CN111593145B - CRISPR/Cas12 one-step nucleic acid detection method and novel coronavirus detection kit - Google Patents
CRISPR/Cas12 one-step nucleic acid detection method and novel coronavirus detection kit Download PDFInfo
- Publication number
- CN111593145B CN111593145B CN202010531548.4A CN202010531548A CN111593145B CN 111593145 B CN111593145 B CN 111593145B CN 202010531548 A CN202010531548 A CN 202010531548A CN 111593145 B CN111593145 B CN 111593145B
- Authority
- CN
- China
- Prior art keywords
- primer
- nucleic acid
- cas12
- detection
- region
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of gene detection, in particular to a CRISPR/Cas12 one-step nucleic acid detection method and a novel coronavirus detection kit, wherein the novel coronavirus detection kit comprises crRNA, cas protein, a primer, a buffer system and a single-stranded DNA reporter molecule; the primer is as follows: searching a 5'-TTTN-3' sequence in a target molecule region to be detected, designing a forward primer in 0-200bp near the 5 'region, designing a reverse primer in 25-200bp near the 3' region, and chemically modifying the obtained primer to prevent degradation of DNase or Cas12 protein after activation. The invention has the beneficial effects that: compared with the existing PCR-based nucleic acid detection technology in the market, the nucleic acid detection method and the novel coronavirus detection kit enable amplification and detection to be carried out synchronously, so that the operation is more convenient.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a one-step method nucleic acid detection method and a novel coronavirus detection kit based on CRISPR/Cas12 and RPA isothermal amplification.
Background
The world health organization names a new coronavirus, namely "2019-nCoV", 1 month 12 of 2020. The virus can be transmitted by respiratory tract droplets or by close contact; the crowd is generally susceptible, and the elderly and people with basic diseases are easy to develop severe infection. With the migration of spring traffic population, the disease evolves into a nationwide large-scale malignant infectious disease, causing huge loss to socioeconomic.
Common signs after coronavirus infection are respiratory symptoms, fever, cough, shortness of breath, dyspnea, and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. However, because the initial symptoms of the novel coronavirus are not obvious, the latent period is as long as 14 days and other relatively hidden factors, patients and carriers cannot be found in time, and the transmission is aggravated.
The rapid and effective means for early and specific diagnosis of the infected person is an important means for timely finding and isolating the infectious source, effectively curing the patient and guaranteeing the social order. This has created new demands on rapid diagnosis of pathogens, but in the event of large-scale outbreaks of virulent infectious diseases, rapid diagnosis of pathogens is very difficult, especially in areas lacking laboratory detection conditions.
Nucleic acid diagnostic techniques have the advantage of being rapid and sensitive, and being able to detect pathogens in a latent phase. At present, most laboratories adopt a PCR method to detect novel coronaviruses, the sensitivity and the specificity are good, but the time consumption is long, the instrument and the equipment are expensive, and the detection is difficult to popularize in basic-level inspection institutions. In recent years, various isothermal amplification technologies such as RPA (Recombinase Polymerase Amplification, recombinase polymerase isothermal amplification), LAMP and the like have appeared, and can be used for on-site detection, but how to rapidly and accurately detect an amplification product has been a development constraint factor. At present, RPA amplification can be combined with a probe to detect an amplification product, and the introduction of the probe can prevent the amplification reaction from proceeding, so that the method has extremely high requirements on primer probe combination screening, and is difficult to achieve the aim of rapidly coping with sudden epidemic situations. And the probe method itself does not change the essence of the single-stage amplification reaction, and does not substantially improve the detection sensitivity compared with the real-time quantitative PCR (Q-PCR) method in the current market. There is therefore a need to establish a detection technique that is applicable on-site, simple, fast, and highly sensitive, and a CRISPR/Cas detection technique developed in recent years is an ideal candidate.
CRISPR (Clustered regularly interspaced short palindromic repeats) is an adaptive immune modality against viral invasion in most bacteria and archaea. Upon viral invasion, the bacteria produce a corresponding crRNA that recognizes the viral genome, which directs the recognition and cleavage of the viral target sequence by Cas protein with endonuclease activity (CRISPR-associated proteins).
The group Zhang Feng subject found a CRISPR effector protein Cas13a, 6 months 2016. The protein is an endonuclease that binds to and degrades the target RNA under the direction of crRNA (Makarova et al 2011). An endonuclease called Leptotrichia buccalis Cas a (LbuCas 13 a) was found by Doudna et al (East-Seletsky et al 2016) at 10 months of the year to have not only cleavage activity for target RNAs but also activity for cleavage of non-target RNAs, which is called accessory cleavage. With this property, this study group used Cas13a for detection of RNA targets, but its detection limit was about 10pmol/L. This is of no application to nucleic acid detection, since most nucleic acid detection methods have detection limits on the order of amol/L (Song et al, 2013). In 2017, zhang Feng, in cooperation with Collins, combined Cas13a with a recombinase polymerase isothermal amplification technique (Recombinase polymerase amplification, RPA), developed a highly Specific and sensitive nucleic acid detection system (specificity High-Sensitivity Enzymatic Reporter UnLOCKing, SHERLOCK) (Gootenberg et al, 2017). In the detection system, after the target is transcribed and amplified by the RPA and T7RNA polymerase, the target RPA molecule can be specifically identified and cut under the guidance of corresponding crRNA, and simultaneously the nonspecific single-stranded RNase activity of LwCas13a is activated, and a fluorescent signal is generated by cutting a substrate. The introduction of RPA obviously improves the sensitivity of the system, and the detection limit of SHERLOCK on the target is as low as 2X10 3 The detection of single molecules was achieved by copies/mL (3.2 amol/L). This breakthrough research effort again demonstrates the value of CRISPR/Cas systems in the field of nucleic acid diagnostics, with the hope of having a significant impact in the public health field. However, ribonuclease (RNase) exists widely and is very stable in the environment, and the key components of the SHERLOCK system are RNA, so that the system has a severe requirement on the operation environment.
