CN111593145A - 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
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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 a region 0-200bp near the 5 'region, designing a reverse primer in a region 25-200bp near the 3' region, and chemically modifying the obtained primer to prevent degradation of the DNA enzyme or activated Cas12 protein. The invention has the beneficial effects that: compared with the existing PCR-based nucleic acid detection technology on the market, the nucleic acid detection method and the novel coronavirus detection kit can synchronously carry out amplification and detection, so that the operation is more convenient and fast.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a one-step nucleic acid detection method based on CRISPR/Cas12 and RPA isothermal amplification and a novel coronavirus detection kit.
Background
On 12.1.2020, the world health organization names a new type of coronavirus, namely "2019-nCoV". The virus can be transmitted by respiratory droplets or by intimate contact; the population is generally susceptible, and the elderly and people with basic diseases are easy to develop severe infection. With the migration of the spring-carried population, the disease develops into a nationwide large-scale malignant infectious disease, which causes huge losses to the social economy.
The common signs after coronavirus infection are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. However, the novel coronavirus has relatively hidden factors such as unobvious initial symptoms, 14-day incubation period and the like, so that patients and carriers cannot find the coronavirus in time, and the spread is aggravated.
The rapid and effective means for early and specific diagnosis of the infected person is an important means for timely finding and isolating the infection source, effectively curing the patient and guaranteeing the social order. This has raised new demands for rapid diagnosis of pathogens, but in large-scale outbreaks of virulent infectious diseases, rapid diagnosis of pathogens is very difficult, especially in certain areas lacking laboratory detection conditions.
Nucleic acid diagnostic techniques have the advantage of being rapid and sensitive and are capable of detecting pathogens in the latent phase. At present, most laboratories adopt a PCR method to detect the novel coronavirus, the sensitivity and the specificity are good, but the time consumption is long, and the price of instruments and equipment is high, so that the popularization of the instruments and the equipment in basic level inspection institutions is difficult. In recent years, various isothermal Amplification technologies such as RPA (recombinase polymerase isothermal Amplification) and LAMP have appeared, and can be used for field detection, but how to rapidly and accurately detect an Amplification product is always a factor of the development restriction. At present, RPA amplification can be combined with a probe to detect an amplification product, and the introduction of the probe can block the progress of an amplification reaction, so that the method has extremely high requirements on primer and probe combination screening, and is difficult to achieve the aim of rapidly coping with the outbreak epidemic situation. And the probe method does not change the essence of the single-stage amplification reaction, and compared with the real-time quantitative PCR (Q-PCR) method on the current market, the detection sensitivity is not substantially improved. Therefore, it is urgently needed to establish a detection technology which can be applied to the field, is simple, quick and high-sensitivity, and the CRISPR/Cas detection technology developed in recent years becomes an ideal candidate scheme.
Crispr (clustered regulated short palindromic repeats) is an acquired immune means against viral invasion in most bacteria and archaea. When a virus invades, the bacteria produce corresponding crRNA capable of recognizing the viral genome, which guides Cas protein (CRISPR-associated proteins) with endonuclease activity to recognize and cleave the viral target sequence.
In 2016, month 6, the Zhang Feng task group discovered a CRISPR effector protein Cas13 a. The protein is an endonuclease that binds to and degrades a target RNA under crRNA guidance (Makarova et al, 2011). Doudna et al (East-Seletsky et al, 2016) discovered in the same year 10 that an endonuclease called Leptotrichia buccalis Cas13a (LluCas 13a) has not only cleavage activity for target RNA, but also cleavage activity for non-target RNA, this non-specific cleavage being referred to as accessory cleavage. Using this property, Cas13a was used by the study group to detect RNA targets, but with a detection limit of about 10 pmol/L. This is of no practical value for nucleic acid detection, since most nucleic acid detection methods have detection limits on the order of amol/L (Song et al, 2013). In 2017, by the cooperation of Zhang Feng and Collins, Cas13a was combined with Recombinase polymerase isothermal amplification (RPA) to develop a nucleic acid detection system with High specificity and Sensitivity (Specific High-Sensitivity enzymatic reporter UnLOCKing, SHELLOK) (Goodenberg et al, 2017). In the detection system, after the target is subjected to transcription and amplification by RPA and T7RNA polymerase, under the guidance of corresponding crRNA, the target RPA molecule can be specifically identified and cut, the non-specific single-stranded RNA enzyme activity of LwCas13a is simultaneously activated, and the substrate is cutA fluorescent signal is generated. The introduction of RPA significantly improves the sensitivity of the system, and the detection limit of SHERELOCK on the target is as low as 2X103copies/mL (3.2amol/L), single molecule detection was achieved. The breakthrough research result proves the value of the CRISPR/Cas system in the field of nucleic acid diagnosis again, and is expected to have great influence in the field of public health. However, the ribonuclease (RNase) in the environment is widely existed and very stable, and the key components of the SHERLOCK system are all RNA, so the system has severe requirements on the operating environment.
In 2015, zhanfeng et al found a class ii type v CRISPR effector protein Cas12(Zetsche et al, 2015). Cas12 can bind to target double-stranded DNA and cleave genomic DNA under crRNA guidance. In 2018, Doudna et al found that Cas12 has the activity of non-specifically cleaving single-stranded DNA (ssDNA) after it specifically binds to and cleaves the target dsDNA, and developed a nucleic acid detection system named DETECTR (DNA Endonuclease Targeted CRISPR Trans reporter) using this activity of Cas 12. The DETECTR combines the RPA isothermal amplification technology with the Cas12, the amplification product activates the accessory cleavage activity of the Cas12, the cleavage substrate generates a fluorescent signal, and single-molecule-level sensitivity is realized. The system has wide application potential in the aspects of single nucleotide polymorphism analysis, cancer screening, bacterial and viral infection detection, drug resistance screening and the like. As the target and the substrate of cas12 are DNA and have strong stability, the system has low requirement on experimental operation environment and can be applied to on-site rapid detection. Meanwhile, after the Cas12 is specifically activated by the target, the single-stranded reporter molecule can be nonspecifically cut, and the step has a signal amplification effect, so that the technology has higher sensitivity compared with the traditional nucleic acid detection technologies such as a probe method and the like.
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 cleaves the RPA amplified target DNA molecule and activated Cas12 cleaves the single-stranded DNA (ssdna) molecule including the primer, thereby hindering the amplification reaction. But the step-by-step operation increases the complexity of the operation, and the whole detection consumes longer time, and meanwhile, the uncovering operation is easy to form aerosol, so that the pollution of a nucleic acid laboratory is caused, and the clinical application is not facilitated.
In addition, the existing CRISPR/cas12 detection system still needs a fluorescence detector, and the result can not be visually interpreted, which brings difficulty to the popularization of the technology in the primary inspection institution.
Disclosure of Invention
The invention aims to: provides a CRISPR/Cas12 one-step nucleic acid detection method and a novel coronavirus detection kit. The nucleic acid amplification and detection are simultaneously carried out, modified primers are adopted, and corresponding crRNA is designed, so that the cas12 protein cutting site is positioned on the modified primer site, an amplification product is not degraded by cas12, and the purpose of nucleic acid molecule exponential amplification is further realized.
The invention provides a technical scheme that: a CRISPR/Cas12 one-step nucleic acid detection method is provided, which comprises the following steps:
step 1) designing and synthesizing an RPA upstream primer and a RPA downstream primer for amplifying a target gene, and carrying out chemical modification on the primers for preventing degradation of DNase and activated Cas protein;
step 2) designing specific crRNA aiming at the target gene, wherein the design principle is that the cleavage site of the crRNA on the target gene (specifically the complementary strand of the target gene and the crRNA) is positioned 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 performing in-vitro transcription and purification by using a T7 transcription kit; designing a single-stranded DNA reporter molecule;
step 3) mixing components including crRNA, Cas protein, single-stranded DNA reporter molecules, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer and target nucleic acid to be detected to obtain a reaction mixture;
and 4) after the reaction mixture reacts, judging the detection result.
Preferably, in the above CRISPR/Cas12 one-step nucleic acid detecting method, the step 4) specifically is: 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 detecting method, the step 4) specifically is: and incubating the reaction mixture for 20min at 37 ℃, and observing the result by naked eyes 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 ° c as12a or Lb4Cas12 a.
The other technical scheme of the invention is as follows: providing a novel coronavirus detection kit, which comprises crRNA, Cas protein, a primer, a buffer system and a single-stranded DNA reporter molecule;
the primer is as follows: 5 '-TTTN-3' sequence is searched in conserved region ORF1ab and N gene region of the novel coronavirus, forward primer is designed in 0-200bp near 5 'region, reverse primer is designed in 25-200bp near 3' region, and the first three and last three phosphodiester bonds of the obtained primers are chemically modified to prevent degradation of DNase or activated Cas protein.
Preferably, in the above novel coronavirus detection kit, the chemical modification is: phosphothioate, 2 '-O-methyl or 2' -O-methoxyhexyl nucleosides.
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:
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 comprises 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, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, B ° ca 12a or 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) fast: the time from the sample to the detection result is only 15-40min according to the amount of the template to be detected, the amplification efficiency and the crRNA cutting efficiency, wherein the sample is processed for 5-10min, and the amplification detection reaction lasts for 10-30 min.
B. And (3) accuracy: 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 single nucleotide mutation in discrimination in a sample.
C. Is convenient and fast: the invention is carried out at the constant temperature of 37 ℃, so the requirement on equipment is extremely low, the reaction can be carried out only by simple constant temperature equipment even by using body temperature, and the popularization of the invention in basic level inspection institutions is facilitated.
D. The cost is low: the invention has the advantages of common materials and enzymes, less consumption and low cost because of the capability of carrying out micro-quantitative test analysis.
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 consumed by the conventional CRISPR/Cas technology from a sample to a result is 1-3h, and the whole process only needs 15-40 min.
B. The operation is simpler and more convenient: because the prior art needs to carry out nucleic acid amplification and detection step by step, the operation is more complicated, and the invention leads the amplification and the detection to be carried out synchronously by the innovative RPA primer and crRNA design, thereby leading the operation to be more convenient and faster.
C. The operation environment can not be polluted: in the prior art, amplification and detection are carried out step by step, and a cover is required to be opened, so that aerosol is easily formed, the operation environment is polluted, false positive of a detection result is caused, and the clinical popularization is not facilitated. The invention adopts a one-step method, avoids uncovering 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 has the following advantages:
1) the reaction is faster: the time consumed by the conventional CRISPR/Cas technology from a sample to a result is 1-3h, the whole detection process using the novel coronavirus detection kit only needs 15-40min, and the visual result interpretation is provided, particularly through direct observation by blue light irradiation or through visual result interpretation by a nucleic acid detection test strip.
2) The operation is simpler and more convenient: because the prior art needs to carry out nucleic acid amplification and detection step by step, the operation is more complicated, and the invention leads the amplification and the detection to be carried out synchronously by the innovative RPA primer and crRNA design, thereby leading the operation to be more convenient and faster.
3) The operation environment can not be polluted: in the prior art, amplification and detection are carried out step by step, and a cover is required to be opened, so that aerosol is easily formed, the operation environment is polluted, false positive of a detection result is caused, and the clinical popularization is not facilitated. The kit can be operated by adopting a one-step method, avoids uncovering operation, does not form aerosol pollution, and is more beneficial to clinical application.
Drawings
FIG. 1 is a graph showing the result of detecting a fluorescent signal of example 1 in an embodiment of the present invention;
FIG. 2 is a graph of blue light excitation detection results of example 1 in an embodiment of the present invention;
FIG. 3 is a diagram showing the result of detection by the nucleic acid test strip of example 1 in the embodiment of the present invention;
FIG. 4 is a graph showing the result of detecting the fluorescent signal of ORF1ab gene in example 2 in the embodiment of the present invention;
FIG. 5 is a graph showing the result of detecting the fluorescent signal of the N gene in example 2 in the embodiment of the present invention;
FIG. 6 is a graph showing the result of blue light excitation detection of ORF1ab gene in example 2 according to the embodiment of the present invention;
FIG. 7 is a graph showing the result of blue light excitation detection of the N gene in example 2 according to the present invention;
FIG. 8 is a diagram showing the result of detection by the ORF1ab gene nucleic acid test paper in example 2 according to the embodiment of the present invention;
FIG. 9 is a diagram showing the result of detection by the N gene nucleic acid test strip of example 2 in the embodiment of the present invention.
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
The most key concept of the invention is as follows: RPA-mediated DNA amplification can be performed at 30-42 deg.C, thus eliminating the need for more sophisticated PCR instruments. Meanwhile, the cleavage of the target molecule by cas12a is also performed under isothermal conditions. Based on the characteristics, the system can be used for on-site rapid detection, has great significance for rapid detection of nucleic acid, and especially has great value for basic clinical examination lacking laboratory conditions.
However, the existing CRISPR/Cas12 technology cannot or hardly achieve the purpose of simultaneously performing nucleic acid amplification and detection, but the universal method for nucleic acid detection and the novel coronavirus detection kit based on CRISPR/Cas12 and isothermal amplification provided by the invention adopt modified primers and design corresponding crRNA so that the cleavage site is positioned on the modified primers, so that the amplification product is not degraded by Cas12, and the purpose of nucleic acid molecule exponential amplification is further achieved.
Example 1
A CRISPR/Cas12 one-step nucleic acid detection method is a novel universal CRISPR/Cas 12-based one-step nucleic acid detection method, and comprises components such as crRNA, Cas protein, primers, a buffer system, a single-stranded DNA reporter molecule and an RNase inhibitor;
the following describes the steps of the method, taking the detection of human beta Actin (ACTB) as an example:
1) 5 '-TTTN-3' sequence is searched in the human beta actin region, a forward primer is designed in the 0-200bp near 5 'region, and a reverse primer is designed in the 25-200bp near 3' region. The first three and the last three phosphodiester bonds of the primer were synthesized and chemically modified. The primers used 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: selecting 17-25N downstream of the 5 '-TTTN-3' sequence in step 1) as the crRNA sequence. The crRNA selected in this example is shown in Table 2 below.
TABLE 2
3) Reporter design and chemical synthesis: the 12-base random single-stranded DNA molecule with FAM and BHQ1 groups at two ends is used for fluorescence signal detection and blue light excitation visualization detection. The single-stranded reporter molecules selected for use in this example are shown in Table 3 below.
TABLE 3
| Single-stranded DNA reporter molecules | 5’-FAM-ACGTCTTAGTCC-BHQ1-3’(SEQ ID NO：10) |
4) Healthy volunteers were sampled with pharyngeal swabs and added to the quick lysates of the following formulation in Table 4 and lysed at 80 ℃ for 5 minutes.
TABLE 4
| Reagent | Concentration of |
| Guanidine hydrochloric | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
5) Taking 2 mu L of lysate product in the step 4), adding a primer combination, crRNA, LbaCas12a, a single-stranded DNA reporter molecule, an RPA upstream primer and a RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer, NEB buffer2.1 and RNaseINHIbitor. The reaction mixture is placed in a constant-temperature fluorescent signal detector at 37 ℃, signals are collected every 30s, and the detection is continuously carried out for two hours. As a result, the signal gradually rises with time as shown in fig. 1.
The proportions of the components used in the present invention are shown in Table 5 below.
TABLE 5
6) Adding the primer combination with the highest amplification efficiency in the step 5) into the reaction mixture in the table 5 above, reacting at a constant temperature of 37 ℃ for 20min, exciting the reactants with blue light, judging the result by naked eyes, and taking a picture by a mobile phone, wherein the result is shown in fig. 2. FIG. 2 Positive PC is very dark in color.
In this embodiment, the preferred one-step nucleic acid detection method for CRISPR/Cas12 for detecting human-derived β Actin (ACTB) is 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: selecting 17-25N downstream of the 5 '-TTTN-3' sequence in step 1) as the crRNA sequence. The crRNA selected in this example is shown in Table 7 below.
TABLE 7
3) 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 below.
TABLE 8
| Single-stranded DNA reporter molecules | 5’-FAM-ACGTCTTAGTCC-Biotin-3’(SEQ ID NO：10) |
4) Healthy volunteers were sampled with pharyngeal swabs and added to the quick lysates according to the formulation in Table 9 and lysed at 80 ℃ for 5 minutes.
TABLE 9
| Reagent | Concentration of |
| Guanidine hydrochloric | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
5) Taking 2 mu L of lysate product in the step 2), adding a primer combination, crRNA, LbaCas12a, a single-stranded DNA reporter molecule, an RPA upstream primer and a RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer, NEB buffer2.1 and RNaseINHIbitor. The reaction mixture was incubated at 37 ℃ for 20 min. The compounding ratio of each component in this example is shown in Table 10.
Watch 10
6) 20 μ L of the reaction solution in the above step is taken, 80 μ L of sample diluent is added, the mixture is subjected to chromatography at room temperature for 2min, 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, and the negative sample is not developed on a detection line, and in the step, a HybriDetect 1 latex flow strips (Milenia) of TwistDx company in England or other nucleic acid test strips with similar characteristics can be adopted.
Example 2
A novel coronavirus detection kit comprises crRNA, Cas protein, a primer, a buffer system and a single-stranded DNA reporter molecule; the comparison of the optimal primer combination of the novel coronavirus detection kit is determined as follows:
1) 5 '-TTTN-3' sequence is searched in the conserved region ORF1ab and N gene region of the novel coronavirus, forward primer is designed in the near 5 'region 0-200bp, and reverse primer is designed in the near 3' region 25-200 bp. The first three and the last three phosphodiester bonds of the primer were synthesized and chemically modified. The primers that can be used 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: selecting 17-25N downstream of the 5 '-TTTN-3' sequence in step 1) as the crRNA sequence. The crrnas selected for use 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 design and chemical synthesis: the 12-base random single-stranded DNA molecule with FAM and BHQ1 groups at two ends is used for fluorescence signal detection and blue light excitation visualization detection. The single-stranded reporter molecules selected for use in this example are shown in Table 13 below.
Watch 13
| Single-stranded DNA reporter molecules | 5’-FAM-ACGTCTTAGTCC-BHQ1-3’(SEQ ID NO：19) |
4) Healthy volunteers were sampled with pharyngeal swabs and added to the quick lysis buffer of the formulation in Table 14 below, followed by the addition of new coronavirus particles and lysis at 80 ℃ for 5 minutes.
TABLE 14
| Reagent | Concentration of |
| Guanidine hydrochloric | 800mM |
| Tween | |
| 20 | 0.5％ |
| Polyethylene glycol octyl phenyl ether | 1％ |
| DEPC water | - |
5) Taking 2 mu L of lysate product in the step 4), adding a primer combination, crRNA, LbaCas12a, a single-stranded DNA reporter molecule, an RPA upstream primer and a RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer, NEB buffer2.1 and RNaseINHIbitor. The reaction mixture is placed in a constant-temperature fluorescent signal detector at 37 ℃, signals are collected every 30s, and the detection is continuously carried out for two hours. The results of ORF1ab are shown in FIG. 4, and the results of the N gene are shown in FIG. 5, in which the signals of the positive samples gradually increased with time, and the signals of the negative samples did not change with time.
The ratio of each component in the novel coronavirus detection kit of the invention is shown in table 15 below.
Watch 15
6) Adding the primer combination with the highest amplification efficiency in the step 5) into the reaction mixture in the table 15 above, placing the mixture at a constant temperature of 37 ℃ for reaction for 20min, exciting the reactant by using blue light, carrying out result interpretation by naked eyes, and taking a picture by using a mobile phone, wherein the result is shown in fig. 6 and 7, a positive sample shows a signal, and a negative sample does not show a signal.
Preferably, the novel coronavirus detection kit comprises crRNA, Cas protein, primers, a buffer system and a single-stranded DNA reporter molecule; the manufacturing method comprises the following steps:
1) 5 '-TTTN-3' sequence is searched in the conserved region ORF1ab and N gene region of the novel coronavirus, forward primer is designed in the region near 5 '0-200 bp, reverse primer is designed in the region near 3' 25-200bp, and the first three and the last three phosphodiester bonds of the obtained primers are chemically modified. Preventing dnase and Cas protein degradation after activation. Such chemical modifications include, but are not limited to, phosphothioate, 2 '-O-methyl or 2' -O-Methoxyethyl (MOE) nucleotides modifications.
The sequences of the primers used 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 crRNA is designed for the target gene. The design principle should be such that the cleavage site for the target gene (specifically, the target gene and the complementary strand of 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 performing in-vitro transcription and purification by using a T7 transcription kit;
selecting 17-25N downstream of the 5 '-TTTN-3' sequence in step 1) as the crRNA sequence. In this example, the crRNA sequence is:
crRNA-ORF1ab：SEQ ID NO：17；
crRNA-nCOV-N：SEQ ID NO：18；
3) design and chemical synthesis of single-stranded DNA reporter: 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 volunteers were sampled with pharyngeal swabs and added to the quick lysates of the formulations in Table 14 and lysed at 80 ℃ for 5 minutes.
5) Taking 2 mu L of lysate product in the step 4), adding a primer combination, crRNA, LbaCas12a, a single-stranded DNA reporter molecule, an RPA upstream primer and a RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer, NEB buffer2.1 and RNaseINHIbitor. The reaction mixture was incubated at 37 ℃ for 20 min. The compounding ratio of each component in this example is shown in Table 10.
6) And (3) taking 20 mu L of the reaction solution in the previous step, adding 80 mu L of sample diluent, carrying out chromatography for 2min at room temperature, and carrying out result judgment by naked eyes, wherein as shown in a figure 8(1ab gene) and a figure 9(N gene), PC is positive, positive develops color on a T line, NTC negative samples do not develop color at a detection line, NTC is negative, and negative samples do not develop color. In this step, HybriDetect 1 latex flow strips (Milenia) from TwistDx, UK or other nucleic acid strips with similar properties can be used.
The use of the above-described novel coronavirus detection kit of this example:
1) clinical samples were taken from throat swabs and added to a quick lysis solution of the formula shown in Table 14 and lysed at 80 ℃ for 5 minutes.
2) Taking 2 mu L of lysate product in the step 2), adding a primer combination, crRNA, LbaCas12a, a single-stranded DNA reporter molecule, an RPA upstream primer and a RPA downstream primer, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer, NEB buffer2.1 and RNaseINHIbitor. The reaction mixture was incubated at 37 ℃ for 20 min. The composition ratio of each component in this example is as shown in table 10 above.
3) And (3) taking 20 mu L of the reaction solution in the previous step, adding 80 mu L of sample diluent, performing chromatography for 2min at room temperature, and performing result interpretation by naked eyes, wherein a positive sample develops color at the detection line T, and a negative sample does not develop color at the line T. In this step, HybriDetect 1 latex flow strips (Milenia) from TwistDx, UK or other nucleic acid strips with similar properties can be used.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
SEQUENCE LISTING
<110> Yaenergetic Biotechnology (Shenzhen) Limited
<120> one-step nucleic acid detection method of CRISPR/Cas12 and novel coronavirus detection kit
<130>2020
<160>19
<170>PatentIn version 3.5
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tgtggatcag caagcaggag tatgacgagt cc 32
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gatgtggatc agcaagcagg agtatgacga gt 32
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aagccatgcc aatctcatct tgttttctgc gc 32
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Claims (10)
1. A CRISPR/Cas12 one-step nucleic acid detection method is characterized by comprising the following steps:
step 1) designing and synthesizing an RPA upstream primer and a RPA downstream primer for amplifying a target gene, and carrying out chemical modification on the primers for preventing degradation of DNase and activated Cas protein;
step 2) designing specific crRNA aiming at a target gene, and designing a single-stranded DNA reporter molecule;
step 3) mixing components including crRNA, Cas protein, single-stranded DNA reporter molecules, RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, RPA hydration buffer and target nucleic acid to be detected to obtain a reaction mixture;
and 4) after the reaction mixture reacts, judging the detection result.
2. The one-step nucleic acid detection method of CRISPR/Cas12 according to claim 1, wherein the step 4) is specifically: 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 one-step nucleic acid detection method of CRISPR/Cas12 according to claim 1, wherein the step 4) is specifically: and incubating the reaction mixture for 20min at 37 ℃, and observing the result by naked eyes 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 according to claim 1, wherein the Cas protein is Cas12, and the Cas12 is one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12 a.
5. A 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: 5 '-TTTN-3' sequence is searched in conserved region ORF1ab and N gene region of the novel coronavirus, forward primer is designed in 0-200bp near 5 'region, reverse primer is designed in 25-200bp near 3' region, and the first three and last three phosphodiester bonds of the obtained primers are chemically modified to prevent degradation of DNase or activated Cas protein.
6. The novel coronavirus detection kit of claim 5, wherein the chemical modification is: phosphothioate, 2 '-O-methyl or 2' -O-methoxyhexyl nucleosides.
7. The novel coronavirus detection kit according to claim 5,
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:
crRNA-ORF1ab：SEQ ID NO：17；
crRNA-nCOV-N：SEQ ID NO：18。
8. the novel coronavirus detection kit of claim 5, wherein the single-stranded DNA reporter molecule has a sequence as follows: SEQ ID NO: 19.
9. the novel coronavirus detection kit of claim 8, wherein the single-stranded DNA reporter molecule comprises FAM and BHQ1 groups.
10. The novel coronavirus detection kit of claim 5, wherein the Cas protein is Cas12, and the Cas12 is one of FnCas12a, AsCas12a, LbCas12a, Lb5Cas12a, HkCas12a, OsCas12a, TsCas12a, BbCas12a, BoCas12a or Lb4Cas12 a.
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