CN114107526B - RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof - Google Patents
RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof Download PDFInfo
- Publication number
- CN114107526B CN114107526B CN202111440174.6A CN202111440174A CN114107526B CN 114107526 B CN114107526 B CN 114107526B CN 202111440174 A CN202111440174 A CN 202111440174A CN 114107526 B CN114107526 B CN 114107526B
- Authority
- CN
- China
- Prior art keywords
- crrna
- rpa
- lbcas12a
- primer pair
- seq
- 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
- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000219109 Citrullus Species 0.000 title claims abstract description 22
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 title claims abstract description 22
- 241000894006 Bacteria Species 0.000 title claims description 9
- 108020004414 DNA Proteins 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 230000003321 amplification Effects 0.000 claims description 10
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 102000053602 DNA Human genes 0.000 claims description 7
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 7
- 229940069446 magnesium acetate Drugs 0.000 claims description 7
- 235000011285 magnesium acetate Nutrition 0.000 claims description 7
- 239000011654 magnesium acetate Substances 0.000 claims description 7
- 230000000813 microbial effect Effects 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 25
- 241001600126 Acidovorax citrulli Species 0.000 abstract description 12
- 244000052616 bacterial pathogen Species 0.000 abstract description 11
- 238000005516 engineering process Methods 0.000 abstract description 7
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 238000011901 isothermal amplification Methods 0.000 abstract description 5
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 108020004707 nucleic acids Proteins 0.000 abstract description 4
- 102000039446 nucleic acids Human genes 0.000 abstract description 4
- 150000007523 nucleic acids Chemical class 0.000 abstract description 4
- 238000012800 visualization Methods 0.000 abstract description 4
- 108091033409 CRISPR Proteins 0.000 abstract description 3
- 238000011895 specific detection Methods 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 244000000010 microbial pathogen Species 0.000 abstract 1
- 238000001917 fluorescence detection Methods 0.000 description 12
- 239000000047 product Substances 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 241000219112 Cucumis Species 0.000 description 2
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 2
- 241000219104 Cucurbitaceae Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 230000001066 destructive effect Effects 0.000 description 2
- 238000012921 fluorescence analysis Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101100385364 Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CCM 3970 / CIP 100100 / NCTC 11856 / SLCC 3954 / 1120) cas13 gene Proteins 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100036546 Salivary acidic proline-rich phosphoprotein 1/2 Human genes 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 101150059443 cas12a gene Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000010206 sensitivity analysis Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000011179 visual inspection Methods 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- 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
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
Abstract
The invention belongs to the field of rapid detection of pathogenic microorganisms, and in particular relates to a method for detecting watermelon acidophilic bacteriaAcidovorax citrulli) And crRNA, and kits and methods of use thereof. The RPA primer pair sequences are shown in SEQ ID No.1 and SEQ ID No.2, and the crRNA target sequence is shown in SEQ ID No. 3. The invention adopts a Crispr/LbCAs12a nucleic acid detection platform, designs a specific detection site aiming at the genome of the watermelon acidophilus (Acidovorax citrulli), and combines a fluorescence visualization technology to develop an easy diagnosis method suitable for detecting the pathogenic bacteria. And simultaneously, the sensitivity is further improved by combining with the RPA isothermal amplification technology.
Description
Technical Field
The invention belongs to the field of rapid detection of pathogenic bacteria, and particularly relates to an RPA primer pair and crRNA for detecting watermelon acidophilus (Acidovorax citrulli), a kit and a use method thereof.
Background
Bacterial pathogens often cause significant reductions in yield and quality in vegetable and fruit production areas worldwide, thus posing a serious threat to the horticultural industry. Wherein, the watermelon acidophilic bacteria isAcidovorax citrulli) Bacterial fruit blotch is caused in cucurbitaceae crops, is a main limited pathogenic bacteria of the cucurbitaceae crops worldwide, and can cause destructive injury to fruit industries such as watermelons, melons and the like in the market. Because of their destructive effect on horticultural crops, watermelon acidophiles are classified as quarantine pests (EPPO global database; https:// gd. EPPO. Int/datasheets /) in agricultural plant pest quarantine tables at home and abroad. At present, watermelon acidophiles become local diseases in most of the global planting areas, and the most main transmission and diffusion modes are that seeds are adopted, and a simple, rapid and sensitive field detection technology is adopted to eliminate batches of pathogenic bacteria polluted seeds, so that very important quarantine measures are adopted.
