CN111534643A - A kit, detection method and application for detecting nucleic acid of respiratory pathogens - Google Patents

Abstract

The present invention provides a kit for detecting nucleic acid of respiratory pathogens, which comprises a CRISPR-Cas detection system: crRNA, primer pair, Cas protein and nucleic acid probe according to the present invention. The invention also provides a method for detecting nucleic acid of respiratory pathogens, a combination of crRNA and primer pair, and application thereof. The kit and the detection method of the present invention do not rely on large-scale instruments, but can directly observe the results with the naked eye, and the detection can be realized under mild conditions, and the detection is more convenient; the kit and the detection method of the present invention can be used for efficient and rapid detection. Detection/diagnosis of respiratory pathogens (such as influenza A, influenza B, and novel coronavirus, etc.), with high specificity and sensitivity, and can be used for the detection and screening of respiratory pathogens, such as rapid differentiation including influenza A and influenza B and various respiratory pathogens including the new coronavirus.

Description

A kit, detection method and application for detecting nucleic acid of respiratory pathogens

technical field

The invention belongs to the field of biotechnology, and in particular relates to a kit, a detection method and an application for detecting nucleic acid of respiratory pathogens.

Background technique

Cases of COVID-19 quickly appeared in countries around the world and were declared a worldwide public health threat by the World Health Organization (WHO). While the median time to onset of COVID-19 is currently reported to be 4-5 days, a small percentage of infected individuals develop symptoms after 14 days of isolation. The most common symptoms of COVID-19 are cough and fever. However, a certain percentage of patients do not show symptoms, including X-ray testing and CT testing. The uncertainty of the onset of symptoms of COVID-19 and the situation of asymptomatic infection have brought great obstacles to the early and accurate detection of the disease, thus making disease prevention and control extremely challenging. This makes the molecular detection of COVID-19 pathogenic nucleic acids particularly important.

The causative agent of COVID-19 has been identified as a beta coronavirus, named SARS-CoV-2. Similar to other coronaviruses, SARS-CoV-2 is a single-stranded, positive-sense RNA virus. SARS-CoV-2 shares 79% and 50% nucleic acid similarity with SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Existing molecular diagnostic methods for COVID-19 are mainly based on reverse transcription PCR (RT-PCR). The E, N and ORF1ab genes of SARS-CoV-2 are commonly used RT-PCR target genes. High-throughput RT-PCR platforms have also been developed for large-scale diagnostics. However, RT-PCR usually relies on special, large-scale instruments, so large-scale RT-PCR testing is often limited to patients in hospitals.

Recent studies have demonstrated that Clustered Regularly Interspaced Short PalindromicrRepeats (CRISPR) technology can be used for low-cost, portable molecular detection of pathogens. CRISPR and CRISPR-associated genes (Cas) genes are bacterial immune systems against foreign viral infections. The modular nature of CRISPR makes this technology widely used in genome engineering. CRISPR-based molecular diagnosis of pathogenic nucleic acids relies on the RNA or DNA targeting activity of Cas nucleases. At the same time, different CRISPR systems can be combined to achieve multiple detection of pathogens. During the recent outbreak of African swine fever in China, many CRISPR-Cas12a-based diagnostic platforms were developed.

At present, as a novel coronavirus that is an emerging infectious disease, its subgenome expression profile has not been clearly elucidated, and the expression of each gene and the sensitivity of each gene fragment to amplification and CRISPR-Cas detection are also unclear. The detection methods for respiratory pathogens such as novel coronavirus, influenza A virus, and influenza B virus with high specificity and no cross-reactivity are still unexplained scientific problems. The traditional RT-qPCR method currently used to detect the new coronavirus is more prone to false detection, and it is not easy to distinguish the new coronavirus from other influenza such as influenza A virus and influenza B virus. These three are clinically manifested The symptoms are very similar, and cases of misdiagnosing the new crown as influenza have also occurred in practical applications (Kong et al., 2020, Nat Microbiol ). In addition, pathogen detection technology based on CRISPR-Cas12a has been reported (Gootenberg et al , 2017, Science ; Gootenberg et al , 2018, Science ; Chen et al , 2017, Science ;), especially the CRISPR detection platform for novel coronavirus It has also been successfully developed (Wang et al , 2020, Sci Bull; Guo et al , 2020, Cell Discov; Broughton et al , 2020, Nat Biotech), but these studies mostly focus on principled technology development, and the technology is pathogen-specific The detection research is not deep enough, for example, the specificity of each pathogen subtype has not been studied, and the cross-reaction between different probe combinations has not been determined (such as whether there is cross-reaction between the pathogen A probe and the pathogen B probe between the two pathogens). reaction).

Therefore, there is an urgent need for primers and crRNA probes that are both efficient and fast, have high specificity and sensitivity, and have no cross-reactivity in the detection of various respiratory pathogens, so as to realize the specific detection of various respiratory pathogens.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the defects of the existing methods for detecting respiratory pathogens (such as novel coronavirus (SARS-CoV-2), influenza A, influenza B) relying on large-scale instruments and inconvenient detection. Disclosed are a kit for detecting nucleic acid of respiratory pathogens, a method for detecting nucleic acid of respiratory pathogens, crRNA, primer pairs and applications thereof. First, the kit and the detection method of the present invention do not rely on large-scale instruments, but can directly observe the results with the naked eye, and at the same time, the detection can be realized under mild conditions (25-42° C.), and the detection is more convenient. The kit and detection method of the present invention can be used for efficient and rapid detection/diagnosis of respiratory pathogens (such as influenza A, influenza B, and new coronavirus, etc.). Secondly, when using the kit and detection method of the present invention to detect multiple respiratory pathogens (such as influenza A, influenza B, new coronavirus, etc.), there is no cross-reaction with each other, and the specificity and sensitivity of the detection are high. At present, there is no patent or literature report that can completely realize the simultaneous detection of new coronavirus, influenza A, and influenza B. This application provides a kit and detection method for the first time, which can realize the detection of new crown virus while ensuring sensitivity. Complete, interactive, specific detection of , A, and B. The kit and detection method of the present invention can be used for the detection and screening of respiratory pathogens, for example, to rapidly distinguish influenza A (including various subtypes), influenza B (including various subtypes) and novel coronaviruses. Various respiratory pathogens. In addition, all current CRISPR-based detections use respiratory samples such as throat swabs or nasal swabs, and the kit and detection method of the present invention can further realize the detection of other types of samples (such as anal swabs, feces, etc.) in addition to respiratory samples. , sputum, sputum supernatant and other secretions); at the same time, it can detect various pathogens in samples derived from living tissue and containing multiple pathogens.

First of all, when designing crRNAs and primers for the detection of new coronaviruses and other influenza such as influenza A and influenza B viruses, on the one hand, it is necessary to consider the prevention of cross-reaction and misdiagnosis when detecting new crowns and other influenzas, and on the other hand, due to new crowns. The subgenomic architecture of the virus itself has not been clearly scientifically explained. Current research shows that the subgenome structure of 2019-nCoV is very complex because it contains 10 discontinuous coding regions, and the expression of each gene and the sensitivity of each gene fragment to amplification and CRISPR-Cas detection are also unclear; There are other unknown transcripts for the 10 known coding regions. When designing primers, it is necessary to find a pathogenic gene fragment with high transcription efficiency for design (for example, the inventors found that, both of the new coronavirus genes, the N gene is easier to obtain efficient amplification primers than the M gene (Figures 14 and 15); also found that The same is M gene, the amplification efficiency of different fragments is very different (Figure 14)), the design of crRNA needs to meet the constraints of the Cas12a PAM sequence, and it is also necessary to consider that it must be located within the amplification region of the primer, and at the same time meet these requirements. The primer pair and crRNA also need to be able to ensure high sensitivity and specificity, and also need to ensure that no cross-reaction occurs when detecting the new coronavirus and other influenza viruses such as influenza A and influenza B at the same time, which is very difficult to design. Second, specificity reflects the ability of screening tests to determine non-positive samples; sensitivity refers to the degree of change in response of a method to changes in unit concentration or unit amount of the substance to be tested; in the process of designing crRNA and primer pairs, The relationship between specificity and sensitivity needs to be balanced, and it is well known in the art that it is very difficult to simultaneously improve both sensitivity and specificity. While ensuring high specificity, the sensitivity will decrease, and while ensuring high sensitivity, the specificity will decrease. When detecting different viruses in the kit to be protected by the present invention, it can not only ensure specificity, but also ensure high sensitivity. In addition, all current CRISPR-based assays use respiratory tract samples such as throat swabs, while the kits and detection methods of the present invention detect a wide range of samples, not limited to respiratory samples (such as throat swabs or nasal swabs). ), for example, it can also be used to detect secretions such as anal swabs, feces, sputum, sputum supernatant, etc., and the interfering bacteria present in the detection of samples from different parts and different environments are different. For example, the detection of anal swabs Escherichia coli and other interfering substances exist in the presence of bacteria, feces, etc., which will affect the sensitivity and specificity of the detection. However, the kit and detection method of the present invention can maintain good specificity and sensitivity in various types of samples without cross-infection, and have a wide range of applications.

