted to prepare AuNPs:
into each clean 250mL Erlenmeyer flask, 100mL of distilled water was added, and 100. Mu.L of HAuCl with a mass concentration of 10% was added 4 Then placing the mixture on a high-temperature magnetic stirrer, heating and vigorously stirring the mixture at a high temperature until the solution is boiled, immediately adding 3mL of 3% sodium citrate solution with mass concentration, continuously heating the mixture for 30min until the solution turns into a reddish wine, concentrating the final volume of the solution to 20mL, cooling the solution to room temperature to obtain AuNPs, and placing the AuNPs into a centrifuge tube wrapped by 50mL of tinfoil paper for preparation of a subsequent experiment;
preparation of the nano probe of AuNPs@4-MBA@DNA:
1mL of 20nmAuNPs is added into each 20 mu L of 1mM 4-mercaptobenzoic acid solution, and the mixture is stirred for 2 hours in a water bath kettle at 37 ℃ to obtain AuNPs@4-MBA;
mu.L of 100. Mu.M of thiol DNA 1 per 25. Mu.L of 100. Mu.M of thiol DNA2 per 25. Mu.L of 100. Mu.M was mixed with 1mL of AuNPs@4-MBA and frozen at-20℃for 2 hours to prepare AuNPs@4-MBA@DNA;
after thawing, centrifuging at 4 ℃ for 30min at a speed of 12,000rpm/min, discarding the supernatant, washing off the excessive DNA by using BufferA, and then dissolving the nanoprobe by using Buffer B; wherein buffer a is 5mM HEPES buffer, ph=7.6, buffer B is 10mM HEPES buffer,300mM NaCl,pH =7.6;
Storing the prepared SERS nano probe in a centrifuge tube wrapped by tinfoil paper, and using a refrigerator at 4 ℃ for the subsequent use;
t7 promoter has the sequence of SEQ ID NO.2, mercapto DNA1 has the sequence of SEQ ID NO.3, and mercapto DNA2 has the sequence of SEQ ID NO.4.
Further, extracting virus RNA and carrying out reverse transcription to obtain single-stranded ss complementary DNA, namely cDNA; after Cas12a-crRNA binary complex specifically recognizes the target cDNA, cas12a will activate the non-specific nucleic acid cleavage (trans-clean) activity of Cas12a, and Cas12a will randomly cleave ssDNA randomly; the designed ssDNA is used as a connector and hybridized with a SERS nano probe prepared in advance; SERS nanoprobes are prepared by linking raman reporter molecules 4-mercaptobenzoic acid and thiolated ssDNA (DNA 1 and DNA 2) to AuNPs via Au-S bonds; when the SARS-CoV-2 target exists, the linker ssDNA is crushed by the activated Cas12a in a non-specific manner, thereby preventing aggregation of the SERS nanoprobes; the dispersed SERS nano-probe can be uniformly distributed in the solution by stable colloid particles even after being centrifuged for 1min at 3000rpm and the relative centrifugal force is 850g, and can penetrate a filter membrane with the aperture of 1.2 mu m; thus, the solution remains red, producing a stronger SERS signal; in contrast, in the absence of SARS-CoV-2 target, the linker ssDNA remained intact, the SERS nanoprobes were crosslinked, and the SERS nanoprobes tended to aggregate and precipitate after centrifugation at 3000rpm for 1min, or remained on the filter membrane after filtration, with a relative centrifugal force of 850 g. The solution thus becomes colorless and negligible SERS signal can be detected in the supernatant or filtrate; the SERS signal of the novel coronavirus target opening can be detected by a portable raman spectrometer.
The novel sleeve device for single-tube detection for implementing the method comprises an inner tube, an outer tube, an inner tube cover and a metal ball with a rod, wherein the inner tube is arranged in the vertical direction, the inner tube can be meshed with the outer tube through a connecting thread and can be detachably connected with the outer tube through the coaxial thread, and the inner tube cover is connected with the inner tube through a connecting hose;
the inner tube and the outer tube are hollow with open tops, the bottom of the outer tube is sealed, the bottoms of the inner tubes are coaxially and tightly connected and provided with an outer tube connecting part, the outer tube connecting part is internally and coaxially provided with a reaction liquid outflow hole, the reaction liquid outflow hole extends from the top of the outer tube connecting part and is arranged at the bottom of the outer tube connecting part, the diameter of the reaction liquid outflow hole from top to bottom is gradually reduced, and the reaction liquid outflow hole is communicated with the hollow interior of the inner tube;
the metal ball with the rod comprises a metal ball part and a rod part which are coaxially connected, the rod part is matched with the middle upper part of the reaction liquid outflow hole, the rod part can be movably arranged at the middle upper part of the reaction liquid outflow hole, the metal ball part is matched with the bottom in the inner tube and the top of the reaction liquid outflow hole, and the lower surface of the metal ball part can seal the top of the reaction liquid outflow hole;
The inner tube cover comprises a cover body and a reaction cavity, the reaction cavity is arranged in the cover body, the reaction cavity can contain a reagent of reverse transcription reaction, and the cover body can be closely and detachably connected with the inner wall of the upper part of the inner tube;
the inner tube can hold a CRISPR reagent and act as a reaction vessel for CRISPR-cas12a cleavage and the outer tube can hold SERS nanoprobes and act as a final reaction vessel.
Further, the inner tube, the outer tube and the inner tube cover are all made of polypropylene, the maximum volume of the reaction cavity is 50 mu L, and the diameter of the foremost part of the reaction liquid outflow hole is 0.6mm.
A method for detecting pathogenic microorganisms using the novel cannula device as described above, comprising the steps of:
under the aseptic operation condition, firstly opening the inner tube cover, then plugging the metal ball with the rod into the reaction liquid outflow hole by using tweezers and the like until the bottom surface of the metal ball part plugs the top of the reaction liquid outflow hole, and then adding 200 mu L of AuNP@4-MBA@ssDNA probe into the outer tube; secondly, nesting the inner pipe in the outer pipe; 180 μl of Cas12a trans-lysis reaction solution was added to the inner tube; thirdly, 20 mu L of reverse transcription reaction liquid containing SARS-CoV-2RNA with different concentrations (pseudovirus or clinical sample) is dripped into the inner tube cover for 20min to finish Reverse Transcription (RT); then inverting the inner tube, mixing the reverse transcription product with a CRISPR reagent, triggering CRISPR/Cas12a to cut in a trans mode, and inverting the inner tube by force during inversion to enable the metal ball with the rod to shake out from the reaction liquid outflow hole; after incubation at 37 ℃ for 20min, transfer CRISPR/Cas12a trans-cleavage reaction into outer tube by vigorous shaking, then incubation with SERS nanoprobe for 4min at 37 ℃; centrifuging the final reaction solution in the outer tube for 1min or filtering with a 1.2 μm polyethersulfone membrane; note that the Relative Centrifugal Force (RCF) is 850g; a portable Raman spectrometer (OptoskyATR 8300) is adopted, the excitation laser power is 150mW, the wavelength is 785nm, the objective lens is 40×, the accumulation time is 2s, and the Raman signal of the supernatant or the filtrate is obtained;
Wherein, cas12a trans-cleavage reaction liquid is: 200nM Cas12a,250nM crRNA and 30nM linker ssDNA were placed in 10mM HEPES buffer and 300mM NaCl, 100mM MgCl2 and 0.5M betaine were added.