In 2015, zhang Feng et al found a class ii v CRISPR effector protein Cas12 (Zetsche et al, 2015). Cas12 can bind to target double-stranded DNA and cleave genomic DNA under the guidance of crRNA. In 2018, doudna et al found that Cas12 has activity of non-specifically cleaving single-stranded DNA (ssDNA) after specifically binding and cleaving target dsDNA, and developed a nucleic acid detection system named DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) using this activity of Cas 12. The detection combines the RPA isothermal amplification technique with Cas12, the amplification product activates the accessory cleavage activity of Cas12, the cleavage substrate generates a fluorescent signal, and single molecule level sensitivity is achieved. The system has wide application potential in the aspects of single nucleotide polymorphism analysis, cancer screening, bacterial and virus infection detection, drug resistance screening and the like. Because the target and the substrate of cas12 are DNA, the stability is strong, so the system has low requirements on experimental operation environment and can be applied to on-site rapid detection. Meanwhile, after the Cas12 is specifically activated by the target, the single-chain reporter molecule can be cut in a non-specific way, and the step has a signal amplification effect, so that the technology has higher sensitivity compared with the traditional nucleic acid detection technology such as a probe method.
Although this technique can achieve rapid and sensitive detection of nucleic acid molecules, RPA nucleic acid amplification and CRISPR/Cas12 signal detection must be performed separately in this system, since Cas12 will cleave the target DNA molecule of RPA amplification and Cas12 after activation will cleave single stranded DNA (ssDNA) molecules including primers, thus impeding the progress of the amplification reaction. But the step-by-step operation increases the complexity of the operation, and the whole detection time is longer, and aerosol is easily formed in the uncapping operation, so that the pollution of a nucleic acid laboratory is caused, and the clinical application is not facilitated.
In addition, existing CRISPR/cas12 detection systems still require the aid of a fluorescence detector and the result cannot be interpreted visually, which presents difficulties in the popularization of this technology in basic level inspection facilities.
Disclosure of Invention
The invention aims at: provides a CRISPR/Cas12 one-step nucleic acid detection method and a novel coronavirus detection kit. The nucleic acid amplification and detection are carried out simultaneously, modified primers are adopted, and corresponding crRNA is designed to enable cas12 protein cleavage sites to be located on the modified primer sites, so that amplified products cannot be degraded by cas12, and the purpose of nucleic acid molecule exponential amplification is achieved.
The technical scheme provided by the invention is as follows: provided is a CRISPR/Cas12 one-step nucleic acid detection method, comprising the following steps:
step 1) designing and synthesizing an RPA upstream primer and an RPA downstream primer for amplifying a target gene, and carrying out chemical modification on the primers for preventing DNase and Cas protein degradation after activation;
step 2) designing specific crRNA for the target gene, wherein the design principle is that the cleavage site of the target gene (specifically, the complementary strand of the target gene and the crRNA) is located on the primer sequence modified in step 1). Directly chemically synthesizing or constructing a crRNA in-vitro transcription vector according to the designed crRNA sequence, and carrying out in-vitro transcription and purification by using a T7 transcription kit; designing a single-stranded DNA reporter molecule;
step 3) mixing components comprising crRNA, cas protein, single-stranded DNA reporter molecules, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer solution and target nucleic acid to be detected to obtain a reaction mixture;
and 4) after the reaction mixture is reacted, judging the detection result.
Preferably, in the above CRISPR/Cas12 one-step nucleic acid detection method, the step 4) specifically includes: the reaction mixture is placed in a constant temperature detector at 37 ℃ for real-time signal monitoring, the reaction time is 10-30min, fluorescent signals are collected every 30s, and the detection result is interpreted.
Preferably, in the above CRISPR/Cas12 one-step nucleic acid detection method, the step 4) specifically includes: incubating the reaction mixture at 37 ℃ for 20min, and then visually observing the result through a nucleic acid test strip or blue light excitation device to judge the detection result.
Preferably, in the CRISPR/Cas12 one-step nucleic acid detection method, the Cas protein is Cas12, and the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, b°as12a, or Lb4Cas12 a.
The other technical scheme of the invention is as follows: providing a novel coronavirus detection kit comprising crRNA, cas protein, a primer, a buffer system and a single-stranded DNA reporter molecule;
the primer is as follows: searching 5'-TTTN-3' sequence in ORF1ab and N gene region of conserved region of novel coronavirus, designing forward primer in 0-200bp near 5 'region, designing reverse primer in 25-200bp near 3' region, and chemically modifying the first three and last three phosphodiester bonds of the obtained primer to prevent degradation of DNase or Cas protein after activation.
Preferably, in the above novel coronavirus detection kit, the chemical modification is: phosphothioate, 2'-O-methyl or 2' -O-methoxyethyl nucleosides modification.
Preferably, in the above novel coronavirus detection kit, the primer sequence is:
ORF1ab-F2：SEQ ID NO：12；
ORF1ab-R：SEQ ID NO：13；
N-F2：SEQ ID NO：15；
N-R：SEQ ID NO：16；
the crRNA sequence is as follows:
crRNA-ORF1ab：SEQ ID NO：17；
crRNA-nCOV-N：SEQ ID NO：18。
preferably, in the above novel coronavirus detection kit, the sequence of the single-stranded DNA reporter molecule is as follows: SEQ ID NO:19.
preferably, in the above novel coronavirus detection kit, the single-stranded DNA reporter molecule has FAM and BHQ1 groups, respectively.
Preferably, in the above novel coronavirus detection kit, the Cas protein is Cas12, and the Cas12 is one of FnCas12a, asccas 12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, bβas12a, and Lb4Cas12 a.
