CN111500792A - Novel coronavirus detection kit - Google Patents
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Abstract
The invention relates to the technical field of gene detection, in particular to a novel coronavirus detection kit, which comprises crRNA, Cas12 protein, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule; the crRNA is a specific crRNA designed aiming at a target gene; the RPA upstream and downstream primers are as follows: aiming at the conserved region ORF1ab and N gene region of the novel coronavirus, 5 '-TTTN-3' sequence is searched, a forward primer is designed in the near 5 'region of 0-200bp, and a reverse primer is designed in the near 3' region of 25-200 bp. The invention has the beneficial effects that: because the requirement of the RPA isothermal amplification on a sample is low, the method can adopt a rapid and simple sample processing mode, and the RPA mediated DNA amplification can be carried out at the temperature of 30-42 ℃, so that the dependence on a more precise PCR instrument is eliminated.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a novel coronavirus detection kit.
Background
The common signs after coronavirus infection are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. However, the novel coronavirus has relatively hidden factors such as unobvious initial symptoms, 14-day incubation period and the like, so that patients and carriers cannot find the coronavirus in time, and the spread is aggravated.
The rapid and effective means for early and specific diagnosis of the infected person is an important means for timely finding and isolating the infection source, effectively curing the patient and guaranteeing the social order. This has put new demands on the rapid diagnosis technique of pathogens, but the rapid diagnosis of pathogens is very difficult in the case of large-scale outbreaks of virulent infectious diseases, and the challenge is especially prominent in some areas lacking laboratory detection conditions.
The existing RPA Amplification can be combined with a probe to detect an Amplification product, and the introduction of the probe can block the progress of an Amplification reaction, so the method has extremely high requirements on primer and probe combination screening, and is difficult to achieve the purpose of rapidly responding to a sudden epidemic situation.
Crispr (clustered regulated short palindromic repeats) is an acquired immune means against viral invasion in most bacteria and archaea. When a virus invades, the bacteria produce corresponding crRNA capable of recognizing the viral genome, which guides Cas protein (CRISPR-associated proteins) with endonuclease activity to recognize and cleave the viral target sequence.
In 2016.6, the group of Zhang-frontier subjects discovered a CRISPR effector protein Cas13a, which is an endonuclease that binds to a target RNA and degrades the target under the guidance of crRNA (Makarova et al, 2011). Doudna et al (East-Seletsky et al,2016) in the same year 10.10 discovered that an endonuclease called L eptrichalis burcals Cas13a (L buCas13a) not only has cleavage activity for the target RNA, but also has cleavage activity for non-target RNA, which is called as accessory cleavage.Using this property, this group used Cas13a for the detection of RNA targets, but its detection limit was about 10 pmol/L. this has no application value for the detection of nucleic acids, because most of the detection methods, the detection limit was of the order of amol/L (Song et al, 2013. 2017, the amplification sensitivity of the polymerase for the amplification of the target RNA by cooperation with Zhang-frontier proteins, the polymerase, the High sensitivity of the cleavage of the polymerase of the RNA, the polymerase, the probe-frontier kinase, the detection system of RNA, the cleavage of the RNA, the polymerase, the probe-frontier kinase, the detection system, the detection of the High sensitivity of the High specificity of the cleavage of the RNA, the High specificity of the RNA-frontier proteins, the High-frontier-targeting RNA cleavage activity of the High-target RNA-targeting RNA cleavage activity of the amplification of the RNA-targeting RNA, the amplificationHas a detection limit as low as 2X103Although the value of the CRISPR/Cas system in the field of nucleic acid diagnosis is proved again by the breakthrough research result, the CRISPR/m L (3.2 amol/L) is expected to produce great influence in the field of public health, the ribonuclease (RNase) in the environment exists widely and is very stable, and the key components of the SHER L OCK system are RNA, so that the system has strict requirements on the operating environment.
In 2015, zhanfeng et al found a class ii type v CRISPR effector protein Cas12(Zetsche et al, 2015). Cas12 can bind to target double-stranded DNA and cleave genomic DNA under crRNA guidance. In 2018, Doudna et al found that Cas12 has the activity of non-specifically cleaving single-stranded DNA (ssDNA) after it specifically binds to and cleaves the target dsDNA, and developed a nucleic acid detection system named DETECTR (DNA Endonuclease Targeted CRISPR Trans reporter) using this activity of Cas 12. The DETECTR combines the RPA isothermal amplification technology with the Cas12, the amplification product activates the accessory cleavage activity of the Cas12, the cleavage substrate generates a fluorescent signal, and single-molecule-level sensitivity is realized. The system has wide application potential in the aspects of single nucleotide polymorphism analysis, cancer screening, bacterial and viral infection detection, drug resistance screening and the like. As the target and the substrate of cas12 are DNA and have strong stability, the system has low requirement on experimental operation environment and can be applied to on-site rapid detection. Meanwhile, after the Cas12 is specifically activated by the target, the single-stranded reporter molecule can be nonspecifically cut, and the step has a signal amplification effect, so that the technology has higher sensitivity compared with the traditional nucleic acid detection technologies such as a probe method and the like.
However, the conventional CRISPR/cas12 detection system requires a fluorescence detector, and the result cannot be visually interpreted, which brings difficulty to the popularization of the technology in the primary inspection institution.
Disclosure of Invention
The invention aims to: the novel coronavirus detection kit can detect new coronavirus more quickly, accurately and conveniently, can be carried out at 30-42 ℃, and gets rid of dependence on a more precise PCR instrument.
The technical scheme of the invention is as follows: provides a novel coronavirus detection kit, which comprises crRNA, Cas12 protein, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule;
the method comprises the following steps that the crRNA is a specific crRNA designed for a target gene, the length of the crRNA is 17-25 nucleotides, a PAM sequence exists at the near 5' end of the crRNA, the PAM sequence is TTTN, and the PAM sequence is used for identifying a compound consisting of the crRNA and cas 12;
the RPA upstream and downstream primers are as follows: aiming at a conserved region ORF1ab and an N gene region of the novel coronavirus, searching a 5 '-TTTN-3' sequence, designing a forward primer in a region 0-200bp near the 5 'region, and designing a reverse primer in a region 25-200bp near the 3' region;
preferably, in the above novel coronavirus detection kit, the RPA upstream and downstream primer sequences are:
2019-nCoV-ORF1ab-F：SEQ ID NO：1；
2019-nCoV-ORF1ab-R：SEQ ID NO：2；
2019-nCoV-N-F：SEQ ID NO：3；
2019-nCoV-N-R：SEQ ID NO：4；
the crRNA sequence is:
crRNA-ORF1ab：SEQ ID NO：5；
crRNA-nCoV-N：SEQ ID NO：6。
preferably, in the above novel coronavirus detection kit, the sequence of the single-stranded DNA reporter molecule is as follows: 5 '-TTATT-3'.
Preferably, in the above novel coronavirus detection kit, the single-stranded DNA reporter molecule comprises FAM and BHQ1 groups, respectively.
Preferably, in the above novel coronavirus detection kit, the single-stranded DNA reporter molecule comprises FAM and Biotin groups, respectively.
Preferably, in the above-mentioned novel coronavirus detection kit, the Cas12 protein is one of FnCas12a, AsCas12a, L bCas12a, L b5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or L b4Cas12 a.
The invention has the beneficial effects that: in the novel coronavirus detection kit, the requirement of RPA isothermal amplification on a sample is low, so that the kit can adopt a rapid and simple sample processing mode, and the RPA-mediated DNA amplification can be carried out at 30-42 ℃, so that the dependence on a precise PCR instrument is eliminated. Meanwhile, the cleavage of the target molecule by cas12a is also performed under isothermal conditions. Based on the characteristics, the system can be used for on-site rapid detection, has great significance for rapid detection of novel coronavirus, and especially has great value for basic clinical examination lacking laboratory conditions. Compared with the existing RT-PCR-based novel coronavirus detection kit on the market at present, the kit has the following advantages: 1) the technical scheme of the invention can detect the new coronavirus more quickly, accurately and conveniently, can distinguish the common fever cold from the new coronavirus in a quicker way, and reduces the possibility of hospital cross infection. 2) The professional level of the operator is not required, and the operator can operate the medical equipment, so that the working pressure of medical workers is relieved.
Drawings
FIG. 1 is a graph showing the results of fluorescence signals of 1ab gene in example 1 according to the embodiment of the present invention;
FIG. 2 is a graph showing the result of fluorescence signals of the N gene in example 1 according to the embodiment of the present invention;
FIG. 3 is a graph showing the result of blue light excitation of 1ab gene in example 2 according to the embodiment of the present invention;
FIG. 4 is a signal result graph of a blue light excitation instrument for N gene in example 2 according to an embodiment of the present invention;
FIG. 5 is a test strip chromatogram result chart of the 1ab gene of example 2 according to the embodiment of the present invention;
FIG. 6 is a test strip chromatography result chart of the N gene of example 2 in the embodiment of the present invention.
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
The traditional nucleic acid detection technology such as fluorescent PCR has better accuracy and rapidity, but has higher requirements on sample processing and instrument equipment, and various isothermal amplification technologies such as RPA, <tttranslation = L "&tttL <t/T &tttAMP which are developed in recent years are combined with corresponding probes and can be used for field detection, but the method has extremely high requirements on primer and probe combination and has certain difficulty in rapid development.
The most key concept of the invention is as follows: the CRISPR/Cas12a technology is adopted, and an RPA isothermal amplification technology is combined, primers and corresponding crRNA are designed aiming at a conserved region of the novel coronavirus, and the novel coronavirus is rapidly and accurately visually detected. Because the requirement of the RPA isothermal amplification on a sample is low, the method adopts a rapid and simple sample processing mode, and the RPA mediated DNA amplification can be carried out at 30-42 ℃, so that the dependence on a more precise PCR instrument is eliminated. Meanwhile, the cleavage of the target molecule by cas12a is also performed under isothermal conditions. Based on the characteristics, the system can be used for on-site rapid detection, has great significance for rapid detection of novel coronavirus, and especially has great value for basic clinical examination lacking laboratory conditions.
Example 1
A novel coronavirus detection kit comprises crRNA, Cas12 protein, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule;
the method comprises the following steps that the crRNA is a specific crRNA designed for a target gene, the length of the crRNA is 17-25 nucleotides, a PAM sequence exists at the near 5' end of the crRNA, the PAM sequence is TTTN, and the PAM sequence is used for identifying a compound consisting of the crRNA and cas 12;
the crRNA and single-stranded DNA reporter molecules designed and chemically synthesized in this example are shown in table 1 below.
TABLE 1
The RPA upstream and downstream primers in this example are shown in table 2 below.
TABLE 2
| Oligo name | Sequence(5'-3') |
| 2019-nCoV-ORF1ab-F | ATGGTGACTTTTTGCATTTCTTACCTAGAGTTT(SEQ ID NO：1) |
| 2019-nCoV-ORF1ab-R | GCTGATGTTGCAAAGTCAGTGTACTCTATAAG(SEQ ID NO：2) |
| 2019-nCoV-N-F | CAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAAT(SEQ ID NO：3) |
| 2019-nCoV-N-R | GTTGGCCTTTACCAGACATTTTGCTCTCAAGCTG(SEQ ID NO：4) |
The use of the kit comprises the following steps:
1) healthy volunteers were sampled with pseudovirions containing the new coronary genomic sequence and with pharyngeal swabs for mock clinical samples, added to quick lysates of the following formulation in table 3, and lysed for 5 min at 80 ℃.
TABLE 3
| Reagent | Concentration of |
| Guanidine | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
2) Adding the lysis solution obtained in the step 1) of 2 mu L into RPA reaction particles, RPA hydration buffer solution, RPA upstream and downstream primers and magnesium acetate according to a proper proportion, placing the reaction mixture at a constant temperature of 42 ℃ for reaction for 30min, adopting an Ampu future isothermal amplification kit (RNA basic type) in the invention, and matching the RPA reaction solution as shown in Table 4.
TABLE 4
3) Adding RT-RPA product 10 mu L into the reaction detection liquid of 40 mu L crRNA, cas12a and single-stranded DNA report molecules, and monitoring fluorescence signals in real time by a microplate reader, wherein the figure 1 is a 1ab fluorescence signal diagram, the figure 2 is an N gene fluorescence signal diagram, positive sample signals show an S-shaped curve, and negative sample signals are kept unchanged.
The formulation of the above-mentioned cas12a test solution is shown in Table 5 below
TABLE 5
Example 2
A novel coronavirus detection kit comprises crRNA, Cas12 protein, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule;
the method comprises the following steps that the crRNA is a specific crRNA designed for a target gene, the length of the crRNA is 17-25 nucleotides, a PAM sequence exists at the near 5' end of the crRNA, the PAM sequence is TTTN, and the PAM sequence is used for identifying a compound consisting of the crRNA and cas 12;
the crRNA and single-stranded DNA reporter molecules designed and chemically synthesized in this example are shown in table 6.
TABLE 6
In this example, the upstream and downstream primers of RPA are shown in Table 2.
The formulation of the detection solution of cas12a used in this example is shown in Table 7 below.
TABLE 7
The use of the kit comprises the following steps:
1) healthy volunteers were sampled with pseudovirions containing the new coronary genomic sequence and with pharyngeal swabs for mock clinical samples, added to quick lysates as formulated in table 3, and lysed for 5 min at 80 ℃.
2) Adding the lysis solution obtained in the step 1) of 2 mu L into RPA reaction particles, RPA hydration buffer solution, RPA upstream and downstream primers and magnesium acetate according to a proper proportion, placing the reaction mixture at a constant temperature of 42 ℃ for reaction for 30min, adopting an Ampu future isothermal amplification kit (RNA basic type) in the invention, and matching the RPA reaction solution as shown in Table 4.
3) Adding an RT-RPA product 10 mu L into a reaction detection solution containing 40 mu L crRNA, cas12a and a reporter molecule, reacting for 20 minutes at 37 ℃, adding the reaction product into a test strip or a blue light instrument, observing the result by naked eyes and taking a picture by a mobile phone, wherein in the figure 3, the figure 4 respectively shows signals of a blue light excitation instrument of 1ab and N genes, a positive sample emits visible light by naked eyes, the color of a negative sample is transparent, in the figure 5, the figure 6 respectively shows the chromatography result of test strips of the 1ab and the N genes, the positive sample has a strip on the detection line, and the negative sample has no strip on the detection line.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
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<110> Yaenergetic Biotechnology (Shenzhen) Limited
<120> a novel coronavirus detection kit
<130>20200529
<160>6
<170>PatentIn version 3.5
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<211>34
<212>DNA
<213> Artificial sequence
<400>3
caggcagcag taggggaact tctcctgcta gaat 34
<210>4
<211>34
<212>DNA
<213> Artificial sequence
<400>4
gttggccttt accagacatt ttgctctcaa gctg 34
<210>5
<211>44
<212>RNA
<213> Artificial sequence
<400>5
uaauuucuac uaaguguaga ugugcaguug guaacaucug uuac 44
<210>6
<211>43
<212>RNA
<213> Artificial sequence
<400>6
uaauuucuac uaaguguaga ucugcugcuu gacagauuga acc 43
Claims (6)
1. A novel coronavirus detection kit is characterized by comprising crRNA, Cas12 protein, RPA upstream and downstream primers, a buffer system and a single-stranded DNA reporter molecule;
the method comprises the following steps that the crRNA is a specific crRNA designed for a target gene, the length of the crRNA is 17-25 nucleotides, a PAM sequence exists at the near 5' end of the crRNA, the PAM sequence is TTTN, and the PAM sequence is used for identifying a compound consisting of the crRNA and cas 12;
the RPA upstream and downstream primers are as follows: aiming at the conserved region ORF1ab and N gene region of the novel coronavirus, 5 '-TTTN-3' sequence is searched, a forward primer is designed in the near 5 'region of 0-200bp, and a reverse primer is designed in the near 3' region of 25-200 bp.
2. The novel coronavirus detection kit according to claim 1,
the sequence of the RPA upstream and downstream primers is as follows:
2019-nCoV-ORF1ab-F：SEQ ID NO：1；
2019-nCoV-ORF1ab-R：SEQ ID NO：2；
2019-nCoV-N-F：SEQ ID NO：3；
2019-nCoV-N-R：SEQ ID NO：4；
the crRNA sequence is:
crRNA-ORF1ab：SEQ ID NO：5；
crRNA-nCoV-N：SEQ ID NO：6。
3. the novel coronavirus detection kit of claim 1, wherein the single-stranded DNA reporter molecule has a sequence as follows: 5 '-TTATT-3'.
4. The novel coronavirus detection kit of claim 1, wherein the single-stranded DNA reporter molecule comprises FAM and BHQ1 groups, respectively.
5. The novel coronavirus detection kit of claim 1, wherein the single-stranded DNA reporter molecule comprises FAM and Biotin groups, respectively.
6. The novel coronavirus detection kit of claim 1, wherein the Cas12 protein is one of FnCas12a, assas 12a, L bCas12a, L b5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or L b4Cas12 a.
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