CN111534643B

CN111534643B - Kit for detecting nucleic acid of respiratory tract pathogen, detection method and application

Info

Publication number: CN111534643B
Application number: CN202010659510.5A
Authority: CN
Inventors: 刘佳
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2020-07-10
Filing date: 2020-07-10
Publication date: 2020-10-09
Anticipated expiration: 2040-07-10
Also published as: CN111534643A

Abstract

The invention provides a kit for detecting nucleic acid of respiratory tract pathogen, which comprises a CRISPR-Cas detection system: crRNA, primer pairs, Cas protein, and nucleic acid probes as described herein. The invention also provides a detection method of nucleic acid of respiratory tract pathogen, a combination of crRNA and a primer pair and application thereof. The kit and the detection method provided by the invention do not depend on a large-scale instrument, can directly observe results through naked eyes, can realize detection under mild conditions, and are more convenient to detect; the kit and the detection method can be used for efficiently and quickly detecting/diagnosing the respiratory tract pathogens (such as influenza A, influenza B, novel coronavirus and the like), have high specificity and sensitivity, and can be used for detecting and screening the respiratory tract pathogens, such as quickly distinguishing various respiratory tract pathogens including influenza A, influenza B and novel coronavirus.

Description

Kit for detecting nucleic acid of respiratory tract pathogen, detection method and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a kit for detecting nucleic acid of respiratory tract pathogens, a detection method and application.

Background

Covi-19 cases soon appeared in World countries and were announced by the World Health Organization (WHO) as a worldwide public health threat. Although the median 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 proportion of patients do not present symptoms, including X-ray and CT tests. The uncertain symptom time of the COVID-19 and the condition of asymptomatic infection bring great obstacles to early accurate detection of diseases, thereby making the disease prevention and control extremely challenging. This makes molecular detection of the COVID-19 pathogenic nucleic acids particularly important.

The pathogen responsible for COVID-19 has been identified as a β coronavirus, designated SARS-CoV-2. Like other coronaviruses, SARS-CoV-2 is a single-stranded, positive-sense RNA virus. SARS-CoV-2 and SARS-CoV as well as the middle east respiratory syndrome coronavirus (MERS-CoV) have nucleic acid similarities of 79% and 50%, respectively. Existing molecular diagnostic methods for COVID-19 are mainly based on reverse transcription PCR (RT-PCR). E, N and ORF1ab genes of SARS-CoV-2 are target genes of commonly used RT-PCR. High throughput RT-PCR platforms have also been developed for large scale diagnostics. However, RT-PCR usually relies on specialized, large-scale instruments, and thus large-scale RT-PCR assays are often limited to patients in hospitals.

Recent studies have shown that the Clustered Regulated Interleaved Short Palindromic Repeats (CRISPR) technology can be used for low-cost, portable molecular detection of pathogens. CRISPR and CRISPR-associated genes (Cas) genes are the immune system of bacteria against foreign viral infection. The modular nature of CRISPR makes this technology widely used in genome engineering. Molecular diagnostics of CRISPR-based pathogenic nucleic acids rely on the targeting activity of Cas nucleases RNA or DNA. Meanwhile, different CRISPR systems can be combined to realize multiple detection of pathogens. In the recent outbreak of african swine fever epidemic in china, many diagnostic platforms based on CRISPR-Cas12a were developed.

At present, the expression spectrum of the subgenomic of the novel coronavirus serving as a new infectious disease is not clearly clarified, the expression condition of each gene and the sensitivity of each gene fragment to amplification and CRISPR-Cas detection are also unclear, and whether a detection method which is high in specificity and free of cross reaction and aims at respiratory pathogens such as the novel coronavirus, the influenza A virus and the influenza B virus can be designed or not is a scientific problem which is not clarified at present. The conventional RT-qPCR method used in the detection of the novel coronavirus is easy to misdetect and is not easy to distinguish the novel coronavirus from other influenzas such as influenza A virus and influenza B virus, the three symptoms on clinical manifestation are very similar, but the misdiagnosis of the novel coronavirus as the influenzas occurs in practical application (Kong et al, 2020,Nat Microbiol). In addition, pathogen detection technology based on CRISPR-Cas12a has been reported (Gootenberg)et al, 2017,Science；Gootenberget al, 2018,Science；Chenet al, 2017,Science(ii) a ) In particular, CRISPR detection platforms for novel coronaviruses have also been successfully developed (Wang)et al, 2020, Sci Bull；Guoet al,2020, Cell Discov；Broughtonet al2020 Nat Biotech), however, these techniques have been developed based on various principles, and the research on the detection of pathogen specificity has not been deep enough, e.g. no study on the specificity among various subtypes of pathogens has been made, as well as the research on the detection of pathogen specificityCross-reactivity between different probe combinations (e.g., the presence or absence of cross-reactivity between pathogen A and pathogen B probes) is not well defined.

Therefore, there is an urgent need for a primer and crRNA probe that is efficient, fast, specific, and sensitive, and does not have cross reaction when detecting various respiratory pathogens, so as to achieve specific detection of various respiratory pathogens.