Successful quarantine measures require an accurate, timely, on-site and simple method of pathogen detection in order to destroy infectious material on site. According to the identification criteria of pathogenic bacteria recommended by guidelines of the European Plant Protection Organization (EPPO),A. citrullithe identification of (C) requires a series of steps such as isolation, purification, culture and physiological biochemical analysis, and further confirmation by molecular techniques such as PCR or RT-PCR. However, these methods are time consuming and require the establishment of a laboratory, the purchase of expensive equipment and the training of specialized operators, which makes them difficult to carry and unsuitable for immediate detection in the primary sector.
Nucleic acid detection systems based on CRISPR-Cas proteins (Cas 12, cas13 and Cas 14) have also had great application prospects in molecular diagnostics in recent years, as they show trans-cleavage on non-specific targets after triggering cis-cleavage in binding to the target. Among them, cas12a systems are particularly suitable for detecting target sites, pathogens or transgenic organisms from viruses by non-specific cleavage of ssDNA by recognition of specific dsDNA sequences.
Disclosure of Invention
The invention provides an RPA primer pair and crRNA for detecting watermelon acidophilus (Acidovorax citrulli), a kit and a use method thereof, and the invention adopts a Crispr/LbCAs12a nucleic acid detection platform aiming at watermelon acidophilusAcidovorax citrulli) The genome of (a) is designed with specific detection sites, and a fluorescent visualization technology is combined to develop an easy diagnosis method suitable for detecting the pathogenic bacteria. And simultaneously, the sensitivity is further improved by combining with the RPA isothermal amplification technology.
The technical scheme of the invention is realized as follows:
an RPA primer pair for detecting watermelon acidophilic bacteria and crRNA are provided, wherein the sequence of the RPA primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the sequence of the crRNA is shown as SEQ ID No. 3.
A kit comprising the RPA primer pair and crRNA target described above, said kit comprising 2 μl of RPA amplification product system and 50 μl of crRNA/LbCas12a mixed system.
The RPA amplification product system included RPA enzyme powder, 29.5. Mu.L of rehydration buffer, 11. Mu.L of nuclease-free water, 280 nM magnesium acetate, 2. Mu.L of genomic DNA, and RPA pre-amplification was performed using a commercial TwistAmp Basic kit (TwistDx).
The crRNA/LbCAs12a mixed system comprises 100 nM LbCAs12a, 120 nM crRNA, 400 nM ssDNA FQ-report, 20U RNase inhibitor, 2. Mu.L RPA amplification product, 5. Mu.L 10 XNEBuffer 3.1 and ddH 2 O was 50. Mu.L in total.
The concentration of the forward primer and the reverse primer in the RPA primer pair in the kit is 10 mu M, and the content is 2.5 mu L.
The FQ-reporter sequence in the kit is 5 '-FAM-TTATT-Quancher-3'.
The application method of the kit comprises the following steps:
(1) Mixing the freeze-dried RPA enzyme powder with 10 mu M of RPA primer pair, rehydration buffer solution, nuclease-free water and microbial genome DNA of the seed to be detected to obtain a reaction system;
(2) Adding 280 nM magnesium acetate into the reaction system of the step (1), centrifugally starting the reaction, and incubating to obtain an initial product;
(3) A crRNA/LbCAs12a mixed system was prepared and 2. Mu.L of the initial product of step (2) was added, and the trans-cleavage reaction was performed on a Bio-Tek FLx800 microplate fluorescence reader and the results were observed.
The volume of the 10 mu M RPA primer pair in the step (1) is 2.5 mu L, the volume of the rehydration buffer is 29.5 mu L, the volume of the nuclease-free water is 11 mu L, and the volume of the microbial genome DNA of the seed to be detected is 2 mu L.
The volume of 280. 280 nM magnesium acetate in the step (2) is 2.5 mu L, and the incubation condition is that the incubation is performed for 30 min at 37 ℃.
The preparation method of the crRNA/LbCAs12a mixed system in the step (3) comprises the following steps: 100 nM LbCAs12a and 120 nM crRNA are pre-mixed and pre-incubated at 25℃for 10 min to promote formation of crRNA/LbCAs12a complex, then 400 nM ssDNA FQ-reporter, 20U RNase inhibitor, 2. Mu.L RPA amplification product, 5. Mu.L 10 XNEBuffer 3.1 are added to make up the reaction system and ddH is added 2 O to 50. Mu.L.
The temperature of the trans-cutting reaction in the step (3) is 37 ℃; the observation mode is that every 30 s is at λex:485 nm; λem:535 Primary fluorescence was measured at nm or macroscopic observation was performed under 254nm uv light.
The invention has the following beneficial effects:
the invention is based on a Crispr/LbCAs12a nucleic acid detection platform and aims at watermelon acidophilic bacteriaAcidovorax citrulli) The genome of (a) is designed with specific detection sites, and a fluorescent visualization technology is combined to develop an easy diagnosis method suitable for detecting the pathogenic bacteria. And simultaneously, the sensitivity is further improved by combining with the RPA isothermal amplification technology. Compared with the conventional PCR detection, the lowest detection bacterial liquid concentration of LbCAs12a fluorescence detection is 40 CFU/reaction (FIG. 2B), which is 100 times of that of PCR (FIG. 2C).
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows acidophilus of watermelonA. citrulliLbCAs12a fluorescence detection procedure (A) and detection effect (B);
FIG. 2 is a chart of specificity and sensitivity analysis of LbCAs12a fluorescence detection, wherein A is the specificity analysis of LbCAs12a fluorescence detection A. Citrulli, B is the sensitivity of LbCAs12a fluorescence detection A. Citrulli, and C is the sensitivity of PCR detection A. Citrulli.
FIG. 3 shows 40 batches of watermelon seeds based on LbCAs12a fluorescence detectionA. citrulliVisual inspection of (c).
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
Examples
Strains and culture conditions
Acidophilic bacteria of watermelonA. citrulli The GDMCC 803619 strain is typically grown on King's medium B. 72 h were grown under aerobic conditions at 28 ℃ and genomic DNA was extracted according to instructions using TIANamp Bacteria DNA Kits kit (Tiangen, beijing, china).
To prepare bacterial suspensions of different concentrations, single colonies of the strain were inoculated into LB and cultured at 28℃under 120 r/min for 24 h. Bacterial cells were collected by centrifugation and resuspended in sterile 0.01M PBS buffer, the concentration was adjusted to od600=0.8. In 1mL of sterile 0.01M PBS bufferSerial dilutions were made 10-fold and 100 μl was plated on agar medium and repeated 3 times to determine cell concentration. After calculation according to the growth conditions on the medium after serial dilution, the primary cell concentration was unified to about 1×10 8 CFU mL -1 。
2、A. citrulliDesign of specific RPA primers and crRNA
Extraction from the software package of Metaphlan (v 3.0) metagenomic analysis softwareA. citrulliThe program pre-analyzed all genomic sequences in the known database for about 13500 bacteria and 3500 viruses, and screened for species-specific gene markers. The extracted gene markers were further aligned to NCBI database by BLAST analysis to confirm their presenceA. citrulliIntra-species conservation and inter-species specificity, the coding gene for hypothetical protein C E07-2517 (NCBI accession number REG 69369) was finally selected, and specific RPA primers and crRNA target sites were designed.
。
Isothermal amplification of RPA and LbCAs12a fluorescence analysis
RPA pre-amplification was performed using a commercial twist amp Basic kit (twist dx) prior to performing LbCas12a fluorescence analysis. The lyophilized RPA enzyme powder was first mixed with 2.5. Mu.L of forward and reverse primers (10. Mu.M), 29.5. Mu.L of rehydration buffer, 11. Mu.L of nuclease-free water, and 2. Mu.L of genomic DNA. Subsequently, 2.5. Mu.L of magnesium acetate (280 nM) was added to the tube cap and the reaction was started by centrifugation, and after incubation at 37℃for 30 min, 2. Mu.L of the product was used for the LbCAS12a detection reaction described below.
The reaction system was composed using 100 nM LbCAs12a, 120 nM crRNA, 400 nM ssDNA FQ-reporter (5 '-FAM-TTATT-Quencher-3', synthesized by the biological organism), 20U RNase inhibitor, 2. Mu.L RPA product, 5. Mu.L NEBuffer 3.1 (10X) and ddH was added 2 The trans-cleavage activity was measured at 50. Mu.L of O. Wherein LbCAs12a and crRNA are pre-mixed and pre-incubated at 25℃for 10 min to promote crR prior to addition of other ingredients to the reaction systemNA/LbCas12a complex formation. Reactions were performed at 37℃on a Bio-Tek FLx800 microplate fluorescence reader, with fluorescence measured every 30. 30 s (λex:485 nm; λem:535 nm). In addition, the fluorescence signal was visually observed with a hand-held ultraviolet lamp (254, nm) to the lowest concentration capable of producing a visible positive green fluorescence as the detection Limit (LOD).
Watermelon acidophilus as shown in figure 1A. citrulliLbCAs12a fluorescence detection procedure and final detection effect. The extracted microbial DNA is first pre-amplified with RPA to amplify and detect the number of target templates. The RPA product was then added to an LbCAS12a fluorescence detection system in the presence of UV ultraviolet light (254 nm)A. citrulliThe EP tube of the genome exhibited a distinct green fluorescence signal, whereas in the blank control tube without the genome, there was no macroscopic green fluorescence. The result shows that the RPA primer and crRNA designed by the research can effectively detect the acidophilic bacteria of the watermelonA. citrulliIs present.
Wherein FIG. 1A is a sample of 2. Mu.LA. citrulliGenomic DNA was subjected to RPA pre-amplification. After 20 min of reaction at 37 ℃, 2. Mu.L of RPA product was taken for LbCAs12a fluorescence detection. FIG. 1B shows the endpoint fluorescence values obtained after 20 min of LbCAs12a reaction. Centrifuge tubes above the bar graph represent the visual signal after uv irradiation. Data shown are mean ± standard deviation from 6 independent reactions. The blank control represents no addition to the reactionA. citrulliIs a DNA of (a) a DNA of (b).
To confirm that the designed RPA primers and crRNA bind specifically onlyA. citrulliOther 5 kinds of and are selectedA. citrulliThe strains with closer relatedness were subjected to specificity analysis. The results showed (fig. 2A) that only a. Citrulli was able to excite the green fluorescent signal. In addition, the microbial community of healthy, non-diseased watermelon and melon leaves also does not cause false positive results, while leaf microbial DNA taken from watermelon fruit blotch is able to excite green fluorescent signals. The results show that the developed A. Citrulli detection method has high specificity and can specifically detect pathogenic bacteria. The detection sensitivity is analyzed and compared. Compared with the conventional PCR detection, the lowest detection bacterial liquid concentration of LbCAs12a fluorescence detection is 40 CFU/inverseShould be 100-fold PCR (FIG. 2B) than PCR (FIG. 2C).
Implementation effect example: detection of watermelon seed A. Citrulli
For detecting pathogenic bacteria transmitted through seedsA. citrulli40 seed samples were randomly selected from the market. 50 watermelon seeds of each product were gently crushed, soaked in 40 mL sterile water (0.1% tween 80 added) and gently vortexed for 10 min to promote bacterial elution. The 1mL soak eluate was centrifuged for 5 min with a small centrifuge (9660×g) to remove the supernatant, and the resulting precipitate was used for subsequent DNA extraction. To enable sample preparation on site, bacterial genomic DNA was rapidly prepared using commercial lysis buffer MightyPrep reagent for DNA (TaKaRa, dally, china). Briefly, the pellet was resuspended in 50 μl extraction buffer and then heated with a small constant temperature metal bath at 95 ℃ for 10 min. After brief centrifugation, 2 μl of supernatant was directly used as substrate for detection of pathogenic bacteria by RPA isothermal amplification and LbCas12a visualization. The DNA template extracted by the kit can also be used for qPCR detection for sensitivity comparison.
