CN117568335A - HRP-biotin double-modified ssDNA reporter and application thereof - Google Patents

HRP-biotin double-modified ssDNA reporter and application thereof Download PDF

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CN117568335A
CN117568335A CN202311493980.9A CN202311493980A CN117568335A CN 117568335 A CN117568335 A CN 117568335A CN 202311493980 A CN202311493980 A CN 202311493980A CN 117568335 A CN117568335 A CN 117568335A
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hrp
nucleic acid
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cas12a
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吴暄
李毅
向荣
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Oral Hospital Affiliated To Zunyi Medical University
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Abstract

The invention relates to an HRP-biotin double-modified ssDNA reporter and application thereof, and relates to the technical field of biological detection. The invention achieves the effect of amplifying the signal output of the CRISPR/Cas detection system by performing HRP coupling on the reporter. Meanwhile, an HRP enhanced CRISPR/Cas12a detection system for detecting target nucleic acid fragments is provided, so that the nucleic acid targets can be detected simply, conveniently and rapidly, nucleic acid pre-amplification is not needed, the result is visualized, special or expensive equipment is not needed for detecting the nucleic acid targets rapidly, and the detection of the nucleic acid of the spike virus is realized by taking the nucleic acid of the spike virus as an example, and the detection system can be applied to the preparation of detection products such as the spike virus and the like.

Description

HRP-biotin double-modified ssDNA reporter and application thereof
Technical Field
The invention relates to the technical field of biological detection, in particular to an HRP-biotin double-modified ssDNA reporter and application thereof.
Background
Currently, as an important tool for genome editing, CRISPR/Cas systems have target sequence specific recognition functions. Certain Cas homologs, such as Cas13 and Cas12a, also activate non-specific endoenzyme activity (trans activity) upon specific recognition of the target sequence. More importantly, as a programmable nuclease, the target specificity of CRISPR/Cas12a can be achieved by simply adjusting the sequence of the gRNA in its reaction system. Essentially the CRISPR/Cas12a reaction can be chosen for essentially any nucleic acid sequence. Based on this principle, CRISPR/Cas systems have been widely used for nucleic acid detection. In earlier developed platforms such as detect and sholock, the binding of isothermal amplification techniques to CRISPR/Cas12a, cas13a and Cas14 not only helps to detect target sequences at the attomole level (aM) with high sensitivity, but also allows for the differentiation of genotypes and Single Nucleotide Polymorphisms (SNPs) in these target sequences. In recent years, CRISPR/Cas systems have been widely used to develop high sensitivity detection methods for various targets, such as ZIKV, dengue virus (DENV), SARS-CoV-2, african Swine Fever Virus (ASFV), and the like. However, achieving high sensitivity in the CRISPR/Cas detection methods described above requires a pre-amplification step of the target sequence. This not only extends the detection timeline, but also introduces a large number of unexpected target sequences, similar to the aforementioned PCR method, and thus there is a risk of aerosol contamination.
Horseradish peroxidase (HRP) is able to catalyze the degradation reaction of its substrate with high efficiency, resulting in a color or fluorescence reaction. The intensity of this color or fluorescence is highly correlated with the amount of HRP present in the reaction. This property makes HRP widely used for output of biological experimental results, even for quantitative analysis of antigens or proteins in ELISA and Western blotting assays. Amplification of Cas13a reaction results using HRP has been reported previously. However, in this report, HRP was conjugated to magnetic beads via reporters, cleaved by Cas13a, released into Cas reaction solution, and color reaction was generated by adding the substrate TMB (3, 3', 5' -tetramethylbenzidine) of HRP to Cas reaction solution. However, this method has very limited amplification effect on the result, and the sensitivity of the detection method is limited when no pre-amplification step is added.