The invention has the beneficial effects that: 1. compared with the existing PCR-based nucleic acid detection technology on the market, the CRISPR/Cas12 one-step nucleic acid detection method has the following advantages:
A. and (3) quick: the amplification efficiency and crRNA cleavage efficiency only need 15-40min from sample to detection result according to the template to be detected, wherein the sample is processed for 5-10min, and the amplification detection reaction is carried out for 10-30min.
B. The accuracy is that: the invention has extremely high sensitivity and can detect single nucleic acid molecules; meanwhile, since crRNA is sensitive to single base mismatch, the invention has excellent specificity and can detect mutation of single nucleotide in distinction in a sample.
C. Convenient: the invention is carried out at the constant temperature of 37 ℃, so the requirement on equipment is extremely low, and the reaction can be carried out by simple constant temperature equipment even by using the body temperature, thereby being beneficial to popularization in basic-level inspection institutions.
D. Low cost: the materials and enzymes used in the invention are common, and the dosage is small, so that the invention can be used for micro-testing analysis, and therefore, the cost is low.
2. Compared with the existing CRISPR/Cas-based nucleic acid detection technology, the CRISPR/Cas12 one-step nucleic acid detection method has the following advantages:
A. the reaction is faster: the time spent from a sample to a result of the existing CRISPR/Cas technology is 1-3 hours, and the whole process of the invention only needs 15-40 minutes.
B. The operation is simpler and more convenient: because the prior art has to carry out nucleic acid amplification and detection step by step, the operation is more complicated, and the invention enables the amplification and the detection to be carried out synchronously by innovative RPA primer and crRNA design, thereby the operation is more convenient.
C. The operating environment is not polluted: in the prior art, the amplification and detection are carried out step by step, and the uncapping operation is necessary, so that aerosol is extremely easy to form, the operation environment is polluted, the false positive of the detection result is caused, and the clinical popularization is not facilitated. The invention adopts a one-step method, avoids the uncapping operation, does not form aerosol pollution, and is more beneficial to clinical application.
3. Compared with the novel coronavirus detection kit of the existing nucleic acid detection technology on the market, the novel coronavirus detection kit of the invention has the following advantages:
1) The reaction is faster: the time consumption of the existing CRISPR/Cas technology from a sample to a result is different from 1-3 hours, the whole detection process using the novel coronavirus detection kit only needs 15-40 minutes, and visual result interpretation is provided, and particularly, visual result interpretation is directly observed by naked eyes or a nucleic acid detection test strip is provided through blue light irradiation.
2) The operation is simpler and more convenient: because the prior art has to carry out nucleic acid amplification and detection step by step, the operation is more complicated, and the invention enables the amplification and the detection to be carried out synchronously by innovative RPA primer and crRNA design, thereby the operation is more convenient.
3) The operating environment is not polluted: in the prior art, the amplification and detection are carried out step by step, and the uncapping operation is necessary, so that aerosol is extremely easy to form, the operation environment is polluted, the false positive of the detection result is caused, and the clinical popularization is not facilitated. The kit can be operated by adopting a one-step method, avoids uncapping operation, does not form aerosol pollution, and is more beneficial to clinical application.
Drawings
FIG. 1 is a graph showing the detection results of fluorescent signals of example 1 in the embodiment of the present invention;
fig. 2 is a graph showing the blue excitation detection result of example 1 according to the embodiment of the present invention;
FIG. 3 is a graph showing the detection results of the nucleic acid test strip of example 1 according to the embodiment of the present invention;
FIG. 4 is a graph showing the detection result of the fluorescence signal of the ORF1ab gene of example 2 in the embodiment of the invention;
FIG. 5 is a graph showing the detection result of N-gene fluorescent signals of example 2 in the embodiment of the present invention;
FIG. 6 is a graph showing the results of blue light excitation detection of ORF1ab gene of example 2 in the embodiment of the invention;
FIG. 7 is a graph showing the results of blue excitation detection of the N gene in example 2 according to the embodiment of the present invention;
FIG. 8 is a graph showing the detection results of ORF1ab gene nucleic acid test strips of example 2 in accordance with an embodiment of the invention;
FIG. 9 is a graph showing the detection results of the N-gene nucleic acid test strip of example 2 in the embodiment of the present invention.
Detailed Description
In order to describe the technical content, the constructional features, the achieved objects and effects of the present invention in detail, the following description is made in connection with the embodiments and the accompanying drawings.
The most critical concept of the invention is as follows: RPA-mediated DNA amplification can be performed at 30-42℃and thus is free of the dependence on more precise PCR instruments. At the same time, the cleavage of the target molecule by cas12a is also performed under isothermal conditions. Based on the characteristics, the system can be used for on-site rapid detection, has great significance for rapid detection of nucleic acid, and has great value for basic clinical examination lacking laboratory conditions.
However, the existing CRISPR/Cas12 technology cannot or hardly realize the purpose of simultaneously carrying out nucleic acid amplification and detection, and the CRISPR/Cas12 and isothermal amplification-based general method for detecting nucleic acid and the novel coronavirus detection kit provided by the invention adopt modified primers and design corresponding crRNAs to enable cleavage sites of the crRNAs to be positioned on the modified primers, so that amplification products cannot be degraded by Cas12, and the purpose of amplifying nucleic acid molecules in an exponential manner is realized.
Example 1
A CRISPR/Cas12 one-step nucleic acid detection method, which is a novel and universal CRISPR/Cas 12-based one-step nucleic acid detection method and uses components such as crRNA, cas protein, primers, a buffer system, a single-stranded DNA reporter molecule, an RNase inhibitor and the like;
the following describes the procedure of the method, taking the detection of human beta Actin (ACTB) as an example:
1) Searching 5'-TTTN-3' sequence in human beta actin region, designing forward primer in 0-200bp near 5 'region and designing reverse primer in 25-200bp near 3' region. The first three and last three phosphodiester linkages of the primer were synthesized and chemically modified. The primers selected in this example are shown in Table 1 below.
