CN116904662A - Raa-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method - Google Patents
Raa-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method Download PDFInfo
- Publication number
- CN116904662A CN116904662A CN202310036091.3A CN202310036091A CN116904662A CN 116904662 A CN116904662 A CN 116904662A CN 202310036091 A CN202310036091 A CN 202310036091A CN 116904662 A CN116904662 A CN 116904662A
- Authority
- CN
- China
- Prior art keywords
- raa
- sequence seq
- crispr
- nucleic acid
- primer sequence
- 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.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 241000700627 Monkeypox virus Species 0.000 title claims abstract description 39
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 28
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 28
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 28
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 26
- 108700004991 Cas12a Proteins 0.000 claims abstract description 19
- 108020004414 DNA Proteins 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 230000003321 amplification Effects 0.000 claims abstract description 16
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 16
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 15
- 101150076749 C10L gene Proteins 0.000 claims abstract description 14
- 102000053602 DNA Human genes 0.000 claims abstract description 11
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 101150117815 D3R gene Proteins 0.000 claims description 7
- 101150091263 E3L gene Proteins 0.000 claims description 7
- 101150016272 A21L gene Proteins 0.000 claims description 5
- 101100222866 Vaccinia virus (strain Ankara) MVA100R gene Proteins 0.000 claims description 5
- 101100119458 Vaccinia virus (strain Copenhagen) F5L gene Proteins 0.000 claims description 5
- 101100119460 Vaccinia virus (strain Western Reserve) VACWR044 gene Proteins 0.000 claims description 5
- 101100332586 Vaccinia virus (strain Western Reserve) VACWR059 gene Proteins 0.000 claims description 5
- 101100222869 Vaccinia virus (strain Western Reserve) VACWR108 gene Proteins 0.000 claims description 5
- 101100119461 Variola virus (isolate Human/India/Ind3/1967) C9L gene Proteins 0.000 claims description 5
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 2
- 101150022701 F5L gene Proteins 0.000 claims description 2
- 238000010791 quenching Methods 0.000 claims description 2
- 230000000171 quenching effect Effects 0.000 claims description 2
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 241001529936 Murinae Species 0.000 abstract description 6
- 241000700618 Vaccinia virus Species 0.000 abstract description 4
- 208000015181 infectious disease Diseases 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 10
- 108091033409 CRISPR Proteins 0.000 description 7
- 208000005871 monkeypox Diseases 0.000 description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000011529 RT qPCR Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 101100000222 Vaccinia virus (strain Western Reserve) VACWR140 gene Proteins 0.000 description 3
- 241000700649 Vaccinia virus Tian Tan Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000012197 amplification kit Methods 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 241000700629 Orthopoxvirus Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000011304 droplet digital PCR Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011901 isothermal amplification Methods 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 238000001921 nucleic acid quantification Methods 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108091079001 CRISPR RNA Proteins 0.000 description 1
- 238000010453 CRISPR/Cas method Methods 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 238000007397 LAMP assay Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000700625 Poxviridae Species 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 208000035472 Zoonoses Diseases 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 101150059443 cas12a gene Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000007847 digital PCR Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting monkey pox virus nucleic acid based on RAA-CRISPR/Cas12a, which comprises the following steps: carrying out RAA amplification by taking nucleic acid in a sample as a template, wherein a RAA amplification primer is selected from any gene of C10L, F5L, E3L, D3R, A L, the upstream primer sequence is sequentially shown as SEQ ID NO.1-5, and the downstream primer sequence is sequentially shown as SEQ ID NO. 6-10; using the RAA amplification product, cas12a protein, crRNA, fluorescently labeled ssDNA, in a CRISPR-Cas12a system detection system; the fluorescence intensity of the reaction system was measured. The sensitivity of the method is 2.5 copies/reaction, and the method has no cross reaction with the murine poxvirus and the vaccinia virus, has high specificity, and provides a powerful tool for efficiently, quickly and specifically detecting the infection of the monkey poxvirus.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a monkey pox virus nucleic acid detection method based on RAA-CRISPR/Cas12 a.
Background
Monkey pox is a zoonotic infectious disease caused by monkey pox virus (MPXV), a linear double stranded DNA virus belonging to the genus orthopoxvirus of the family poxviridae. MPXV was first found in experimental monkeys in 1958 and was first reported in 1970 to infect humans. Based on genomic sequence differences, MPXV is split into two branches: branch I (once called congo strain) and branch II (once called western strain), where branch I is strongly infectious and causes severe disease. Infection can be initiated by human exposure to or biting by MPXV infected animals, while MPXV also exists in a human-to-human transmission pattern.