The Langehead virus (LayV) belongs to the Henipah virus (Henipah virus), and the more well-known viruses include Henipah virus and Nipa virus. Current nucleic acid detection methods for henipa virus mainly include virus isolation, immunological methods, and nucleic acid detection methods. Among them, virus isolation is a gold standard for virus detection. However, this method is time-consuming and laborious, and for highly pathogenic pathogens, virus isolation is performed in the BSL-4 laboratory (biosafety class IV laboratory); ELISA (Enzyme-Linked Immunosorbent Assays, ELISA, enzyme-linked immunosorbent assay) in immunological methods has been widely used in Hendela virus and Nipa virus pandemics, but ELISA methods are poorly sensitive; PCR (polymerase chain reaction) is the most commonly used method at present, and the detection method based on nucleic acid amplification has high sensitivity and specificity. However, the PCR method requires a precise and expensive temperature cycling apparatus (PCR instrument), and is difficult to be widely popularized in areas with different experimental conditions. And the PCR-based nucleic acid amplification method is extremely easy to cause aerosol pollution of target nucleic acid fragments in an experimental environment, and causes false positive results for subsequent detection. Meanwhile, the existing nucleic acid detection method is limited to other members of henipavirus, and a specific nucleic acid diagnosis method of the wolf virus is not available.
Disclosure of Invention
In order to solve the problems, the invention provides an HRP-biotin double-modified ssDNA reporter and application thereof.
In a first aspect, the present invention provides an HRP-biotin double-modified ssDNA reporter, wherein the HRP-biotin double-modified ssDNA reporter is obtained by coupling DBCO-biotin double-modified ssDNA reporter and HRP by click chemistry.
In a second aspect, the present invention provides the use of the HRP-biotin double-modified ssDNA reporter of the first aspect in the preparation of a CRISPR/Cas detection system, said HRP-biotin double-modified ssDNA reporter acting as a signal output amplification for the CRISPR/Cas detection system.
Further, the CRISPR/Cas detection system does not perform a nucleic acid pre-amplification loop.
In a third aspect, the invention provides an HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection, the HRP enhanced CRISPR/Cas12a detection system comprising a Cas12a reaction system, an HRP modified reporter and a coated reaction vessel thereof, and a chromogenic reagent;
the HRP modified reporter coating reaction vessel is obtained by specifically combining an avidin coated reaction vessel and an HRP-biotin double modified ssDNA reporter through a biotin-avidin system;
the HRP-biotin double-modified ssDNA reporter is obtained by coupling DBCO-biotin double-modified ssDNA reporter and HRP in a click chemistry mode.
Further, the Cas12a reaction system includes the following components: reaction buffers such as NEBuffer 2.1 (10X), cas12a protein, gRNA, dithiothreitol and ddH 2 O。
Further, the preparation method of the HRP-biotin double-modified ssDNA reporter comprises the following steps:
dissolving HRP in sodium bicarbonate solution to obtain HRP solution;
adding the Azido-PEG-NHS Ester into the HRP solution to perform room temperature stirring reaction, and filtering and washing after the reaction is finished to obtain an azide modified HRP;
and (3) carrying out room-temperature shaking reaction on the azide-modified HRP and DBCO-biotin double-modified ssDNA reporter, and filtering and washing after the reaction is finished to obtain the HRP-biotin double-modified ssDNA reporter.
Further, the avidin-coated reaction vessel comprises an avidin-coated 96-well plate.
Further, the target nucleic acid fragments include, but are not limited to, viruses such as wolf tooth virus, influenza A virus and Porphyromonas gingivalis, and bacterial pathogenic nucleic acids, etc., and the chromogenic reagent includes 3,3', 5' -Tetramethylbenzidine (TMB).
Furthermore, when the HRP enhanced CRISPR/Cas12a detection system is applied to pathogenic microorganism detection, a nucleic acid pre-amplification link is not needed.
In a fourth aspect, the invention provides the use of the HRP enhanced CRISPR/Cas12a detection system of any of the first aspects in the preparation of a virus detection product, said virus comprising a wolf tooth virus, a zika virus, a dengue virus, SARS-CoV-2 and african swine fever virus.
In a fifth aspect, the present invention provides a biosensor for detecting a target nucleic acid fragment comprising the HRP-enhanced CRISPR/Cas12a detection system of any one of the first aspects, wherein the target nucleic acid fragment comprises, but is not limited to, viruses such as wolf tooth virus, influenza a virus and porphyromonas gingivalis, and bacterial pathogenic nucleic acids, and the like.
In a sixth aspect, the present invention provides a kit for detecting a target nucleic acid fragment, the kit for detecting a target nucleic acid fragment comprising but not limited to a virus such as a wolf tooth virus, influenza a virus and porphyromonas gingivalis, and a bacterial pathogen nucleic acid, etc., according to any one of the HRP enhanced CRISPR/Cas12a detection system of the first aspect.
In a seventh aspect, the present invention provides a method for detecting a target nucleic acid fragment comprising, but not limited to, a viral and bacterial pathogenic nucleic acid such as a spike virus, an influenza a virus, and a porphyromonas gingivalis, etc., using the HRP enhanced CRISPR/Cas12a detection system of any one of the first aspects.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has at least the following advantages:
1. the invention provides an HRP-biotin double-modified ssDNA reporter and application thereof, and achieves the effect of amplifying signal output of a CRISPR/Cas detection system by performing HRP coupling on the reporter. Specifically:
the principle of CRISPR/Cas system specific recognition of target sequences is: the gRNA is a segment of about 20nt specifically designed according to the target sequence and can be specifically combined with the target sequence through the base complementation principle, and RNPs formed by the Cas enzyme and the gRNA can specifically recognize the target sequence under the guidance of the gRNA and further activate trans enzyme cleavage activity after the target sequence is combined. The versatility of CRISPR/Cas systems as detection tools is also based on this principle. When the detection target needs to be changed, only the gRNA in the system needs to be redesigned and synthesized according to the target sequence, and the flexibility is the inherent property of the Cas enzyme and is also a great advantage when CRISPR/Cas is used for nucleic acid detection. By designing different gRNA sequences, CRISPR/Cas systems have been applied to the specific detection of a variety of nucleic acid target sequences. And as a programmable nuclease, the target specificity of the CRISPR/Cas12a can be achieved by simply adjusting the sequence of the gRNA in its reaction system, the CRISPR/Cas12a reaction can be directed against essentially any nucleic acid sequence for any nucleic acid target detection.
The invention does not change the characteristic of the Cas enzyme, but only achieves the effect of signal amplification output by performing HRP coupling on a reporter at a signal output end. Thus, its versatility is consistent with the common CRISPR/Cas system. The present invention subsequently provides detection results for a variety of additional targets to further demonstrate the versatility of the HRP-biotin double modified ssDNA reporters of this design for detecting different targets when applied to CRISPR/Cas systems.
2. Based on the HRP-biotin double-modified ssDNA reporter, the invention provides an HRP enhanced CRISPR/Cas12a detection system for detecting target nucleic acid fragments, and establishes a spike virus nucleic acid rapid detection system which is simple, convenient and rapid, does not need nucleic acid pre-amplification, has visualized results and does not need special or expensive equipment, and can be applied to preparation of detection products such as spike viruses and the like. Specifically, the HRP enhanced CRISPR/Cas12a detection system for detecting target nucleic acid fragments has the following characteristics:
1) The operation is simple, and expensive equipment is not needed: the detection method is implemented by only basic nucleic acid extraction related equipment and an enzyme-labeled instrument, and is simple and convenient to operate.
2) No nucleic acid amplification is required, and no aerosol pollution is caused: the existing high-sensitivity nucleic acid detection methods are all based on a nucleic acid amplification principle (such as a PCR method), and a large amount of target sequences are amplified for a long time, so that aerosol pollution of target fragments is inevitably caused to an experimental environment, and false positive results are caused in the future nucleic acid detection. The method can detect the target nucleic acid fragments with low concentration in the sample by the thought of amplifying the output signal without amplifying the nucleic acid.
3) The results can be observed by naked eyes or read by an enzyme-labeling instrument: the result can be visualized by using HRP as signal output. When the sample contains high-concentration target fragments, the experimental result can be judged only by naked eyes, and the detection result can be more objectively judged through the numerical value by matching with simple light absorption value reading equipment (such as an enzyme label instrument).
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the description of the embodiments or the prior art will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
Fig. 1 is a schematic diagram of detection of an HRP enhanced CRISPR/Cas12a detection system for detecting a target nucleic acid fragment according to an embodiment of the present invention.
FIG. 2 is a flow chart of operation of an HRP enhanced CRISPR/Cas12a detection system employing detection of target nucleic acid fragments in an embodiment of the invention.
FIG. 3 shows the results of a study of the molar ratio of avidin to biotin-modified HRP-reporter in the examples of the present invention.
FIG. 4 shows the results of a specific study of the HRP-enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection in accordance with the present invention applied to Langya virus detection.
FIG. 5 is a graph showing the results of a sensitivity study of an HRP-enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection in accordance with an embodiment of the present invention applied to Langya virus detection.
FIG. 6 is a study result of HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection applied to NP gene detection of influenza A virus H1N1 subtype strains in the embodiment of the present invention.
Fig. 7 is a study result of an HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection applied to NP gene detection of influenza a virus H3N2 subtype strains in an embodiment of the present invention.
FIG. 8 is a study result of an HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection applied to Porphyromonas gingivalis 16s gene detection in an embodiment of the present invention.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
In a first aspect, the present invention provides an HRP-biotin double-modified ssDNA reporter, wherein the HRP-biotin double-modified ssDNA reporter is obtained by coupling DBCO-biotin double-modified ssDNA with HRP using click chemistry.
In a second aspect, based on the same inventive concept, the present invention provides the use of the HRP-biotin double-modified ssDNA reporter of the first aspect in the preparation of a CRISPR/Cas detection system, said HRP-biotin double-modified ssDNA reporter functioning as a signal output amplification of the CRISPR/Cas detection system.
In some embodiments, the CRISPR/Cas detection system may not perform a nucleic acid pre-amplification loop.
In a third aspect, based on the same inventive concept, the invention provides an HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection, the HRP enhanced CRISPR/Cas12a detection system comprising a Cas12a reaction system, an HRP modified reporter coated vessel, and a chromogenic reagent;
the HRP modified reporter coating vessel is obtained by specifically combining an avidin coated reaction vessel and an HRP-biotin double-modified ssDNArepter through a biotin-avidin system;
the HRP-biotin double-modified ssDNA reporter is obtained by coupling DBCO-biotin double-modified ssDNA and HRP in a click chemistry mode.
Cas12a can specifically recognize a target sequence under the guidance of guide RNA (gRNA), thereby activating a non-specific (trans) single stranded deoxyribonucleic acid (ssDNA) endonuclease activity. Horseradish peroxidase (HRP) has high-efficiency degradation activity on a substrate, and the substrate can generate color or fluorescence reaction after degradation; and the intensity of the color or fluorescence is highly related to the amount of HRP in the reaction, the characteristic is widely applied to quantitative analysis of antigens or proteins in various molecular biology experiments. Applicants found that Cas reaction system has significant inhibitory effect on HRP degradation substrate activity, which may be the reason why similar methods have been previously low in sensitivity.
The patent combines HRP and Cas12a technology to establish a quick detection technology for the nucleic acid of the Langerhans virus, which is simple, convenient and quick, does not need nucleic acid pre-amplification, results are visualized, and special or expensive equipment is not needed. As shown in fig. 1, the principle of the present technology is: coupling ssDNA reporters with HRP by click chemistry means, followed by immobilization in vessels such as 96-well plates; cas12a reaction solution, with or without the sequence of interest, is then added to a 96-well plate; when the sample contains a target sequence (such as spike virus nucleic acid), the trans activity of Cas12a is activated, then HRP modified ssDNA reporters fixed in the holes are cut, the number of HRP in the holes is reduced, then the cut-off free HRP and residual Cas12a reaction solution in the holes are removed through a plate washing step, so that the inhibition of the Cas reaction system on the HRP enzyme activity is eliminated, then a substrate TMB of HRP is added into the holes for color development, and whether the spike virus nucleic acid is contained in the sample is judged by detecting OD450nm after color development or directly observing the color depth of an air color and a negative control through naked eyes. Meanwhile, the problem that sensitivity is low in the prior art is solved by fixing the HRP modified reporter and avoiding the inhibiting effect of the Cas reaction system on the enzyme activity of the HRP.
In some specific embodiments, the Cas12a reaction system comprises the following components: NEBuffer 2.1 (10X) buffer, cas12a protein, gRNA, dithiothreitol, and ddH 2 O。
In some embodiments, the HRP-biotin double modified ssDNA reporter preparation method comprises the steps of:
dissolving HRP in sodium bicarbonate solution to obtain HRP solution;
adding the Azido-PEG-NHS Ester into the HRP solution to perform room temperature stirring reaction, and filtering and washing after the reaction is finished to obtain an azide modified HRP;
and (3) carrying out room-temperature shaking reaction on the HRP modified and DBCO-biotin double-modified ssDNA, and filtering and washing after the reaction is finished to obtain the HRP-biotin double-modified ssDNA reporter.
In some embodiments, the avidin-coated reaction vessel comprises an avidin-coated 96-well plate.
In some embodiments, the target nucleic acid fragments include, but are not limited to, wolf's virus, influenza a virus, porphyromonas gingivalis, and the like, as well as bacterial pathogenic nucleic acids, and the like, and the chromogenic reagent comprises 3,3', 5' -tetramethylbenzidine.
In a fourth aspect, based on the same inventive concept, the present invention provides the use of the HRP enhanced CRISPR/Cas12a detection system of any one of the first aspects in the preparation of a virus detection product, said virus comprising a wolf tooth virus, a zika virus, a dengue virus, SARS-CoV-2 and african swine fever virus.
In a fifth aspect, based on the same inventive concept, the present invention provides a biosensor for detecting a target nucleic acid fragment, the biosensor for detecting a target nucleic acid fragment comprising but not limited to a virus such as a wolf tooth virus, influenza a virus and porphyromonas gingivalis, and a bacterial pathogen nucleic acid, etc., according to any one of the first aspect of HRP-enhanced CRISPR/Cas12a detection system.
In a sixth aspect, based on the same inventive concept, the present invention provides a kit for detecting a target nucleic acid fragment, the kit for detecting a target nucleic acid fragment comprising, but not limited to, virus and bacterial pathogenic nucleic acids such as wolf tooth virus, influenza a virus and porphyromonas gingivalis, etc., according to any one of the first aspect of HRP-enhanced CRISPR/Cas12a detection system.
In a seventh aspect, based on the same inventive concept, the present invention provides a method for detecting a target nucleic acid fragment, wherein the detection method adopts the HRP enhanced CRISPR/Cas12a detection system according to any one of the first aspects, and the target nucleic acid fragment comprises but is not limited to virus and bacterial pathogenic nucleic acids such as spike virus, influenza a virus and porphyromonas gingivalis, and the like.
In summary, the invention provides a preparation method and application of an HRP-biotin double-modified ssDNA reporter, and simultaneously provides an HRP enhanced CRISPR/Cas12a detection system for detecting target nucleic acid fragments based on the HRP-biotin double-modified ssDNA reporter, which establishes a nucleic acid rapid detection system capable of simply, conveniently, rapidly, without nucleic acid pre-amplification and result visualization and without special or expensive equipment, and can be applied to preparation of detection products such as wolf tooth viruses. Specifically, the HRP enhanced CRISPR/Cas12a detection system for detecting target nucleic acid fragments has the following characteristics:
1) The operation is simple, and expensive equipment is not needed: the detection method is implemented by only basic nucleic acid extraction related equipment and an enzyme-labeled instrument, and is simple and convenient to operate.
2) No nucleic acid amplification is required, and no aerosol pollution is caused: the existing high-sensitivity nucleic acid detection methods are all based on a nucleic acid amplification principle (such as a PCR method), and a large amount of target sequences are amplified for a long time, so that aerosol pollution of target fragments is inevitably caused to an experimental environment, and false positive results are caused in the future nucleic acid detection. According to the method, through the thought that the Cas12a is subjected to trans-enzyme digestion and HRP amplification output signals are adopted, the target nucleic acid fragments with low concentration in a sample can be detected without amplifying nucleic acid.
3) The results can be observed by naked eyes or read by an enzyme-labeling instrument: the result can be visualized by using HRP as signal output. When the sample contains high-concentration target fragments, the experimental result can be judged only by naked eyes, and the detection result can be more objectively judged through the numerical value by matching with simple light absorption value reading equipment (such as an enzyme label instrument).
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer.
Example 1
The present example provides an HRP enhanced CRISPR/Cas12a detection system for detecting a target nucleic acid fragment, and further applies the same to the detection of the spike virus, examine the specificity and sensitivity of the spike virus detection, and establish and apply an HRP enhanced CRISPR/Cas12a detection system for detecting a target nucleic acid fragment, and an operation flow chart of the HRP enhanced CRISPR/Cas12a detection system is shown in fig. 2.
1. Material method
Reverse transcription polymerase chain reaction (RT-PCR) assay: PCR primers were designed using Primer Premier 6 software. The components of the PCR reaction mixture included 12.5. Mu.L of 2 XM 5 Pfu PCRMastermix, 10pmol of each primer, 1. Mu.L of cDNA as a template, and double distilled water (ddH 2 O) the total reaction volume was 25. Mu.L. PCR cycle parameters are included inAn initial denaturation step at 94℃for 5 min, followed by 32 cycles of denaturation at 94℃for 50 sec, annealing for 50 sec, an extension step at 72℃for a duration varying between 10 and 30 sec depending on the product size, and finally a final extension step at 72℃for 10 min.
2. Synthesis of HRP-biotin double modified ssDNA reporter
HRP 0.05. Mu. Mol dissolved in 1mL of 0.1M NaHCO 3 In the above, a 50. Mu.M HRP solution was prepared. Subsequently, 50mmol of azido-PEG4-NHS ester was added to the solution and reacted with HRP at room temperature for 2 hours. After sufficient reaction, the azide-modified HRP was washed three times (4,000 rcf centrifuged for 5 minutes) through a 30kDa MWCO centrifuge filter. Subsequently, 200. Mu.L of 10. Mu.M azide-modified HRP was mixed with 2nmol of DBCO-biotin double-modified ssDNA reporter (commercially available from the company Kyoto Biotechnology Co., ltd.) and reacted with shaking at room temperature for 15 hours to form HRP-biotin double-modified ssDNA reporter. The HRP-biotin double modified ssDNA reporter was washed three times (4,000 rcf centrifugation for 5 minutes) through a 30kDa MWCO centrifuge filter.
3. Establishment and application of HRP enhanced CRISPR/Cas12a detection system
3.1, first, an HRP-reporter coated plate was prepared. Avidin was prepared as a 1 ng/. Mu.L solution using 0.05M carbonate buffer (pH 9.6). Avidin solution was added to high affinity 96-well plates at 100 μl per well and incubated overnight at 4 ℃. The wells were then discarded and rinsed well with 0.02M PBS containing 0.05% (v/v) Tween-20, 200. Mu.L each for 5 minutes. Subsequently, 100. Mu.L of Bovine Serum Albumin (BSA) solution was added to each well, and incubated at room temperature for 2 hours, and blocked. The wells were discarded and three washes were performed following the same procedure as described above. Subsequently, 0 to 5-fold molar amounts of HRP-biotin double-modified ssDNA reporters corresponding to avidin were added to each well, and the plates were incubated at room temperature for 2 hours, allowing biotin on the HRP-biotin double-modified ssDNA reporters to bind well to immobilized avidin within the wells. After incubation, the wells were discarded and washed three times. The prepared HRP-reporter coated plate was stored at 4deg.C for subsequent use.
The Cas12a reaction system used for this step includes the following components: 10. Mu.L of 10 XNEB 2.1 buffer, 6pmol of Cas12a protein, 6pmol of gRNA, 1. Mu. Mol of DTT, variable amounts of target nucleic acid fragment, and the necessary amount of ddH 2 O to achieve a total volume of 100. Mu.L. After mixing well, the mixture was transferred to HRP-reporter coated plate. Incubation for 30 min at room temperature caused cleavage of the immobilized HRP-reporters in the wells by the RNP complex. After incubation, the wells were discarded and washed three times. Then 50. Mu.L of TMB substrate was added to each well and reacted at 37℃for 15 minutes. Subsequently, 50. Mu.L of 2M H was added to each well 2 SO 4 To terminate the reaction. OD450nm was measured quantitatively using ELISA reader.
3.2, investigating the specificity and sensitivity of HRP enhanced CRISPR/Cas12a detection System
In the specificity evaluation, six other Paramyxoviruses N genes were used in the present invention to evaluate the detection methods developed in the present invention with three other different viral cDNAs.
In sensitivity studies, in view of the fact that the actual LayV genome consists of RNA, the present invention synthesizes the target RNA fragment by an in vitro transcription process: the DNA sequence comprising the fragment of interest was synthesized downstream of the T7 promoter sequence. Subsequently, hiScribe was used TM T7 High Yield RNA Synthesis Kit is transcribed in vitro. The concentration of RNA was quantified by means of a Qubit 4 fluorometer. Subsequently, the target RNA was diluted by a 10-fold gradient. Using PrimeScript TM The RT kit reverse transcribes different concentrations of the target RNA into cDNA, which is then used in subsequent Cas reactions or PCR assays.
3.3 experimental results
Preparation of HRP-reporter coated plate: theoretically, one avidin can bind to 4 HRP-, biotin-double modified reporters, but in practice the molar ratio of avidin to HRP-, biotin-double modified reporters actually bound needs to be explored due to steric hindrance. As a result, as shown in FIG. 3, the avidin actually coated on the 96-well plate can bind to at most 3 HRP-, biotin-double modified reporters.
Specificity experimental results: to examine the specificity of the HRP-Cas detection method, the present invention synthesizes the N genes of the Mojiang virus (MojV), cedar virus (celv), hendra virus (HeV), nipah virus (NiV), daryong virus (DARV), gamak virus (GAKV), while cdnas of strains Human Respiratory Syncytial Virus (RSV), influhenza Avirus (IAV) H1N1 and H3N2 are also used to examine the specificity. The results show that HRP-Cas reactions are able to generate positive signals specifically to LayV nucleic acid fragments without non-specific reactions to other unrelated viruses (as shown in fig. 4).
Sensitivity test results: the invention uses 10-fold gradient diluted RNA to test the sensitivity of HRP-Cas detection method. The results show that the method can detect 1+E4 copies of the LayV N gene fragment at the lowest, and the sensitivity is 100 times higher than that of RT-PCR, as shown in FIG. 5, and in FIG. 5: (a) is an HRP-Cas response sensitivity study; (B) is a RT-PCT detection method.