The use of the method as described above in the detection of pathogenic microorganisms.
The invention has the advantages and beneficial effects that:
1. the method is a detection method of a Surface Enhanced Raman Scattering (SERS) biosensor based on CRISPR-Cas12a drive, and can detect the concentration of target genome RNA only by simple pretreatment of a sample, CRISPR-Cas12a system reaction and 3 steps of handheld microscopic Raman instant detection, more importantly, the method can realize detection of different microorganisms only by changing a nucleic acid sequence, can be used as a universal nucleic acid detection platform under various conditions, and greatly widens the application of CRISPR-Cas12a in the field of molecular detection.
2. The method of the invention avoids the need for pre-amplifying a plurality of enzymes, complex primers or precise instruments which are commonly used, and reduces the complexity of the procedure and the chance of aerosol contamination. High sensitivity and selectivity detection without nucleic acid amplification is realized.
3. The detection method established by the invention has low detection limit, the minimum detection limit is 200copies/mL, and the detection can be carried out up to 2X 10 2 ～10 8 The detection range is wide and the sensitivity is high due to the copies/mL.
4. The detection method established by the invention has strong specificity, takes the detection of novel coronavirus (SARS-CoV-2) as an example, has no obvious Raman signal increase to microorganisms such as SARS-CoV, MERS-CoV, MHV, IAV and the like, and proves that the invention has good selectivity to the novel coronavirus and is not influenced by other pathogenic microorganisms.
5. The steps of the biosensor established by the invention can be further integrated to realize single tube detection, thereby enhancing applicability. A low-cost tube-in-tube reactor can perform RNA reverse transcription, cas12a trans-cleavage and nanoprobe depolymerization step by step in one pot, so as to realize amplification-free and anti-interference detection. The work opens up a new path for CRISPR-based diagnosis (CRISPR Dx), provides a new platform for the detection of SARS-CoV-2 and variants thereof, and is expected to be popularized in practical application.
6. Compared with other traditional detection methods, the design of the invention has the advantages of simple operation method, high sensitivity and the like, and greatly reduces the detection cost, thereby being convenient for detecting a large number of samples.
7. The invention designs a novel sleeve device for performing CRISPR-Cas12a driven SERS nano biosensing, which is used for detecting pathogenic microorganisms without amplification, eliminates a nucleic acid pre-amplification step, and has sensitivity and specificity compared with a real-time PCR technology. On one hand, the programmability, single base recognition capability and target-induced nonspecific DNA cleavage capability of crRNA-mediated CRISPR-Cas12a are explored, and a self-made nano probe is used for constructing a SERS biosensing strategy so as to obtain higher detection sensitivity; on the other hand, a low cost tube-in-tube reactor was designed to accommodate reverse transcription of RNA, cas12a reverse cleavage and stepwise decomposition of nanoprobes in a novel cannula to achieve amplification-free and tamper-resistant detection.
8. The method is a single-tube enhanced RNA detection based on a CRISPR-Cas12a system, and particularly can integrate a CRISPR-Cas12a driven biological nano sensor in a novel sleeve device to detect novel coronaviruses and variants thereof in an ultrasensitive, accurate and portable manner, and the detection method and the device application do not need to perform nucleic acid amplification in advance.
9. The novel sleeve device is a tube-in-tube container consisting of an inner tube and an outer tube, and is designed to avoid uncapping operation. The inner tube with the cover is fixed inside the outer tube through threaded engagement. The cap of the inner tube serves as a reaction vessel for reverse transcription, the inner tube serves as a storage vessel for the CRISPR reagent and a reaction vessel for CRISPR-Cas12a cleavage, and the outer tube serves as a storage vessel for SERS nanoprobes and a final reaction vessel. The bottom of the inner tube is provided with a hydrophobic hole with the diameter of 0.6mm, and a metal ball with a rod is used as a valve closing hole. The device integrates the steps of RNA reverse transcription, cas12a trans-cleavage, SERS nano-probe crosslinking and the like into a novel sleeve device to develop amplification-free detection. The proposed biosensor has high selectivity and can specifically identify different SARS-CoV-2 variants. The detection result can be measured by portable raman spectroscopy or identified with naked eyes, which can be adapted to instant detection. Furthermore, the proposed biosensor also allows sensitive detection of SARS-CoV-2 from an actual sample and enables detection of SARS-CoV-2 mutants in a novel cannula device.
Drawings
FIG. 1 is a schematic diagram of a CRISPR-Cas12a driven SERS biosensor for use in a SARS-CoV-2 ultrasensitive detection scheme without nucleic acid amplification in accordance with the present invention;
FIG. 2 is a representation of SERS nanoprobes of the present invention; wherein 2A: raman spectral diagram, 2B: ultraviolet-visible absorption spectrum, 2C: dynamic laser scattering diagram, 2D: TEM characterization;
FIG. 3 is a graph of detection feasibility analysis of a Surface Enhanced Raman Scattering (SERS) biosensor based on CRISPR-Cas12a actuation in accordance with the present invention;
FIG. 4 is a graph of a detection selectivity analysis of a CRISPR-Cas12a driven Surface Enhanced Raman Scattering (SERS) based biosensor in accordance with the present invention;
FIG. 5 is a graph of detection results of a centrifugation method of a Surface Enhanced Raman Scattering (SERS) biosensor based on CRISPR-Cas12a system actuation in the present invention; wherein, 5A: raman spectra of different concentrations of novel coronavirus pseudoviruses after centrifugation, 5B: the wave number is 1075cm -1 Is a corresponding raman signal histogram of 5C: the established standard curve of the new coronavirus pseudovirus;
FIG. 6 is a graph of detection results of a filtration method based on a CRISPR-Cas12a system driven Surface Enhanced Raman Scattering (SERS) biosensor in accordance with the present invention; wherein, 6A: raman spectra of SARS-CoV-2 pseudoviruses at different concentrations after filtration, 5B: the wave number is 1075cm -1 Is a corresponding raman signal histogram of (C), 6C: the established standard curve of the new coronavirus.
FIG. 7 is a graph of detection of novel coronavirus mutants based on the CRISPR-Cas12a system in the present invention; wherein, 7A: key mutant EF156-157del variant map, 7B: key mutant N501Y variant map, 7C: key mutant L452R variant pattern, 7D: key mutant L452R variant map;
FIG. 8 is a schematic diagram of the application of a nano-biosensor based on CRISPR-Cas12a system drive in detecting SARS-CoV-2 clinical samples in the present invention; wherein, a diagram: workflow schematic for detecting clinical samples using the proposed nanosensor, panel B: nanosensor and RT-qPCR detection results (ordered by Ct values) for 50 COVID-19 positive clinical samples, panel C, D: comparison of all clinical samples (50 covd-19 positive and 50 negative) with RT-qPCR assay results summarised, E panel: for 100 clinical samples, the proposed nanosensor and RT-qPCR were consistent. F. Graph G: correlation of RT-qPCR Ct value and Raman intensity in clinical sample detection, RT-qPCR positive sample (Ct range: 20-40), H picture RT-qPCR negative sample;
FIG. 9 is a schematic illustration of the development of amplification-free detection by integrating RNA reverse transcription, cas12a reverse cleavage, and SERS nanoprobe cross-linking into a novel sleeve device;
FIG. 10 is a front view of one structural attachment of the novel sleeve device of the present invention;
fig. 11 is a schematic top view of the inner tube of fig. 10.
Detailed Description
The present invention will be further described in detail with reference to examples, but the scope of the present invention is not limited to the examples.
The raw materials used in the invention are conventional commercial products unless specified otherwise, the methods used in the invention are conventional methods in the art unless specified otherwise, and the mass of each substance used in the invention is conventional.
A method for detecting pathogenic microorganisms by using a novel sleeve device and a tube method based on a CRISPR-Cas12a driven surface enhanced Raman scattering biological nano sensor comprises the following steps:
(1) Extracting pathogenic microorganism RNA, and then carrying out reverse transcription on the inner tube cover of the novel sleeve device to reversely transcribe the pathogenic microorganism RNA into cDNA, wherein the cDNA is used as a Target for subsequent experiments;
(2) Specific crrnas designed in the CRISPR-Cas12a system Target-recognize Target, when present, triggering the nucleic acid-specific recognition-induced accessory cleavage ability of Cas12a-crRNA complex, i.e., cleavage of unrelated single-stranded DNA, wherein the reactive accessory cleavage in the triggered CRISPR-Cas12a system is performed in the novel cannula device inner tube;
(3) The system formed by the cutting reaction of the AuNPs@4-MBA@DNA nano probe and the CRISPR-Cas12a is uniformly mixed in a system of an outer tube of the novel sleeve device, the sleeve device is placed at 37 ℃ for 10min, the sleeve device is centrifuged at 3000rpm for 1min, the relative centrifugal force is 850g or the sleeve device is filtered by a 1.2 mu m polyether sulfone membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a micro Raman spectrometer.
As can be seen in fig. 1.
Preferably, the raman spectrometer in the step (3) is a handheld micro-raman spectrometer.
Preferably, the method comprises the following steps:
(1) Firstly, extracting viral RNA from pseudoviruses of novel coronaviruses by using a viral extraction kit, and then carrying out reverse transcription on an inner tube cover of a novel sleeve device to carry out reverse transcription on the viral RNA into cDNA, wherein the cDNA is used as a Target for subsequent experiments;
(2) Specific crrnas designed in the CRISPR-Cas12a system Target-recognize Target, when present, triggering the nucleic acid-specific recognition-induced accessory cleavage ability of Cas12a-crRNA complex, i.e., cleavage of unrelated single-stranded DNA, wherein the reactive accessory cleavage in the triggered CRISPR-Cas12a system is performed in the novel cannula device inner tube;
(3) The system formed by the cutting reaction of the AuNPs@4-MBA@DNA nano probe and the CRISPR-Cas12a is uniformly mixed in a system of an outer tube of the novel sleeve device, the sleeve device is placed at 37 ℃ for 10min, the sleeve device is centrifuged at 3000rpm for 1min, the relative centrifugal force is 850g or the sleeve device is filtered by a 1.2 mu m polyether sulfone membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a handheld micro-Raman spectrometer.
Preferably, the virus is a novel coronavirus SARS-CoV-2.
Preferably, the specific steps are as follows:
(1) Design and Synthesis of crRNA
Designing a crRNA sequence specific to SARS-CoV-2N gene sequence and carrying out gene synthesis, wherein the crRNA synthesis sequence is SEQ ID NO.1:
the crRNA template was centrifuged at 12000rpm for 5min, then dissolved and diluted to 4. Mu.M using DEPC water; then polymerizing the prepared solution with a T7 master in a PCR instrument to form a DNA double-strand, and performing a transcription step after annealing is finished; taking out a transcribed sample incubated for 16 hours at 37 ℃ in a PCR instrument, supplementing 50 mu L of the transcribed sample with RNase-free water, then adding 2 mu L of DNase I with the concentration of 2000U/mL into the sample, gently shaking and uniformly mixing, instantly centrifuging to ensure that the solution is completely collected at the bottom of a tube, placing the tube in a metal bath for incubation reaction at 37 ℃ for 30 minutes so as to thoroughly eliminate redundant DNA templates in a reaction system, and then carrying out RNA purification; after purification, determining the concentration of RNA for subsequent experiments, and storing the rest in a refrigerator at-80 ℃;
(1) Preparation of SERS nano-probe-AuNPs@4-MBA@DNA
The sodium citrate reduction method is adopted to prepare AuNPs:
into each clean 250mL Erlenmeyer flask, 100mL of distilled water was added, and 100. Mu.L of HAuCl with a mass concentration of 10% was added 4 Then placing the mixture on a high-temperature magnetic stirrer, heating and vigorously stirring the mixture at a high temperature until the solution is boiled, immediately adding 3mL of 3% sodium citrate solution with mass concentration, continuously heating the mixture for 30min until the solution turns into a reddish wine, concentrating the final volume of the solution to 20mL, cooling the solution to room temperature to obtain AuNPs, and placing the AuNPs into a centrifuge tube wrapped by 50mL of tinfoil paper for preparation of a subsequent experiment;
preparation of the nano probe of AuNPs@4-MBA@DNA:
1mL of 20nmAuNPs is added into each 20 mu L of 1mM 4-mercaptobenzoic acid solution, and the mixture is stirred for 2 hours in a water bath kettle at 37 ℃ to obtain AuNPs@4-MBA;
mu.L of 100. Mu.M of thiol DNA 1 per 25. Mu.L of 100. Mu.M of thiol DNA2 per 25. Mu.L of 100. Mu.M was mixed with 1mL of AuNPs@4-MBA and frozen at-20℃for 2 hours to prepare AuNPs@4-MBA@DNA;
after thawing, centrifuging at 4 ℃ for 30min at a speed of 12,000rpm/min, discarding the supernatant, washing off the excessive DNA by using BufferA, and then dissolving the nanoprobe by using Buffer B; wherein buffer a is 5mM HEPES buffer, ph=7.6, buffer B is 10mM HEPES buffer,300mM NaCl,pH =7.6;
Storing the prepared SERS nano probe in a centrifuge tube wrapped by tinfoil paper, and using a refrigerator at 4 ℃ for the subsequent use;
t7 promoter has the sequence of SEQ ID NO.2, mercapto DNA1 has the sequence of SEQ ID NO.3, and mercapto DNA2 has the sequence of SEQ ID NO.4.
Preferably, viral RNA is extracted and reverse transcribed to obtain single-stranded ss complementary DNA, i.e., cDNA; after Cas12a-crRNA binary complex specifically recognizes the target cDNA, cas12a will activate the non-specific nucleic acid cleavage (trans-clean) activity of Cas12a, and Cas12a will randomly cleave ssDNA randomly; the designed ssDNA is used as a connector and hybridized with a SERS nano probe prepared in advance; SERS nanoprobes are prepared by linking raman reporter molecules 4-mercaptobenzoic acid and thiolated ssDNA (DNA 1 and DNA 2) to AuNPs via Au-S bonds; when the SARS-CoV-2 target exists, the linker ssDNA is crushed by the activated Cas12a in a non-specific manner, thereby preventing aggregation of the SERS nanoprobes; the dispersed SERS nano-probe can be uniformly distributed in the solution by stable colloid particles even after being centrifuged for 1min at 3000rpm and the relative centrifugal force is 850g, and can penetrate a filter membrane with the aperture of 1.2 mu m; thus, the solution remains red, producing a stronger SERS signal; in contrast, in the absence of SARS-CoV-2 target, the linker ssDNA remained intact, the SERS nanoprobes were crosslinked, and the SERS nanoprobes tended to aggregate and precipitate after centrifugation at 3000rpm for 1min, or remained on the filter membrane after filtration, with a relative centrifugal force of 850 g. The solution thus becomes colorless and negligible SERS signal can be detected in the supernatant or filtrate; the SERS signal of the novel coronavirus target opening can be detected by a portable raman spectrometer.
As shown in fig. 10 and 11, the novel sleeve device for single tube detection for implementing the method comprises an inner tube 4, an outer tube 6, an inner tube cover l and a metal ball with a rod 2, wherein the inner tube is arranged along the vertical direction, the inner tube can be detachably connected with the outer tube through coaxial thread engagement of a connecting thread 3, and the inner tube cover is connected with the inner tube through a connecting hose 7;
the inner tube and the outer tube are hollow with open tops, the bottom of the outer tube is sealed, the bottom of the inner tube is coaxially and tightly connected with an outer tube connecting part 5, a reaction liquid outflow hole 51 is coaxially arranged in the outer tube connecting part, the reaction liquid outflow hole extends from the top of the outer tube connecting part to the bottom of the outer tube connecting part, the diameter of the reaction liquid outflow hole from top to bottom is gradually reduced, and the reaction liquid outflow hole is communicated with the hollow interior of the inner tube;
the metal ball with the rod comprises a metal ball part and a rod part which are coaxially connected, the rod part is matched with the middle upper part of the reaction liquid outflow hole, the rod part can be movably arranged at the middle upper part of the reaction liquid outflow hole, the metal ball part is matched with the bottom in the inner tube and the top of the reaction liquid outflow hole, and the lower surface of the metal ball part can seal the top of the reaction liquid outflow hole;
The inner tube cover comprises a cover body 11 and a reaction cavity 12, wherein the reaction cavity is arranged in the cover body, can contain a reagent of reverse transcription reaction, and can be closely and detachably connected with the inner wall of the upper part of the inner tube;
the inner tube can hold a CRISPR reagent and act as a reaction vessel for CRISPR-Cas12a cleavage, and the outer tube can hold SERS nanoprobes and act as a final reaction vessel.
The device is a tube-in-tube container consisting of an inner tube and an outer tube, designed to circumvent the uncapping operation. The inner tube with the cover is fixed inside the outer tube through threaded engagement. The cap of the inner tube serves as a reaction vessel for reverse transcription, the inner tube serves as a storage vessel for the CRISPR reagent and a reaction vessel for CRISPR-Cas12a cleavage, and the outer tube serves as a storage vessel for SERS nanoprobes and a final reaction vessel. The bottom of the inner tube is provided with a hydrophobic hole with the diameter of 0.6mm, and a metal ball with a rod is used as a valve closing hole.
Preferably, the inner tube, the outer tube and the inner tube cover are all made of polypropylene (PP), and the maximum volume of the reaction cavity is 50 mu L, so that the reaction liquid cannot naturally separate from the tube cover due to gravity when the novel sleeve device is used.
Preferably, the diameter of the reaction solution outlet hole is 0.6mm.
The novel sleeve device is a tube-in-tube container consisting of an inner tube and an outer tube, and is designed to avoid uncapping operation. The cap of the inner tube serves as a reaction vessel for reverse transcription, the inner tube serves as a storage vessel for the CRISPR reagent and a reaction vessel for CRISPR-Cas12a cleavage, and the outer tube serves as a storage vessel for SERS nanoprobes and a final reaction vessel. The bottom of the inner tube is provided with a hydrophobic hole with the diameter of 0.6mm, and a metal ball with a rod is used as a valve closing hole.
A method for detecting pathogenic microorganisms using the novel cannula device as described above, comprising the steps of:
under the aseptic operation condition, firstly opening the inner tube cover, then plugging the metal ball with the rod into the reaction liquid outflow hole by using tweezers and the like until the bottom surface of the metal ball part plugs the top of the reaction liquid outflow hole, and then adding 200 mu L of AuNP@4-MBA@ssDNA probe into the outer tube; secondly, nesting the inner pipe in the outer pipe; 180 μl of Cas12a trans-lysis reaction solution was added to the inner tube; thirdly, 20 mu L of reverse transcription reaction liquid containing SARS-CoV-2RNA with different concentrations (pseudovirus or clinical sample) is dripped into the inner tube cover for 20min to finish Reverse Transcription (RT); then inverting the inner tube, mixing the reverse transcription product with a CRISPR reagent, triggering CRISPR/Cas12a to cut in a trans mode, and inverting the inner tube by force during inversion to enable the metal ball with the rod to shake out from the reaction liquid outflow hole; after incubation at 37 ℃ for 20min, transfer CRISPR/Cas12a trans-cleavage reaction into outer tube by vigorous shaking, then incubation with SERS nanoprobe for 4min at 37 ℃; centrifuging the final reaction solution in the outer tube for 1min or filtering with a 1.2 μm polyethersulfone membrane; note that the Relative Centrifugal Force (RCF) is 850g; a portable Raman spectrometer (OptoskyATR 8300) is adopted, the excitation laser power is 150mW, the wavelength is 785nm, the objective lens is 40×, the accumulation time is 2s, and the Raman signal of the supernatant or the filtrate is obtained;
Wherein, cas12a trans-cleavage reaction liquid is: 200nM Cas12a,250nM crRNA and 30nM linker ssDNA were placed in 10mM HEPES buffer and 300mM NaCl, 100mM MgCl2 and 0.5M betaine were added.
The invention designs and designs a smart container device, and the steps of RNA reverse transcription, cas12a trans-cleavage and SERS nano-probe crosslinking can be integrated into one container. As can be seen in fig. 9.
The use of the method as described above in the detection of pathogenic microorganisms.
Specifically, the related preparation and detection are as follows:
a CRISPR-Cas12a driven Surface Enhanced Raman Scattering (SERS) based biosensor utilizes a novel cannula device that is capable of a one-tube method in situ detection of a novel coronavirus, comprising the steps of:
(1) Firstly, extracting viral RNA from pseudoviruses of novel coronaviruses by using a viral extraction kit, and then carrying out reverse transcription on an inner tube cover of a novel sleeve device to reversely transcribe the viral RNA into cDNA, wherein the cDNA is used as a Target for subsequent experiments.
(2) The specific crrnas designed in the CRISPR-Cas12a system Target recognizes the Target, which when present, triggers the nucleic acid specific recognition of the Cas12a-crRNA complex to trigger the accessory cleavage ability, i.e. cleavage of unrelated single stranded DNA, wherein the reactive accessory cleavage in the triggered CRISPR-Cas12a system is performed in the novel cannula device inner tube.
(3) The system of AuNPs@4-MBA@DNA nano probe and CRISPR-Cas12a after the completion of the cutting reaction is uniformly mixed in a system of an outer tube of a novel sleeve device, the mixture is placed for 10min at 37 ℃, the Relative Centrifugal Force (RCF) of slight centrifugation (3000 rpm for 1 min) is 850g or the mixture is filtered by a 1.2 mu m Polyethersulfone (PES) membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a handheld micro Raman spectrometer.
Preferably, the virus is a novel coronavirus SARS-CoV-2.
The proposed nanosensor has two functional components, trans-cleavage that activates CRISPR/Cas12a and SERS-based detection. Briefly, viral RNA is extracted and reverse transcribed to obtain single stranded (ss) complementary DNA (cDNA). After the Cas12a-crRNA binary complex specifically recognizes the target cDNA, the non-specific nucleic acid cleavage activity of Cas12a will be activated, and Cas12a will randomly cleave ssDNA irregularly. The designed ssDNA served as a linker hybridized with the pre-prepared SERS nanoprobe. SERS nanoprobes are prepared by linking the raman reporter 4-mercaptobenzoic acid (4-MBA) and the thiolated ssDNA (DNA 1 and DNA 2) to AuNPs via Au-S bonds. When the SARS-CoV-2 target is present, the linker ssDNA will be broken down by the activated Cas12a in a non-specific manner, thereby preventing aggregation of the SERS nanoprobes. The dispersed SERS nanoprobe can be uniformly distributed in a solution as stable colloidal particles even after light centrifugation (3000 rpm for 1 min) with a Relative Centrifugal Force (RCF) of 850g, and can penetrate a filter membrane having a pore size of 1.2 μm. Thus, the solution remains red, producing a stronger SERS signal. In contrast, in the absence of SARS-CoV-2 target, the linker ssDNA remained intact and the SERS nanoprobes crosslinked, tended to aggregate and precipitate after light centrifugation (3000 rpm for 1 min) at 850g Relative Centrifugal Force (RCF), or remained on the filter membrane after filtration. The solution thus becomes colorless and negligible SERS signal can be detected in the supernatant or filtrate. The SERS signal of the novel coronavirus target spot which is turned on can be detected by a portable Raman spectrometer, and the detection method is easy to adapt to field detection.
Design and Synthesis of crRNA
1.1 design SARS-CoV-2N gene sequence specific crRNA sequence and make gene synthesis. The crRNA synthesis sequence is shown in SEQ ID NO.1.
1.2 first, crRNA template (sequence: 5' -TCTGTACCGTCTGCGGTATGTGATCTACACTTAGTAGAAATTACCCTATAGTGAGTC GTATTAATTTC) obtained after synthesis by the company was centrifuged at 12000rpm for 5min using a centrifuge, and then dissolved and diluted to 4. Mu.M using DEPC water. The prepared solution was then combined with T7 master (sequence:
GAAATTAATACGACTCACTATAGGG) to form a DNA double strand, and after annealing, a transcription step was performed using a HiScribe T7 rapid and efficient RNA synthesis kit. Taking out a transcribed sample incubated for 16h at 37 ℃ in a PCR instrument, supplementing 50 mu L of the transcribed sample with RNase-free water, adding 2 mu LDNase I (2000U/mL) into the sample, gently shaking and mixing, performing instantaneous centrifugation to ensure that the solution is completely collected at the bottom of a tube, placing the tube in a metal bath for incubation reaction for 30min at 37 ℃ to thoroughly eliminate redundant DNA templates in a reaction system, and then performing RNA purification according to the steps of a Monarch RNA purification kit instruction book. Pure waterUsed after chemical treatmentThe RNA concentration was measured by basic ultra-micro nucleic acid quantitative instrument for the subsequent experiments, and the remainder was stored in a-80℃refrigerator.
Preparation of SERS nanoprobe (AuNPs@4-MBA@DNA)
2.1 preparation of AuNPs by sodium citrate reduction method
(1) Firstly, preparing a 250mL conical flask; 3% sodium citrate; 10% HAuCl 4 The method comprises the steps of carrying out a first treatment on the surface of the A high temperature magnetic stirrer; distilled water; measuring cylinder, tinfoil paper.
(2) A clean 250mL Erlenmeyer flask was charged with 100mL distilled water followed by 100. Mu.L of 10% HAuCl 4 Then placing the solution on a high-temperature magnetic stirrer, heating and vigorously stirring at high temperature until the solution is boiled, immediately adding 3mL of 3% sodium citrate, continuously heating for 30min until the solution turns into wine red, concentrating the final volume of the solution to 20mL, cooling to room temperature, and placing the solution into a 50mL tin foil paper wrapped centrifuge tube for preparation of subsequent experiments.
2.2 preparation of nanoprobe of AuNPs@4-MBA@DNA
(1) 20 mu L of 4-MBA (1 mM) solution is added into 1mL of 20 nmAuNPs, and the mixture is stirred for 2 hours in a water bath kettle at 37 ℃ to obtain AuNPs@4-MBA;
(2) 25. Mu.L of thiol DNA 1 (100. Mu.M) (order)
Column 5 '-C6-SH-AAAAAAAAACCCAGGTTCTCT-3') or 25. Mu.L of thiol DNA2 (100. Mu.M) (sequence: 5 '-TCACAGATGCGTAAAAAAAAA-C6-SH-3') was mixed with 1mL of 4-MBA labeled AuNPs and frozen at-20℃for 2 hours to prepare AuNPs@4-MBA@DNA;
(3) After thawing, the solution was centrifuged at 12,000rpm/min for 30min at 4℃and the supernatant was discarded, and excess DNA was washed off by Buffer A, after which the nanoprobe was dissolved by Buffer B. Wherein BufferA is 5mM HEPES buffer,pH 7.6,Buffer B and 10mM HEPES buffer,300mM NaCl,pH 7.6.
(4) Storing the prepared SERS nano probe in a centrifuge tube wrapped by tinfoil paper for subsequent experiments at the temperature of 4 ℃.
Characterization of sers nanoprobes
To verify whether the prepared SERS nanoprobe can be subjected to subsequent experiments, we performed the following characterization on the prepared SERS nanoprobe in different forms as shown in fig. 2: raman characterization, ultraviolet characterization, DLS characterization, TEM characterization, the specific experimental method is as follows:
raman characterization: taking 50 mu L of each of the prepared AuNPs, 4-MBA, auNP+4-MBA and AuNP@4-MBA solutions, and respectively measuring the Raman signal intensities of different samples on a micro Raman spectrometer base, wherein parameters of the micro Raman spectrometer are set as follows: the integration time was 20000ms and the excitation light intensity was 150mW.
Uv appearance: and taking 100 mu L of each of the prepared AuNPs, auNP@4-MBA and AuNP@4-MBA@DNA sample solutions, respectively dripping the solutions into a white transparent 96-well plate, and scanning ultraviolet full spectrum by using an enzyme-labeled instrument in a scanning range of 400-800nm and a scanning interval of 2nm by using each sample in three groups of parallelism.
DLS characterization: the prepared AuNPs, auNP@4-MBA, auNP@4-MBA@DNA sample solution and Cross-linked samples (after complementary pairing of linker ssDNA and AuNP@4-MBA@DNA1/2) were taken and the water and particle size of each sample were respectively determined by using a dynamic light scattering instrument.
TEM characterization: taking 10 mu L of each of prepared AuNPs, auNP@4-MBA, auNP@4-MBA@DNA sample solution and Cross-linked samples (after complementary pairing of linker ssDNA and AuNP@4-MBA@DNA1/2), respectively dripping the solution onto a transmission electron microscope supporting film, drying the samples in an oven, and observing the images of the samples by using a transmission electron microscope (talos G2200X).
4. Surface Enhanced Raman Scattering (SERS) biosensor detection feasibility experiment based on CRISPR-Cas12a drive
In the detection process based on the CRISPR/Cas12a system, only when the Cas12a and the crRNA recognize target DNA, the three form a ternary complex, the auxiliary cutting activity of the Cas12a can be activated to non-specifically cut ssDNA. The Linker ssDNA (sequence: ACGCATCTGTGAAGAGAACCTGGG) designed in the experiment can carry out base complementary pairing on two different types of SERS nano probes (AuNP@4-MBA@DNA1 and AuNP@4-MBA@DNA2), so that the SERS nano probes are changed from a uniform colloid state to an aggregation state, aggregated nano gold particles are precipitated after simple centrifugation, and the supernatant is colorless, so that SERS signal molecules in the supernatant are lower. As shown in fig. 3, when the SARS-CoV-2 target is present, the activated Cas12a non-specifically cleaves linker ssDNA, the nano gold particles are in a dispersed state, the solution is mauve, and after centrifugation, still mauve, and the SERS signal molecules in the supernatant solution are higher. The feasibility of the method of the invention is preliminarily proved, and the method has specific recognition capability. The experiment was conducted in a single experimental group and four negative control groups (100. Mu.L of each reaction system, less than 100. Mu.L of the negative control system was filled with RNase-free water), and the specific reaction systems are shown in Table 1, and the concentrations in the tables are the final concentrations of the reaction. Each sample was subjected to three repeated experiments at 37 ℃ for 20min, 50 μl of the reacted sample was placed in a 1.5mL EP tube, then 25 μl of SERS nanoprobes (aunp@4-mba@dna1 and aunp@4-mba@dna2) were added respectively, and after mixing, the reaction was carried out at 37 ℃ for 5min, after that, the mixture was centrifuged at 3000rpm for 2min, and finally 50 μl of the supernatant was obtained to measure raman signals.
TABLE 1
5. Surface Enhanced Raman Scattering (SERS) biosensor detection selectivity experiment based on CRISPR-Cas12a drive
In order to verify whether the Cas12a detects the novel coronavirus with the better selectivity, a selectivity verification experiment is performed, as shown in fig. 4, when Cas12a, crRNA and different target DNAs are added, only the novel coronavirus cDNA has higher raman signals, and when other targets SARS-CoV, MERS-CoV, MHV, IAV and other microorganisms are respectively added in the system, no obvious raman signal increase exists, and the raman signals are lower, which indicates that the Cas12a has good selectivity in detecting the novel coronavirus.
6. Novel coronavirus nanosensor detection
First 200. Mu.L of AuNP@4-MBA@ssDNA probe was added to the outer tube. Second, the inner sleeve is nested within the outer sleeve. 180 μl of Cas12a trans-cleavage reaction (200 nM Cas12a,250nM crRNA and 30nM linker ssDNA, placed in 10mM HEPES buffer and 300mM NaCl,100mM MgCl2 and 0.5M betaine added) was added to the inner tube. Thirdly, 20 mu L of reverse transcription reaction liquid containing SARS-CoV-2RNA with different concentrations (pseudovirus or clinical sample) is dripped on a test tube cover for 20min, and Reverse Transcription (RT) is completed. The tube is then inverted, and the reverse transcription product is mixed with the CRISPR reagent, triggering CRISPR/Cas12a trans-cleavage. After incubation at 37 ℃ for 20min, the CRISPR/Cas12a trans-cleavage reaction was transferred into the outer tube by vigorous shaking, then incubated with SERS nanoprobe for 4min at 37 ℃. The final reaction solution in the outer tube was centrifuged for 1min or filtered with a 1.2 μm Polyethersulfone (PES) membrane. Note that the Relative Centrifugal Force (RCF) is 850g. Raman signals of the supernatant or filtrate were obtained using a portable raman spectrometer (OptoskyATR 8300), with an excitation laser power of 150mW, a wavelength of 785nm, an objective lens of 40×, and a cumulative time of 2 s.
In FIG. 5A, SERS spectra show that Raman intensity increases with increasing SARS-CoV-2 pseudovirus concentration, as shown in the corresponding histogram (1075 cm -1 Raman intensity at). The sensitivity of the detection method can reach 200 copies/mL as shown in FIG. 5C. At 10 2 ～10 8 In the copy/mL range, raman intensity is linearly related to pseudovirus concentration (R 2 =0.964). In addition, to meet the need of POC detection without equipment, we have adopted a non-centrifugal filtration method to separate SERS nanoprobes in different dispersion states. We propose that dispersed SERS nanoprobes will penetrate the filter (pore size 1.2 μm) and that aggregated SERS probes will be captured on the filter. The raman signal of the filtered liquid is detected. As shown in FIG. 6A, the Raman intensity increases with increasing concentration of SARS-CoV-2RNA, and there is a better linear relationship between the Raman intensity and the concentration of SARS-CoV-2RNA (R 2 =0.953) (fig. 6C). The detection limit is about 10 3 copy/mL, slightly higher than the detection by centrifugation (fig. 6B). The relatively low sensitivity and SERS intensity of the filtration method may be due to excessive retention of SERS nanoprobes on the filter membrane. Nonetheless, the sensitivity of the proposed nano-biosensors is comparable to and superior to those CRISPR detection methods based on pre-amplification steps, and those without amplification previously reported.
We have further studied the proposed nano-biosensor for clinical sample detection. 100 clinical nasopharynx swab samples are detected, wherein 50 samples are COVID-19 positive case samples, and 50 samples are healthy crowd samples, and are formally verified by the Hubei province disease prevention control center. We detected viral RNA extracted from a swab sample using the proposed nano-biosensor (fig. 8A). All positive samples tested by the OVER-SARS-CoV-2 centrifugation method and 49 positive samples tested by the OVER-SARS-CoV-2 filtration method (except for 1 sample ct=36.6) were higher in raman intensity, and all 50 negative samples were lower in raman intensity (i.e., 100% in specificity) (fig. 8B, 8C). The nano-biosensor proposed by the invention can sensitively detect positive samples with threshold (Ct) values between 35 and 40, which is generally considered as a gray area, indicating that the nano-biosensor proposed by us has ultrahigh sensitivity for identifying samples with ultralow viral load. In centrifugation and filtration, the clinical sensitivity of the proposed biosensor was 100% and 98%, respectively (fig. 8D). In addition, there is a clear correlation between raman intensity and RT-qPCR Ct values (fig. 8E-G).
7. Specific detection of SARS-CoV-2 mutant strain based on CRISPR-Cas12a driven nano biosensor
During the evolution of SARS-CoV-2, the viral genome was mutated to produce different variants. To assess the ability of the proposed nanosensor to detect SARS-CoV-2 variants, we designed detection methods against a set of key spike protein mutations (EF 156-157del, L452R, N Y and D614G), which can be found in some of the key SARS-CoV-2 variants, including B.1.1.7 (Alpha), P1.351 (Beta), P1 (Gama), B.1.617.2 (Delta) and BA.2 (Omicron). For each mutation we designed mutation-specific crrnas and mismatched crrnas for detection of wild-type (WT) SARS-CoV-2 or Mutant (MT) SARS-CoV-2. In fig. 7, when crrnas were used to detect their corresponding cognate targets, significantly higher crrnas were observed than were mutated.
EF156-157 crRNA wild type sequence is SEQ ID NO.6, EF156-157 crRNA mutant type sequence is SEQ ID NO.7, crRNAY501 mutant type sequence is SEQ ID NO.8, crRNAD614-1m wild type sequence is SEQ ID NO.9, crRNAG614-1m mutant type sequence is SEQ ID NO.10, crRNAL452 wild type sequence is SEQ ID NO.11, crRNAR452 mutant type sequence is SEQ ID NO.12, crRNAR452-1m mutant type sequence is SEQ ID NO.13,
8. Compared with other detection methods commonly used in the prior art, the method for detecting pathogenic microorganisms by using the novel sleeve device based on the CRISPR-Cas12a driven surface enhanced Raman scattering biological nano sensor
The comparison results are shown in Table 2.
TABLE 2 advantages and disadvantages of the New coronavirus detection methods of the prior art
As can be seen from table 2, the method for detecting pathogenic microorganisms based on the CRISPR-Cas12a driven surface enhanced raman scattering biosensor of the present invention has obvious advantages compared with other detection methods commonly used in the prior art, namely: high specificity, high Shan Jian base region division, high sensitivity, low cost, no need of professional technicians, detection time less than 45min and field detection.
The sequences used in the invention are:
SEQ ID NO.1：
5’-TCTGTACCGTCTGCGGTATGTGATCTACACTTAGTAGAAATTACCCTATAGTGAGTC GTATTAATTTC-3’
SEQ ID NO.2：
5’-GAAATTAATACGACTCACTATAGGG-3’
SEQ ID NO.3：
5’-C6-SH-AAAAAAAAACCCAGGTTCTCT-3’
SEQ ID NO.4：
5’-TCACAGATGCGTAAAAAAAAA-C6-SH-3’
SEQ ID NO.5：
5’-ACGCATCTGTGAAGAGAACCTGGG-3’
SEQ ID NO.6：
5’-CTGAACTCACTTTCCATCATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTA TTAATTTC-3’
SEQ ID NO.7：
5’-TAAACTCTACTTTCCATCATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTA TTAATTTC-3’
SEQ ID NO.8：
5’-GTATGGTTGGTAACCAACACCAATCTACACTTAGTAGAAATTAcccTATAGTGAGT CGTATTAATTTC-3’
SEQ ID NO.9：
5’-TTCTGTGCAGTTAACATCGTGAATCTACACTTAGTAGAAATTACCCTATAGTGAGT CGTATTAATTTC-3’
SEQ ID NO.10：
5’-TTCTGTGCAGTTAACACCGTGAATCTACACTTAGTAGAAATTACCCTATAGTGAG TCGTATTAATTTC-3’
SEQ ID NO.11：
5’-CTTCCTAAACAATCTATACAGGATCTACACTTAGTAGAAATTACCCTATAGTGAG TCGTATTAATTTC-3’
TCGTATTAATTTC-3’
SEQ ID NO.12：
5’-CTTCCTAAACAATCTATACCGGATCTACACTTAGTAGAAATTACCCTATAGTGAG TCGTATTAATTTC-3’
SEQ ID NO.13：
5’-CTTCCTAAACAATCTATAGCGGATCTACACTTAGTAGAAATTACCCTATAGTGAG TCGTATTAATTTC-3’
although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (10)
1. A method for detecting pathogenic microorganisms based on a CRISPR-Cas12a driven surface enhanced Raman scattering biological nano sensor is characterized by comprising the following steps: the method comprises the following steps:
(1) Extracting pathogenic microorganism RNA, and then reversely transcribing the pathogenic microorganism RNA into cDNA by reverse transcription, wherein the cDNA is used as a Target for subsequent experiments;
(2) Specific crrnas designed in the CRISPR-Cas12a system Target-recognize Target, which when present, triggers the nucleic acid-specific recognition-induced accessory cleavage ability of the Cas12a-crRNA complex, i.e., cleavage of unrelated single-stranded DNA;
(3) The system of AuNPs@4-MBA@DNA nano probe and CRISPR-Cas12a after the completion of the cleavage reaction is uniformly mixed in one system, the mixture is placed at 37 ℃ for 10min, centrifuged at 3000rpm for 1min, the relative centrifugal force is 850g or filtered by a 1.2 mu m polyethersulfone membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a micro Raman spectrometer.
2. The method according to claim 1, characterized in that: the Raman spectrometer in the step (3) is a handheld micro-Raman spectrometer.
3. The method according to claim 1, characterized in that: the method comprises the following steps:
(1) Firstly, extracting viral RNA from pseudoviruses of novel coronaviruses by using a viral extraction kit, and then carrying out reverse transcription on the viral RNA into cDNA (complementary deoxyribonucleic acid) by reverse transcription, wherein the cDNA is used as a Target for subsequent experiments;
(2) Specific crrnas designed in the CRISPR-Cas12a system Target-recognize Target, which when present, triggers the nucleic acid-specific recognition-induced accessory cleavage ability of the Cas12a-crRNA complex, i.e., cleavage of unrelated single-stranded DNA;
(3) The system of AuNPs@4-MBA@DNA nano probe and CRISPR-Cas12a after the cutting reaction is uniformly mixed in one system, the mixture is placed at 37 ℃ for 10min, the mixture is centrifuged at 3000rpm for 1min, the relative centrifugal force is 850g or the mixture is filtered by a 1.2 mu m polyethersulfone membrane, the supernatant is taken, the color change is recorded by a smart phone, and the data analysis is carried out by a handheld micro-Raman spectrometer.
4. A method according to claim 3, characterized in that: the virus is novel coronavirus SARS-CoV-2.
5. A method according to claim 3, characterized in that: the method comprises the following specific steps:
(1) Design and Synthesis of crRNA
Designing a crRNA sequence specific to SARS-CoV-2N gene sequence and carrying out gene synthesis, wherein the crRNA synthesis sequence is SEQ ID NO.1:
the crRNA template was centrifuged at 12000rpm for 5min, then dissolved and diluted to 4. Mu.M using DEPC water; then polymerizing the prepared solution with a T7 master in a PCR instrument to form a DNA double-strand, and performing a transcription step after annealing is finished; taking out a transcribed sample incubated for 16 hours at 37 ℃ in a PCR instrument, supplementing 50 mu L of the transcribed sample with RNase-free water, then adding 2 mu L of DNase I with the concentration of 2000U/mL into the sample, gently shaking and uniformly mixing, instantly centrifuging to ensure that the solution is completely collected at the bottom of a tube, placing the tube in a metal bath for incubation reaction at 37 ℃ for 30 minutes so as to thoroughly eliminate redundant DNA templates in a reaction system, and then carrying out RNA purification; after purification, determining the concentration of RNA for subsequent experiments, and storing the rest in a refrigerator at-80 ℃;
(1) Preparation of SERS nano-probe-AuNPs@4-MBA@DNA
The sodium citrate reduction method is adopted to prepare AuNPs:
into each clean 250mL Erlenmeyer flask, 100mL of distilled water was added, and 100. Mu.L of HAuCl with a mass concentration of 10% was added 4 Then placing the mixture on a high-temperature magnetic stirrer, heating and vigorously stirring the mixture at a high temperature until the solution is boiled, immediately adding 3mL of 3% sodium citrate solution with mass concentration, continuously heating the mixture for 30min until the solution turns into a reddish wine, concentrating the final volume of the solution to 20mL, cooling the solution to room temperature to obtain AuNPs, and placing the AuNPs into a centrifuge tube wrapped by 50mL of tinfoil paper for preparation of a subsequent experiment;
preparation of the nano probe of AuNPs@4-MBA@DNA:
1mL of 20nmAuNPs is added into each 20 mu L of 1mM 4-mercaptobenzoic acid solution, and the mixture is stirred for 2 hours in a water bath kettle at 37 ℃ to obtain AuNPs@4-MBA;
mu.L of 100. Mu.M of thiol DNA 1 per 25. Mu.L of 100. Mu.M of thiol DNA2 per 25. Mu.L of 1mLAuNPs@4-MBA was mixed with 1mLAuNPs@4-MBA and frozen at-20℃for 2 hours to prepare AuNPs@4-MBA@DNA;
after thawing, centrifuging at 4 ℃ for 30min at a speed of 12,000rpm/min, discarding the supernatant, washing off the excessive DNA by using BufferA, and then dissolving the nanoprobe by using Buffer B; wherein buffer a is 5mM HEPES buffer, ph=7.6, buffer B is 10mM HEPES buffer,300mM NaCl,pH =7.6;
Storing the prepared SERS nano probe in a centrifuge tube wrapped by tinfoil paper, and using a refrigerator at 4 ℃ for the subsequent use;
t7 promoter has the sequence of SEQ ID NO.2, mercapto DNA 1 has the sequence of SEQ ID NO.3, and mercapto DNA2 has the sequence of SEQ ID NO.4.
6. The method according to any one of claims 1 to 5, wherein: extracting virus RNA and carrying out reverse transcription to obtain single-stranded ss complementary DNA (cDNA); after the Cas12a-crRNA binary complex specifically recognizes the target cDNA, the non-specific nucleic acid cleavage activity of Cas12a will be activated, and Cas12a will randomly cleave ssDNA irregularly; the designed ssDNA is used as a connector and hybridized with a SERS nano probe prepared in advance; SERS nanoprobes are prepared by linking the raman reporter 4-mercaptobenzoic acid and the thiolated ssDNA to AuNPs via Au-S bonds; when the SARS-CoV-2 target exists, the linker ssDNA is crushed by the activated Cas12a in a non-specific manner, thereby preventing aggregation of the SERS nanoprobes; the dispersed SERS nano-probe can be uniformly distributed in the solution by stable colloid particles even after being centrifuged for 1min at 3000rpm and the relative centrifugal force is 850g, and can penetrate a filter membrane with the aperture of 1.2 mu m; thus, the solution remains red, producing a stronger SERS signal; in contrast, in the absence of SARS-CoV-2 target, the linker ssDNA remained intact, the SERS nanoprobes were crosslinked, and the SERS nanoprobes tended to aggregate and precipitate after centrifugation at 3000rpm for 1min, or remained on the filter membrane after filtration, with a relative centrifugal force of 850 g. The solution thus becomes colorless and negligible SERS signal can be detected in the supernatant or filtrate; the SERS signal of the novel coronavirus target opening can be detected by a portable raman spectrometer.
7. A novel cannula device for single tube detection implementing the method of any one of claims 1 to 5, characterized in that: the device comprises an inner pipe, an outer pipe, an inner pipe cover and a metal ball with a rod, wherein the inner pipe is arranged in the vertical direction, the inner pipe can be meshed with the outer pipe through a connecting thread and can be detachably connected with the outer pipe through a coaxial thread, and the inner pipe cover is connected with the inner pipe through a connecting hose;
the inner tube and the outer tube are hollow with open tops, the bottom of the outer tube is sealed, the bottoms of the inner tubes are coaxially and tightly connected and provided with an outer tube connecting part, the outer tube connecting part is internally and coaxially provided with a reaction liquid outflow hole, the reaction liquid outflow hole extends from the top of the outer tube connecting part and is arranged at the bottom of the outer tube connecting part, the diameter of the reaction liquid outflow hole from top to bottom is gradually reduced, and the reaction liquid outflow hole is communicated with the hollow interior of the inner tube;
the metal ball with the rod comprises a metal ball part and a rod part which are coaxially connected, the rod part is matched with the middle upper part of the reaction liquid outflow hole, the rod part can be movably arranged at the middle upper part of the reaction liquid outflow hole, the metal ball part is matched with the bottom in the inner tube and the top of the reaction liquid outflow hole, and the lower surface of the metal ball part can seal the top of the reaction liquid outflow hole;
The inner tube cover comprises a cover body and a reaction cavity, the reaction cavity is arranged in the cover body, the reaction cavity can contain a reagent of reverse transcription reaction, and the cover body can be closely and detachably connected with the inner wall of the upper part of the inner tube;
the inner tube can hold a CRISPR reagent and act as a reaction vessel for CRISPR-cas12a cleavage and the outer tube can hold SERS nanoprobes and act as a final reaction vessel.
8. The novel cannula device of claim 7, wherein: the inner tube, the outer tube and the inner tube cover are all made of polypropylene, the maximum volume of the reaction cavity is 50 mu L, and the diameter of the foremost part of the reaction liquid outflow hole is 0.6mm.
9. A method for detecting pathogenic microorganisms using a novel cannula device according to claim 7 or 8, characterized in that: the method comprises the following steps:
under the aseptic operation condition, firstly opening the inner tube cover, then plugging the metal ball with the rod into the reaction liquid outflow hole by using tweezers and the like until the bottom surface of the metal ball part plugs the top of the reaction liquid outflow hole, and then adding 200 mu L of AuNP@4-MBA@ssDNA probe into the outer tube; secondly, nesting the inner pipe in the outer pipe; 180 μl of Cas12a trans-lysis reaction solution was added to the inner tube; thirdly, 20 mu L of reverse transcription reaction liquid containing SARS-CoV-2RNA with different concentrations is dripped into the inner tube cover for 20min to complete reverse transcription; then inverting the inner tube, mixing the reverse transcription product with a CRISPR reagent, triggering CRISPR/Cas12a to cut in a trans mode, and inverting the inner tube by force during inversion to enable the metal ball with the rod to shake out from the reaction liquid outflow hole; after incubation at 37 ℃ for 20min, transfer CRISPR/Cas12a trans-cleavage reaction into outer tube by vigorous shaking, then incubation with SERS nanoprobe for 4min at 37 ℃; centrifuging the final reaction solution in the outer tube for 1min or filtering with a 1.2 μm polyethersulfone membrane; note that the Relative Centrifugal Force (RCF) is 850g; a portable Raman spectrometer is adopted, the excitation laser power is 150mW, the wavelength is 785nm, the objective lens is 40×, the accumulation time is 2s, and the Raman signal of the supernatant or filtrate is obtained;
Wherein, cas12a trans-cleavage reaction liquid is: 200nM Cas12a,250nM crRNA and 30nM linker ssDNA were placed in 10mM HEPES buffer and 300mM NaCl, 100mM MgCl2 and 0.5M betaine were added.
10. Use of a method according to any one of claims 7 to 9 for the detection of pathogenic microorganisms.
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