In the final method validation, a total of 40 batches of watermelon seed samples were collected from the market to verify the new method pairsA. citrulliDetection effect of pathogen. The LbCAs12a fluorescence test results showed (FIG. 3), most samples were negative and 6 samples were positive. The PCR result is the same as LbCAs12a fluorescence detection, which shows that the diagnosis results of the two methods are consistent, but the LbCAs12a fluorescence detection does not need to use a complex instrument, and the detection time is shorter and only needs about 40 minutes.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
<110> Henan agricultural university
<120> an RPA primer pair and crRNA for detecting watermelon acidophilus, and kit and use method thereof
<141> 2021-11-30
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> Acidovorax citrulli
<400> 1
gagatgtgct ttggagccct tgaggaattg tt 32
<210> 2
<211> 32
<212> DNA
<213> Acidovorax citrulli
<400> 2
gaacgcatgg atgatgttga agttgtgttc tg 32
<210> 3
<211> 41
<212> DNA/RNA
<213> Acidovorax citrulli
<400> 3
uaauuucuac uaaguguaga ugccgaaagg uugcgcgacg u 41
Claims (6)
1. A kit for detecting acidophilic bacteria of watermelons is characterized in that: comprises an RPA primer pair, 2 mu L of an RPA amplification product system and 50 mu L of a crRNA/LbCAs12a mixed system; the RPA amplification product system comprises RPA enzyme powder, 29.5 mu L of rehydration buffer, 11 mu L of nuclease-free water, 280 nM magnesium acetate and 2 mu L of genome DNA; the crRNA/LbCAs12a mixture comprises 100 nM LbCAs12a, 120 nM crRNA, 400 nM ssDNA FQ-report, 20U RNase inhibitor, 5 μL 10 XNEBuffer 3.1 and ddH 2 O is 50 mu L in total;
the RPA primer pair sequences are shown in SEQ ID No.1 and SEQ ID No.2, and the crRNA sequence is shown in SEQ ID No. 3; the concentration of the forward primer and the reverse primer in the RPA primer pair is 10 mu M, and the content is 2.5 mu L; FQ-reporter sequence is 5'-FAM-TTATT-Quencher-3'.
2. The method of using the kit of claim 1, comprising the steps of:
(1) Mixing the freeze-dried RPA enzyme powder with 10 mu M of RPA primer pair, rehydration buffer solution, nuclease-free water and microbial genome DNA of the seed to be detected to obtain a reaction system;
(2) Adding 280 nM magnesium acetate into the reaction system of the step (1), centrifugally starting the reaction, and incubating to obtain an initial product;
(3) A crRNA/LbCAs12a mixed system was prepared and 2. Mu.L of the initial product of step (2) was added, and the trans-cleavage reaction was performed on a Bio-Tek FLx800 microplate fluorescence reader and the results were observed.
3. The method of use according to claim 2, wherein: the volume of the 10 mu M RPA primer pair in the step (1) is 2.5 mu L, the volume of the rehydration buffer is 29.5 mu L, the volume of the nuclease-free water is 11 mu L, and the volume of the microbial genome DNA of the seed to be detected is 2 mu L.
4. The method of use according to claim 2, wherein: the volume of 280. 280 nM magnesium acetate in the step (2) is 2.5 mu L, and the incubation condition is that the incubation is performed for 30 min at 37 ℃.
5. The method of claim 2, wherein the crRNA/LbCas12a mixed system in step (3) is prepared by the steps of: 100 nM LbCAs12a and 120 nM crRNA are pre-mixed and pre-incubated at 25℃for 10 min to promote formation of crRNA/LbCAs12a complex, then 400 nM ssDNA FQ-reporter, 20U RNase inhibitor, 2. Mu.L RPA amplification product, 5. Mu.L 10 XNEBuffer 3.1 are added to make up the reaction system and ddH is added 2 O to 50. Mu.L.
6. The method of use according to claim 2, wherein: the temperature of the trans-cutting reaction in the step (3) is 37 ℃; the observation mode is that every 30 s is at λex:485 nm; λem:535 Primary fluorescence was measured at nm or macroscopic observation was performed under 254nm uv light.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111440174.6A CN114107526B (en) | 2021-11-30 | 2021-11-30 | RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111440174.6A CN114107526B (en) | 2021-11-30 | 2021-11-30 | RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114107526A CN114107526A (en) | 2022-03-01 |
| CN114107526B true CN114107526B (en) | 2024-04-16 |
Family
ID=80368315
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111440174.6A Active CN114107526B (en) | 2021-11-30 | 2021-11-30 | RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114107526B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6146834A (en) * | 1999-09-10 | 2000-11-14 | The United States Of America As Represented By The Secretary Of Agriculture | PCR primers for detection of plant pathogenic species and subspecies of acidovorax |
| JP2017023084A (en) * | 2015-07-24 | 2017-02-02 | 国立大学法人徳島大学 | Method for detecting bacteria causing bacterial fruit blotch disease and bacteria causing bacterial brown stripe disease in cucurbitaceae |