The establishment of MPXV nucleic acid detection methods is critical for the monitoring and diagnosis of monkey pox.
Clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats, CRISPR) and related protein (Cas) systems are an adaptive immune system of bacteria that recognizes foreign substances and eliminates them by the endoenzyme activity of Cas proteins. The CRISPR/Cas system has the functions of target recognition and trans-cleavage activity, and is beneficial to developing an in-vitro nucleic acid detection technology, which is called a next-generation molecular detection technology. Cas12a is an RNA-guided DNA-targeting enzyme capable of binding and cleaving target DNA. The guide RNA, namely clustered regularly interspaced short palindromic repeated sequence RNA (CRISPR RNA, crRNA), guides double-stranded DNA or single-stranded DNA (ssDNA) complementary to specific recognition bases of the Cas12a protein, activates trans-cleavage activity of the guide RNA, can non-specifically cleave free ssDNA reporter molecules, and a corresponding detection system is established by utilizing the characteristic.
Currently, methods such as real-time fluorescent quantitative polymerase chain reaction (quantitative polymerase chain reaction, qPCR), virus isolation and culture, electron microscope observation, polymerase chain reaction (polymerase chain reaction, PCR) and the like are used for monkey pox diagnosis, wherein qPCR is the gold standard for current monkey pox detection. But qPCR has long detection time, and requires special instruments and equipment, high-standard laboratory environment and professional technicians, which restricts the wide application in underdeveloped or underdeveloped areas. At present, loop-mediated isothermal amplification (LAMP-mediated isothermal amplification) has been used for MPXV detection, the detection limit of the method is 20 copies/reaction, 55 minutes are required for detection, six pairs of primers are required in detection, and the detection principle is complex and is not easy to popularize. It is therefore highly desirable to establish a simple, rapid and accurate method for detecting MPXV nucleic acid.
Disclosure of Invention
In view of the above-mentioned shortcomings in the prior art, the present invention aims to provide a method for detecting monkey poxvirus nucleic acid based on RAA-CRISPR/Cas12 a. The sensitivity is 2.5 copies/reaction, the specificity is better, the detection time is about 50 minutes, and the method has important significance for developing monkey pox epidemic situation prevention and control.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
there is provided a method for detecting a monkey poxvirus nucleic acid based on RAA-CRISPR/Cas12a, the method comprising the steps of:
step 1, carrying out RAA amplification by taking nucleic acid in a sample as a template, wherein a RAA amplification primer is selected from any gene of C10L, F5L, E3L, D3R, A L, the upstream primer sequence is sequentially shown as SEQ ID NO.1-5, and the downstream primer sequence is sequentially shown as SEQ ID NO. 6-10;
the RAA amplification kit is selected from one or more of the following: basic DNA isothermal rapid amplification reagent (goods number: S001 ZC) produced by Hangzhou public detection biotechnology Co., ltd., basic DNA isothermal rapid amplification KIT (goods number: WLB8201 KIT) produced by Anpu future biotechnology Co., ltd., basic RAA nucleic acid amplification KIT (goods number: B00100) produced by Jiangsu Qishi gene biotechnology Co., ltd., twistAmp Basic KIT (goods number: TABAS03 KIT) produced by TwistDx Co., ltd., most preferably, basic DNA isothermal rapid amplification reagent (goods number: S001 ZC) produced by Hangzhou public detection biotechnology Co., ltd.
Step 2, using the RAA amplification product obtained in the step 1, reacting Cas12a protein, crRNA and fluorescent-labeled ssDNA in a CRISPR-Cas12a system detection system;
cas12a is selected from one or more of the following: lbaCas12a (cat# 32108-03) manufactured by Shanghai Tuber harbor Biotechnology Co., ltd., lba Cas12a (cat# M0653T) manufactured by U.S. New England Biolabs.
And 3, detecting fluorescence intensity of the reaction system in the step 2.
Further, in step 2, the sequence of crRNA is selected from any one of SEQ ID NO.11-15, wherein:
the sequence SEQ ID NO.11 is matched with the upstream primer sequence SEQ ID NO.1 and the downstream primer sequence SEQ ID NO.6 of the C10L gene;
the sequence SEQ ID NO.12 is matched with the upstream primer sequence SEQ ID NO.2 and the downstream primer sequence SEQ ID NO.7 of the F5L gene;
the sequence SEQ ID NO.13 is matched with the upstream primer sequence SEQ ID NO.3 and the downstream primer sequence SEQ ID NO.8 of the E3L gene;
the sequence SEQ ID NO.14 is matched with the upstream primer sequence SEQ ID NO.4 and the downstream primer sequence SEQ ID NO.9 of the D3R gene;
the sequence SEQ ID NO.15 is matched with the upstream primer sequence SEQ ID NO.5 and the downstream primer sequence SEQ ID NO.10 of the A21L gene.
Further, the fluorophore is selected from one or more of the following: FAM, CY5, VIC, HEX, most preferably FAM.
Further, the fluorescence quenching group is selected from one or more of the following: BHQ1, BHQ2, TAMRA, most preferably BHQ1.
The beneficial effects of the invention are as follows:
the invention establishes a monkey pox virus nucleic acid detection method based on RAA-CRISPR/Cas12a, has the sensitivity of 2.5 copies/reaction, has no cross reaction with the murine pox virus and the vaccinia virus, has high specificity, and provides a powerful tool for efficiently, quickly and specifically detecting the infection of the monkey pox virus.
Drawings
FIG. 1 is a schematic diagram showing the synthesis of crRNA of the C10L gene provided in example 1 of the present invention; wherein, the C10L gene crRNA synthesizes a template sequence (fig. 1A), italics letters: a CRISPR/Cas12a-crRNA specific recognition sequence; bold letters: CRISPR/Cas12a-crRNA immobilization backbone sequences; underlined letters: a T7 promoter sequence; C10L Gene crRNA preparation flow chart (Panel B), grey: a T7 promoter sequence; white: CRISPR/Cas12a-crRNA immobilization backbone sequences; black: a CRISPR/Cas12a-crRNA specific recognition sequence;
fig. 2 is a schematic diagram of a detection method of RAA-CRISPR/Cas12a provided in embodiment 1 of the present invention;
FIG. 3 is a schematic diagram of the result of detecting MPXV by RAA-CRISPR/Cas12a provided in example 1 of the present invention; NC, negative control; a dashed line, a threshold line;
FIG. 4 is a schematic diagram showing the evaluation of the specificity of the MPXV RAA-CRISPR/Cas12a nucleic acid detection method provided in example 2 of the present invention; wherein, MPXV: monkey poxvirus; VTT: vaccinia virus Tiantan strain; ECTV: a murine poxvirus; NC: a negative control; dotted line: a threshold line;
FIG. 5 is a schematic diagram showing the sensitivity and reproducibility evaluation of the MPXV RAA-CRISPR/Cas12a nucleic acid detection method provided in example 3 of the present invention; wherein, the dashed line, the threshold line.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Example 1
1.1 sample A monkey pox virus nucleic acid sample is obtained by extracting a monkey pox virus strain MPXV-B.1-China-C-Tan-CQ01 cell culture separated from a first input monkey pox case sample in the laboratory, and murine poxvirus nucleic acid and vaccinia virus nucleic acid used in RAA-CRISPR/Cas12a specific detection are prepared by the laboratory.
1.2RAA amplification
The RAA primers (Table 1) were synthesized by the company Highway, inc. of biological engineering (Shanghai). RAA amplification was performed according to the instructions of the basic DNA isothermal rapid amplification reagent (cat# S001 ZC) manufactured by Hangzhou public detection Biotechnology Co., ltd. 25 μl of A buffer,2 μl of upstream and downstream primers (10 μM), 2 μl of sample, 16.5 μl of sterilized nuclease-free water were mixed and added to a basic reaction unit containing lyophilized powder to allow complete reconstitution of the lyophilized powder. Then 2.5 mu l of B buffer is added on the tube cover of each reaction tube, the tube cover is covered, the mixture is evenly mixed for 5 to 6 times in an upside down way, and the mixture is rapidly centrifuged for 10s, and then the reaction is carried out for 30min at 42 ℃ to finish the RAA amplification.
TABLE 1 RAA primer and crRNA and template sequence
1.3 preparation of crRNA
Referring to FIG. 1, this example specifically illustrates the synthesis of the C10L gene crRNA. The crRNA is 39 or 41nt in length, consists of two parts, the first 21nt is CRISPR/Cas12a-crRNA fixed backbone sequence UAAUUUCUACUAAGUGUAGAU, the last 18 or 20nt is CRISPR/Cas12a-crRNA specific recognition sequence (fig. 1B), the recognition region is located after the prospacer sequence adjacent motif (protospacer adjacent motif, PAM) site. Preparation of crRNA the required DNA oligonucleotide templates (Table 1) were synthesized in the Shanghai Co., ltd. crRNA was prepared according to the kit instructions, the specific procedure (fig. 1) was: 1) Synthesis of double-stranded DNA: the reaction system was prepared and reacted according to Table 2. 2) Preparation of crRNA by in vitro transcription: the reaction system was prepared and reacted according to Table 3 to obtain crRNA. The harvested crRNA was then purified using the RNeasy Mini Kit (Qiagen, hilden, germany, cat# 74104), the crRNA purity and concentration were measured by a Nanodrop 2000 spectrophotometer (America Thermo Fisher Scientific Co.), and the crRNA was sub-packaged and frozen at-80℃for use.
TABLE 2 double-stranded DNA Synthesis procedure and reaction System
Primer sequence: TAACGACTCACTTAGAGG (SEQ ID NO. 21)
Phi29 MAX DNA Polymerase is manufactured by Nanjinouzan biotechnology, inc., cat No.: n106-01).
Deoxynucleotide (dNTP) Solution Mix is manufactured by New England Biolabs company, U.S. Pat. No.: N0447S).
TABLE 3 in vitro transcription reaction System of crRNA
The in vitro transcription kit RiboMAXTM Large Scale RNA Production System-T7 is manufactured by Promega corporation, USA, product number: p1300).
1.4CRISPR/Cas12a fluorescence detection
A CRISPR-Cas12a system detection system was prepared, 2. Mu.l of RAA amplification product, 1. Mu.l of LbacAS12a protein (cpf 1) (Shanghai Korea biosciences, inc. with a final concentration of 32108-03, 50 nM), 2. Mu.l of crRNA (with a final concentration of 100 nM), 2. Mu.l of fluorescence-labeled ssDNA (5 '-FAM-TTATT-BHQ 1-3') (with a final concentration of 250 nM), 2. Mu.l of NEB buffer 2.1, 10. Mu.l of sterilized nuclease-free water were added to the same tube, and after mixing, the detection system was placed in a qPCR apparatus (Kunpro Gene technology Co., ltd.) for reaction at 37℃and fluorescence was collected once every 2min for 40 times.
1.5 Simian poxvirus nucleic acid quantification
The monkey poxvirus nucleic acid quantification was performed using a digital PCR instrument (sonafu medical science limited, su zhou) and the detection method was established by the present laboratory. The detection system was prepared first according to the instructions of the 2X dPCR Probe Master Mix kit (Japanese Takara Co., ltd., product No.: RR 391A), and 22. Mu.l of the detection system, 11. Mu.l of 2X dPCR Probe Master Mix, 1. Mu.l of the upstream and downstream primers (10. Mu.M), 0.5. Mu.l of the probe (10. Mu.M), 2. Mu.l of the template DNA, and RNase-Free ddH2O were filled in. The reaction procedure was set as follows: 60 ℃/5min-95 ℃/15 min/(95 ℃/30S-60 ℃/45S). Times.40 cycles-60 ℃/1min.
1.6 data analysis and result interpretation
When the RAA-CRISPR/Cas12a is detected, the fluorescence value of the detection sample in the 40 th period is 2 times greater than that of the negative control fluorescence value, and the detection sample is judged to be positive; otherwise, the result is negative. Data processing and mapping were performed using Graphpad Prism.
MPXV nucleic acid was detected simultaneously using 5 sets of RAA primers and crRNA for the C10L, F5L, E3L, D3R and A21L genes, respectively, of MPXV (FIG. 3), with the detection system for the C10L gene having the strongest MPXV signal followed by D3R, A21L, F5L, E3L.
Example 2
Specificity evaluation of detection method
The specificity of the established method was evaluated using DNA of vaccinia virus Tiantan strain (vaccinia virus TianTan strain, VTT) and murine poxvirus (ECTV). The detection system for C10L, F5L, E3L, D3R and A21L genes can distinguish MPXV from VTT, ECTV, and the detection system for C10L gene shows the best detection efficiency (FIGS. 4A-E).
Example 3
MPXV RAA-CRISPR/Cas12a nucleic acid detection method sensitivity and repeatability evaluation
This example further carried out sensitivity and specificity evaluations of the C10L gene detection system for MPXV. The initial MPXV nucleic acid concentration was set at 80 copies/. Mu.L, diluted in sequence to 1.25 copies/. Mu.L, and 2. Mu.L was added to each reaction system for detection, and the result indicated that the detection limit of the method was 2.5 copies/. Mu.L (FIG. 5A). The fluorescent readings of the 3 repeated experiments are similar, the detection limits are consistent, and the repeatability is good (figure 5B).
The invention establishes the isothermal amplification detection method of the monkey pox virus by utilizing RAA-CRISPR/Cas12a, and the method not only has higher sensitivity, can detect the monkey pox virus nucleic acid with the copy/reaction as low as 2.5, but also has no cross reaction with vaccinia virus and murine poxvirus in orthopoxvirus genus, and has better specificity.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (4)
1. A method for detecting a monkey poxvirus nucleic acid based on RAA-CRISPR/Cas12a, comprising the steps of:
step 1, carrying out RAA amplification by taking nucleic acid in a sample as a template, wherein a RAA amplification primer is selected from any gene of C10L, F5L, E3L, D3R, A L, the upstream primer sequence is sequentially shown as SEQ ID NO.1-5, and the downstream primer sequence is sequentially shown as SEQ ID NO. 6-10;
step 2, using the RAA amplification product obtained in the step 1, reacting Cas12a protein, crRNA and fluorescent-labeled ssDNA in a CRISPR-Cas12a system detection system;
and 3, detecting fluorescence intensity of the reaction system in the step 2.
2. The RAA-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method according to claim 1, wherein in step 2 the sequence of crRNA is selected from any one of SEQ ID nos. 11-15, wherein:
the sequence SEQ ID NO.11 is matched with the upstream primer sequence SEQ ID NO.1 and the downstream primer sequence SEQ ID NO.6 of the C10L gene;
the sequence SEQ ID NO.12 is matched with the upstream primer sequence SEQ ID NO.2 and the downstream primer sequence SEQ ID NO.7 of the F5L gene;
the sequence SEQ ID NO.13 is matched with the upstream primer sequence SEQ ID NO.3 and the downstream primer sequence SEQ ID NO.8 of the E3L gene;
the sequence SEQ ID NO.14 is matched with the upstream primer sequence SEQ ID NO.4 and the downstream primer sequence SEQ ID NO.9 of the D3R gene;
the sequence SEQ ID NO.15 is matched with the upstream primer sequence SEQ ID NO.5 and the downstream primer sequence SEQ ID NO.10 of the A21L gene.
3. The RAA-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method according to claim 1, wherein in step 2 the fluorescent moiety is selected from one or more of the following: FAM, CY5, VIC, HEX.
4. The RAA-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method according to claim 1, wherein in step 2, the fluorescence quenching group is selected from one or more of the following: BHQ1, BHQ2, TAMRA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310036091.3A CN116904662A (en) | 2023-01-10 | 2023-01-10 | Raa-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310036091.3A CN116904662A (en) | 2023-01-10 | 2023-01-10 | Raa-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116904662A true CN116904662A (en) | 2023-10-20 |
Family
ID=88363506
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310036091.3A Pending CN116904662A (en) | 2023-01-10 | 2023-01-10 | Raa-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116904662A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839224A (en) * | 2012-06-21 | 2012-12-26 | 浙江国际旅行卫生保健中心 | Reagent and method for detecting monkeypox virus through isothermal amplification |
| CN103468830A (en) * | 2013-09-29 | 2013-12-25 | 浙江国际旅行卫生保健中心 | NASBA kit for detecting monkey pox virus and application thereof |
| CN111108220A (en) * | 2017-03-15 | 2020-05-05 | 博德研究所 | CRISPR-Effector System-based diagnostics for Virus detection |
| US20210269858A1 (en) * | 2018-11-19 | 2021-09-02 | The Regents Of The University Of California | Methods for detecting and sequencing a target nucleic acid |
| CN114752711A (en) * | 2022-06-15 | 2022-07-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting and parting monkeypox virus and application thereof |
| CN115161414A (en) * | 2022-06-10 | 2022-10-11 | 艾康生物技术(杭州)有限公司 | Specificity detection target of monkeypox virus, oligonucleotide and kit thereof |
| CN115466800A (en) * | 2022-06-20 | 2022-12-13 | 浙江大学医学院附属第一医院 | Multiplex fluorescence quantitative PCR kit for detecting monkeypox virus and subtype thereof |
-
2023
- 2023-01-10 CN CN202310036091.3A patent/CN116904662A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839224A (en) * | 2012-06-21 | 2012-12-26 | 浙江国际旅行卫生保健中心 | Reagent and method for detecting monkeypox virus through isothermal amplification |
| CN103468830A (en) * | 2013-09-29 | 2013-12-25 | 浙江国际旅行卫生保健中心 | NASBA kit for detecting monkey pox virus and application thereof |
| CN111108220A (en) * | 2017-03-15 | 2020-05-05 | 博德研究所 | CRISPR-Effector System-based diagnostics for Virus detection |
| US20210269858A1 (en) * | 2018-11-19 | 2021-09-02 | The Regents Of The University Of California | Methods for detecting and sequencing a target nucleic acid |
| CN115161414A (en) * | 2022-06-10 | 2022-10-11 | 艾康生物技术(杭州)有限公司 | Specificity detection target of monkeypox virus, oligonucleotide and kit thereof |
| CN114752711A (en) * | 2022-06-15 | 2022-07-15 | 圣湘生物科技股份有限公司 | Composition, kit and method for detecting and parting monkeypox virus and application thereof |
| CN115466800A (en) * | 2022-06-20 | 2022-12-13 | 浙江大学医学院附属第一医院 | Multiplex fluorescence quantitative PCR kit for detecting monkeypox virus and subtype thereof |
Non-Patent Citations (2)
| Title |
|---|
| LINGJING MAO ET AL.: "Development and Characterization of Recombinase-Based Isothermal Amplification Assays (RPA/RAA) for the Rapid Detection of Monkeypox Virus", VIRUSES, vol. 14, 31 December 2022 (2022-12-31), pages 1 * |
| 蔡鹏 等: "猴痘病原学、流行病学特征及其防控策略", 上海预防医学, vol. 34, no. 10, 30 October 2022 (2022-10-30), pages 1044 - 1051 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108486234B (en) | CRISPR (clustered regularly interspaced short palindromic repeats) typing PCR (polymerase chain reaction) method and application thereof | |
| CN104164488B (en) | A kind of nucleic acid constant-temperature amplification method that single primer causes | |
| CN108192956A (en) | It is a kind of based on the DNA determination methods of Cas9 nucleases and its application | |
| KR870007427A (en) | How to detect viruses by amplification and hybridization | |
| CN113355459B (en) | Method and kit for detecting and screening N501Y mutation of new coronavirus | |
| CN116656850B (en) | Sequence combination for rapidly detecting rice bacterial leaf blight bacteria based on CRISPR/Cas12a-RPA and application thereof | |
| CN111118211A (en) | Bovine sarcoidosis detection kit and method based on loop-mediated isothermal amplification technology | |
| CN115725789A (en) | Nucleic acid composition, kit and method for detecting monkeypox virus | |
| CN115747353A (en) | Primer group, reagent, kit and detection method for detecting Listeria monocytogenes | |
| CN115044710A (en) | Primer group, kit and application for detecting pangolin beta coronavirus | |
| CN112029838B (en) | CRISPR/Cas9 typing PCR method for DNA homogeneous phase detection and application thereof | |
| CN104911275A (en) | Bacterial vaginitis detection kit | |
| NL2031160B1 (en) | Primer Set, Probe and Application for Distinguishing Brucella S2 Vaccine Strain from Wild Strain | |
| CN116904662A (en) | Raa-CRISPR/Cas12 a-based monkey poxvirus nucleic acid detection method | |
| CN114134218B (en) | Fluorescent detection method based on CRISPR-Cas12a | |
| CN113528686B (en) | Reagent and kit for detecting nucleic acid of brucella | |
| DK2753710T3 (en) | MOLECULAR DETECTION ASSAY | |
| CN115029345A (en) | Nucleic acid detection kit based on CRISPR and application thereof | |
| CN112195257A (en) | Primer group, reagent, kit and detection method for detecting vibrio parahaemolyticus | |
| CN111534634A (en) | Visual isothermal amplification detection reagent for type II canine adenovirus and application thereof | |
| CN114134254B (en) | Molecular logic gate intelligent detection system for detecting coronavirus and application thereof | |
| CN117512076B (en) | RNA reverse transcription-free detection method based on split Cas9 system | |
| CN113322353B (en) | RPA kit for detecting sweet potato pinnate mottle virus and sweet potato chlorotic stunt virus | |
| CN110106231B (en) | Method for detecting methylation modification of adenine N6 or N1 bit in nucleic acid by using dUTP or dTTP | |
| CN118308351A (en) | CRRNA (ribonucleic acid) for detecting novel coronavirus BA.5 by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas 12a, detection method and kit |
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 |