Disclosure of Invention

The invention aims to overcome the defects that the existing method for detecting respiratory tract pathogens (such as novel coronavirus (SARS-CoV-2), influenza A and influenza B) depends on a large instrument and is inconvenient to detect, and provides a kit for detecting nucleic acid of the respiratory tract pathogens, a method for detecting nucleic acid of the respiratory tract pathogens, crRNA, a primer pair and application thereof. Firstly, the kit and the detection method of the invention do not depend on large instruments, can directly observe results by naked eyes, can realize detection under mild conditions (25-42 ℃) and are more convenient to detect. The kit and the detection method can be used for efficiently and quickly detecting/diagnosing respiratory tract pathogens (such as influenza A, influenza B, new coronavirus and the like). Secondly, when the kit and the detection method are used for detecting various respiratory tract pathogens (such as influenza A, influenza B, new coronavirus and the like), cross reaction does not occur, and the specificity and the sensitivity of detection are high. At present, no patent or literature report can completely realize the simultaneous detection of novel coronavirus, influenza A and influenza B, and the application provides a kit, a detection method and the like for the first time, and the kit, the influenza A and the influenza B can completely and interactively detect the specificity of the novel coronavirus, the influenza A and the influenza B while ensuring the sensitivity. The kit and the detection method can be used for detecting and screening respiratory tract pathogens, such as rapidly distinguishing various respiratory tract pathogens including influenza A (including various subtypes), influenza B (including various subtypes) and novel coronavirus. In addition, all CRISPR-based assays currently employ respiratory tract samples such as pharyngeal swabs or nasal swabs, and the kits and assays of the invention further allow for the detection of other types of samples (e.g., anal swabs, feces, sputum supernatant, etc.) in addition to respiratory tract samples; meanwhile, the method can realize detection of each pathogen in a sample which is from living tissue and contains various pathogens.

First, in designing crRNA and primers for detecting a novel coronavirus and other influenza viruses such as influenza a virus and influenza b virus, it is necessary to consider the prevention of cross-reaction and misdiagnosis in detecting a novel corona and other influenza viruses on the one hand, and on the other hand, since the subgenomic structure (subgenomic architecture) of the novel corona virus itself has not been clearly and scientifically elucidated. The current research shows that the subgenomic structure of the new coronavirus is very complex due to the fact that the subgenomic structure contains 10 discontinuous coding regions, and the expression condition of each gene and the sensitivity of each gene fragment to amplification and CRISPR-Cas detection are not clear; also, there are other unknown transcripts besides the 10 coding regions known. When designing a primer, a pathogen gene segment with high transcription efficiency needs to be searched for design (for example, the inventor finds that the N gene is a new coronavirus gene and is easier to obtain a primer with high-efficiency amplification than the M gene (figures 14 and 15), also finds that the amplification efficiency of different segments is greatly different (figure 14) when the N gene is the M gene, the restriction of a Cas12a PAM sequence needs to be met when designing crRNA, meanwhile, the fact that the CRRNA needs to be positioned in a primer amplification region needs to be considered, meanwhile, a primer pair meeting the requirements and the crRNA need to be capable of ensuring higher sensitivity and specificity, and the fact that cross reaction does not occur when simultaneously detecting the new coronavirus and other influenza such as influenza A virus and influenza B virus needs to be ensured, so that the design difficulty is very high. Secondly, specificity reflects the ability of the screening test to determine non-positive samples; the sensitivity refers to the degree of change of response quantity caused by the change of a unit concentration or unit quantity of a substance to be detected by a certain method; in designing crRNA and primer pairs, it is necessary to balance the relationship between specificity and sensitivity, and it is well known in the art that it is very difficult to simultaneously improve sensitivity and specificity. Sensitivity is reduced while high specificity is ensured, and specificity is reduced while high sensitivity is ensured. When different viruses are detected in the kit to be protected, the specificity can be ensured, and meanwhile, the high sensitivity can be ensured. In addition, all CRISPR-based detection currently uses respiratory tract samples such as pharyngeal swabs, but the scope of the samples detected by the kit and the detection method of the present invention is large, and the kit and the detection method are not limited to respiratory tract samples (such as pharyngeal swabs or nasal swabs), and can also be used for detecting secretions such as anal swabs, feces, sputum, and sputum supernatant, etc., whereas interfering bacteria existing in different parts and samples in different environments are different, and interfering substances such as escherichia coli, etc., existing in the detection of the anal swabs, feces, etc., have influence on both sensitivity and specificity of detection. The kit and the detection method can keep good specificity and sensitivity in various samples of different types, do not cause cross infection and have wide application range.

In order to solve the above technical problems, the present invention provides in a first aspect a target site for detecting a nucleic acid of a respiratory tract pathogen, the respiratory tract pathogen being a novel coronavirus, an influenza a virus and/or an influenza b virus; wherein,

detecting the sequence of the target site of the novel coronavirus as shown in one or more of SEQ ID NO 1-20;

detecting the sequence of the target site of the influenza A virus is shown as one or more of SEQ ID NO 21-24;

detecting the sequence of the target site of the influenza B virus as shown in one or more of SEQ ID NO 25-28 and 103;

these target sites described in the present invention are capable of specifically distinguishing 3 respiratory pathogens (novel coronaviruses, influenza a viruses and influenza b viruses) and their corresponding DNA sequences comprise a PAM sequence recognized by a Cas protein (e.g., Cas12 a). Furthermore, it will be understood by those skilled in the art that the RNA sequences complementary to SEQ ID NOs 1-28, 103, and the target sites indicated by the sequence of either strand of the double-stranded DNA after reverse transcription of SEQ ID NOs 1-28, 103 are also within the scope of the present invention.

Wherein, the sequence of the target site for detecting ORF1ab gene of the novel coronavirus is shown as one or more of SEQ ID NO 1-4.

Wherein, the sequence of the target site for detecting the S gene of the novel coronavirus is shown as one or more of SEQ ID NO 5-8.

Wherein, the sequence of the target site for detecting the E gene of the novel coronavirus is shown as one or more of SEQ ID NO 9-12.

Wherein, the sequence of the target site for detecting the M gene of the novel coronavirus is shown as one or more of SEQ ID NO 13-16.

Wherein, the sequence of the target site for detecting the N gene of the novel coronavirus is shown as one or more of SEQ ID NO 17-20.

In a preferred embodiment of the present invention, higher signal (up to 3 × 10) can be generated by detecting the target sites of S gene and E gene of the novel coronavirus⁷) The signal is strong when ORF1ab, M gene and N gene of the novel coronavirus are detected, but the signal is not more than 3 × 10⁷。

In the invention, the detection of influenza A virus is mainly to detect the M gene of the influenza A virus. Among them, the Influenza A Virus (IAV) can be a conventional one in the art, and is generally classified into a plurality of subtypes according to the difference between H and N antigens, H can be classified into 18 subtypes (H1 to H18), and N has 11 subtypes (N1 to N11), including, for example, subtypes such as H1N1, H2N2, H3N2, H5N1, H7N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N8, and can be, for example, H1N1, H3N2, and H9N 2.

In the invention, the detection of the influenza B virus is mainly to detect the HA gene of the influenza B virus. The Influenza B Virus (IBV) may be conventional in the art, and includes, for example, Yamagata (Yamagata) or Victoria (Victoria) Influenza B virus. Wherein, the sequence of the target site for detecting the Yamagata line (mountain line) of the influenza B virus can be shown as one or more of SEQ ID NO: 25-28. Wherein, the sequence of the target site for detecting Victoria line (Victoria line) of the influenza B virus can be shown as one or more of

SEQ ID NO

25, 26, 103 and 28.

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

In the invention, the 5 ' end of the obtained targeting sequence has a 5 ' -TTTN-3 ' sequence, and a stable secondary structure is not formed between the targeting sequence and the rest sequences.

In order to solve the above technical problems, the second aspect of the present invention provides a combination of a crRNA and a primer pair for detecting nucleic acids of respiratory pathogens, which 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 QPCR, LAMP (Loop-mediated isothermal amplification) or RPA (recombinase polymerase mediated isothermal amplification) to amplify the nucleic acid (target site of the target site) of the respiratory tract pathogen.

In the present invention, the RPA is used in the conventional sense in the art, which also generally includes RPA-based derivatization reactions. It may be a common RPA including a DNA template, or RT-RPA (RT is an abbreviation for reverse transcription) combined with reverse transcription of an RNA template.

In the invention, the primer pair can be used for amplifying target site sequences of novel coronavirus, influenza A virus and influenza B virus so as to realize fluorescence detection of Cas12 a. The application of the primer pairs in other reactions (such as QPCR) and other CRISPR systems (such as Cas12b or Cas 13) is also in the protection scope of the invention.

In order to solve the above technical problems, a third aspect of the present invention provides a kit for detecting a nucleic acid of a respiratory tract pathogen, comprising a CRISPR-Cas detection system comprising: 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 invention.

Preferably, the CRISPR-Cas detection system further comprises a metal ion and/or a buffer.

In the present invention, the Cas protein may be conventional in the art, including, for example, Cas9, Cas12a, Cas12b, and/or Cas 13. Wherein, the Cas12a protein is a Cas protein with endonuclease activity and accessory cleavage activity. Such as Cas12a, Cas12b, etc. The amino acid sequence of Cas12a is preferably shown as SEQ ID number 57, and the nucleotide sequence thereof is preferably shown as SEQ ID number 102.

In the invention, the nucleic acid probe included in the CRISPR-Cas detection system can be a single-stranded DNA probe. It will be appreciated by those skilled in the art that nucleic acid probes conventionally used in the art should be capable of carrying out the present invention. In a preferred embodiment of the present invention, the nucleic acid probe may be a nucleic acid probe available from general biology systems (Anhui) Inc. under model number RX 012179.

In the present invention, the content of the Cas protein may be generally 100 ng. In a preferred embodiment, the kit comprises (suitable for 20 μ L reaction): 100 ng Cas protein, 25 pM nucleic acid probe such as single-stranded DNA probe, and Cas12a in equimolar ratio of crRNA. For detecting 2. mu.L of a viral nucleic acid to be detected (e.g., a product of RT-RPA reaction using the above primer pair or a double-stranded DNA mimic substrate reverse-transcribed to a target site of a respiratory pathogenic RNA nucleic acid), the kit may further comprise 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl₂,100μg/mL BSA。

In order to solve the above technical problems, the fourth aspect of the present invention provides a method for detecting a nucleic acid of a respiratory tract pathogen, wherein the nucleic acid is detected by using the CRISPR-Cas detection system as described in the kit of the third aspect of the present invention (for example, CRISPR nucleic acid detection using Cas12a protein and crRNA corresponding to the target site), and/or the nucleic acid is amplified by using the primer pair as described in the kit of the fifth aspect of the present invention.

In the present invention, the method for detecting nucleic acid is usually not for diagnostic purposes, such as performing an experiment with an ex vivo sample for research and development, or detecting a sample collected from an environment contaminated by secretions such as sputum and feces of a patient infected with a new coronavirus.

In order to solve the above technical problems, a fifth aspect of the present invention provides a use of the crRNA and the primer pair according to the second aspect of the present invention in detecting nucleic acids of respiratory tract pathogens, or in preparing a reagent or a kit for detecting nucleic acids of respiratory tract pathogens.

Preferably, the respiratory pathogens are novel coronaviruses, influenza a viruses and/or influenza b viruses.

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

In order to solve the above technical problems, a sixth aspect of the present invention provides the use of a crRNA according to the second aspect of the present invention 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.

Preferably, the crRNA disrupts respiratory pathogens in the subject using the CRISPR-Cas system.

The invention also provides a CRISPR-Cas12a specific detection system, which comprises a Cas12a protein and a crRNA, wherein the crRNA is used for detecting the crRNA of the target site according to the first aspect of the invention and is used for detecting 3 respiratory tract pathogens (novel coronavirus, influenza A and influenza B).

Preferably, the sequence of the crRNA for detecting the novel coronavirus is shown as one or more of SEQ ID NO 29-40, 42, 43, 45-48.

Preferably, the sequence of the crRNA for detecting the influenza A virus is shown as one or more of SEQ ID NO 50-52.

Preferably, the sequence of the crRNA for detecting the influenza B virus is shown as one or more of SEQ ID NO: 53-56.

The invention also provides a composition comprising a crRNA according to the second aspect of the invention and/or a primer pair according to the third aspect of the invention.

In the invention, the detection method of the nucleic acid can be based on a CRISPR-Cas12a system, in particular on the CRISPR-Cas12a single-stranded DNA activity activation reaction.

Interpretation of terms

The term crRNA refers to CRISPR RNA, a short RNA that directs Cas12a (Cpf 1) to bind to a target DNA sequence.

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

The term Cas protein refers to a CRISPR-associated protein, which is a related protein in CRISPR systems.

The term Cas12a (also known as Cpf 1) refers to a crRNA-dependent endonuclease, which is a type V (type V) enzyme in the classification of CRISPR systems.

The term PAM refers to the pro-spacer-adjacencies motif (protospacer-adjacent motif) necessary for cleavage by Cas12a, and PAM for LbCas12a is TTTN sequence.

In the present invention, the pathogen (also called pathogen) is a general term for microorganisms and parasites that can cause diseases. The majority of microorganisms comprise viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi and the like; the parasites mainly comprise source insects, insects and the like.

In the present invention, the detection may be to detect whether the sample contains the respiratory tract pathogen, that is, the detected sample may be a sample containing the respiratory tract pathogen, or may be a sample not containing the respiratory tract pathogen. The test may be for screening a sample for the presence of the respiratory pathogen, for example for screening a sample ex vivo.

In the present invention, "a plurality" of said "one or more" may mean 2, 3, 4 or more.

In the present invention, the term "comprising, including or containing" may mean that other components exist in addition to the components listed below; it may also mean "consisting of … …", i.e. including only the ingredients listed later without the presence of other ingredients.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

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

The positive progress effects of the invention are as follows:

(1) the kit and the detection method provided by the invention do not depend on a large instrument, can directly observe results by naked eyes, can realize detection under mild conditions (25-42 ℃) and are more convenient to detect. The kit and the detection method can be used for efficiently and quickly detecting/diagnosing respiratory tract pathogens (such as influenza A, influenza B, new coronavirus and the like).

(2) When the kit and the detection method are used for detecting various respiratory tract pathogens (such as influenza A, influenza B, new coronavirus and the like), cross reaction does not exist, and the specificity and the sensitivity of detection are high. At present, no patent or literature report can completely realize the simultaneous detection of novel coronavirus, influenza A and influenza B, but the application provides a kit, a detection method and the like for the first time, and the kit, the influenza A and the influenza B can completely and interactively detect the specificity of the novel coronavirus, the influenza A and the influenza B while ensuring the sensitivity. The kit and the detection method can be used for detecting and screening respiratory tract pathogens, such as rapidly distinguishing various respiratory tract pathogens including influenza A (including various subtypes), influenza B (including various subtypes) and novel coronavirus.

(3) In addition, all CRISPR-based assays currently employ respiratory tract samples such as pharyngeal swabs or nasal swabs, and the kits and assays of the invention further allow for the detection of other types of samples (e.g., anal swabs, feces, sputum supernatant, etc.) in addition to respiratory tract samples; meanwhile, the method can realize detection of each pathogen in a sample which is from living tissue and contains various pathogens.

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

Drawings

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

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

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

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

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

Fig. 6 is Cas12a fluorescence detection against influenza a virus (H1N 1) M gene target, corresponding target site sequence and crRNA sequence are shown.

Fig. 7 is a Cas12a fluorescence assay for HA gene targets of influenza b virus (Victoria subtype (Victoria line) and Yamagata subtype (Yamagata line)). The corresponding target site sequence and crRNA sequence are shown in the figure. Wherein the CRRNA SEQ ID No. 55 is a specific probe capable of detecting Yamagata subtype (mountain line).

FIG. 8 shows that Cas12a crRNA probe SEQ ID number 34 aiming at novel coronavirus S gene target can specifically detect novel coronavirus, and has no cross reaction with influenza A virus and influenza B virus. Meanwhile, SEQ ID number 34 can distinguish different types of coronaviruses.

FIG. 9 shows that Cas12a crRNA probe SEQ ID number 51 aiming at 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.

FIG. 10 shows that Cas12a crRNA probe SEQ ID number 56 aiming at influenza B virus HA gene target can specifically detect influenza B virus, and HAs no cross reaction with novel coronavirus and influenza A virus.

FIG. 11 shows RT-RPA primer screening against the new coronavirus ORF1ab gene target. Forward documents 1-4 are SEQ ID numbers 58-61, respectively, and Reverse documents 5-8 are SEQ ID numbers 62-65, respectively.

FIG. 12 shows RT-RPA primer screening for novel coronavirus S gene targets. Forward documents 1-4 are SEQ ID numbers 66-69, respectively, and Reverse documents 5-8 are SEQ ID numbers 70-73, respectively.

FIG. 13 shows RT-RPA primer screening for novel coronavirus E gene targets. Forward primers1-4 are SEQ ID numbers 74-77, respectively, and Reverse primers 5-8 are SEQ ID numbers 78-81, respectively.

FIG. 14 is RT-RPA primer screening for novel coronavirus M gene targets. Forward primers1-4 are SEQ ID numbers 82-85, respectively, and Reverse primers 5-8 are SEQ ID numbers 86-89, respectively.

FIG. 15 shows RT-RPA primer screening for novel coronavirus N gene targets. Forward primers1-4 are SEQ ID numbers 90-93, respectively, and Reverse primers 5-8 are SEQ ID numbers 94-97, respectively.

Fig. 16 is a naked eye data read based on Cas12a fluorescence detection, a novel coronavirus sample. White lighted tubes correspond to viral nucleic acid samples that produce fluorescent signals.

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

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

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

Fig. 20 shows the results of the tests performed on different types of samples.

FIG. 21 shows the results of detection of each pathogen in a sample containing multiple pathogens for an animal biopsy source.

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

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1

(1) Expression and purification of Cas12a protein

Lachnospiraceae bacteriumCas12a (LbCas 12a, amino acid sequence shown as SEQ ID NO: 57) gene from ND2006 is subjected to codon optimization (the optimized sequence is shown as SEQ ID NO: 102), and then is connected into pET28a plasmid (Thermo Fisher Scientific, Massachusetts, USA), and protein expression is regulated by a T7 promoter. The C-terminal of LbCas12a contains His6 tag for affinity purification, and TEV cleavage site is inserted between Cas12a and His6 sequences. The resulting recombinant plasmid (hereinafter referred to as pET28a-LbCas12 a) was transformed intoEscherichia coliBL21(DE3) cells. The next day, the single clones were inoculated into Luria-Bertani (LB) medium containing 50. mu.g/mL of kanamycin, and shake-cultured at 37 ℃. At OD₆₀₀When 0.8 was reached, 1 mM isoproyl- β -D-1-thiogalactopyranoside (IPTG) was added to induce protein expression, and the cells were cultured at 37 ℃ for 16 hours, and the cells were cultured by incubation at 4 ℃ for 5,000 hoursgCollected by centrifugation for 10 minutes.

The collected cells were resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, 0.5 mM phenylmethylisolfonyl fluoride (PMSF)). The expressed Cas12a protein was purified by Ni-NTA column (Qiagen, Shanghai, China). The purified protein was purified using fastprotein liquid chromatography (FPLC) using a Superdex200 filter column (GEhealthcare Life Sciences, Connecticut, USA). The purified protein was stored in storage buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 2 mM Dithiotreitol (DTT)), dispensed and stored at-80 ℃. Protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher scientific, Massachusetts, USA).

(2) Preparation of crRNA and DNA substrates

crRNA (detailed in table 2) of double-stranded DNA substrate after reverse transcription against target sites of respiratory pathogenic RNA nucleic acids (detailed in table 1) was synthesized by GenScript Biotech (Nanjing, Jiangsu, China). Substrate DNA mimicking respiratory pathogenic RNA nucleic acid was synthesized by TSINGKE Biological Technology (Shanghai, China) and cloned into pUC57 vector as a template to obtain a product mimicking isothermal amplification by PCR amplification.

TABLE 1

TABLE 2

(3) Isothermal amplification

Isothermal amplification was accomplished by a commercial RT-RPA kit (Qiaian Gene Biotech, Wuxi, Jiangsu, China). The procedure is briefly as follows: mu.L of the pathogenic nucleic acid sample, 0.4. mu.M of forward and Reverse primers (see Table 3 for details) was added to 50. mu.L of RT-RPA (Reverse transcription and polymerase mediated isothermal amplification). Finally, 14 mM magnesium acetate was added to initiate the reaction and incubated at 42 ℃ for 20 minutes to obtain RT-RPA reaction product.

TABLE 3

(4) CRISPR-Cas12 a-based fluorescence activation reaction

The fluorescence activation reaction was performed in a 20 μ L system, comprising: 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl₂100. mu.g/mL of Bone Serum Album (BSA), 0.05 unit (corresponding to 100 ng) of purified LbCas12a, 25 pM single-stranded DNA probe (purchased from general biosystems (Anhui) Ltd., model RX 012179), and Cas12a in equimolar ratio of crRNA, 2. mu.L of the RT-RPA reaction product described above (or double-stranded DNA mimic substrate after reverse transcription against a target site of a respiratory tract pathogenic RNA nucleic acid). The reaction was incubated at 37 ℃ for 30 minutes. The fluorescence signal can be read by a microplate reader or can be judged by naked eyes by a gel reader. The excitation wavelength and the emission wavelength of the fluorescent probe are 485 nm and 520 nm respectively.

(5) Results of the experiment

FIGS. 1-5 show the results of specific detection of novel coronavirus nucleic acid, wherein FIG. 1 shows the fluorescence detection of Cas12a against the novel coronavirus ORF1ab gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure, wherein the crRNA mixture (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 29-32), as can be seen from FIG. 1, both crRNA1-4 (SEQ ID NO: 29-32) and crRNA mixture (mix) can efficiently detect the corresponding gene fragment, and the signal intensity is 1 × 10⁷The above.

FIG. 2 shows fluorescence detection of Cas12a for S gene target of novel coronavirus, wherein the sequence of the corresponding target site and the sequence of crRNA are shown in the figure, wherein the mixture of crRNA (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 33-36). As shown in FIG. 2, both crRNA1-4 (SEQ ID NO: 33-36) and the mixture of crRNA (mix) can efficiently detect the corresponding gene fragment, and the signal intensity is 1 × 10⁷The signal intensity of the crRNA1, 2, 4 (SEQ ID NOS: 33, 34, 36) and the crRNA mixture (mix) was 2 × 10⁷The above.

FIG. 3 shows fluorescence detection of Cas12a against the E gene target of the novel coronavirus, and the sequence of the corresponding target site and the sequence of crRNA are shown in the figure. Wherein the crRNA mixture (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 37-40). As can be seen in FIG. 3, crRNA1-4 (SEQ ID NO: 37-40) and crRNA mixture (mix) can both efficiently detect corresponding gene fragments, and the signal intensity is 1 × 10⁷The signal intensity of both the crRNA2-4 (SEQ ID NO: 38-40) and the crRNA mixture (mix) was 2 × 10⁷The above.

FIG. 4 shows fluorescence detection of Cas12a for M gene target of novel coronavirus, the corresponding target site sequence and crRNA sequence are shown in the figure, wherein the crRNA mixture (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 41-44). As shown in FIG. 4, crRNA2 and crRNA3 (SEQ ID NO: 42, 43) can detect the corresponding gene segment, wherein the detection effect of crRNA3 (SEQ ID NO: 43) is better, and the signal intensity is 2 × 10⁷The above.

FIG. 5 shows fluorescence detection of Cas12a for N gene target of novel coronavirus, wherein the sequence of the corresponding target site and the sequence of crRNA are shown in the figure, wherein the mixture of crRNA (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 45-48). As shown in FIG. 5, the crRNA1-4 (SEQ ID NO: 45-48) and the mixture of crRNA (mix) can efficiently detect the corresponding gene fragment, and the signal intensity is mostly 1 × 10⁷Left and right or more.

FIG. 6 shows the result of specific detection of Influenza A Virus (IAV) nucleic acid, specifically Cas12a fluorescence detection of influenza A virus (H1N 1) M gene target, and the corresponding target site sequence and crRNA sequence are shown in the figure, wherein the crRNA mixture (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 49-52), and it can be seen from FIG. 6 that crRNA2-4 (SEQ ID NO: 50-52) and crRNA mixture (mix) can efficiently detect the corresponding gene segments, and the signal intensity is 5 × 10⁶Above, the signal intensity of crRNA3 (SEQ ID NO: 51) among them was as high as 2 × 10⁷The above.

Specific detection of nucleic acids of different subtypes of Influenza B Virus (IBV) (Victoria subtype (Victoria line) and Yamagata subtype (Yamagata line)), in particular Cas12a fluorescence detection against the HA gene target of influenza b virus, is shown in fig. 7. The corresponding target site sequence and crRNA sequence are shown in the figure, wherein the Yamagata subtype (mountain line) specific target site sequence is shown in SEQ ID NO: 27, and the Victoria subtype (Victoria line) specific target site sequence is shown in SEQ ID NO: 103. Wherein the crRNA mixture (mix) is obtained by mixing crRNA1-4 (SEQ ID NO: 53-56). As can be seen from FIG. 7, the crRNA1-4 (SEQ ID NOS: 53-56) and the crRNA mixture (mix) were able to efficiently detect the corresponding gene fragments, wherein the crRNA3 (SEQ ID number 55) is a specific probe capable of detecting Yamagata subtype (mountain line).

FIG. 8 shows that Cas12a crRNA probe SEQ ID number 34 aiming at novel coronavirus S gene target can specifically detect novel coronavirus, and has no cross reaction with influenza A virus and influenza B virus. Meanwhile, SEQ ID number 34 can distinguish different types of coronaviruses. FIG. 9 shows that Cas12a crRNA probe SEQ ID No. 51 aiming at M gene target of influenza A virus can specifically detect influenza A virus, and has no cross reaction with various coronaviruses including novel coronaviruses and influenza B virus. FIG. 10 shows that Cas12a crRNA probe SEQ ID No.56 aiming at HA gene target of influenza B virus can specifically detect influenza B virus, and HAs no cross reaction with various coronaviruses including novel coronaviruses and influenza A virus. As can be seen in FIGS. 8-10, the specific crRNA probe did not cross-react to the novel coronavirus, influenza A virus, and influenza B virus.

FIG. 11 shows RT-RPA primer screening for novel coronavirus ORF1ab gene targets, Forward primers1-4 and Reverse primers SEQ ID number 58-61 and Reverse primers 5-8 and SEQ ID number 62-65, respectively, which can detect signals after amplification, wherein the Forward and Reverse primers SEQ ID NO 58 and SEQ ID NO 62, 63, 64, or 65, respectively, the Forward and Reverse primers SEQ ID NO 59 and SEQ ID NO 64, respectively, the Forward and Reverse primers SEQ ID NO 60 and SEQ ID NO 62, the Forward and Reverse primers SEQ ID NO 60 and SEQ ID NO 64, respectively, and the Forward and Reverse primers SEQ ID NO 61 and SEQ ID NO 62, respectively, the fluorescence signal intensities are 0.5 × 10⁸The above. Wherein the CRRNA probe is SEQ ID NO: 32.

FIG. 12 shows RT against the S gene target of novel coronavirusForward primers1-4 are respectively SEQ ID numbers 66-69 and Reverse primers 5-8 are respectively SEQ ID numbers 70-73, the primers can basically detect signals after amplification, wherein when a Forward primer and a Reverse primer are respectively SEQ ID NO 66 and SEQ ID NO 70, when the Forward primer and the Reverse primer are respectively SEQ ID NO 66 and SEQ ID NO 72, when the Forward primer and the Reverse primer are respectively SEQ ID NO 66 and SEQ ID NO 73, when the Forward primer and the 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 is obviously higher than 0.5 × 10⁸. Wherein the CRRNA probe is SEQ ID NO. 34.

FIG. 13 shows RT-RPA primer screening for novel coronavirus E gene targets, Forward primers1-4 are respectively SEQ ID numbers 74-77, Reverse primers 5-8 are respectively SEQ ID numbers 78-81, and these primer pairs can detect signals after amplification, wherein the Forward primer and Reverse primer are respectively SEQ ID NO 74 and SEQ ID NO 78, 79, 80 or 81, the Forward primer and Reverse primer are respectively SEQ ID NO 75 and SEQ ID NO 78, 79 or 81, the Forward primer and Reverse primer are respectively SEQ ID NO 76 and SEQ ID NO 78 or 81, and the Forward primer and Reverse primer are respectively SEQ ID NO 77 and SEQ ID NO 78, 79 or 81, the fluorescence signal intensities are stronger and are both significantly higher than 0.5 × 10⁸. Wherein the probe used for crRNA is SEQ ID NO 38.

FIG. 14 is RT-RPA primer screening for novel coronavirus M gene targets. Forward primers1-4 are SEQ ID numbers 82-85, respectively, and Reverse primers 5-8 are SEQ ID numbers 86-89, respectively. The primer pairs can basically detect signals after amplification, wherein when the forward primer and the reverse primer are respectively SEQ ID NO. 82 and SEQ ID NO. 86; when the forward primer and the reverse primer are SEQ ID NO 83 and SEQ ID NO 86, respectively; when the forward primer and the reverse primer are SEQ ID NO 83 and SEQ ID NO 89, respectively; when the forward primer and the reverse primer are SEQ ID NO. 85 and SEQ ID NO. 87 respectively, the fluorescence signal intensity is stronger. Wherein the probe used for crRNA is SEQ ID NO 43.

FIG. 15 shows RT-RPA primer screening for novel coronavirus N gene targets. ForwaThe rd primers1-4 are respectively SEQ ID numbers 90-93, the Reverse primers 5-8 are respectively SEQ ID numbers 94-97, the primer pairs can detect signals after amplification, and the signal intensity is strong and is respectively 0.5 × 10⁸The above. Wherein, the adopted crRNA probe is SEQID NO. 45.

As can be seen in fig. 11-15, specific RT-RPA can amplify the target site sequence of the novel coronavirus for fluorescence detection of Cas12 a.

A nucleic acid sample of a standard strain of a novel laboratory coronavirus is adopted, concentration gradient dilution is carried out (the virus copy number after sample dilution is 3200, 650, 130, 26, 5, 1 and 0.2 virus copies respectively), and then detection is carried out to observe whether a positive signal appears. FIG. 17 shows the results of the detection of the sensitivity to the novel coronavirus, which is shown to be 5 virus copies per reaction.

A nucleic acid sample of a standard strain of laboratory influenza A virus is adopted, concentration gradient dilution is carried out (the virus copy numbers after sample dilution are 3500, 700, 140, 28, 5, 1 and 0.2 virus copies respectively), and then detection is carried out to observe whether a positive signal appears. FIG. 18 shows the results of detection of sensitivity to influenza A virus, which is 5 virus copies per reaction.

A nucleic acid sample of a standard strain of laboratory influenza B virus is adopted, concentration gradient dilution is carried out (the virus copy numbers after sample dilution are respectively 2000, 400, 80, 16, 3.2, 0.6 and 0.1 virus copy), and then detection is carried out to observe whether a positive signal appears. FIG. 19 shows the results of detection of sensitivity to influenza B virus, which is known to be 3 virus copies per reaction.

Example 2

The results obtained in the tests performed on the different types of samples are shown in FIG. 20, and the test method is the same as in example 1. It can be seen from the figure that the crRNA and the primer pair in example 1 can detect different sample types (including sputum supernatant, nasal swab, pharyngeal swab, sputum, anal swab, feces, etc.), and the detection specificity and sensitivity are high.

Example 3 detection of Each pathogen in samples containing multiple pathogens, derived from Living tissue

An Adenovirus encoding ACE2 (Adenovirus serotype 5, Ad 5) was transfected into BALB/c mice by nasal drip (IN); after 5 days, the new coronavirus was then transfected into mice by nasal drip, and samples were collected for virus titer and nucleic acid detection for 1 to 7 days (detection method same as example 1). As a result, as shown in FIG. 21, it can be seen that adenovirus was detected in addition to the new coronaviruses (N and S genes). It can be seen that the crRNA and primer pair of example 1 can be used to detect various pathogens in a sample containing multiple pathogens and derived from living tissue.

Example 4 dependence of Cas12a protein concentration on detection efficiency

The results are shown in FIG. 22. In fig. 22, the DNA substrate concentration is fixed in a and increases in B in equal proportion to Cas12a concentration.

Through research on three batches of Cas12a protein, it is found that the higher the protein concentration is, the better the protein concentration is, and the high-concentration Cas12a can inhibit the detection efficiency (fluorescence intensity) instead under the condition of fixing substrate DNA; while the inhibitory effect of high concentration of Cas12a protein on the reaction is also dependent on the concentration (content) of substrate DNA, i.e. the inhibitory effect disappears if the DNA concentration is increased. Comparing a and B, it can be concluded that an excess of Cas12a protein inhibits the reaction at very low substrate DNA concentrations.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

SEQUENCE LISTING

<110> Shanghai science and technology university

<120> kit for detecting nucleic acid of respiratory tract pathogen, detection method and application

<130>P20012763C

<160>103

<170>PatentIn version 3.5

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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

340345 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

500505 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

660665 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 825830

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 985990

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 gtccttttta 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 tgaaggagat 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

1. A combination of crRNA and a primer pair, wherein the crRNA and the primer pair are for detecting nucleic acids of respiratory pathogens, and the respiratory pathogens are COVID-19 virus, influenza a virus and influenza b virus; wherein,

the sequence of the crRNA for detecting ORF1ab gene of the COVID-19 virus is shown as SEQ ID NO. 32; the sequence of crRNA for detecting the S gene of the COVID-19 virus is shown as SEQ ID NO. 34; the sequence of the crRNA for detecting the E gene of the COVID-19 virus is shown as SEQ ID NO. 38; the sequence of the crRNA for detecting the M gene of the COVID-19 virus is shown as SEQ ID NO. 43; the sequence of the crRNA for detecting the N gene of the COVID-19 virus 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 the ORF1ab gene of the COVID-19 virus 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 COVID-19 virus 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 COVID-19 virus 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 COVID-19 virus 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 COVID-19 virus 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 NO. 99; 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 NO. 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, and the Cas protein is Cas12 a.

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

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 for non-diagnostic purposes, or in the preparation of a reagent or kit for the detection of nucleic acids of respiratory tract pathogens; wherein the respiratory tract pathogen is COVID-19 virus, influenza A virus and influenza B virus.

7. The use of claim 6, wherein the detection is a detection using a CRISPR-Cas detection system and/or RPA.

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