TABLE 1
| Oligo name | Sequence(5'-3') |
| ACTB-F1 | TGTGGATCAGCAAGCAGGAGTATGACGAGTCC(SEQ ID NO：1) |
| ACTB-F2 | TGGATCAGCAAGCAGGAGTATGACGAGTCCGG(SEQ ID NO：2) |
| ACTB-F3 | ATCAGCAAGCAGGAGTATGACGAGTCCGGCCC(SEQ ID NO：3) |
| ACTB-F4 | GATGTGGATCAGCAAGCAGGAGTATGACGAGT(SEQ ID NO：4) |
| ACTB-R1 | CAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAAT(SEQ ID NO：5) |
| ACTB-R2 | CATCTTGTTTTCTGCGCAAGTTAGGTTTTGTC(SEQ ID NO：6) |
| ACTB-R3 | TGCCAATCTCATCTTGTTTTCTGCGCAAGTTA(SEQ ID NO：7) |
| ACTB-R4 | AAGCCATGCCAATCTCATCTTGTTTTCTGCGC(SEQ ID NO：8) |
2) Design and chemical synthesis of crRNA: 17-25N was selected downstream of the 5'-TTTN-3' sequence in step 1) as crRNA sequence. The crrnas selected in this example are shown in table 2 below.
TABLE 2
3) Reporter molecule design and chemical synthesis: a12-base random single-stranded DNA molecule with FAM and BHQ1 groups at two ends is used for fluorescence signal detection and blue light excitation visual detection. The single-stranded reporter molecules selected for use in this example are shown in Table 3 below.
TABLE 3 Table 3
| Single-stranded DNA reporter | 5’-FAM-ACGTCTTAGTCC-BHQ1-3’(SEQ ID NO：10) |
4) Healthy volunteer samples were taken with throat swabs and added to quick lysates formulated as follows in Table 4, and lysed at 80℃for 5 minutes.
TABLE 4 Table 4
| Reagent(s) | Concentration of |
| Guanidine | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
5) 2 mu L of the lysate in the step 4) is added into a primer combination, crRNA, lbacAS12a, a single-stranded DNA reporter molecule, an RPA upstream primer and an RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer2.1 and RNase Inhibitor. The reaction mixture is placed in a constant temperature fluorescent signal detector at 37 ℃, signals are collected every 30s, and the reaction mixture is continuously detected for two hours. The result is shown in fig. 1, where the signal gradually rises over time.
The proportions of the components used in the present invention are shown in Table 5 below.
TABLE 5
6) The primer combination with the highest amplification efficiency in the step 5) is added into the reaction mixture in the table 5, the reaction is carried out for 20min at the constant temperature of 37 ℃, then the reactant is excited by blue light and the result is judged by naked eyes, and the mobile phone is used for photographing, and the result is shown in figure 2. FIG. 2 positive PC is very dark.
In this embodiment, the CRISPR/Cas12 one-step nucleic acid detection method for detecting human β Actin (ACTB) is preferably as follows:
1) The primers selected are shown in Table 6 below.
TABLE 6
| Oligo name | Sequence(5′-3′) |
| ACTB-F1 | TGTGGATCAGCAAGCAGGAGTATGACGAGTCC(SEQ ID NO：1) |
| ACTB-R1 | CAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAAT(SEQ ID NO：5) |
2) Design and chemical synthesis of crRNA: 17-25N was selected downstream of the 5'-TTTN-3' sequence in step 1) as crRNA sequence. The crrnas selected in this example are shown in table 7 below.
TABLE 7
3) Reporter molecule design and chemical synthesis: a12-base random single-stranded DNA molecule with FAM and Biotin groups at two ends is used for visual detection of a nucleic acid test strip. The single-stranded reporter molecules selected for use in this example are shown in Table 8 below.
TABLE 8
| Single-stranded DNA reporter | 5’-FAM-ACGTCTTAGTCC-Biotin-3’(SEQ ID NO：10) |
4) Healthy volunteer samples were taken with throat swabs and added to quick lysates formulated as in table 9, and lysed at 80 ℃ for 5 minutes.
TABLE 9
| Reagent(s) | Concentration of |
| Guanidine | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
5) 2 mu L of the lysate in the step 2) is added into a primer combination, crRNA, lbacAS12a, a single-stranded DNA reporter molecule, an RPA upstream primer and an RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer2.1 and RNase Inhibitor. The reaction mixture was incubated at 37℃for 20min. The proportions of the components in this example are shown in Table 10.
Table 10
6) In the step, 80 mu L of sample diluent is added, chromatography is carried out for 2min at room temperature, and the result is judged by naked eyes, as shown in FIG. 3, NTC is negative, PC is positive, positive is developed on a T line, a negative sample is not developed on a detection line, and in the step, twit Dx company Hybridetect 1lateralflow strips (Milenia) or other nucleic acid test strips with similar characteristics can be adopted.
Example 2
A novel coronavirus detection kit, comprising crRNA, cas protein, a primer, a buffer system and a single-stranded DNA reporter molecule; the comparison of the optimal primer combinations for the novel coronavirus detection kit is determined as follows:
1) Searching 5'-TTTN-3' sequence in the conserved region ORF1ab and N gene region of the novel coronavirus, designing forward primer in 0-200bp near 5 'region, and designing reverse primer in 25-200bp near 3' region. The first three and last three phosphodiester linkages of the primer were synthesized and chemically modified. The primers selected in this example are shown in Table 11 below.