In addition, on the basis of the experiment of the spike virus detection, in order to verify the universality of the HRP-Cas12a system in the patent, the invention establishes a nucleic acid detection method of IAV H1N1, H3N2 and PG by designing the gRNA sequence aiming at NP genes of influenza A virus (influenza A virus, IAV) H1N1 and H3N2 subtype strains and porphyromonas gingivalis (Porphyromonas gingivalis, PG) 16s genes and replacing gRNA aiming at the spike virus N genes in the original HRP-Cas12a system. The detection result proves that the HRP-Cas12a system can be used for detecting nucleic acid of the three pathogens; moreover, the HRP-Cas12a system can simply switch detection targets through simple gRNA redesign and replacement, and the detection results are shown in FIG. 6, FIG. 7 and FIG. 8.
Various embodiments of the invention may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that a description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as1, 2, 3, 4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. An HRP-biotin double-modified ssDNA reporter, which is characterized in that the HRP-biotin double-modified ssDNA reporter is obtained by coupling DBCO-biotin double-modified ssDNA reporter and HRP in a click chemistry mode.
2. The use of HRP-biotin double-modified ssDNA reporter in the preparation of a CRISPR/Cas detection system of claim 1, wherein the HRP-biotin double-modified ssDNA reporter acts as a signal output amplification of the CRISPR/Cas detection system.
3. The use of claim 2, wherein the CRISPR/Cas detection system does not perform a nucleic acid pre-amplification loop.
4. An HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection, characterized in that the HRP enhanced CRISPR/Cas12a detection system comprises a Cas12a reaction system, an HRP modified reporter coated reaction vessel, and a chromogenic reagent;
the HRP modified reporter coating reaction vessel is obtained by specifically combining an avidin coated reaction vessel and an HRP-biotin double modified ssDNA reporter through a biotin-avidin system;
the HRP-biotin double-modified ssDNA reporter is obtained by coupling DBCO-biotin double-modified ssDNA reporter and HRP in a click chemistry mode.
5. The HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection of claim 4, wherein the Cas12a reaction system comprises the following components: reaction buffer, cas12a protein, gRNA, dithiothreitol, and ddH 2 O；
The preparation method of the HRP-biotin double-modified ssDNA reporter comprises the following steps:
dissolving HRP in sodium bicarbonate solution to obtain HRP solution;
adding the Azido-PEG-NHS Ester into the HRP solution to perform room temperature stirring reaction, and filtering and washing after the reaction is finished to obtain an azide modified HRP;
performing room-temperature shaking reaction on the HRP modified and DBCO-biotin double-modified ssDNA, and filtering and washing after the reaction is finished to obtain the HRP-biotin double-modified ssDNA reporter;
the avidin coated reaction vessel comprises an avidin coated 96-well plate;
the target nucleic acid fragment comprises, but is not limited to, viruses such as wolf tooth virus, influenza A virus, porphyromonas gingivalis and the like, bacterial pathogenic nucleic acid and the like;
the chromogenic reagent comprises 3,3', 5' -tetramethylbenzidine.
6. The HRP enhanced CRISPR/Cas12a detection system for target nucleic acid fragment detection of claim 4, wherein the HRP enhanced CRISPR/Cas12a detection system does not require a nucleic acid pre-amplification step when applied to pathogenic microorganism detection.
7. Use of the HRP enhanced CRISPR/Cas12a detection system of any one of claims 4 to 6 in the preparation of a pathogenic microorganism detection product.
8. A biosensor for detection of a target nucleic acid fragment, comprising the HRP enhanced CRISPR/Cas12a detection system of any one of claims 4-6.
9. A kit for detection of a target nucleic acid fragment, comprising the HRP enhanced CRISPR/Cas12a detection system of any one of claims 4 to 6.
10. A method for detection of a target nucleic acid fragment, characterized in that the detection method employs the HRP enhanced CRISPR/Cas12a detection system of any one of claims 4 to 6.
CN202311493980.9A 2023-11-08 2023-11-08 HRP-biotin double-modified ssDNA reporter and application thereof Pending CN117568335A (en)

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