In order to solve the above technical problems, the first aspect of the present invention provides a target site for detecting nucleic acid of respiratory pathogens, and the respiratory pathogens are novel coronavirus, influenza A virus and/or influenza B virus; wherein,

Detect the sequence of the target site of the novel coronavirus as shown in one or more of SEQ ID NOs: 1-20;

Detecting the sequence of the target site of the influenza A virus is shown in one or more of SEQ ID NOs: 21-24;

Detecting the sequence of the target site of the influenza B virus is shown in one or more of SEQ ID NOs: 25-28, 103;

These target sites described in the present invention can specifically distinguish three respiratory pathogens (new coronavirus, influenza A virus and influenza B virus) and their corresponding DNA sequences contain PAM sequences recognized by Cas proteins (eg Cas12a) . In addition, those skilled in the art should understand that the RNA sequence complementary to SEQ ID NO: 1-28, 103, the sequence of any strand in the double-stranded DNA after reverse transcription of SEQ ID NO: 1-28, 103 is shown in The target site should also be within the scope of protection of the present invention.

Wherein, the sequence of the target site of the ORF1ab gene of the novel coronavirus is detected as shown in one or more of SEQ ID NOs: 1-4.

Wherein, the sequence of the target site of the S gene of the novel coronavirus is detected as shown in one or more of SEQ ID NOs: 5-8.

Wherein, the sequence of detecting the target site of the E gene of the novel coronavirus is shown in one or more of SEQ ID NOs: 9-12.

Wherein, the sequence of detecting the target site of the M gene of the novel coronavirus is shown in one or more of SEQ ID NOs: 13-16.

Wherein, the sequence of detecting the target site of the N gene of the novel coronavirus is shown in one or more of SEQ ID NOs: 17-20.

In a preferred embodiment of the present invention, detecting the target sites of the S gene and E gene of the new coronavirus can generate a relatively high signal (up to 3×10 ⁷ ), and detecting the ORF1ab, M gene and N of the new coronavirus The gene signal was also stronger, but did not exceed 3×10 ⁷ .

In the present invention, the detection of influenza A virus is mainly to detect the M gene of the influenza A virus. Wherein, the described influenza A virus (Influenza A virus, IAV) can be conventional in the field, and is usually divided into many subtypes according to different H and N antigens, and H can be divided into 18 subtypes (H1～H18), There are 11 subtypes of N (N1 to N11), including, for example, H1N1, H2N2, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N8 subtypes, such as H1N1, H3N2, and H9N2.

In the present invention, the detection of influenza B virus is mainly to detect the HA gene of the influenza B virus. Wherein, the influenza B virus (Influenza B virus, IBV) can be conventional in the art, for example, including Yamagata series (Yamagata series) or Victoria series (Victoria series) influenza B virus and the like. Wherein, the sequence of the target site of the Yamagata line (Yamagata line) for detecting the influenza B virus can be as shown in one or more of SEQ ID NOs: 25-28. Wherein, the sequence of the target site of the Victoria line (Victoria line) for detecting the influenza B virus can be as shown in one or more of SEQ ID NOs: 25, 26, 103, and 28.

In the present invention, the target site can be preferably used in the CRISPR-Cas detection system.

In the present invention, the 5' end of the obtained targeting sequence has a 5'-TTTN-3' sequence, and no stable secondary structure is formed between the targeting sequence itself and the rest of the sequences.

In order to solve the above-mentioned technical problems, the second aspect of the present invention provides a combination of crRNA and primer pair, the crRNA and primer pair are crRNA and primer pair for detecting nucleic acid of respiratory pathogens, and the respiratory pathogens are novel coronavirus, Influenza virus type and influenza B virus; of which,

Detect the sequence of the crRNA of the ORF1ab gene of the novel coronavirus as shown in SEQ ID NO: 32; Detect the sequence of the crRNA of the S gene of the novel coronavirus as shown in SEQ ID NO: 34; Detect the novel coronavirus The sequence of the crRNA of the E gene is as shown in SEQ ID NO: 38; The sequence of the crRNA of the M gene of the detection described novel coronavirus is as shown in SEQ ID NO: 43; Detect the crRNA of the N gene of the novel coronavirus as shown in SEQ ID NO: 43. Sequence is as shown in SEQ ID NO: 48; The sequence of the crRNA that detects the M gene of the influenza A virus is as shown in SEQ ID NO: 51; And, the sequence of the crRNA that detects the HA gene of the influenza B virus is as shown in SEQ ID NO: 51. shown in SEQ ID NO: 56;

The nucleotide sequence of the primer pair for amplifying the ORF1ab gene of the novel coronavirus is as shown in SEQ ID NO: 58 and SEQ ID NO: 62; the nucleotide sequence of the primer pair for amplifying the S gene of the novel coronavirus As shown in SEQ ID NO: 66 and SEQ ID NO: 70; the nucleotide sequences of the primer pairs for amplifying the E gene of the novel coronavirus are as shown in SEQ ID NO: 74 and SEQ ID NO: 78; The nucleotide sequence of the primer pair of the M gene of the novel coronavirus is as shown in SEQ ID NO: 82 and SEQ ID NO: 86; the nucleotide sequence of the primer pair of the N gene of the novel coronavirus is as shown in SEQ ID NO: 86; ID NO: 90 and SEQ ID NO: 94; the nucleotide sequences of the primer pairs for amplifying the M gene of the influenza A virus are shown in SEQ ID NO: 98 and SEQ ID NO. 99; and, the amplification The nucleotide sequences of the primer pairs for increasing the HA gene of the influenza B virus are shown in SEQ ID NO: 100 and SEQ ID NO. 101;

The crRNA is used for CRISPR-Cas detection system, and the primer pair is used for QPCR, LAMP (Loop-mediated isothermal amplification, loop-mediated isothermal amplification reaction) or RPA (recombinase polymeraseamplification, recombinase polymerase-mediated isothermal amplification) in order to amplify the nucleic acid (target site) of the respiratory pathogen.

In the present invention, the RPA is the conventional meaning in the art, which generally also includes the derivatization reaction based on RPA. Usually, it may be a general RPA including a DNA template, or an RT-RPA (RT is an abbreviation for reverse transcription) that binds reverse transcription to an RNA template.

In the present invention, the primer pair can be used to amplify the target site sequences of novel coronavirus, influenza A virus and influenza B virus so as to realize the fluorescence detection of Cas12a. The application of these primer pairs in other reactions (such as QPCR) and other CRISPR systems (such as Cas12b or Cas13, etc.) is also within the protection scope of the present invention.

In order to solve the above technical problems, a third aspect of the present invention provides a kit for detecting nucleic acid of respiratory pathogens, which includes a CRISPR-Cas detection system, and the CRISPR-Cas detection system includes: crRNA, primer pairs, Cas protein and nucleic acid probes; wherein, the crRNA and the primer pair are as described in the second aspect of the present invention.

Preferably, the CRISPR-Cas detection system further includes metal ions and/or buffers.

In the present invention, the Cas protein can be conventional in the art, for example, including Cas9, Cas12a, Cas12b and/or Cas13. Wherein, the Cas12a protein is a Cas protein with endonuclease activity and accessory cleavage activity. Such as Cas12a, Cas12b and so on. The amino acid sequence of Cas12a is preferably shown in SEQ ID No. 57, and the nucleotide sequence thereof is preferably shown in SEQ ID No. 102.

In the present invention, the nucleic acid probes included in the CRISPR-Cas detection system can be single-stranded DNA probes. Those skilled in the art should understand that any nucleic acid probes routinely used in the art should be able to accomplish the present invention. In a preferred embodiment of the present invention, the nucleic acid probe may be a nucleic acid probe purchased from Universal Biosystems (Anhui) Co., Ltd., model number RX012179.

In the present invention, the content of the Cas protein can usually be 100 ng. In a preferred embodiment, the kit includes (suitable for 20 μL system reaction): 100 ng Cas protein, 25 pM nucleic acid probe such as single-stranded DNA probe, and equimolar ratio of Cas12a crRNA. To be used to detect 2 μL of the viral nucleic acid to be detected (for example, it can be the product of RT-RPA reaction using the above-mentioned primer pair or a double-stranded DNA mimic substrate after reverse transcription for the target site of respiratory pathogenic RNA nucleic acid), the described The kit may also include 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM _MgCl2 , 100 μg/mL BSA.

In order to solve the above technical problems, the fourth aspect of the present invention provides a method for detecting nucleic acid of respiratory pathogens, using the CRISPR-Cas detection system described in the kit according to the third aspect of the present invention to detect the nucleic acid (for example, Using Cas12a protein and crRNA corresponding to the target site for CRISPR nucleic acid detection), and/or using the primer pair in the kit according to the fifth aspect of the present invention to amplify the nucleic acid.

In the present invention, the nucleic acid detection method is usually for non-diagnostic purposes, such as conducting experiments with in vitro samples for R&D use, etc., or after the sputum, feces and other secretions of patients infected with the new coronavirus cause pollution to the environment, Test samples taken from these environments.

In order to solve the above-mentioned technical problems, the fifth aspect of the present invention provides the application of the crRNA and primer pair as described in the second aspect of the present invention in detecting nucleic acid of respiratory pathogens, or preparing a reagent or kit for detecting nucleic acid of respiratory pathogens.

Preferably, the respiratory pathogen is novel coronavirus, influenza A virus and/or influenza B virus.

Preferably, the detection is performed using a CRISPR-Cas detection system (eg, CRISPR nucleic acid detection using Cas12a protein and crRNA corresponding to the target site), QPCR, LAMP and/or RPA (eg RT-RPA).

In order to solve the above-mentioned technical problems, the sixth aspect of the present invention provides the application of the crRNA as described in the second aspect of the present invention in the preparation of a medicine for preventing and/or treating respiratory tract pathogens, wherein the respiratory tract pathogens are novel coronavirus, influenza A virus and/or influenza B virus.

Preferably, the crRNA utilizes the CRISPR-Cas system to destroy respiratory pathogens in the subject.

The present invention also provides a CRISPR-Cas12a specific detection system, which includes Cas12a protein and crRNA, the crRNA is the crRNA for detecting the target site according to the first aspect of the present invention, which is used to detect three respiratory pathogens ( novel coronavirus, influenza A, influenza B).

Preferably, the sequence of the crRNA for detecting the novel coronavirus is shown in one or more of SEQ ID NOs: 29-40, 42, 43, 45-48.

Preferably, the sequence of the crRNA for detecting influenza A virus is shown in one or more of SEQ ID NOs: 50-52.

Preferably, the sequence of the crRNA for detecting influenza B virus is shown in one or more of SEQ ID NOs: 53-56.

The present invention also provides a composition comprising the crRNA described in the second aspect of the present invention and/or the primer pair described in the third aspect of the present invention.

In the present invention, the nucleic acid detection method can be based on the CRISPR-Cas12a system, especially based on the CRISPR-Cas12a single-stranded DNA activity activation reaction.

Terminology Explanation

The term crRNA refers to CRISPR RNA, which is a short RNA that guides Cas12a (Cpf1) to bind to a target DNA sequence.

The term CRISPR refers to the clustered regularly interspaced short palindromic repeats that are part of the immune system of many prokaryotes.

The term Cas protein refers to CRISPR-associated proteins, which are associated proteins in the CRISPR system.

The term Cas12a (also known as Cpf1) refers to the crRNA-dependent endonuclease, which is a type V enzyme in the CRISPR system classification.

The term PAM refers to the protospacer-adjacent motif, which is necessary for Cas12a cleavage, and the PAM of LbCas12a is the TTTN sequence.

In the present invention, the pathogen (also known as pathogen, pathogen) is usually a general term for microorganisms and parasites that can cause diseases. Microorganisms account for the vast majority, including viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes and fungi; parasites mainly include source worms and worms.

In the present invention, the detection may be to detect whether the sample contains the respiratory pathogen, that is, the detected sample may be a sample containing a respiratory pathogen or a sample that does not contain a respiratory pathogen. The detection may be used to screen samples for the presence of the respiratory pathogens, for example, to screen ex vivo samples.

In the present invention, the "multiple" in the "one or more" may refer to 2, 3, 4 or more.

In the present invention, the "comprising, comprising or containing" may mean that there are other components in addition to the components listed below; it may also mean "consisting of", that is, only the components listed below are included without Other ingredients are present.

On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progressive effect of the present invention is:

(1) The kit and the detection method of the present invention do not rely on large-scale instruments, and can directly observe the results with the naked eye, and at the same time, the detection can be realized under mild conditions (25-42° C.), and the detection is more convenient. The kit and detection method of the present invention can be used for efficient and rapid detection/diagnosis of respiratory pathogens (such as influenza A, influenza B, and new coronavirus, etc.).

(2) Using the kit and detection method of the present invention to detect multiple respiratory pathogens (such as influenza A, influenza B, new coronavirus, etc.) without cross-reaction with each other, and the detection specificity and sensitivity are high . At present, there is no patent or literature report that can completely realize the simultaneous detection of new coronavirus, influenza A, and influenza B, but this application provides a kit and detection method for the first time, which can ensure the sensitivity while realizing the detection of Complete, interactive, specific detection of COVID-19, influenza A and B. The kit and detection method of the present invention can be used for the detection and screening of respiratory pathogens, for example, to rapidly distinguish influenza A (including various subtypes), influenza B (including various subtypes) and novel coronaviruses. Various respiratory pathogens.

(3) In addition, all current CRISPR-based detections use respiratory samples such as throat swabs or nasal swabs, and the kit and detection method of the present invention can further realize the detection of other types of samples (such as anal swabs) in addition to respiratory samples. At the same time, it can detect various pathogens in samples derived from living tissue and containing multiple pathogens.

In a preferred embodiment of the present invention, when using the kit and detection method of the present invention to simultaneously detect different subtypes of novel coronavirus, influenza A virus and different subtypes of influenza B virus, there is no cross-reaction, Specificity is high. In a preferred embodiment of the present invention, using the kit and the detection method of the present invention, the sensitivity of detecting novel coronavirus and influenza A virus can reach 5 virus copies; the sensitivity of detecting influenza B virus can reach 3 virus copies .

Description of drawings

Figure 1 shows the Cas12a fluorescence detection of the novel coronavirus ORF1ab gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure.

Figure 2 shows the Cas12a fluorescence detection of the new coronavirus S gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure.

Figure 3 shows the Cas12a fluorescence detection of the new coronavirus M gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure.

Figure 4 shows the Cas12a fluorescence detection of the new coronavirus E gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure.

Figure 5 shows the Cas12a fluorescence detection for the new coronavirus N gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure.

Figure 6 shows the Cas12a fluorescence detection of the M gene target of influenza A virus (H1N1), and the corresponding target site sequence and crRNA sequence are shown in the figure.

Figure 7 shows the Cas12a fluorescence detection of the HA gene target of influenza B viruses (Victoria subtype (Victoria line) and Yamagata subtype (Yamagata line)). The corresponding target site sequences and crRNA sequences are shown in the figure. Wherein crRNA SEQ ID No. 55 is a specific probe that can detect Yamagata subtype (Yamagata line).

Figure 8 shows that the Cas12a crRNA probe SEQ ID No. 34 for the new coronavirus S gene target can specifically detect the new coronavirus, and has no cross-reaction with influenza A virus and influenza B virus. Meanwhile, SEQ ID No. 34 can distinguish different types of coronaviruses.

Figure 9 shows that the Cas12a crRNA probe SEQ ID No. 51 for the M gene target of influenza A virus can specifically detect influenza A virus, and has no cross-reaction with novel coronavirus and influenza B virus.

Figure 10 shows that the Cas12a crRNA probe SEQ ID No. 56 for the HA gene target of influenza B virus can specifically detect influenza B virus, and has no cross-reaction with novel coronavirus and influenza A virus.

Figure 11 shows the screening of RT-RPA primers for the novel coronavirus ORF1ab gene target. Forward primers 1-4 are respectively SEQ ID No. 58-61, Reverse primers 5-8 are SEQ ID No. 62-65 respectively.

Figure 12 shows the screening of RT-RPA primers for the new coronavirus S gene target. Forward primers 1-4 are SEQ ID No. 66-69, respectively, and Reverse primers 5-8 are SEQ ID No. 70-73, respectively.

Figure 13 shows the screening of RT-RPA primers for the new coronavirus E gene target. Forward primers 1-4 are SEQ ID No. 74-77, respectively, and Reverse primers 5-8 are SEQ ID No. 78-81, respectively.

Figure 14 shows the screening of RT-RPA primers for the M gene target of the novel coronavirus. Forward primers 1-4 are SEQ ID No. 82-85, respectively, and Reverse primers 5-8 are SEQ ID No. 86-89, respectively.

Figure 15 shows the screening of RT-RPA primers against the N gene target of the novel coronavirus. Forward primers 1-4 are SEQ ID No. 90-93, respectively, and Reverse primers 5-8 are SEQ ID No. 94-97, respectively.

Figure 16 shows naked-eye data reading based on Cas12a fluorescence detection, a new coronavirus sample. The white glowing tube corresponds to the viral nucleic acid sample that produces a fluorescent signal.

Figure 17 shows the detection results of the sensitivity to the novel coronavirus.

Figure 18 shows the results of detection of sensitivity to influenza A virus.

Figure 19 shows the detection results of sensitivity to influenza B virus.

Figure 20 shows the assay results for different types of samples.

Figure 21 shows the detection results for each pathogen in a sample containing multiple pathogens derived from animal tissue.

Figure 22 shows the dependence of Cas12a protein concentration on detection efficiency.

Detailed ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.

Example 1

(1) Expression and purification of Cas12a protein

The Cas12a (LbCas12a, amino acid sequence shown in SEQ ID NO: 57) gene derived from Lachnospiraceae bacterium ND2006 was codon-optimized (the optimized sequence was shown in SEQ ID NO: 102), and then ligated into pET28a plasmid (Thermo Fisher Scientific, Massachusetts) , USA), protein expression was regulated by the T7 promoter. The C-terminus of LbCas12a contains a His6 tag for affinity purification, and a TEV restriction site is inserted between the sequences of Cas12a and His6. The resulting recombinant plasmid (hereinafter referred to as pET28a-LbCas12a) was transformed into Escherichia coli BL21(DE3) cells. The next day, monoclones were inoculated into Luria-Bertani (LB) medium containing 50 μg/mL kanamycin, and cultured at 37°C with shaking. When the OD ₆₀₀ reached 0.8, protein expression was induced by adding 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and incubated at 37°C for 16 hours. Cells were harvested by centrifugation at 5,000 g for 10 minutes at 4°C.

Harvested cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)). The expressed Cas12a protein was purified by Ni-NTA column (Qiagen, Shanghai, China). The purified protein was purified by fastprotein liquid chromatography (FPLC) using a Superdex200 filter column (GE Healthcare Life Sciences, Connecticut, USA). Purified proteins were stored in storage buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 2 mM dithiolthreitol (DTT)) in aliquots and stored at -80°C. Protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA).

(2) Preparation of crRNA and DNA substrates

The target site of the respiratory pathogenic RNA nucleic acid (see Table 1 for the sequence) and the reverse-transcribed double-stranded DNA substrate crRNA (see Table 2) was synthesized by GenScript Biotech (Nanjing, Jiangsu, China). The substrate DNA that mimics respiratory pathogenic RNA nucleic acid was synthesized by TSINGKE Biological Technology (Shanghai, China), cloned into pUC57 vector as a template, and the product of simulated isothermal amplification was obtained by PCR amplification.

Table 1

Table 2

(3) Isothermal amplification

Isothermal amplification was accomplished by a commercial RT-RPA kit (Qitian Gene Biotech, Wuxi, Jiangsu, China). The steps are as follows: add 2 μL of pathogenic nucleic acid sample, 0.4 μM forward and reverse primers to 50 μL of RT-RPA (Reverse transcription recombinase polymerase amplification, reverse transcription recombinase polymerase-mediated isothermal amplification) reaction solution (see Table for details). 3). Finally, 14 mM magnesium acetate was added to initiate the reaction and incubated at 42°C for 20 minutes to obtain the RT-RPA reaction product.

table 3

(4) Fluorescence activation based on CRISPR-Cas12a

Fluorescence activation was performed in a 20 μL system containing: 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl ₂ , 100 μg/mL bovine serum albumin (BSA), 0.05 units (corresponding to 100 ng) of purified LbCas12a, 25 pM single-stranded DNA probe (purchased from Universal Biosystems (Anhui) Co., Ltd., model RX012179), and crRNA in an equimolar ratio of Cas12a, 2 μL of the above RT-RPA reaction product (or a target site reaction for respiratory pathogenic RNA nucleic acids) post-transcriptional double-stranded DNA mimics the substrate). Reactions were incubated at 37°C for 30 minutes. The fluorescent signal can be read by a microplate reader, or judged by the naked eye of a gel meter. The excitation wavelength and emission wavelength of the fluorescent probe are 485 nm and 520 nm, respectively.

(5) Experimental results

Figures 1-5 show the results of the specific detection of novel coronavirus nucleic acid. Among them, Figure 1 shows the Cas12a fluorescence detection of the new coronavirus ORF1ab gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure. Wherein crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 29-32). It can be seen from Figure 1 that both crRNA1-4 (SEQ ID NO: 29-32) and crRNA mix (mix) can efficiently detect the corresponding gene fragments, and the signal intensity is above 1×10 ⁷ .

Figure 2 shows the Cas12a fluorescence detection of the new coronavirus S gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure. Wherein crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 33-36). It can be seen from Figure 2 that both crRNA1-4 (SEQ ID NO: 33-36) and crRNA mix (mix) can efficiently detect the corresponding gene fragments, and the signal intensities are all above 1×10 ⁷ , among which crRNA1, 2 , 4 (SEQ ID NO: 33, 34, 36) and the signal intensity of crRNA mix (mix) were all above 2×10 ⁷ .

Figure 3 shows the Cas12a fluorescence detection of the new coronavirus E gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure. The crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 37-40). It can be seen from Figure 3 that both crRNA1-4 (SEQ ID NO: 37-40) and crRNA mix (mix) can efficiently detect the corresponding gene fragments, and the signal intensity is above 1×10 ⁷ , among which crRNA2-4 (SEQ ID NO: 38-40) and the signal intensities of the crRNA mix (mix) were all above 2×10 ⁷ .

Figure 4 shows the Cas12a fluorescence detection for the new coronavirus M gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure. Wherein crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 41-44). As can be seen from Figure 4, crRNA2 and crRNA3 (SEQ ID NO: 42, 43) can detect the corresponding gene fragments, and the detection effect of crRNA3 (SEQ ID NO: 43) is better, and the signal intensity is above 2 × 10 ⁷ .

Figure 5 shows the Cas12a fluorescence detection of the new coronavirus N gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure. Wherein the crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 45-48). It can be seen from Figure 5 that crRNA1-4 (SEQ ID NO: 45-48) and crRNA mix (mix) can efficiently detect the corresponding gene fragments, and most of the signal intensities are around 1×10 ⁷ or above.

Figure 6 shows the results of the specific detection of influenza A virus (IAV) nucleic acid, specifically the Cas12a fluorescence detection of the M gene target of influenza A virus (H1N1), and the corresponding target site sequence and crRNA sequence are shown in the figure shown. Wherein crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 49-52). As can be seen from Figure 6, crRNA2-4 (SEQ ID NO: 50-52) and crRNA mix (mix) can efficiently detect the corresponding gene fragments, and the signal intensities are all above 5 × 10 ⁶ , wherein crRNA3 (SEQ ID NO: 50-52) NO: 51) the signal strength is as high as 2×10 ⁷ or more.

Figure 7 shows the specific detection of nucleic acids of influenza B virus (IBV) different subtypes (Victoria (Victoria line) and Yamagata (Yamagata line)), specifically for the HA gene target of influenza B virus Cas12a fluorescence detection. The corresponding target site sequence and crRNA sequence are shown in the figure, wherein the Yamagata subtype (Yamagata line) specific target site sequence is shown in SEQ ID NO: 27, and the Victoria subtype (Victoria line) specific target site The sequence is shown in SEQ ID NO:103. Wherein crRNA mix (mix) is obtained after mixing crRNA1-4 (SEQ ID NO: 53-56). As can be seen from Figure 7, crRNA1-4 (SEQ ID NO: 53-56) and crRNA mix (mix) can efficiently detect the corresponding gene fragments, and crRNA3 (SEQ ID No. 55) can detect Yamagata subtypes. (Yamagata series) specific probe.

Figure 8 shows that the Cas12a crRNA probe SEQ ID No. 34 for the new coronavirus S gene target can specifically detect the new coronavirus, and has no cross-reaction with influenza A virus and influenza B virus. Meanwhile, SEQ ID No. 34 can distinguish different types of coronaviruses. Figure 9 shows that the Cas12a crRNA probe SEQ ID No. 51 for the M gene target of influenza A virus can specifically detect influenza A virus, and has no cross-reaction with various coronaviruses including new coronaviruses and influenza B virus . Figure 10 shows that the Cas12a crRNA probe SEQ ID No. 56 for the HA gene target of influenza B virus can specifically detect influenza B virus, and has no crossover with various coronaviruses including new coronavirus and influenza A virus reaction. It can be seen from Figures 8-10 that the specific crRNA probes do not cross-react to the new coronavirus, influenza A virus and influenza B virus.

Figure 11 shows the screening of RT-RPA primers for the novel coronavirus ORF1ab gene target. Forward primers 1-4 are respectively SEQ ID No. 58-61, Reverse primers 5-8 are SEQ ID No. 62-65 respectively. Signals can be detected after amplification of these primer pairs, wherein the forward primer and the reverse primer are respectively SEQ ID NO: 58 and SEQ ID NO: 62, 63, 64 or 65; the forward primer and the reverse primer are respectively SEQ ID NO: 58 and SEQ ID NO: 62, 63, 64 or 65. When ID NO: 59 and SEQ ID NO: 64; when forward primer and reverse primer are respectively SEQ ID NO: 60 and SEQ ID NO: 62; when forward primer and reverse primer are respectively SEQ ID NO: 60 and SEQ ID NO: 62 When the ID NO: 64; and when the forward primer and the reverse primer are respectively SEQ ID NO: 61 and SEQ ID NO: 62, 63 or 64, the fluorescence signal intensity is above 0.5×10 ⁸ . Wherein the crRNA probe used is SEQ ID NO:32.

Figure 12 shows the screening of RT-RPA primers for the new coronavirus S gene target. Forward primers 1-4 are SEQ ID No. 66-69, respectively, and Reverse primers 5-8 are SEQ ID No. 70-73, respectively. These primers can basically detect the signal after amplification, wherein the forward primer and the reverse primer are respectively SEQ ID NO: 66 and SEQ ID NO: 70; the forward primer and the reverse primer are respectively SEQ ID NO: 66 and SEQ ID NO: 70. When SEQ ID NO: 72; when forward primer and reverse primer are respectively SEQ ID NO: 66 and SEQ ID NO: 73; when forward primer and reverse primer are respectively SEQ ID NO: 69 and SEQ ID NO: 72 ; and when the forward primer and the reverse primer are respectively SEQ ID NO: 69 and SEQ ID NO: 73, the fluorescence signal intensity is stronger, and both are significantly higher than 0.5×10 ⁸ . Wherein the crRNA probe used is SEQ ID NO: 34.

Figure 13 shows the screening of RT-RPA primers for the new coronavirus E gene target. Forward primers 1-4 are SEQ ID No. 74-77, respectively, and Reverse primers 5-8 are SEQ ID No. 78-81, respectively. Signals can be detected after amplification of these primer pairs, wherein the forward primer and the reverse primer are respectively SEQ ID NO: 74 and SEQ ID NO: 78, 79, 80 or 81; the forward primer and the reverse primer are respectively When SEQ ID NO: 75 and SEQ ID NO: 78, 79 or 81; the forward primer and the reverse primer are respectively SEQ ID NO: 76 and SEQ ID NO: 78 or 81; the forward primer and the reverse primer are respectively When SEQ ID NO: 77 is compared with SEQ ID NO: 78, 79 or 81, the fluorescence signal intensity is stronger, which is significantly higher than 0.5×10 ⁸ . Wherein the crRNA probe used is SEQ ID NO: 38.

Figure 14 shows the screening of RT-RPA primers for the M gene target of the novel coronavirus. Forward primers 1-4 are SEQ ID No. 82-85, respectively, and Reverse primers 5-8 are SEQ ID No. 86-89, respectively. These primers can basically detect the signal after amplification, wherein the forward primer and the reverse primer are respectively SEQ ID NO: 82 and SEQ ID NO: 86; the forward primer and the reverse primer are respectively SEQ ID NO: 83 and SEQ ID NO: 86. When SEQ ID NO: 86; when forward primer and reverse primer are respectively SEQ ID NO: 83 and SEQ ID NO: 89; when forward primer and reverse primer are respectively SEQ ID NO: 85 and SEQ ID NO: 87 , the fluorescence signal intensity is strong. Wherein the crRNA probe used is SEQ ID NO: 43.

Figure 15 shows the screening of RT-RPA primers against the N gene target of the novel coronavirus. Forward primers 1-4 are SEQ ID No. 90-93, respectively, and Reverse primers 5-8 are SEQ ID No. 94-97, respectively. These primer pairs can detect the signal after amplification, and the signal intensity is very strong, all above 0.5×10 ⁸ . Wherein the crRNA probe used is SEQ ID NO: 45.

As can be seen from Figures 11-15, specific RT-RPA can amplify the target site sequence of the new coronavirus for the fluorescence detection of Cas12a.

Figure 16 shows naked-eye data reading based on Cas12a fluorescence detection, a new coronavirus sample. The white glowing tube corresponds to the viral nucleic acid sample that produces a fluorescent signal.

The nucleic acid samples of the standard strain of the new coronavirus in the laboratory are used for concentration gradient dilution (the number of virus copies after dilution of the samples is 3200, 650, 130, 26, 5, 1, and 0.2 virus copies, respectively) and then tested to observe whether A positive signal appears. Figure 17 shows the detection results of the sensitivity to the novel coronavirus, and it can be seen from the figure that the sensitivity is 5 virus copies per reaction.

The nucleic acid samples of the standard strain of influenza A virus in the laboratory were used for concentration gradient dilution (the number of virus copies after dilution of the samples were 3500, 700, 140, 28, 5, 1, and 0.2 virus copies, respectively) and then detected. whether there is a positive signal. FIG. 18 shows the detection results of the sensitivity to influenza A virus, and it can be seen from the figure that the sensitivity is 5 virus copies per reaction.

The nucleic acid samples of the standard strain of influenza B virus in the laboratory were used for concentration gradient dilution (the number of virus copies after dilution of the samples were 2000, 400, 80, 16, 3.2, 0.6, and 0.1 virus copies, respectively) for detection and observation. whether there is a positive signal. FIG. 19 shows the detection results of the sensitivity to influenza B virus, and it can be seen from the figure that the sensitivity is 3 virus copies per reaction.

Example 2

The results obtained in the detection of different types of samples are shown in Figure 20, and the detection method is the same as that in Example 1. It can be seen from the figure that the crRNA and primer pairs in Example 1 can be detected for different sample types (including sputum supernatant, nasal swab, throat swab, sputum, anal swab, stool, etc.). The specificity and sensitivity are high.

Example 3 Detection of each pathogen in a sample containing multiple pathogens derived from biopsies

Adenovirus serotype 5 (Ad5) encoding ACE2 was transfected into BALB/c mice by intranasal (IN); 5 days later, the new coronavirus was transfected into mice by intranasal, 1 To 7 days, samples were collected for virus titer and nucleic acid detection (the detection method was the same as that of Example 1). The results are shown in Figure 21. It can be seen from the figure that in addition to the new coronavirus (N and S genes), adenovirus can also be detected. It can be seen that the crRNA and primer pairs in Example 1 can detect each pathogen in a sample containing multiple pathogens derived from living tissue.

Example 4 Dependence of Cas12a protein concentration and detection efficiency

The results are shown in Figure 22. In Figure 22, the DNA substrate concentration is fixed in A, and the DNA substrate concentration is proportionally increased with the Cas12a concentration in B.

Through the study of three batches of Cas12a protein, it is found that the protein concentration is not as high as possible. In the case of immobilizing substrate DNA, high concentration of Cas12a will inhibit the detection efficiency (fluorescence intensity); and high concentration of Cas12a protein will inhibit the reaction. The effect also depends on the concentration (content) of the substrate DNA, that is, if the DNA concentration increases, the inhibitory effect disappears. Comparing A and B, it can be speculated that at very low substrate DNA concentrations, excess Cas12a protein inhibits the reaction.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form or substance. It should be pointed out that for those skilled in the art, without departing from the method of the present invention, the Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. All those skilled in the art, without departing from the spirit and scope of the present invention, can utilize the above-disclosed technical content to make some changes, modifications and equivalent changes of evolution, all belong to the present invention. Equivalent embodiments; at the same time, any modification, modification and evolution of any equivalent changes made to the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> ShanghaiTech University

<120> A kit, detection method and application for detecting nucleic acid of respiratory pathogens

<130> P20012763C

<160> 103

<170> PatentIn version 3.5

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<212> RNA

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<212> RNA

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<223> Novel coronavirus E gene, target site 4

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<212> RNA

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<223> Novel coronavirus M gene, target site 2

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<212> RNA

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<223> Novel coronavirus M gene, target site 3

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<212> RNA

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<223> Novel coronavirus M gene, target site 4

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<212> RNA

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<212> RNA

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<220>

<223> Novel coronavirus N gene, target site 2

<400> 18

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<212> RNA

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<223> Novel coronavirus N gene, target site 3

<400> 19

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<212> RNA

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<220>

<223> Novel coronavirus N gene, target site 4

<400> 20

uuuguaugcg ucaauaugcu uauucag 27

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<211> 27

<212> RNA

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<223> Influenza A virus M gene, target site 1

<400> 21

uuugcaggga aaaacaccga uuugcaggga 27

<210> 22

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<212> RNA

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<223> Influenza A virus M gene, target site 2

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ucuugaggcg cucauggaau ggcuaaa 27

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<212> RNA

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<223> Influenza A virus M gene, target site 3

<400> 23

uuuguguuca cgcucaccgu gcccagu 27

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<212> RNA

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<220>

<223> Influenza A virus M gene, target site 4

<400> 24

uuuuguauuca cgcucaccgu gcccagu 27

<210> 25

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<212> RNA

<213> Artificial Sequence

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<223> Influenza B virus HA gene, target site 1

<400> 25

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<210> 26

<211> 27

<212> RNA

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<223> Influenza B virus HA gene, target site 2

<400> 26

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<212> RNA

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<223> Influenza B virus HA gene, target site 3

<400> 27

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<223> Influenza B virus HA gene, target site 4

<400> 28

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<210> 29

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<212> RNA

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<223> Novel coronavirus ORF1ab gene, crRNA1, targeting positive strand

<400> 29

guggugcauc guguugucug uac 23

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<212> RNA

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<223> Novel coronavirus ORF1ab gene, crRNA2, targeting positive strand

<400> 30

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<223> Novel coronavirus ORF1ab gene, crRNA3, targeting negative strand

<400> 31

uacauacuua ccuuuuaagu cac 23

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<212> RNA

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<223> Novel coronavirus ORF1ab gene, crRNA4, targeting positive strand

<400> 32

cacuuaaaaa cacagucugu acc 23

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<223> Novel coronavirus S gene, crRNA1, targeting positive strand

<400> 33

acacguggug uuuauuaccc uga 23

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<223> Novel coronavirus S gene, crRNA2, targeting positive strand

<400> 34

agauccucag uuuuacauuc aac 23

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<223> Novel coronavirus S gene, crRNA3, targeting positive strand

<400> 35

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<223> Novel coronavirus S gene, crRNA4, targeting positive strand

<400> 36

caauguuacu ugguuccaug cua 23

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<223> Novel coronavirus E gene, crRNA1, targeting positive strand

<400> 37

uugcuuucgu gguauucuug cua 23

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<212> RNA

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<223> Novel coronavirus E gene, crRNA2, targeting positive strand

<400> 38

gugguauucu ugcuaguuac acu 23

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<223> Novel coronavirus E gene, crRNA3, targeting negative strand

<400> 39

caagacucac guuaacaaua uug 23

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<212> RNA

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<220>

<223> Novel coronavirus E gene, crRNA4, targeting positive strand

<400> 40

cucucguguu aaaaaucuga auu 23

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<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, crRNA1, targeting positive strand

<400> 41

agacuguuug cgcguacgcg uuc 23

<210> 42

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<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, crRNA2, targeting positive strand

<400> 42

cgcguacgcg uuccaugugg uca 23

<210> 43

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, crRNA3, targeting negative strand

<400> 43

uggauugaau gaccacaugg aac 23

<210> 44

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<212> RNA

<213> Artificial Sequence

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<223> Novel coronavirus M gene, crRNA4, targeting negative strand

<400> 44

uagaagcggu cuggucagaa uag 23

<210> 45

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, crRNA1, targeting negative strand

<400> 45

auggcaccug uguaggucaa cca 23

<210> 46

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<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, crRNA2, targeting negative strand

<400> 46

ucauccaauu ugauggcacc ugu 23

<210> 47

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, crRNA3, targeting positive strand

<400> 47

aaagaucaag ucauuuugcu gaa 23

<210> 48

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, crRNA4, targeting positive strand

<400> 48

uaugcgucaa uaugcuuauu cag 23

<210> 49

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza A virus M gene, crRNA1, targeting positive strand

<400> 49

cagggaaaaa caccgaucuu gag 23

<210> 50

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza A virus M gene, crRNA2, targeting minus strand

<400> 50

gccauuccau gagcgccuca aga 23

<210> 51

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza A virus M gene, crRNA3, targeting positive strand

<400> 51

uguucacgcu caccgugccc agu 23

<210> 52

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza A virus M gene, crRNA4, targeting positive strand

<400> 52

uauucacgcu caccgugccc agu 23

<210> 53

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza B virus HA gene, crRNA1, targeting negative strand

<400> 53

cauguuccccc uguguaguaa ggc 23

<210> 54

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza B virus HA gene, crRNA2, targeting negative strand

<400> 54

cuauggccuu ugcauguucc ccu 23

<210> 55

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza B virus HA gene, crRNA3, targeting positive strand

<400> 55

aagcuugcca auggaaccaa aua 23

<210> 56

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza B virus HA gene, crRNA4, targeting negative strand

<400> 56

cuuuaauagu uuugcaggag guc 23

<210> 57

<211> 1227

<212> PRT

<213> Lachnospiraceae bacterium ND2006

<400> 57

Ser Lys Leu Glu Lys Phe Thr Asn Cys Tyr Ser Leu Ser Lys Thr Leu

1 5 10 15

Arg Phe Lys Ala Ile Pro Val Gly Lys Thr Gln Glu Asn Ile Asp Asn

20 25 30

Lys Arg Leu Leu Val Glu Asp Glu Lys Arg Ala Glu Asp Tyr Lys Gly

35 40 45

Val Lys Lys Leu Leu Asp Arg Tyr Tyr Leu Ser Phe Ile Asn Asp Val

50 55 60

Leu His Ser Ile Lys Leu Lys Asn Leu Asn Asn Tyr Ile Ser Leu Phe

65 70 75 80

Arg Lys Lys Thr Arg Thr Glu Lys Glu Asn Lys Glu Leu Glu Asn Leu

85 90 95

Glu Ile Asn Leu Arg Lys Glu Ile Ala Lys Ala Phe Lys Gly Asn Glu

100 105 110

Gly Tyr Lys Ser Leu Phe Lys Lys Asp Ile Ile Glu Thr Ile Leu Pro

115 120 125

Glu Phe Leu Asp Asp Lys Asp Glu Ile Ala Leu Val Asn Ser Phe Asn

130 135 140

Gly Phe Thr Thr Ala Phe Thr Gly Phe Phe Asp Asn Arg Glu Asn Met

145 150 155 160

Phe Ser Glu Glu Ala Lys Ser Thr Ser Ile Ala Phe Arg Cys Ile Asn

165 170 175

Glu Asn Leu Thr Arg Tyr Ile Ser Asn Met Asp Ile Phe Glu Lys Val

180 185 190

Asp Ala Ile Phe Asp Lys His Glu Val Gln Glu Ile Lys Glu Lys Ile

195 200 205

Leu Asn Ser Asp Tyr Asp Val Glu Asp Phe Phe Glu Gly Glu Phe Phe

210 215 220

Asn Phe Val Leu Thr Gln Glu Gly Ile Asp Val Tyr Asn Ala Ile Ile

225 230 235 240

Gly Gly Phe Val Thr Glu Ser Gly Glu Lys Ile Lys Gly Leu Asn Glu

245 250 255

Tyr Ile Asn Leu Tyr Asn Gln Lys Thr Lys Gln Lys Leu Pro Lys Phe

260 265 270

Lys Pro Leu Tyr Lys Gln Val Leu Ser Asp Arg Glu Ser Leu Ser Phe

275 280 285

Tyr Gly Glu Gly Tyr Thr Ser Asp Glu Glu Val Leu Glu Val Phe Arg

290 295 300

Asn Thr Leu Asn Lys Asn Ser Glu Ile Phe Ser Ser Ile Lys Lys Leu

305 310 315 320

Glu Lys Leu Phe Lys Asn Phe Asp Glu Tyr Ser Ser Ala Gly Ile Phe

325 330 335

Val Lys Asn Gly Pro Ala Ile Ser Thr Ile Ser Lys Asp Ile Phe Gly

340 345 350

Glu Trp Asn Val Ile Arg Asp Lys Trp Asn Ala Glu Tyr Asp Asp Ile

355 360 365

His Leu Lys Lys Lys Ala Val Val Thr Glu Lys Tyr Glu Asp Asp Arg

370 375 380

Arg Lys Ser Phe Lys Lys Ile Gly Ser Phe Ser Leu Glu Gln Leu Gln

385 390 395 400

Glu Tyr Ala Asp Ala Asp Leu Ser Val Val Glu Lys Leu Lys Glu Ile

405 410 415

Ile Ile Gln Lys Val Asp Glu Ile Tyr Lys Val Tyr Gly Ser Ser Glu

420 425 430

Lys Leu Phe Asp Ala Asp Phe Val Leu Glu Lys Ser Leu Lys Lys Asn

435 440 445

Asp Ala Val Val Ala Ile Met Lys Asp Leu Leu Asp Ser Val Lys Ser

450 455 460

Phe Glu Asn Tyr Ile Lys Ala Phe Phe Gly Glu Gly Lys Glu Thr Asn

465 470 475 480

Arg Asp Glu Ser Phe Tyr Gly Asp Phe Val Leu Ala Tyr Asp Ile Leu

485 490 495

Leu Lys Val Asp His Ile Tyr Asp Ala Ile Arg Asn Tyr Val Thr Gln

500 505 510

Lys Pro Tyr Ser Lys Asp Lys Phe Lys Leu Tyr Phe Gln Asn Pro Gln

515 520 525

Phe Met Gly Gly Trp Asp Lys Asp Lys Glu Thr Asp Tyr Arg Ala Thr

530 535 540

Ile Leu Arg Tyr Gly Ser Lys Tyr Tyr Leu Ala Ile Met Asp Lys Lys

545 550 555 560

Tyr Ala Lys Cys Leu Gln Lys Ile Asp Lys Asp Asp Val Asn Gly Asn

565 570 575

Tyr Glu Lys Ile Asn Tyr Lys Leu Leu Pro Gly Pro Asn Lys Met Leu

580 585 590

Pro Lys Val Phe Phe Ser Lys Lys Trp Met Ala Tyr Tyr Asn Pro Ser

595 600 605

Glu Asp Ile Gln Lys Ile Tyr Lys Asn Gly Thr Phe Lys Lys Gly Asp

610 615 620

Met Phe Asn Leu Asn Asp Cys His Lys Leu Ile Asp Phe Phe Lys Asp

625 630 635 640

Ser Ile Ser Arg Tyr Pro Lys Trp Ser Asn Ala Tyr Asp Phe Asn Phe

645 650 655

Ser Glu Thr Glu Lys Tyr Lys Asp Ile Ala Gly Phe Tyr Arg Glu Val

660 665 670

Glu Glu Gln Gly Tyr Lys Val Ser Phe Glu Ser Ala Ser Lys Lys Glu

675 680 685

Val Asp Lys Leu Val Glu Glu Gly Lys Leu Tyr Met Phe Gln Ile Tyr

690 695 700

Asn Lys Asp Phe Ser Asp Lys Ser His Gly Thr Pro Asn Leu His Thr

705 710 715 720

Met Tyr Phe Lys Leu Leu Phe Asp Glu Asn Asn His Gly Gln Ile Arg

725 730 735

Leu Ser Gly Gly Ala Glu Leu Phe Met Arg Arg Ala Ser Leu Lys Lys

740 745 750

Glu Glu Leu Val Val His Pro Ala Asn Ser Pro Ile Ala Asn Lys Asn

755 760 765

Pro Asp Asn Pro Lys Lys Thr Thr Thr Leu Ser Tyr Asp Val Tyr Lys

770 775 780

Asp Lys Arg Phe Ser Glu Asp Gln Tyr Glu Leu His Ile Pro Ile Ala

785 790 795 800

Ile Asn Lys Cys Pro Lys Asn Ile Phe Lys Ile Asn Thr Glu Val Arg

805 810 815

Val Leu Leu Lys His Asp Asp Asn Pro Tyr Val Ile Gly Ile Asp Arg

820 825 830

Gly Glu Arg Asn Leu Leu Tyr Ile Val Val Val Asp Gly Lys Gly Asn

835 840 845

Ile Val Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Asn Phe Asn Gly

850 855 860

Ile Arg Ile Lys Thr Asp Tyr His Ser Leu Leu Asp Lys Lys Glu Lys

865 870 875 880

Glu Arg Phe Glu Ala Arg Gln Asn Trp Thr Ser Ile Glu Asn Ile Lys

885 890 895

Glu Leu Lys Ala Gly Tyr Ile Ser Gln Val Val His Lys Ile Cys Glu

900 905 910

Leu Val Glu Lys Tyr Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser

915 920 925

Gly Phe Lys Asn Ser Arg Val Lys Val Glu Lys Gln Val Tyr Gln Lys

930 935 940

Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Met Val Asp Lys Lys

945 950 955 960

Ser Asn Pro Cys Ala Thr Gly Gly Ala Leu Lys Gly Tyr Gln Ile Thr

965 970 975

Asn Lys Phe Glu Ser Phe Lys Ser Met Ser Thr Gln Asn Gly Phe Ile

980 985 990

Phe Tyr Ile Pro Ala Trp Leu Thr Ser Lys Ile Asp Pro Ser Thr Gly

995 1000 1005

Phe Val Asn Leu Leu Lys Thr Lys Tyr Thr Ser Ile Ala Asp Ser

1010 1015 1020

Lys Lys Phe Ile Ser Ser Phe Asp Arg Ile Met Tyr Val Pro Glu

1025 1030 1035

Glu Asp Leu Phe Glu Phe Ala Leu Asp Tyr Lys Asn Phe Ser Arg

1040 1045 1050

Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys Leu Tyr Ser Tyr Gly

1055 1060 1065

Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys Asn Asn Val Phe

1070 1075 1080

Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys Glu Leu Phe

1085 1090 1095

Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg Ala Leu

1100 1105 1110

Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met Ala

1115 1120 1125

Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg

1130 1135 1140

Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly

1145 1150 1155

Ile Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile

1160 1165 1170

Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg

1175 1180 1185

Lys Val Leu Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu

1190 1195 1200

Lys Leu Asp Lys Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu

1205 1210 1215

Glu Tyr Ala Gln Thr Ser Val Lys His

1220 1225

<210> 58

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, forward primer 1

<400> 58

gcaataacag ttacaccgga agccaatatg 30

<210> 59

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, forward primer 2

<400> 59

caggcaataa cagttacacc ggaagccaat a 31

<210> 60

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, forward primer 3

<400> 60

tggtactggt caggcaataa cagttacacc g 31

<210> 61

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, forward primer 4

<400> 61

tacacacact ggtactggtc aggcaataac 30

<210> 62

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, reverse primer 1

<400> 62

atcacaacta cagccataac ctttccacat 30

<210> 63

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, reverse primer 2

<400> 63

actacagcca taacctttcc acataccgca ga 32

<210> 64

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, reverse primer 3

<400> 64

tcacaactac agccataacc tttccacata c 31

<210> 65

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus ORF1ab gene, isothermal amplification, reverse primer 4

<400> 65

actacagcca taacctttcc acataccgca 30

<210> 66

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, forward primer 1

<400> 66

atgtttgttt ttcttgtttt attgccacta gtc 33

<210> 67

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, forward primer 2

<400> 67

agtgtgttaa tcttacaacc agaactcaat 30

<210> 68

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, forward primer 3

<400> 68

aatcttacaa ccagaactca attaccccct gc 32

<210> 69

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, forward primer 4

<400> 69

ctctagtcag tgtgttaatc ttacaaccag aac 33

<210> 70

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, reverse primer 1

<400> 70

agtaccattg gtcccagaga catgtatagc 30

<210> 71

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, reverse primer 2

<400> 71

atcaaacctc ttagtaccat tggtcccaga gac 33

<210> 72

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, reverse primer 3

<400> 72

gttatcaaac ctcttagtac cattggtccc ag 32

<210> 73

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus S gene, isothermal amplification, reverse primer 4

<400> 73

cttagtacca ttggtcccag agacatgtat agc 33

<210> 74

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, forward primer 1

<400> 74

ttcggaagag acaggtacgt taatagttaa tagcg 35

<210> 75

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, forward primer 2

<400> 75

tcgtttcgga agagacaggt acgttaatag ttaatagc 38

<210> 76

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, forward primer 3

<400> 76

tcggaagaga caggtacgtt aatagttaat agcgtac 37

<210> 77

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, forward primer 4

<400> 77

tcgtttcgga agagacaggt acgttaatag ttaatag 37

<210> 78

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, reverse primer 1

<400> 78

agaccagaag atcaggaact ctagaagaat 30

<210> 79

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, reverse primer 2

<400> 79

tttagaccag aagatcagga actctagaag aattc 35

<210> 80

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, reverse primer 3

<400> 80

tcaggaactc tagaagaatt cagattttta acacgag 37

<210> 81

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus E gene, isothermal amplification, reverse primer 4

<400> 81

agaccagaag atcaggaact ctagaagaat tc 32

<210> 82

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, forward primer 1

<400> 82

tcttgtaggc ttgatgtggc tcagctactt 30

<210> 83

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, forward primer 2

<400> 83

gtaggcttga tgtggctcag ctacttcatt 30

<210> 84

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, forward primer 3

<400> 84

tacttcattg cttctttcag actgtttgcg 30

<210> 85

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, forward primer 4

<400> 85

gctacttcat tgcttctttc agactgtttg cg 32

<210> 86

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, reverse primer 1

<400> 86

tcacagctcc gattacgagt tcactttcta 30

<210> 87

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, reverse primer 2

<400> 87

aggatcacag ctccgattac gagttcactt 30

<210> 88

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, reverse primer 3

<400> 88

gtgtccagca atacgaagat gtccacgaag 30

<210> 89

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus M gene, isothermal amplification, reverse primer 4

<400> 89

gtcctagatg gtgtccagca atacgaagat gt 32

<210> 90

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, forward primer 1

<400> 90

catggaagtc acaccttcgg gaacgtggtt 30

<210> 91

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, forward primer 2

<400> 91

atggaagtca caccttcggg aacgtggttg 30

<210> 92

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, forward primer 3

<400> 92

aagtcacacc ttcgggaacg tggttgacct 30

<210> 93

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, forward primer 4

<400> 93

gaagtcacac cttcgggaac gtggttgacc ta 32

<210> 94

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, reverse primer 1

<400> 94

gtttcatcag ccttcttctt tttgtccttt 30

<210> 95

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, reverse primer 2

<400> 95

ggctctgttg gtgggaatgt tttgtatgcg 30

<210> 96

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, reverse primer 3

<400> 96

tcatcagcct tcttcttttt gtcctttttta g 31

<210> 97

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> Novel coronavirus N gene, isothermal amplification, reverse primer 4

<400> 97

cttctttttg tcctttttag gctctgttgg tg 32

<210> 98

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> Influenza A virus M gene, isothermal amplification, forward primer 1

<400> 98

atgagycttc taacygargt cgaaacgtac gttc 34

<210> 99

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Influenza A virus M gene, isothermal amplification, reverse primer 1

<400> 99

cgtctacgct gcagtccycg ctcactgggc 30

<210> 100

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Influenza B virus HA gene, isothermal amplification, forward primer 1

<400> 100

caygaaaaat acggtggatt aaacaaaagc 30

<210> 101

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> Influenza B virus HA gene, isothermal amplification, reverse primer 1

<400> 101

cgtgccarcc tgcaatcatt ccttcccatc 30

<210> 102

<211> 3684

<212> DNA

<213> Artificial Sequence

<220>

<223> Optimized Cas12a nucleic acid sequence

<400> 102

atgagcaagc tggaaaaatt taccaactgc tacagcctga gcaagaccct gcgtttcaaa 60

gcgatcccgg ttggcaagac ccaggaaaac attgacaaca aacgtctgct ggttgaggac 120

gaaaagcgtg cggaggatta taaaggtgtg aagaaactgc tggatcgtta ctatctgagc 180

tttatcaacg acgtgctgca cagcattaag ctgaaaaacc tgaacaacta catcagcctg 240

ttccgtaaga aaacccgtac cgagaaggaa aacaaagagc tggaaaacct ggaaatcaac 300

ctgcgtaagg agattgcgaa ggcgttcaag ggtaacgagg gctacaagag cctgttcaag 360

aaagatatca tcgaaaccat cctgccggag ttcctggacg ataaggacga aattgcgctg 420

gttaacagct tcaacggttt taccaccgcg ttcaccggct tctttgataa ccgtgagaac 480

atgtttagcg aggaagcgaa aagcaccagc atcgcgttcc gttgcattaa cgaaaacctg 540

acccgttaca tcagcaacat ggacattttc gagaaggttg acgcgatctt tgataaacac 600

gaggtgcagg aaatcaagga gaaaattctg aacagcgact atgatgttga agatttcttt 660

gagggtgaat tctttaactt tgttctgacc caagagggca tcgacgtgta caacgcgatc 720

attggtggct tcgtgaccga aagcggcgag aagatcaaag gcctgaacga gtacattaac 780

ctgtataacc agaagaccaa acaaaagctg ccgaaattta agccgctgta taagcaggtg 840

ctgagcgatc gtgaaagcct gagcttctac ggcgagggct ataccagcga cgaggaagtt 900

ctggaagtgt ttcgtaacac cctgaacaaa aacagcgaga tcttcagcag cattaagaaa 960

ctggaaaagc tgttcaaaaa ctttgacgag tacagcagcg cgggtatctt tgttaagaac 1020

ggcccggcga tcagcaccat tagcaaagat atcttcggtg aatggaacgt gattcgtgac 1080

aagtggaacg cggagtatga cgatatccac ctgaagaaaa aggcggtggt taccgaaaag 1140

tacgaggacg atcgtcgtaa aagcttcaaa aagattggca gctttagcct ggaacagctg 1200

caagagtacg cggacgcgga tctgagcgtg gttgaaaaac tgaaggat cattatccag 1260

aaggttgatg aaatctacaa agtgtatggt agcagcgaga agctgttcga cgcggatttt 1320

gttctggaga agagcctgaa aaagaacgac gcggtggttg cgatcatgaa ggacctgctg 1380

gatagcgtga aaagcttcga aaactacatt aaggcgttct ttggtgaagg caaagagacc 1440

aaccgtgacg agagcttcta tggcgatttt gttctggcgt acgacatcct gctgaaggtg 1500

gaccacatct acgatgcgat tcgtaactat gttacccaaa aaccgtacag caaggataag 1560

ttcaagctgt acttccagaa cccgcaattc atgggtggct gggacaagga taaagagacc 1620

gactatcgtg cgaccatcct gcgttacggt agcaagtact atctggcgat tatggataaa 1680

aagtacgcga aatgcctgca gaagatcgac aaagacgatg ttaacggtaa ctacgaaaag 1740

atcaactaca agctgctgcc gggcccgaac aagatgctgc cgaaagtgtt ctttagcaaa 1800

aagtggatgg cgtactataa cccgagcgag gacatccaaa agatctacaa gaacggtacc 1860

ttcaaaaagg gcgatatgtt taacctgaac gactgccaca agctgatcga cttctttaaa 1920

gatagcatta gccgttatcc gaagtggagc aacgcgtacg atttcaactt tagcgagacc 1980

gaaaagtata aagacatcgc gggtttttac cgtgaggttg aggaacaggg ctataaagtg 2040

agcttcgaaa gcgcgagcaa gaaagaggtg gataaactgg tggaggaagg taaactgtac 2100

atgttccaaa tctacaacaa ggacttcagc gataagagcc acggcacccc gaacctgcac 2160

accatgtact tcaagctgct gtttgacgaa aacaaccatg gtcagatccg tctgagcggt 2220

ggcgcggagc tgttcatgcg tcgtgcgagc ctgaagaaag aggagctggt tgtgcacccg 2280

gcgaacagcc cgattgcgaa caaaaacccg gataacccga aaaagaccac caccctgagc 2340

tacgacgtgt ataaggataa acgttttagc gaagaccaat acgagctgca cattccgatc 2400

gcgattaaca agtgcccgaa aaacatcttc aagattaaca ccgaagttcg tgtgctgctg 2460

aaacacgacg ataacccgta tgttatcggt attgaccgtg gcgagcgtaa cctgctgtac 2520

atcgtggttg tggacggtaa aggcaacatt gtggaacagt atagcctgaa cgagattatc 2580

aacaacttta acggtatccg tattaagacc gattaccaca gcctgctgga caaaaaggag 2640

aaggaacgtt tcgaggcgcg tcagaactgg accagcatcg aaaacattaa ggagctgaaa 2700

gcgggctata tcagccaagt tgtgcacaag atttgcgaac tggttgagaa atacgatgcg 2760

gtgatcgcgc tggaggacct gaacagcggt tttaagaaca gccgtgttaa ggtggaaaag 2820

caggtttacc aaaagttcga gaagatgctg atcgataagc tgaactacat ggtggacaaa 2880

aagagcaacc cgtgcgcgac cggtggcgcg ctgaaaggtt atcagattac caacaagttc 2940

gaaagcttta aaagcatgag cacccaaaac ggcttcatct tttacattcc ggcgtggctg 3000

accagcaaaa tcgatccgag caccggtttt gttaacctgc tgaagaccaa atataccagc 3060

attgcggata gcaaaaagtt catcagcagc tttgaccgta ttatgtacgt gccggaggaa 3120

gacctgttcg agtttgcgct ggactataag aacttcagcc gtaccgacgc ggactacatc 3180

aaaaagtgga aactgtacag ctatggtaac cgtatccgta ttttccgtaa cccgaaaaag 3240

aacaacgttt ttgactggga ggaagtgtgc ctgaccagcg cgtataagga actgttcaac 3300

aaatacggta tcaactatca gcaaggcgat attcgtgcgc tgctgtgcga gcagagcgac 3360

aaggcgttct acagcagctt tatggcgctg atgagcctga tgctgcaaat gcgtaacagc 3420

atcaccggtc gtaccgatgt tgattttctg atcagcccgg tgaaaaacag cgacggcatt 3480

ttctacgata gccgtaacta tgaagcgcag gagaacgcga ttctgccgaa gaacgcggac 3540

gcgaacggtg cgtataacat cgcgcgtaaa gttctgtggg cgattggcca gttcaaaaag 3600

gcggaggacg aaaagctgga taaggtgaaa atcgcgatta gcaacaaaga atggctggag 3660

tacgcgcaaa ccagcgttaa gcac 3684

<210> 103

<211> 27

<212> RNA

<213> Artificial Sequence

<220>

<223> Influenza B virus (IBV-Victoria) HA gene, target site 5

<400> 103

cuugaagcug gccaauggaa ccaaaua 27

Claims (10)

1. A combination of crRNA and a primer pair, wherein the crRNA and the primer pair are used for detecting nucleic acid of respiratory pathogens, and the respiratory pathogens are novel coronavirus, influenza a virus and influenza b virus; wherein,

the sequence of crRNA for detecting ORF1ab gene of the novel coronavirus is shown as SEQ ID NO. 32; the sequence of crRNA for detecting the S gene of the novel coronavirus is shown as SEQ ID NO. 34; the sequence of the crRNA for detecting the E gene of the novel coronavirus is shown as SEQ ID NO 38; the sequence of crRNA for detecting the M gene of the novel coronavirus is shown as SEQ ID NO. 43; the sequence of the crRNA for detecting the N gene of the novel coronavirus is shown as SEQ ID NO. 48; the sequence of the crRNA for detecting the M gene of the influenza A virus is shown as SEQ ID NO. 51; and, detecting the sequence of the crRNA of the HA gene of the influenza B virus is shown in SEQ ID NO 56;

the nucleotide sequence of a primer pair for amplifying ORF1ab gene of the novel coronavirus is shown as SEQ ID NO. 58 and SEQ ID NO. 62; the nucleotide sequence of the primer pair for amplifying the S gene of the novel coronavirus is shown as SEQ ID NO. 66 and SEQ ID NO. 70; the nucleotide sequence of the primer pair for amplifying the E gene of the novel coronavirus is shown as SEQ ID NO. 74 and SEQ ID NO. 78; the nucleotide sequence of the primer pair for amplifying the M gene of the novel coronavirus is shown as SEQ ID NO. 82 and SEQ ID NO. 86; the nucleotide sequence of the primer pair for amplifying the N gene of the novel coronavirus is shown as SEQ ID NO. 90 and SEQ ID NO. 94; the nucleotide sequence of the primer pair for amplifying the M gene of the influenza A virus is shown as SEQ ID NO. 98 and SEQ ID number 99; and the nucleotide sequence of the primer pair for amplifying the HA gene of the influenza B virus is shown as SEQ ID NO 100 and SEQ ID number 101;

the crRNA is used in a CRISPR-Cas detection system, and the primer pair is used in RPA to amplify the nucleic acid of the respiratory tract pathogen.

2. The combination of crRNA and primer pair of claim 1, wherein the primer pair is used in RT-RPA to amplify nucleic acids of the respiratory tract pathogen.

3. A kit for detecting a nucleic acid of a respiratory pathogen comprising a CRISPR-Cas detection system, wherein the CRISPR-Cas detection system comprises: crRNA, primer pairs, Cas protein, and nucleic acid probes; wherein the crRNA and the primer pair are as set forth in claim 1 or 2.

4. The kit of claim 3, wherein the CRISPR-Cas detection system further comprises a metal ion and/or a buffer; and/or, the Cas protein comprises Cas9, Cas12a, Cas12b, and/or Cas 13.

5. A method for detecting nucleic acids of respiratory pathogens of non-diagnostic interest, characterized in that said nucleic acids are detected using the CRISPR-Cas detection system as described in the kit of claim 3 or 4.

6. Use of a combination of crRNA and a primer pair according to claim 1 or 2 in the detection of nucleic acids of respiratory tract pathogens, or in the preparation of a reagent or kit for the detection of nucleic acids of respiratory tract pathogens.

7. The use according to claim 6, wherein the respiratory pathogens are novel coronaviruses, influenza A viruses and/or influenza B viruses; and/or the detection is performed using a CRISPR-Cas detection system and/or RPA.

8. The use of claim 7, wherein said RPA is RT-RPA.

9. Use of a crRNA as claimed in claim 1 or 2 for the preparation of a medicament for the prevention and/or treatment of infection by respiratory pathogens which are novel coronaviruses, influenza a viruses and/or influenza b viruses.

10. The use of claim 9, wherein the crRNA disrupts a respiratory tract pathogen in the subject using a CRISPR-Cas system.

Images