TABLE 11
| Oligo name | Sequence(5′-3′) |
| ORF1ab-F1 | TTGCCTGGCACGATATTACGCACAACTAATGGT(SEQ ID NO：11) |
| ORF1ab-F2 | ATGGTGACTTTTTGCATTTCTTACCTAGAGTTT(SEQ ID NO：12) |
| ORF1ab-R | AAGTCAGTGTACTCTATAAGTTTTGATGGTGTGT(SEQ ID NO：13) |
| N-F1 | CCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAAT(SEQ ID NO：14) |
| N-F2 | CAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAAT(SEQ ID NO：15) |
| N-R | GTTGGCCTTTACCAGACATTTTGCTCTCAAGCTGG(SEQ ID NO：16) |
2) Design and chemical synthesis of crRNA: 17-25N was selected downstream of the 5'-TTTN-3' sequence in step 1) as crRNA sequence. The crrnas selected in this example are shown in table 12 below.
Table 12
| crRNA-ORF1ab | UAAUUUCUACUAAGUGUAGAUgugcaguugguaacaucuguuac(SEQ ID NO：17) |
| crRNA-nCOV-N | UAAUUUCUACUAAGUGUAGAUcugcugcuugacagauugaacc(SEQ ID NO：18) |
3) Reporter molecule design and chemical synthesis: a12-base random single-stranded DNA molecule with FAM and BHQ1 groups at two ends is used for fluorescence signal detection and blue light excitation visual detection. The single-stranded reporter molecules selected for use in this example are shown in Table 13 below.
TABLE 13
| Single-stranded DNA reporter | 5’-FAM-ACGTCTTAGTCC-BHQ1-3’(SEQ ID NO：19) |
4) Healthy volunteer samples were taken with throat swabs and added to the quick lysates formulated in table 14 below, followed by addition of new coronavirus particles and lysis at 80 ℃ for 5 minutes.
TABLE 14
| Reagent(s) | Concentration of |
| Guanidine | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
5) 2 mu L of the lysate in the step 4) is added into a primer combination, crRNA, lbacAS12a, a single-stranded DNA reporter molecule, an RPA upstream primer and an RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer2.1 and RNase Inhibitor. The reaction mixture is placed in a constant temperature fluorescent signal detector at 37 ℃, signals are collected every 30s, and the reaction mixture is continuously detected for two hours. ORF1ab results are shown in FIG. 4, and N gene results are shown in FIG. 5, showing that positive sample signals gradually increase with time and negative sample signals do not change with time.
The proportions of the components in the novel coronavirus detection kit of the present invention are shown in Table 15 below.
TABLE 15
6) The primer combination with the highest amplification efficiency in the step 5) is added into the reaction mixture in the table 15, the reaction mixture is placed at the constant temperature of 37 ℃ for 20min, then the reactant is excited by blue light and the result is judged by naked eyes, and the mobile phone is used for photographing, so that the positive sample shows a signal and the negative sample shows no signal as shown in the figures 6 and 7.
Preferably, the novel coronavirus detection kit comprises crRNA, cas protein, primers, buffer system, and single-stranded DNA reporter; the manufacturing method comprises the following steps:
1) Searching 5'-TTTN-3' sequence in ORF1ab and N gene region of conserved region of novel coronavirus, designing forward primer in 0-200bp near 5 'region, designing reverse primer in 25-200bp near 3' region, and chemically modifying the first three and last three phosphodiester bonds of the obtained primer. Preventing degradation of dnase and Cas proteins after activation. Such chemical modifications include, but are not limited to, phosphothioate, 2'-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides modifications.
The primer sequences selected in this example are shown in Table 16 below.
Table 16
| Oligo name | Sequence(5′-3′) |
| ORF1ab-F2 | TTGCCTGGCACGATATTACGCACAACTAATGGT(SEQ ID NO：12) |
| ORF1ab-R | AAGTCAGTGTACTCTATAAGTTTTGATGGTGTGT(SEQ ID NO：13) |
| N-F2 | AGTTCAAGAAATTCAACTCCAGGCAGCAGTAGG(SEQ ID NO：15) |
| N-R | CCTTTACCAGACATTTTGCTCTCAAGCTGGTTC(SEQ ID NO：16) |
2) Specific crrnas were designed for target genes. The design principle is that the cleavage site of the target gene (specifically, the complementary strand of the target gene and crRNA) is located on the primer sequence modified in the step 1). Directly chemically synthesizing or constructing a crRNA in-vitro transcription vector according to the designed crRNA sequence, and carrying out in-vitro transcription and purification by using a T7 transcription kit;
17-25N was selected downstream of the 5'-TTTN-3' sequence in step 1) as crRNA sequence. The crRNA sequence in this example is:
crRNA-ORF1ab：SEQ ID NO：17；
crRNA-nCOV-N：SEQ ID NO：18；
3) Single-stranded DNA reporter design and chemical synthesis: a12-base random single-stranded DNA molecule with FAM and Biotin groups at two ends is used for visual detection of a nucleic acid test strip. The single-stranded reporter molecules selected for use in this example are shown in Table 8.
4) Healthy volunteer samples were taken with throat swabs and added to the quick lysates of the formulations of Table 14, and lysed at 80℃for 5 minutes.
5) 2 mu L of the lysate in the step 4) is added into a primer combination, crRNA, lbacAS12a, a single-stranded DNA reporter molecule, an RPA upstream primer and an RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer2.1 and RNase Inhibitor. The reaction mixture was incubated at 37℃for 20min. The proportions of the components in this example are shown in Table 10.
6) Taking 20 mu L of the reaction solution in the step, adding 80 mu L of sample diluent, carrying out chromatography for 2min at room temperature, and judging the result by naked eyes, wherein as shown in FIG. 8 (1 ab gene) and FIG. 9 (N gene), PC is positive, positive is developed on a T line, NTC negative samples are not developed on a detection line, NTC is negative, and negative samples are not developed. In this step, a hybridization detection 1lateralflow strips (Milenia) or other nucleic acid strips having similar properties may be used by Twit-Twit company.
The use of the novel coronavirus detection kit described above in this embodiment:
1) Clinical samples were taken with throat swabs and added to quick lysates of the formulation shown in Table 14, and lysed at 80℃for 5 minutes.
2) 2 mu L of the lysate in the step 2) is added into a primer combination, crRNA, lbacAS12a, a single-stranded DNA reporter molecule, an RPA upstream primer and an RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer2.1 and RNase Inhibitor. The reaction mixture was incubated at 37℃for 20min. The proportions of the components in this example are shown in Table 10.
3) Taking 20 mu L of the reaction solution in the step, adding 80 mu L of sample diluent, carrying out chromatography for 2min at room temperature, judging and reading the result by naked eyes, wherein a positive sample is colored at a detection line T, and a negative sample is not colored at the T line. In this step, a hybridization detection 1lateralflow strips (Milenia) or other nucleic acid strips having similar properties may be used by Twit-Twit company.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.
SEQUENCE LISTING
<110> sub-energy biotechnology (Shenzhen Co., ltd.)
<120> one-step CRISPR/Cas12 nucleic acid detection method and novel coronavirus detection kit
<130> 2020
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 32
<212> DNA
<213> artificial sequence
<400> 1
tgtggatcag caagcaggag tatgacgagt cc 32
<210> 2
<211> 32
<212> DNA
<213> artificial sequence
<400> 2
tggatcagca agcaggagta tgacgagtcc gg 32
<210> 3
<211> 32
<212> DNA
<213> artificial sequence
<400> 3
atcagcaagc aggagtatga cgagtccggc cc 32
<210> 4
<211> 32
<212> DNA
<213> artificial sequence
<400> 4
gatgtggatc agcaagcagg agtatgacga gt 32
<210> 5
<211> 34
<212> DNA
<213> artificial sequence
<400> 5
caggcagcag taggggaact tctcctgcta gaat 34
<210> 6
<211> 32
<212> DNA
<213> artificial sequence
<400> 6
catcttgttt tctgcgcaag ttaggttttg tc 32
<210> 7
<211> 32
<212> DNA
<213> artificial sequence
<400> 7
tgccaatctc atcttgtttt ctgcgcaagt ta 32
<210> 8
<211> 32
<212> DNA
<213> artificial sequence
<400> 8
aagccatgcc aatctcatct tgttttctgc gc 32
<210> 9
<211> 22
<212> DNA
<213> artificial sequence
<400> 9
cggtggacga tggaggggcc gg 22
<210> 10
<211> 12
<212> DNA
<213> artificial sequence
<400> 10
acgtcttagt cc 12
<210> 11
<211> 33
<212> DNA
<213> artificial sequence
<400> 11
ttgcctggca cgatattacg cacaactaat ggt 33
<210> 12
<211> 33
<212> DNA
<213> artificial sequence
<400> 12
atggtgactt tttgcatttc ttacctagag ttt 33
<210> 13
<211> 34
<212> DNA
<213> artificial sequence
<400> 13
aagtcagtgt actctataag ttttgatggt gtgt 34
<210> 14
<211> 35
<212> DNA
<213> artificial sequence
<400> 14
ccaggcagca gtaggggaac ttctcctgct agaat 35
<210> 15
<211> 34
<212> DNA
<213> artificial sequence
<400> 15
caggcagcag taggggaact tctcctgcta gaat 34
<210> 16
<211> 35
<212> DNA
<213> artificial sequence
<400> 16
gttggccttt accagacatt ttgctctcaa gctgg 35
<210> 17
<211> 12
<212> DNA
<213> artificial sequence
<400> 17
acgtcttagt cc 12
<210> 18
<211> 43
<212> RNA
<213> artificial sequence
<400> 18
uaauuucuac uaaguguaga ucugcugcuu gacagauuga acc 43
<210> 19
<211> 44
<212> RNA
<213> artificial sequence
<400> 19
uaauuucuac uaaguguaga ugugcaguug guaacaucug uuac 44
Claims (8)
1. A CRISPR/Cas12 one-step nucleic acid detection method for non-disease diagnosis purposes, comprising the steps of:
step 1) designing and synthesizing an RPA upstream primer and an RPA downstream primer for amplifying a target gene, and carrying out chemical modification on the primers for preventing DNase and Cas protein degradation after activation;
the method comprises the following steps: searching a 5'-TTTN-3' sequence in a target gene, designing a forward primer within 0-200bp of a 5'-TTTN-3' region close to the 5 'region, designing a reverse primer within 25-200bp of a 5' -TTTN-3 'region close to the 3' region, synthesizing and chemically modifying the first three and the last three phosphodiester bonds of the primer;
the chemical modification is as follows: thio modification, 2 '-O-methylation modification or 2' -O-methoxyethyl modification;
step 2) designing a single-stranded DNA reporter molecule aiming at the target gene and designing specific crRNA; the method comprises the following steps: selecting 17-25nt as crRNA sequence at the downstream of 5'-TTTN-3' sequence in step 1), and designing single-stranded DNA reporter molecule;
step 3) mixing components comprising crRNA, cas protein, single-stranded DNA reporter molecules, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer solution and target nucleic acid to be detected to obtain a reaction mixture; the Cas protein is Cas12;
and 4) after the reaction mixture is reacted, judging the detection result.
2. The method for detecting the CRISPR/Cas12 one-step nucleic acid for non-disease diagnosis purposes according to claim 1, wherein the step 4) specifically comprises: the reaction mixture is placed in a constant temperature detector at 37 ℃ for real-time signal monitoring, the reaction time is 10-30min, fluorescent signals are collected every 30s, and the detection result is interpreted.
3. The method for detecting the CRISPR/Cas12 one-step nucleic acid for non-disease diagnosis purposes according to claim 1, wherein the step 4) specifically comprises: incubating the reaction mixture at 37 ℃ for 20min, and then visually observing the result through a nucleic acid test strip or blue light excitation device to judge the detection result.
4. The CRISPR/Cas12 one-step nucleic acid detection method for non-disease diagnosis purposes according to claim 1, wherein the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a or Lb4Cas12 a.
5. The novel coronavirus detection kit is characterized by comprising crRNA, cas protein, a primer, a buffer system and a single-stranded DNA reporter molecule;
the primer is as follows: searching a 5'-TTTN-3' sequence in a conserved region ORF1ab and an N gene region of the novel coronavirus, designing a forward primer in a region 0-200bp close to a 5 'region of the 5' -TTTN-3 'sequence, designing a reverse primer in a region 25-200bp close to a 3' region of the 5'-TTTN-3' sequence, and chemically modifying the first three and the last three phosphodiester bonds of the obtained primers to prevent degradation of DNase or Cas protein after activation; the Cas protein is Cas12;
the chemical modification is as follows: thio modification, 2 '-O-methylation modification or 2' -O-methoxyethyl modification;
the primer sequence is as follows:
ORF1ab-F2：SEQ ID NO：12；
ORF1ab-R：SEQ ID NO：13；
N-F2 ：SEQ ID NO：15；
N-R：SEQ ID NO：16；
the crRNA sequence is as follows:
crRNA-ORF1ab ：SEQ ID NO：17；
crRNA-nCOV-N ：SEQ ID NO：18。
6. the novel coronavirus detection kit of claim 5, wherein the sequence of the single stranded DNA reporter molecule is as follows: SEQ ID NO:19.
7. the novel coronavirus detection kit according to claim 6, wherein the single-stranded DNA reporter molecule has FAM and BHQ1 groups at both ends, respectively.
8. The novel coronavirus detection kit of claim 5, wherein the Cas12 is one of FnCas12a, asCas12a, lbCas12a, lb5Cas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, boCas12a, or Lb4Cas12 a.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010531548.4A CN111593145B (en) | 2020-06-11 | 2020-06-11 | CRISPR/Cas12 one-step nucleic acid detection method and novel coronavirus detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010531548.4A CN111593145B (en) | 2020-06-11 | 2020-06-11 | CRISPR/Cas12 one-step nucleic acid detection method and novel coronavirus detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111593145A CN111593145A (en) | 2020-08-28 |
| CN111593145B true CN111593145B (en) | 2023-05-30 |
Family
ID=72188432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010531548.4A Active CN111593145B (en) | 2020-06-11 | 2020-06-11 | CRISPR/Cas12 one-step nucleic acid detection method and novel coronavirus detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111593145B (en) |
Families Citing this family (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507716A (en) * | 2020-11-16 | 2022-05-17 | 北京迅识科技有限公司 | Method for detecting target nucleic acid in sample |
| CN112813195B (en) * | 2020-12-09 | 2023-04-18 | 广州市第一人民医院（广州消化疾病中心、广州医科大学附属市一人民医院、华南理工大学附属第二医院） | Novel quantitative detection kit for coronavirus nucleic acid based on micro-droplet digital analysis |
| CN112458210B (en) * | 2020-12-09 | 2021-12-10 | 上海伯杰医疗科技有限公司 | Gene conserved sequence, primer probe combination, kit and application for detecting new coronavirus |
| CN112553378B (en) * | 2020-12-29 | 2022-02-18 | 广州白云山拜迪生物医药有限公司 | Reagent and kit for detecting 2019-nCoV and application |
| CN112695073A (en) * | 2020-12-30 | 2021-04-23 | 上海理工大学 | CRISPR/Cas12a one-step nucleic acid detection method |
| WO2022148450A1 (en) * | 2021-01-08 | 2022-07-14 | Wuhan University | Compositions and methods for instant nucleic acid detection |
| CN113151584A (en) * | 2021-01-11 | 2021-07-23 | 南通大学 | SARS-CoV-2 detection kit and detection method |
| CN112725539B (en) * | 2021-02-24 | 2023-05-23 | 遵义医科大学珠海校区 | RPA/Cas12a/IF kit for respiratory syncytial virus and detection method thereof |
| CN112980929B (en) * | 2021-04-13 | 2021-07-20 | 广州海思医疗科技有限公司 | Kit for rapidly detecting HLA-B1502 |
| CN113005230B (en) * | 2021-04-28 | 2024-02-20 | 南通海关综合技术中心(江苏国际旅行卫生保健中心南通分中心、南通海关口岸门诊部) | Asia-type Zika virus RT-RPA/CRISPR-Cas12a detection kit and detection method thereof |
| CN113403424B (en) * | 2021-05-28 | 2024-01-09 | 华南理工大学 | Method and kit for rapidly detecting novel coronaviruses and mutant strains based on CRISPR/Cas12a technology |
| CN113373223A (en) * | 2021-06-01 | 2021-09-10 | 武汉大学 | CRISPR-Cas system for FLT3-D835Y mutation detection and application thereof |
| CN113373264B (en) * | 2021-06-10 | 2022-04-12 | 安徽医科大学 | Novel coronavirus double-target rapid detection method and kit based on CRISPR/Cas system |
| CN113355459B (en) * | 2021-06-10 | 2022-04-01 | 安徽医科大学 | Method and kit for detecting and screening N501Y mutation of new coronavirus |
| CN113308575B (en) * | 2021-06-10 | 2022-04-01 | 安徽医科大学 | Method and kit for detecting and screening N439K mutation of new coronavirus |
| CN113215164B (en) * | 2021-06-21 | 2022-06-07 | 南方医科大学 | Method for detecting novel coronavirus variant and subtype |
| CN113512548A (en) * | 2021-07-16 | 2021-10-19 | 江门市妇幼保健院 | Novel coronavirus detection kit based on CRISPR-Cas12a system and application thereof |
| CN113684314B (en) * | 2021-08-27 | 2022-10-28 | 中国农业科学院作物科学研究所 | Kit and method for detecting RBSDV (radial basis sequence-associated protein sequence) based on CRISPR-Cas (clustered regularly interspaced short palindromic repeats) system |
| CN114410752A (en) * | 2022-01-24 | 2022-04-29 | 华南师范大学 | CRISPR-Cas nucleic acid detection kit based on light control and detection method |
| CN114634972B (en) * | 2022-05-19 | 2022-08-26 | 舜丰生物科技(海南)有限公司 | Method for detecting nucleic acid by using Cas enzyme |
| CN115927747B (en) * | 2022-08-26 | 2023-11-10 | 深圳市卓润生物科技有限公司 | Novel coronavirus detection reagent, detection kit and detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894557A (en) * | 2019-09-20 | 2020-03-20 | 武汉大学 | CRRNA (crribonucleic acid) for detecting African swine fever virus based on CRISPR (clustered regularly interspaced short palindromic repeats) mode and kit |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10253365B1 (en) * | 2017-11-22 | 2019-04-09 | The Regents Of The University Of California | Type V CRISPR/Cas effector proteins for cleaving ssDNAs and detecting target DNAs |
| CN110066865B (en) * | 2019-04-30 | 2023-03-03 | 常州桐树生物科技有限公司 | Detection method and probe of specific nucleic acid fragment based on CRISPR-Cas13a |
| CN110184329A (en) * | 2019-05-31 | 2019-08-30 | 华南理工大学 | A kind of one-step method nucleic acid detection method and kit based on CRISPR/Cas and constant-temperature amplification |
| CN111057798A (en) * | 2020-01-20 | 2020-04-24 | 复旦大学附属华山医院 | LAMP primer combination and kit for detecting novel coronavirus |
| CN111187856B (en) * | 2020-02-13 | 2020-11-13 | 上海科技大学 | Cpf1 kit for rapid detection of new coronavirus nucleic acid and preparation method and application thereof |
| CN111118226B (en) * | 2020-03-25 | 2021-04-02 | 北京微未来科技有限公司 | Novel coronavirus whole genome capture method, primer group and kit |
-
2020
- 2020-06-11 CN CN202010531548.4A patent/CN111593145B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110894557A (en) * | 2019-09-20 | 2020-03-20 | 武汉大学 | CRRNA (crribonucleic acid) for detecting African swine fever virus based on CRISPR (clustered regularly interspaced short palindromic repeats) mode and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111593145A (en) | 2020-08-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111593145B (en) | CRISPR/Cas12 one-step nucleic acid detection method and novel coronavirus detection kit | |
| CN111187856B (en) | Cpf1 kit for rapid detection of new coronavirus nucleic acid and preparation method and application thereof | |
| CN111560482B (en) | Detection method based on CRISPR/Cas and nucleic acid test paper and human papilloma virus detection kit | |
| CN111394490B (en) | CRISPR-Cas12a detection primer group for eupolyphaga and application thereof | |
| JP6739339B2 (en) | Covered sequence-converted DNA and detection method | |
| JP6773651B2 (en) | Sequence conversion and signal amplification DNA having a poly DNA spacer sequence and detection methods using them | |
| Kikuchi et al. | Split dapoxyl aptamer for sequence-selective analysis of nucleic acid sequence based amplification amplicons | |
| CN111500792A (en) | Novel coronavirus detection kit | |
| Wang et al. | CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples | |
| Newsome et al. | Presence of bacterial phage-like DNA sequences in commercial Taq DNA polymerase reagents | |
| CN111521781B (en) | Detection kit for SARS-CoV-2 nucleic acid of new coronary pneumonia virus and detection method thereof | |
| JP2018504898A (en) | Detection method based on signal-amplified DNA cascade reaction with target extension | |
| Mota et al. | Recombinase polymerase amplification in the molecular diagnosis of microbiological targets and its applications | |
| KR101158934B1 (en) | Nucleic acid detection | |
| Agrawal et al. | Rapid detection of SARS-CoV-2 with Cas13 | |
| WO2023207909A1 (en) | Crispr-based nucleic acid detection kit and use thereof | |
| CN115838834A (en) | Human rhinovirus type A and type C detection system based on RT-RAA and CRISPR/Cas12a | |
| CN113337638A (en) | Method and kit for detecting novel coronavirus (SARS-CoV-2) | |
| CN111676272A (en) | Cas12 a/crRNA-based food-borne pathogenic bacteria detection method | |
| CN114214455B (en) | Quick quantitative primer probe for hepatitis B virus DNA and CRISPR/Cas12b detection system thereof | |
| CN114480732B (en) | Primer pair, crRNA, detection method and kit for detecting and identifying hepatitis C virus | |
| Artika et al. | Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis. Genes 2022, 13, 2387 | |
| Sahel et al. | CRISPR/Cas System: Applications in Diagnosis of Infectious Diseases | |
| CN116024316A (en) | Nucleic acid test strip detection method capable of replacing CRISPR system and novel coronavirus detection kit | |
| CN116479093A (en) | Rhinoceros nucleic acid rapid detection method and detection kit based on CRISPR fluorescence method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Tang Guanghui Inventor after: Xue Liang Inventor after: Gong Jiaojiao Inventor after: Dai Wen Jun Inventor before: Tang Guanghui Inventor before: Xue Liang Inventor before: Dai Wen Jun |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |