CN113025749A - Visual virus detection method based on CRISPR-Cas12a system and application - Google Patents
Visual virus detection method based on CRISPR-Cas12a system and application Download PDFInfo
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Abstract
本发明公开一种基于CRISPR‑Cas12a系统的可视化病毒检测方法，所述方法通过将病毒基因组反转录或者直接进行PCR扩增获得dsDNA，即target DNA。设计特异靶向病毒基因的crRNA。当target DNA存在时，Cas12a‑crRNA特异识别目标DNA并激活Cas12a非特异ssDNase活性，降解连接AuNPs‑DNA探针的linker ssDNA，诱导AuNPs‑DNA聚集‑分散状态的改变，纳米金聚集‑分散状态的改变产生明显的颜色变化。该颜色变化可通过肉眼直接判断，也可通过智能手机App颜色识别器读取和分析检测结果。
The invention discloses a visual virus detection method based on the CRISPR-Cas12a system. The method obtains dsDNA, namely target DNA, by reverse transcription of the virus genome or direct PCR amplification. Design crRNAs that specifically target viral genes. When the target DNA exists, Cas12a-crRNA specifically recognizes the target DNA and activates the non-specific ssDNase activity of Cas12a, degrades the linker ssDNA connected to the AuNPs-DNA probe, induces the change of the aggregation-dispersed state of AuNPs-DNA, and the aggregation-dispersed state of gold nanoparticles. Alteration produces a noticeable color change. The color change can be directly judged by the naked eye, or the detection result can be read and analyzed through the smartphone App color recognizer.
Description
Technical Field
The invention belongs to the technical field of virus detection, and particularly relates to a visual virus detection method based on a CRISPR-Cas12a system and application thereof.
Background
Viral infection seriously harms human health, virus detection is crucial to diagnosis, prevention and treatment of diseases, and development of effective, accurate, rapid and sensitive detection tools plays a crucial role in disease control. Although the development of detection methods in the laboratory has made great progress, there is still a need to enhance the development of detection methods in non-laboratory conditions, and particularly to promote the establishment of wide applicability in point-of-care testing (POCT). The nucleic acid detection technology based on CRISPR-Cas provides a novel mode for developing sensitive and rapid POCT detection. At present, virus detection methods mainly include nucleic acid detection, antibody detection, and antigen detection. However, the detection rate of antigen detection is low, and the new crown detection is mainly focused on antibody and nucleic acid detection at present. Wherein, the nucleic acid detection has the characteristics of early diagnosis, high sensitivity and specificity and the like, and is considered as the 'gold standard' for detecting virus infection; although the nucleic acid amplification test (PCR/RT-PCR) is currently the most sensitive and specific method for early detection of pathogens, it is not suitable for rapid and timely diagnosis due to the need for specialized laboratory equipment and highly trained technicians. In addition, highly contagious pathogens require real-time monitoring in order to prevent their transmission from person to person. The antibody detection is convenient and fast to operate and rapid to detect, can be used as a supplementary means for nucleic acid diagnosis, but is not suitable for screening common people such as repeated work, repeated production, repeated study and the like due to the limitation of false positive and false negative of antibody detection, and is also not suitable for epidemiological investigation background technology in low-prevalence areas.
The CRISPR-cas (clustered regulated interstitial vaccine repeat) system is an acquired immune system present in most bacteria and archaea. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) gene codes constitute an RNA-mediated immune system that can protect bacteria and archaea from viral and foreign DNA invasion. In recent years, some CRISPR-Cas systems, including Cas13a and Cas12a, were found to have non-specific nucleic acid cleavage activity (also referred to as trans-cleavage) when crrna (CRISPR rna) recognizes a target sequence. The trans-cleavage activity provides a basis for nucleic acid detection with high sensitivity and high selectivity.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a visual virus detection method based on a CRISPR-Cas12a system and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a visual virus detection method based on a CRISPR-Cas12a system comprises the steps of carrying out reverse transcription on virus genome RNA to obtain cDNA, carrying out reverse transcription on DNA genome, carrying out PCR amplification to obtain dsDNA, designing a product called Target DNA, namely Target DNA, designing crRNA specifically targeting a virus gene sequence, and detecting the Target DNA by using a Cas12a-crRNA compound.
Furthermore, the virus is a novel coronavirus SARS-CoV-2.
The method comprises the following specific steps:
when Cas12a-crRNA detects corresponding Target DNA, the fluorescent probe can be cut, so that a fluorescent group FAM is far away from a quenching group BHQ1, the quenching of BHQ1 on FAM fluorescence is relieved, and the fluorescence value of FAM is increased to detect novel coronavirus SARS-CoV-2; in addition, by utilizing the property of nanogold aggregation color change, the depth of the nanogold color has a linear relation with the Target RNA concentration, namely the concentration of the novel coronavirus genome RNA, and the concentration of the novel coronavirus genome RNA, namely the novel coronavirus copy number, can be converted into a specific numerical value through the intelligent mobile phone color recognizer APP to be output.
The method comprises the following specific steps:
the presence of target DNA can excite the auxiliary cleavage capability triggered by the nucleic acid specific recognition of the Cas12a-crRNA complex, namely, the cleavage of unrelated single-stranded DNA; in this case, if a single-stranded DNA is synthesized, one end of the single-stranded DNA is modified with a fluorescent group, and the other end of the single-stranded DNA is modified with a quenching group to prepare a reporter DNA, i.e., a report DNA, the reporter DNA can be digested by Cas12a, and an enhanced fluorescent signal is generated for output; the change of the fluorescence signal and the concentration of the novel coronavirus to be detected can form a linear relation, and the novel coronavirus can be detected and quantified; in the nano-gold color development experiment, two ends of a linker-ssDNA sequence are designed into a DNA sequence which can be cross-linked with the nano-gold particles, namely DNA1/2-AuNPs are subjected to complementary pairing; the Cas12a/crRNA system was subsequently mixed with equal volumes of DNA1/2-AuNPs, and when linker-ssDNA was degraded by Cas12a, the DNA1/2-AuNPs remained dispersed and appeared red; the shade of the color can be analyzed and read through an App gray scale of a color recognizer of the smart phone; the numerical value obtained by gray level analysis and the concentration of the novel coronavirus to be detected can form a linear relation, and the novel coronavirus can be detected and quantified.
The method comprises the following specific steps:
the target DNA is obtained by detecting the RNA of the novel coronavirus through a two-step method, when the Cas12a-crRNA detects the corresponding target DNA, namely the target DNA and the crRNA can be complementarily paired, the fluorescent probe can be cut, a fluorescent group FAM is separated from a quenching group BHQ1, the quenching of BHQ1 on the FAM fluorescence is relieved, the increase of the FAM fluorescence value can be linearly related to the concentration of the novel coronavirus, and the specific novel coronavirus can be specifically detected and quantified; in addition, when linker-ssDNA is degraded by Cas12a, DNA1/2-AuNPs remain dispersed and thus appear red; the shade of the color can be read through the gray level analysis of an App color recognizer of the smart phone or through Tecan
The OD value of the 200Pro plate reader is measured, the measured value is in linear relation with the concentration of the novel coronavirus, and the specific novel coronavirus can be specifically detected and quantified.
The method comprises the following specific steps:
(1) construction of SARS-CoV-2 plasmid: synthesizing double-stranded DNA containing a novel coronavirus N gene fragment NC-045512.2, performing enzyme digestion by BamHI EcoR I and linking with T4 DNA ligase to construct into a PLVX-AcGFP-N1 lentiviral vector so as to obtain a SARS-CoV-2 plasmid, wherein the constructed plasmid is named as a plasmid of lentiSARS-CoV-2-N;
the sequence information (5 '-3') of the synthesized SARS-CoV-2 gene fragment is SEQ ID NO. 1: catcgtgttgtctgtactgccgttgccacatagatcatccaaatcctaaaggattttgtgacttaaaaggtaagtatgtacaaatacctacaacttgtgctaatgaccctgtgggttttacacttaaaaacacagtctgtaccgtctgcggtatgtggaaaggttatggctgtagttgtgatcaactccgcgaacccatgcttcagtcagctgatgcacaatcgtttttaaacgggtttgcggtgtaagtgcagcccgtcttacaccgtgcggcacaggcactagtactgatgtcgtaagctggacttccctatggtgctaacaaagacggcatcatatgggttgcaactgagggagccttgaatacaccaaaagatcacattggcacccgcaatcctgctaacaatgctgcaatcgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcagaagggagcagaggcggcagtcaagcctcttctcgttcctcatcacgtagtcgcaacagttcaagaaattcaactccaggcagcagtaggggaacttctcctgctagaatggctggcaatggcggtgatgctgctcttgctttgctgctgcttgacagattgaaccagcttgagagcaaaatgtctggtaaaggccaacaacaacaaggccaaactgtcactaagaaatctgctgctgaggcttctaagaagcctcggcaaaaacgtactgccac
(2) SARS-CoV-2 pseudovirus packaging: cotransfecting the constructed plasmid lentiSARS-CoV-2-N containing SARS-CoV-2N gene and ORF1ab gene fragment, psPAX2 and pMD2.G plasmid into 293T cell, after 48 hours of transfection, collecting cell culture supernatant, filtering through 0.45 μm filter membrane to obtain lentivirus containing SARS-CoV-2 gene fragment, namely SARS-CoV-2 pseudovirus;
(3) SARS-CoV-2N fragment gene amplification: the N fragment is amplified by using SARS-CoV-2 specific primers, and the sequence of the amplified primers is as follows:
F：SEQ ID NO.2：GGGGAACTTCTCCTGCTAGAAT
R：SEQ ID NO.3：CAGACATTTTGCTCTCAAGCTG
(4) transcribing the novel coronavirus plasmid to obtain RNA, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification to obtain a product for fluorescence detection of a Cas12a/crRNA system;
(5) transcribing the novel coronavirus plasmid to obtain RNA, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification to obtain a product for color development detection of a Cas12a/crRNA system combined with nanogold;
(6) extracting lentiviral RNA containing a novel coronavirus N gene segment, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification on the product to be used for fluorescence detection of a Cas12a/crRNA system;
(7) extracting lentiviral RNA containing a novel coronavirus N gene segment, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification on the product to be used for combined nanogold color development detection of a Cas12a/crRNA system.
Application of the visual virus detection method based on the CRISPR-Cas12a system in the aspect of detection of microorganisms.
The application of the visual virus detection method based on the CRISPR-Cas12a system in the detection of novel coronavirus is disclosed.
The invention has the following advantages and positive effects:
1. the method is a fluorescence detection method and a nanogold color development method based on detection of a CRISPR-Cas12a system, the method can detect the concentration of the target genome RNA only by 4 steps of simple sample pretreatment, PCR amplification, fluorescence detection and nanogold color development, more importantly, the method can realize the detection of different microorganisms only by changing a nucleic acid sequence, and the application of the CRISPR-Cas12a in the field of molecular detection is greatly widened.
2. The invention is a method for detecting novel coronavirus through a CRISPR-Cas12a system established through a series of experiments and detecting the novel coronavirus through combining nanogold color development and reading a numerical value through an intelligent mobile phone App, and a correlation coefficient R of a detected standard curve2The values of (A) and (B) are 0.996 and 0.991, respectively, and the linearity is good, so that the copy number of the microorganism detected by the standard curve is accurate.
3. The detection method established by the invention has low detection limit, the minimum detection limit is 1 copy/mu L, and the virus 100To 108The copies/mu L has a linear relation with the detection signal intensity.
4. The established detection method has strong specificity, takes the detection of the novel coronavirus (SARS-CoV-2) as an example, has no obvious fluorescence increase on the microorganisms such as SARS-CoV, MERS-CoV, MHV, IAV and the like and the novel coronavirus, and proves that the invention has good selectivity on the novel coronavirus and is influenced by other pathogenic microorganisms.
5. The fluorescent probe designed by the invention can accurately detect the novel coronavirus, and can be used for detecting other pathogens only by simply designing a primer sequence. The detection method is accurate, wide in detection range, simple, fast and easy to operate.
6. Compared with other traditional detection methods, the design of the invention has the advantages of simple operation method, high sensitivity and the like. Meanwhile, compared with other isothermal amplification methods (RPA, LAMP and the like), the design of the invention greatly reduces the detection cost, thereby being convenient for detecting a large number of samples.
7. The method is based on a CRISPR-Cas12a (LbCas12a) system, combined with a PCR amplification technology, detects the novel coronavirus with high sensitivity and specificity, combined with a nanogold developing method for visual detection, and simultaneously combined with a smart phone to read a value according to the color depth of the nanogold developing, wherein the detection limit is as low as1 copy/mu L. Furthermore, the practical applicability of this detection strategy for the detection of novel coronaviruses in complex biological samples was demonstrated. The method has the characteristics of excellent reliability, sensitivity, specificity and easiness in implementation, and is expected to provide great potential for detecting the novel coronavirus in the seafood sample.
8. The principle of the method for detecting the novel coronavirus in the invention is as follows: the Cas12a-crRNA complex will only trigger accessory cleavage activity of Cas12a, i.e., can cleave other single-stranded unrelated DNA (ssdna), in the presence of specific Target DNA that can complementarily bind to crRNA. If one end of the unrelated DNA is modified with FAM fluorescent group, and the other end is modified with quenching group BHQ1 (namely reporter RNA), the cutting effect can be converted into a fluorescent signal to be output. When Cas12a-crRNA detects corresponding Target DNA, the fluorescent probe can be cut, so that the fluorescent group FAM is far away from the quenching group BHQ1, the quenching of the BHQ1 on FAM fluorescence is released, and the FAM fluorescence value is increased. Briefly, the change in fluorescence value has a linear relationship with Target DNA concentration, i.e. the concentration of the novel coronavirus DNA (gdna), such that the concentration of the novel coronavirus gRNA, i.e. the novel coronavirus copy number (copies/. mu.l), can be converted into a corresponding fluorescence signal. In addition, the Cas12a/AuNPs detection platform developed by the research mixes a Cas12a/crRNA system and DNA1/2-AuNPs in equal volume, when linker-ssDNA is degraded by Cas12a, the DNA1/2-AuNPs keep a dispersed state so as to be red (positive), and on the contrary, when no virus target sequence exists in the system, ssDNase cleavage activity of Cas12a protein cannot be activated, and the complete linker-ssDNA can lead DNA1/2-AuNPs to precipitate and aggregate, so that red color is faded. The change of the color shade has a linear relation with the concentration of Target DNA, namely the concentration of the novel coronavirus DNA, and the concentration of the novel coronavirus genome RNA, namely the copy number (copies/mu L) of the novel coronavirus can be converted into an enzyme-labeling instrument to read the OD value of the instrument or a smart phone App to read the value.
9. The invention provides a novel coronavirus (SARS-CoV-2) detection method which combines the ultrasensitiveness, high specificity and visualization of a CRISPR-Cas12a system and a smart phone based on the CRISPR-Cas12a nanogold chromogenic detection result and has sensitivity and specificity which are comparable to those of a real-time PCR technology, and the method can provide a new idea for detecting pathogenic microorganisms, particularly novel coronavirus (SARS-CoV-2).
10. The method firstly carries out pre-inactivation pretreatment on the virus, extracts the genome RNA of the virus, carries out reverse transcription on the RNA into cDNA by M-MLV reverse transcriptase, and carries out PCR amplification to obtain double-stranded DNA (dsDNA), namely target dsDNA. The accessory cleavage ability of Cas12a-crRNA complex, i.e., cleavage of unrelated single stranded DNA (ssDNA), can be stimulated when target dsDNA is present. On one hand, the non-specific cleavage of the ssDNA can be detected by a fluorescence signal, namely, one end is modified with a fluorescent group, and the other end is modified with a quenching group. When the Cas12a-crRNA compound detects corresponding Target DNA, the fluorescent probe can be cut, so that the fluorescence value is increased, and the increase of the fluorescence value has a linear corresponding relation with the virus to be detected, thereby detecting the novel coronavirus; on the other hand, the non-specific cleavage of ssDNA can be detected by gold nano-chromogenic detection, and the linker DNA is subjected to base complementary pairing with ssDNA1 or ssDNA2 bound on the surface of the gold nano-particle. Namely, when target DNA exists, linker-ssDNA is degraded by Cas12a, DNA1-AuNPs and DNA2-AuNPs are separated, irrelevant single-stranded DNA is cut, and the DNA1 and the DNA2 which are combined with the surface of the gold nanoparticles can not be subjected to complementary pairing, so that the gold nanoparticles are in a dispersed non-aggregated state, the gold nanoparticle solution is red, and the gold nanoparticle solution is red after centrifugation; when the target is not present, the irrelevant single-stranded DNA cannot be cut, and the irrelevant single-stranded DNA is subjected to base complementary pairing with the DNA1 and the DNA2, so that the gold nanoparticles are aggregated, the color is blue-purple, and the supernatant of the solution is colorless after centrifugation. The color of the image of the gold nanoparticle solution photo can be read and the signal value is reported by utilizing the color recognizer APP in the smart phone. The larger the amount of target virus, the stronger the detection signal. The method has extremely high sensitivity, the detection line is as low as1 copy/mu L, the specificity is high, the method can be distinguished from other coronavirus and influenza virus, the visualization is realized by naked eyes, the signal can be read by combining an intelligent mobile phone, and the real-time detection on site is convenient.
11. The invention designs an intelligent mobile phone integrated biosensor based on CRISPR-Cas12a combined gold nanoparticles (AuNPs) visual detection technology by utilizing CRISPR-Cas12a trans-cutting activity, and can carry out sensitive quantitative detection on SARS-CoV-2. The method utilizes CRISPR-Cas12a to perform linker DNA trans-cutting when identifying SARS-CoV-2 specific gene amplicon, and triggers the aggregation-dispersion of AuNPs. The color change can be identified by naked eyes and a smart phone and is used for semi-quantitative or quantitative field detection of SARS-CoV-2. The biosensor designed by the invention is applied to real clinical samples, and the positive rate and the negative rate are 100 percent and 100 percent respectively. The detection method established by the invention can be used for visually detecting the novel coronavirus nucleic acid in the food sample with high sensitivity and high specificity. The invention researches a method for detecting SARS-CoV-2 by using CRISPR-Cas12a, and the detection method is characterized in that a detection value is directly read by combining nanogold color development and smart phone APP software, and the detection result can be read in an environment outside a laboratory without a complex instrument.
Drawings
FIG. 1 is a diagram of the feasibility analysis of SARS-CoV-2 based on the CRISPR-Cas12a system for detection in the present invention;
fig. 2 is a diagram of a selectivity assay based on CRISPR-Cas12a system detection in the present invention;
fig. 3 is a graph of RNA concentration dependence analysis based on CRISPR-Cas12a system in the present invention; wherein, 3A: time-scan, 3B: a plot of RNA concentration versus fluorescence intensity;
FIG. 4 is a standard graph for detecting novel coronavirus genome SARS-CoV-2 based on CRISPR-Cas12a system in the present invention; wherein, 4A: time-scan, 4B: novel coronavirus genome RNA concentration and fluorescence intensity dot line graph, 4C: qPCR method for detecting novel coronavirus genome amplification curve, 4D: carrying out histogram analysis on the genome of the novel coronavirus detected by a qPCR method;
FIG. 5 is a time scan of detection of bound nanogold in SARS-CoV-2 by the CRISPR-Cas12a system of the invention; wherein, 5A: visual detection of novel coronavirus genome SARS-CoV-2, 5B: time-scan, 5C: linear fit standard curve, 5D: the smart phone APP reads a nanogold color development gray scale map according to the color depth of the novel coronavirus genome with different concentrations, 5E: gradation analysis graph, 5F: a gray scale analysis line graph;
FIG. 6 is a graph showing the results of detecting SARS-CoV-2 in a pork sample using the CRISPR-Cas12a system of the present invention; wherein, 6A: qPCR method for detecting new coronavirus amplification curve in pork, 6B: histogram analysis of genome of new coronavirus in pork detected by qPCR method, 6C: time-scan of CRISPR-Cas12a system for detection of novel coronavirus genomes in pork, 6D: detecting a new coronavirus genome fluorescence intensity histogram in pork by using a CRISPR-Cas12a system; 6E: visualization detection of SARS-CoV-2 color development map of novel coronavirus genome in pork by combination of CRISPR-Cas12a system and nanogold, 6F: detecting a SARS-CoV-2 time scanning picture of a novel coronavirus genome in pork by combining a CRISPR-Cas12a system with nanogold; 6G: a smart phone APP gray level analysis graph;
FIG. 7 is a diagram comparing the detection of novel coronavirus by Cas12 in the present invention with other methods; wherein: 7A: the qPCR method is used for carrying out heat map comparison on the novel coronavirus detected based on the fluorescent intensity of the CRISPR-Cas12a system and the novel coronavirus detected by combining the CRISPR-Cas12a system with nanogold visualization, and the heat map comparison is 7B: the qPCR method is based on a CRISPR-Cas12a system fluorescence intensity analysis method, and three method heat map comparisons of novel coronavirus detection and CRISPR-Cas12a system combined with nanogold visual detection in different cold chains are as follows: 7C: ROC curve analysis diagram, 7D: wien diagram analysis;
FIG. 8 is a clinical sample experimental chart of the developing test of nanogold in the invention; wherein: 8A: visually detecting a new coronavirus genome positive group in a clinical sample; 8B: visually detecting a new coronavirus genome negative group in a clinical sample; 8C: CRISPR-Cas12a system binding nanogold time scan histogram.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The raw materials used in the invention are all conventional commercial products if no special description is provided, the method used in the invention is all conventional methods in the field if no special description is provided, and the mass of all the materials used in the invention is the conventional use mass.
The experimental method of the present invention, in which specific conditions are not specified, is generally carried out under conventional conditions such as molecular cloning: the conditions described in the Laboratory Manual (New York Cold Spring Harbor Laboratory Press, 1989). Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
A visual virus detection method based on a CRISPR-Cas12a system comprises the steps of carrying out reverse transcription on virus genome RNA to obtain cDNA, carrying out reverse transcription on DNA genome, carrying out PCR amplification to obtain dsDNA, designing a product called Target DNA, namely Target DNA, designing crRNA specifically targeting a virus gene sequence, and detecting the Target DNA by using a Cas12a-crRNA compound.
Preferably, the virus is a novel coronavirus SARS-CoV-2.
Preferably, the specific steps are as follows:
when Cas12a-crRNA detects corresponding Target DNA, the fluorescent probe can be cut, so that a fluorescent group FAM is far away from a quenching group BHQ1, the quenching of BHQ1 on FAM fluorescence is relieved, and the fluorescence value of FAM is increased to detect novel coronavirus SARS-CoV-2; in addition, by utilizing the property of changing the aggregation color of the nanogold, the depth of the color of the nanogold has a linear relation with the concentration of Target RNA, namely the concentration of the novel coronavirus genome RNA, and the concentration of the novel coronavirus genome RNA, namely the copy number of the novel coronavirus, can be converted into a specific numerical value through the smartphone color recognizer App and then output.
Preferably, the specific steps are as follows:
the presence of target DNA can excite the auxiliary cleavage capability triggered by the nucleic acid specific recognition of the Cas12a-crRNA complex, namely, the cleavage of unrelated single-stranded DNA; in this case, if one is selected to be synthesized, one end of the selected strand is modified with a fluorescent group, and the other end of the selected strand is modified with a quenching group to prepare reporter DNA, i.e., report DNA, the reporter DNA can be digested by Cas12a, and an enhanced fluorescent signal is generated for output; the change of the fluorescence signal and the concentration of the novel coronavirus to be detected can form a linear relation, and the novel coronavirus can be detected and quantified; in the nano-gold color development experiment, the two ends of the linker-ssDNA sequence are designed to be complementary matched with the DNA sequence which can be crosslinked with the nano-gold particles, namely DNA 1/2-AuNPs; the Cas12a/crRNA system was subsequently mixed with equal volumes of DNA1/2-AuNPs, and when linker-ssDNA was degraded by Cas12a, the DNA1/2-AuNPs remained dispersed and appeared red; the shade of the color can be analyzed and read through the APP gray scale of the color identifier of the smart phone; the numerical value obtained by gray level analysis and the concentration of the novel coronavirus to be detected can form a linear relation, and the novel coronavirus can be detected and quantified.
Preferably, the specific steps are as follows:
the target DNA is obtained by detecting the RNA of the novel coronavirus through a two-step method, when the Cas12a-crRNA detects the corresponding target DNA, namely the target DNA and the crRNA can be complementarily paired, the fluorescent probe can be cut, a fluorescent group FAM is separated from a quenching group BHQ1, the quenching of BHQ1 on the FAM fluorescence is relieved, the increase of the FAM fluorescence value can be linearly related to the concentration of the novel coronavirus, and the specific novel coronavirus can be specifically detected and quantified; furthermore, when linker-sWhen the sDNA is degraded by Cas12a, the DNA1/2-AuNPs keep a dispersed state and thus show red color; the shade of the color can be read through the grayscale analysis of the APP color recognizer of the smart phone or through the Tecan
The OD value of the 200Pro plate reader is measured, the measured value is in linear relation with the concentration of the novel coronavirus, and the specific novel coronavirus can be specifically detected and quantified.
Preferably, the specific steps are as follows:
(1) construction of SARS-CoV-2 plasmid: synthesizing double-stranded DNA containing a novel coronavirus N gene fragment NC-045512.2, performing enzyme digestion by BamHI EcoR I and linking with T4 DNA ligase to construct into a PLVX-AcGFP-N1 lentiviral vector so as to obtain a SARS-CoV-2 plasmid, wherein the constructed plasmid is named as a plasmid of lentiSARS-CoV-2-N;
the sequence information (5 '-3') of the synthesized SARS-CoV-2 gene fragment is SEQ ID NO. 1:
catcgtgttgtctgtactgccgttgccacatagatcatccaaatcctaaaggattttgtgacttaaaaggtaagtatgtacaaatacctacaacttgtgctaatgaccctgtgggttttacacttaaaaacacagtctgtaccgtctgcggtatgtggaaaggttatggctgtagttgtgatcaactccgcgaacccatgcttcagtcagctgatgcacaatcgtttttaaacgggtttgcggtgtaagtgcagcccgtcttacaccgtgcggcacaggcactagtactgatgtcgtaagctggacttccctatggtgctaacaaagacggcatcatatgggttgcaactgagggagccttgaatacaccaaaagatcacattggcacccgcaatcctgctaacaatgctgcaatcgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcagaagggagcagaggcggcagtcaagcctcttctcgttcctcatcacgtagtcgcaacagttcaagaaattcaactccaggcagcagtaggggaacttctcctgctagaatggctggcaatggcggtgatgctgctcttgctttgctgctgcttgacagattgaaccagcttgagagcaaaatgtctggtaaaggccaacaacaacaaggccaaactgtcactaagaaatctgctgctgaggcttctaagaagcctcggcaaaaacgtactgccac
(2) SARS-CoV-2 pseudovirus packaging: cotransfecting the constructed plasmid lentiSARS-CoV-2-N containing SARS-CoV-2N gene and ORF1ab gene fragment, psPAX2(Addge plasma #12260) and pMD2.G (Addge plasma #12259) plasmid into 293T cell, after 48 hours of transfection, collecting cell culture supernatant, filtering through 0.45 μm filter membrane to obtain lentivirus containing SARS-CoV-2 gene fragment, namely SARS-CoV-2 pseudovirus;
(3) SARS-CoV-2N fragment gene amplification: the N fragment is amplified by using SARS-CoV-2 specific primers, and the sequence of the amplified primers is as follows:
F：SEQ ID NO.2：GGGGAACTTCTCCTGCTAGAAT
R：SEQ ID NO.3：CAGACATTTTGCTCTCAAGCTG
(4) transcribing the novel coronavirus plasmid to obtain RNA, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification to obtain a product for fluorescence detection of a Cas12a/crRNA system;
(5) transcribing the novel coronavirus plasmid to obtain RNA, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification to obtain a product for color development detection of a Cas12a/crRNA system combined with nanogold;
(6) extracting lentiviral RNA containing a novel coronavirus N gene segment, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification on the product to be used for fluorescence detection of a Cas12a/crRNA system;
(7) extracting lentiviral RNA containing a novel coronavirus N gene segment, performing gradient dilution on the obtained RNA, performing reverse transcription on the RNA subjected to gradient dilution to obtain cDNA, and performing PCR amplification on the product to be used for combined nanogold color development detection of a Cas12a/crRNA system.
The application of the visual virus detection method based on the CRISPR-Cas12a system in the detection of microorganisms is disclosed.
The application of the visual virus detection method based on the CRISPR-Cas12a system in the detection of novel coronavirus is disclosed.
Specifically, the preparation and detection are as follows:
the method comprises the following specific steps:
design and Synthesis of crRNA
1.1 designing SARS-CoV-2N gene sequence specific crRNA sequence and making gene synthesis. The sequence for crRNA synthesis is shown in NO 1.
1.2 first, a crRNA template (sequence:
5' -TCTGTACCGTCTGCGGTATGTGATCTACACTTAGTAGAAATTACCCTATAGTGAGTCGTATTAATTTC), using a centrifuge at 12000rpm for 10min, then dissolving and diluting to 4 μ M using DEPC water. The prepared solution was then mixed with T7 promoter (sequence:
GAAATTAATACGACTCACTATAGGG) into a double strand of DNA, and after annealing, performing a transcription step using a HiScribe T7 Rapid high Performance RNA Synthesis kit. Taking out the transcription sample which is incubated overnight at 37 ℃ in the PCR instrument, adding 2 mu L of DNase I (2000U/mL) into the reaction system in the previous step, uniformly mixing by using a pipette, instantly centrifuging to ensure that the solution is completely gathered at the bottom of the tube, placing the tube on a thermal cycler for incubation reaction at 37 ℃ for 30min to completely eliminate the DNA template in the system, and then carrying out RNA purification according to the instruction steps of a Monarch RNA purification kit. Purified RNA was used with Eppendorf
The RNA concentration is measured by a basic ultramicro nucleic acid quantifier and finally stored in a refrigerator at the temperature of 80 ℃ below zero.
CRISPR-LbCas12a assay for novel coronavirus selectivity
In order to verify whether the Cas12a has the described better selectivity for detecting the novel coronavirus, a selective verification experiment was performed, as shown in fig. 3, when Cas12a, crRNA and different target targt DNAs are used, only the target DNA of the novel coronavirus is added to have a higher fluorescence value, and when other targets SARS-CoV are added to the system respectively; MERS-CoV; MHV; an IAV; (S.aureus); top 10; hela and the like have low fluorescence values, which indicates that Cas12a has good selectivity for detecting the novel coronavirus.
Feasibility experiment for detecting novel coronavirus by CRISPR-LbCas12a
3.1 PCR amplification of N Gene fragments of novel coronavirus
Double-stranded DNA containing a novel coronavirus N gene fragment (NC-045512.2) is synthesized by Shanghai Jie biological engineering Limited according to a SARS-CoV-2 gene sequence, a proper restriction endonuclease sequence is selected, a novel coronavirus plasmid pGH 1abN constructed by a novel coronavirus N gene upstream primer (GGGGAACTTCTCCTGCTAGAAT) and a novel coronavirus N gene downstream primer (CAGACATTTTGCTCTCAAGCTG) is designed, and the extracted pGH 1abN plasmid is used as a PCR template to carry out PCR reaction.
In order to verify whether Cas12a has the described accessory cleavage ability triggered by nucleic acid specific recognition, a feasibility verification experiment was performed, as shown in fig. 1, and a strong fluorescence signal was generated if and only if Cas12a, crRNA and target DNA complementary to crRNA were present at the same time, which preliminarily proved the feasibility of the method of the present invention, having specific recognition ability.
3.2CRISPR-LbCas12a assay for RNA dependency of novel coronavirus
To verify whether Cas12a has the described high nucleic acid detection sensitivity, a sensitivity verification experiment was performed, as shown in fig. 2, to detect 1aM if and only if Cas12a, crRNA, and different gradients of RNA were bound by the PCR-amplified products after reverse transcription, preliminary demonstrating the high sensitivity of the method of the invention. And the linearity is good.
4. Experiment for detecting novel coronavirus genome based on CRISPR-LbCas12a
4.1 cell Resuscitation
(1) 10mL of freshly cultured DMEM medium was previously taken in a sterile biosafety cabinet and placed in a 15mL centrifuge tube.
(2) The frozen 293T cells were removed and placed in a pre-warmed water bath that had been raised to 37 ℃ and the cells were thawed by shaking rapidly.
(3) The cell suspension was quickly transferred to 10mL of fresh medium prepared in advance, gently mixed by inverting the upper and lower sides, and centrifuged at 800r/min for 3 minutes.
(4) The centrifuged supernatant was discarded, and the cell pellet was blown off with 2mL of the culture medium, transferred to a clean cell culture dish, and cultured in an incubator at 37 ℃.
(5) The next day, a judgment is made on the growth of the cells to allow for fresh medium replacement or passaging of the cells.
4.2 cell passages
(1) The cells grow well and can be passaged when the density of the cells accounts for about 90-100% of the bottom of the culture dish. In a sterile biosafety cabinet, the original medium in the dish was carefully discarded, then 2mL of PBS was slowly added to the cell surface using a pipette gun, and the dish was carefully gently shaken to wash out the waste metabolites and residual medium on the cell surface.
(2) After discarding PBS, about 1mL of trypsin digest containing EDTA was added to allow sufficient coverage of the cells and allowed to stand for about 6 s.
(3) After observing the rounding of the cells under the mirror, the digestion solution was immediately discarded, and about 1-2mL of fresh medium was added to stop the digestion, and the cells were gently suspended by gently blowing the medium using a pipette.
(4) And collecting and transferring the cells into a 15mL centrifuge tube, centrifuging at 800r/min for 4min, removing supernatant, adding a fresh DMEM culture medium to blow off the cells, and transferring a proper amount of cell suspension into a new culture dish.
(5) The 293T cell culture dish was placed in a 37 ℃ cell culture chamber for culture.
4.3 cell transfection and lentivirus packaging
4.3.1LentiSARS-CoV-2-N1 lentivirus using HEK293T cells
(1) Well-grown HEK293T cells were spread evenly onto 46 cm cell culture dishes and placed in a cell incubator for culture.
(2) When the growth density of HEK293T cells reaches 80% -90%, transfecting a three-plasmid system by using Turbofect, wherein the target plasmid is as follows: packaging plasmid: the transfection mixture containing the plasmid of interest was left to stand for about 18-20 minutes and then added dropwise to the surface of HEK293T cells, after which the plates were gently shaken by hand until the mixture was evenly distributed in 6cm cell plates and returned to the incubator for 12 hours.
(3) Cells after 12 hours of culture were removed and carefully replaced with fresh medium in a sterile biosafety cabinet to prevent blowing off of semi-adherent HEK293T cells and cultured overnight again.
(4) Observing the cell state in a 6cm cell culture dish from about 48 hours after the culture of the fresh culture medium is replaced, if the cell survival rate is about 30 percent, gently sucking the culture medium containing the virus in the cell culture dish by a liquid transfer machine, transferring the culture medium into a 15mL centrifuge tube wrapped with clean tinfoil paper in advance, collecting supernatant in a 4-degree centrifuge (precooled centrifuge in advance), filtering the collected supernatant by a 0.45 mu M filter membrane for 10min, and then storing each tube in a-80-degree refrigerator in a 500 mu M dark place.
(5) Experiment of viral infection
a) HEK293T cell lentivirus infection by using cell
b) HEK293T cells in good growth state were uniformly plated on a 3cm cell culture dish and cultured in a cell culture chamber at 37 ℃.
c) When the growth density of the HEK293T cells reached about 30-40%, lentivirus infection was performed by adding 1.5mL of fresh medium to a sterile centrifuge tube in advance, mixing polybrene (polybrene) at a final concentration of 8. mu.g/mL, and slowly adding the polybrene to the HEK293T cells after mixing well. After 48 hours, the expression level of GFP fluorescent protein in the cells was observed under an inverted fluorescence microscope.
4.3.2 extraction of novel coronavirus genomic RNA
(1) The novel coronavirus genome was extracted using the TIANamp Virus rnalit kit. Briefly, 280. mu.L of the collected virus solution was added with 2. mu.L of Carrier RNA and 1.12mL of Buffer PL, vortexed for 15s, mixed well, and briefly centrifuged to collect the fluid adhering to the tube wall and the tube cap. Incubation was carried out at room temperature (15-20 ℃) for 10 min. 1.12mL of absolute ethanol was added thereto. Mixing well. The subsequent steps are to extract column RNA according to the instruction.
(2) Preparation of novel coronavirus genomic cDNA and amplification of cDNA by PCR
And carrying out 10-fold gradient dilution on the extracted novel coronavirus genome RNA, adding the diluted sample into a system as a template, carrying out reverse transcription on the novel coronavirus gene according to the instruction to obtain cDNA, and amplifying by a PCR instrument.
4.4 CRISPR-LbCas12 a-based assay for detecting fluorescence intensity of novel coronavirus genome
Each 100. mu.L reaction system included 15nM of LbCas12a purchased at 15nM, 12.5nM crRNA, 200nM quenched reporter DNA, and 5. mu.L of different concentrations of in vitro PCR amplification product with a buffer composition of 10 XNEB 2.1buffer used at 37 deg.C
Pro plate reader (Tecan, Switzerland) measures the change in fluorescence value with parameters set to an excitation wavelength of 484nm and an emission wavelength of 529nm, and the fluorescence intensity is measured every 30s for a total of 62 Cyele. The results are shown in FIGS. 4A and 4B with the lowest detection line of 11 copy/. mu.L.
4.5qPCR Probe method for detecting novel coronavirus
The new coronavirus was tested by designing the forward (GGGGAACTTCTCCTGCTAGAAT) and the reverse (CAGACATTTTGCTCTCAAGCTG) primers and the probe (5 '-FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3') for the N gene of the new coronavirus according to the Chinese CDC standard, and the results are shown in FIGS. 4C and 4D.
5. CRISPR-LbCas12 a-based color development experiment for detecting novel coronavirus genome combined with nanogold
5.1 preparation of Nanogold and connection of Nanogold and DNA
(1) Process for preparing nano gold
All glassware used in the process of preparing the nano-gold needs aqua regia (HNO)3Soaking in HCl 3:1) for more than 30min, and rinsing with ultrapure water. To a 250mL flask was added 100mL (1mM) of HAuCl4The solution was heated until boiling. 38.8mM sodium citrate solution was then added to the solution, with constant stirring, and the solution changed from pale yellow to wine red (20 min). Then stopping heating and naturally cooling the solution to room temperature, thus obtaining the nano gold with the particle size of 13nm and the maximum absorption peak at the position of 520 nm. The prepared nanogold can be identified by TEM (JEM-2100HR), and finally, the prepared nanogold is stored at 4 ℃ in the dark for future use.
5.2 ligation of DNA and Nanogold
Two types of DNA-functionalized nanogold were prepared using a freezing-based labeling method. The DNA fragment was obtained from thiol DNA1 (100. mu.M, 3. mu.L) (SEQ ID NO: 5 '-C6-SH-AAAAAAAAACCCAGGTTCTCT-3')) or thiol DNA2 (SEQ ID NO: 5 '-TCACAGATGCGTAAAAAAAAA-C6-SH-3')) and nanogold (13nm, 100. mu.L) were mixed together and placed in a laboratory freezer (temperature set at-20 ℃) for 2 hours and then thawed at room temperature. After thawing, it was centrifuged at 12,000rpm/min at 4 ℃ for 30min, the supernatant was discarded, and it was suspended by blowing with buffer solution A (5mM HEPES buffer, pH 7.6), and this operation was repeated three times. Finally, the pellet was resuspended in buffer B (10mM HEPES, pH 7.6, 300mM NaCl) and the resulting DNA-AuNPs probe was stored at 4 ℃ in the dark
5.3 detection of novel coronavirus by Nanogold color development experiment
Reaction mixtures containing 15nM LbCas12a, 12.5nM crRNA, 46nM Linker DNA (SEQ ID NO: ACGCATCTGTGAAGAGAACCTGGG) and varying concentrations of target DNA were incubated in HEPES buffer at 37 ℃ for 25min followed by inactivation at 65 ℃ for 8 min. Then 50. mu.L of this reaction mixture was mixed with 25. mu.L of DNA1-AuNPs and 25. mu.L of DNA2-AuNPs solution and incubated at 37 ℃ for 10 min. After centrifugation (5000rpm, 3min), the supernatant was aspirated, and the pellet was discarded. The color of the supernatant was then recorded by taking a picture on a cell phone and the absorbance value was measured (A526). The results are shown in FIGS. 5A, 5B and 5C. The shade of the color is shown in fig. 5D, 5E and 5F through the grayscale analysis of the smart phone APP.
6. Food labeling experiment
First, 100mg of pork is weighed and 300. mu.L of new coronavirus solution with different gradients is added. These confirmed new coronavirus liquid negative pork samples were then spiked with different copy numbers of new coronavirus liquid (copy number of the new coronavirus was calculated using qPCR). The sample was ground by a tissue grinder, centrifuged at 8000rpm for 5 minutes at 4 ℃ and then 280. mu.L of the supernatant was taken, 2. mu.L of Carrier RNA and 1.12mL of Buffer PL were added, vortexed for 15s, mixed, and briefly centrifuged to collect the liquid adhering to the tube wall and the tube cap. Incubation was carried out at room temperature (15-20 ℃) for 10 min. 1.12mL of absolute ethanol was added thereto. Mixing well. The subsequent steps are to extract column RNA according to the instruction. The extracted RNA sample is subjected to reverse transcription according to the previous steps to obtain cDNA, and a part of the obtained cDNA is used for qPCR detection, as shown in FIGS. 6A and 6B; one portion was amplified by RCR for Cas12a fluorescence detection, as shown in fig. 6C and 6D; the other part is used for nano-gold color development experiments. The results are shown in FIGS. 6E and 6F; the smartphone App grayscale analysis histogram is shown in fig. 6G.
Cas12a graph comparing detection of novel coronavirus with other methods
7A and 7B: qPCR, Cas12a detection of novel coronavirus and nanogold chromogenic detection of novel coronavirus genome. 7C: ROC curve analysis, 7D: analysis of wien diagram.
8. Detecting clinical sample experiments by a nanogold color development experiment.
8.1 preparation of 50 clinical samples of cDNA and amplification of cDNA by PCR the extracted genomic RNA of the novel coronavirus (provided by Tianjin disease control center) was used as a template, 0.66. mu.M primer (CAGACATTTTGCTCTCAAGCTG) was added to a 15. mu.L system, and after 5min at 75 ℃, the system was quickly placed on ice and ice-washed for 5 min. Then, 5. mu. L M-MLV 5 XBuffer, 0.5mM dNTP MiX, 8Units M-MLV, 1unit RNase inhibitor were added thereto and reverse transcription was performed for 1 hour in a 25. mu.L system at 42 ℃. The cDNA obtained was used for the next PCR amplification.
8.2 clinical 50 samples cDNAPCR amplification.
5uL of each of the above reverse-transcribed cDNAs was used as a template, and 0.25. mu.M of the forward primer (GGGGAACTTCTCCTGCTAGAAT), 0.25. mu.M of the reverse primer (CAGACATTTTGCTCTCAAGCTG) and 10uL of 2 XTaq PCR MasterMix II were added thereto and amplified in a PCR instrument to obtain dsDNA (target DNA).
8.3 visual detection of novel coronaviruses.
8.3.1 Process for preparing Nano gold
All glassware used in the process of preparing the nano-gold needs aqua regia (HNO)3Soaking in HCl 3:1) for more than 30min, and rinsing with ultrapure water. To a 250mL flask was added 100mL (1mM) of HAuCl4The solution was heated until boiling. 38.8mM sodium citrate solution was then added to the solution, with constant stirring, and the solution changed from pale yellow to wine red (20 min). Then stopping heating and naturally cooling the solution to room temperature to obtain 13nm nanometerGold, having a maximum absorption peak at 520 nm. The prepared nanogold can be identified by TEM (JEM-2100HR), and finally the prepared nanogold is stored at 4 ℃ in a dark place.
8.3.2 ligation of DNA and Nanogold
Two types of DNA-functionalized nanogold were prepared using a freezing-based labeling method. The DNA fragment was obtained from thiol DNA1 (100. mu.M, 3. mu.L) (SEQ ID NO: 5 '-C6-SH-AAAAAAAAACCCAGGTTCTCT-3')) or thiol DNA2 (SEQ ID NO: 5 '-TCACAGATGCGTAAAAAAAAA-C6-SH-3')) and nanogold (13nm, 100. mu.L) were mixed together and placed in a laboratory freezer (temperature set at-20 ℃) for 2 hours and then thawed at room temperature. After thawing, it was centrifuged at 12,000rpm/min at 4 ℃ for 30min, the supernatant was discarded, and it was suspended by blowing with buffer solution A (5mM HEPES buffer, pH 7.6), and this operation was repeated three times. Finally, the pellet was resuspended in buffer B (10mM HEPES, pH 7.6, 300mM NaCl) and the resulting DNA-AuNPs probe was stored at 4 ℃ in the dark
8.3.3 detection of clinical samples by Nanogold color development experiment
To the different target DNAs amplified above were added 15nM LbCas12a, 12.5nM crRNA, 46nM Linker DNA (SEQ ID NO: ACGCATCTGTGAAGAGAACCTGGG) respectively, and incubated in HEPES buffer at 37 ℃ for 25min, followed by inactivation at 65 ℃ for 8 min. Then 50. mu.L of this reaction mixture was mixed with 25. mu.L of DNA1-AuNPs and 25. mu.L of DNA2-AuNPs solution and incubated at 37 ℃ for 10 min. After centrifugation (5000rpm, 3min) the samples were pelleted. The supernatant was aspirated, and then the color of the supernatant was recorded by taking a picture with a mobile phone and measuring the absorbance value thereof (A526). The results are shown in FIGS. 8A, 8B and 8C.
Although the 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 embodiments disclosed.
Sequence listing
<110> Tianjin science and technology university
<120> visual virus detection method based on CRISPR-Cas12a system and application
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ctggacttcc ctatggtgct aacaaagacg gcatcatatg ggttgcaact gagggagcct 360
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gcagtcaagc ctcttctcgt tcctcatcac gtagtcgcaa cagttcaaga aattcaactc 540
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Claims (8)
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