| CN108588250A (en) * | 2018-06-26 | 2018-09-28 | 浙江大学 | A kind of LAMP primer and its detection method for detecting Acidovorax Avenae Subsp |
| CN113151524A (en) * | 2021-05-19 | 2021-07-23 | 浙江大学 | Primer pair for detecting bacterial fruit blotch of watermelon and application thereof |
| CN113234840A (en) * | 2021-04-29 | 2021-08-10 | 华中农业大学 | Kit for rapidly diagnosing peach bacterial perforation disease |
-
2021
- 2021-11-30 CN CN202111440174.6A patent/CN114107526B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6146834A (en) * | 1999-09-10 | 2000-11-14 | The United States Of America As Represented By The Secretary Of Agriculture | PCR primers for detection of plant pathogenic species and subspecies of acidovorax |
| JP2017023084A (en) * | 2015-07-24 | 2017-02-02 | 国立大学法人徳島大学 | Method for detecting bacteria causing bacterial fruit blotch disease and bacteria causing bacterial brown stripe disease in cucurbitaceae |
| CN108588250A (en) * | 2018-06-26 | 2018-09-28 | 浙江大学 | A kind of LAMP primer and its detection method for detecting Acidovorax Avenae Subsp |
| CN113234840A (en) * | 2021-04-29 | 2021-08-10 | 华中农业大学 | Kit for rapidly diagnosing peach bacterial perforation disease |
| CN113151524A (en) * | 2021-05-19 | 2021-07-23 | 浙江大学 | Primer pair for detecting bacterial fruit blotch of watermelon and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| "A sensitive visual method for onsite detection of quarantine pathogenic bacteria from horticultural crops using an LbCas12a variant system";Jian Jiao et al.;《Journal of Hazardous Materials》;第426卷;第1-9页及补充数据第1-6页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114107526A (en) | 2022-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109486970B (en) | Loop-mediated isothermal amplification detection primer group, detection method and detection kit for xanthomonas oryzae | |
| CN104774955B (en) | The detection method of a kind of grape seat chamber bacterium | |
| CN111534621A (en) | Primer and detection method for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of colletotrichum gloeosporioides | |
| CN113151522A (en) | LFD-RPA technology-based rice bacterial leaf streak germ detection kit, primer probe composition and application thereof | |
| CN109868329B (en) | Screening, quarantine and identification method of colletotrichum specific primers | |
| CN114107526B (en) | RPA primer pair and crRNA for detecting acidophilic bacteria of watermelons, kit and use method thereof | |
| CN108384881B (en) | Fluorescent quantitative PCR detection method of cercospora fuscogilva | |
| CN116516058A (en) | Method and kit for visually detecting phytophthora sojae | |
| CN114277166B (en) | RPA detection primer, probe and detection method for melon bacterial fruit blotch | |
| WO2006123423A1 (en) | Primer for detecting fungus of the genus fusarium | |
| CN113930543A (en) | Real-time fluorescent PCR (polymerase chain reaction) kit for detecting fusarium graminearum toxigenic fungi in food | |
| CN114645096A (en) | Micro-drop type digital PCR (polymerase chain reaction) kit for detecting fusarium solani | |
| JP2006238838A (en) | Method for assaying existence of bacterium | |
| CN106676193B (en) | Molecular marker, primer and probe for identifying penicillium | |
| CN111944919B (en) | Banana fusarium wilt tropical No.4 small species visual detection technology system capable of being operated in field and at normal temperature | |
| Allawi | Isolation and Identification of Penicillium rubens from the Local Strain in Mosul, Iraq, and Investigation of Potassium Phosphate Effect on its Growth | |
| CN108949908A (en) | The method of two step genome comparison methods design 4742 quantitative PCR specific primer of Guizhou trichoderma NJAU | |
| CN107723380A (en) | A kind of stem rot of sweet potato bacterium LAMP detection primer and its application | |
| CN114107532B (en) | Molecular target for identifying pseudomonas aeruginosa and quantitative detection method thereof | |
| JP2005245257A (en) | Primer for detecting bacterium of genus fusarium | |
| CN111705159B (en) | Real-time fluorescent RPA detection primer for sweet potato black spot germs and application thereof | |
| CN114592079B (en) | LAMP detection primer group, reagent and detection method for detecting pathogenic bacteria of potato scab and application of LAMP detection primer group | |
| CN103060926A (en) | Enzyme digestion based preparation method of bacteria nucleic acid fingerprint characteristic spectrums and application thereof | |
| CN108977557B (en) | Specific quantitative PCR (polymerase chain reaction) primer for methylotrophic bacillus strain and design method thereof | |
| CN111020055B (en) | LAMP primer and kit for detecting Lasiodipia gonubiensis |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |