CN116121408A - Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application - Google Patents
Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application Download PDFInfo
- Publication number
- CN116121408A CN116121408A CN202211090936.9A CN202211090936A CN116121408A CN 116121408 A CN116121408 A CN 116121408A CN 202211090936 A CN202211090936 A CN 202211090936A CN 116121408 A CN116121408 A CN 116121408A
- Authority
- CN
- China
- Prior art keywords
- listeria monocytogenes
- crispr
- rpa
- cas12a
- detection
- 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
- 241000186779 Listeria monocytogenes Species 0.000 title claims abstract description 55
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 42
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 42
- 108700004991 Cas12a Proteins 0.000 title claims abstract description 37
- 238000012800 visualization Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 53
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 35
- 230000003321 amplification Effects 0.000 claims abstract description 34
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 27
- 108020004414 DNA Proteins 0.000 claims description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 238000011144 upstream manufacturing Methods 0.000 claims description 10
- 102000053602 DNA Human genes 0.000 claims description 9
- 235000013305 food Nutrition 0.000 claims description 9
- 239000011535 reaction buffer Substances 0.000 claims description 8
- 239000008176 lyophilized powder Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000002834 transmittance Methods 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 10
- 238000012864 cross contamination Methods 0.000 abstract description 2
- 238000003912 environmental pollution Methods 0.000 abstract 1
- 239000013642 negative control Substances 0.000 description 11
- 230000035945 sensitivity Effects 0.000 description 10
- 239000007788 liquid Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 6
- 230000001717 pathogenic effect Effects 0.000 description 6
- 241000588747 Klebsiella pneumoniae Species 0.000 description 5
- 241000607762 Shigella flexneri Species 0.000 description 5
- 235000015277 pork Nutrition 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000607447 Yersinia enterocolitica Species 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 101150093386 prfA gene Proteins 0.000 description 3
- 101150027417 recU gene Proteins 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 229940098232 yersinia enterocolitica Drugs 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010022004 Influenza like illness Diseases 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 206010024641 Listeriosis Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 241000235013 Yarrowia Species 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150059443 cas12a gene Proteins 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- SPTYHKZRPFATHJ-HYZXJONISA-N dT6 Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)CO)[C@@H](O)C1 SPTYHKZRPFATHJ-HYZXJONISA-N 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000003670 easy-to-clean Effects 0.000 description 1
- 208000010227 enterocolitis Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 244000078673 foodborn pathogen Species 0.000 description 1
- 235000014106 fortified food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—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
- 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application thereof, wherein the kit comprises an RPA amplification reagent and a CRISPR/Cas12a detection reagent. The invention also discloses a method for detecting listeria monocytogenes by using the field visualization kit, all reagents are placed in the same centrifuge tube for detection, the operation is simple, and the sample and the environmental pollution are avoided. By using the kit and the detection method for detecting the listeria monocytogenes, constant-temperature detection at 37 ℃ can be realized, RPA reaction volume is greatly reduced, bacterial detection of 4.4CFU/g in 25 minutes can be realized, and the result is visualized. The method has the advantages of reducing the loss of the RPA system, completely separating the amplification system from the detection system, avoiding cross contamination, visualizing the result and the like.
Description
Technical Field
The invention relates to a CRISPR/Cas12a field visualization kit for detecting listeria monocytogenes and application thereof, belonging to the technical field of molecular biological diagnosis.
Background
Listeria monocytogenes (Listeria monocytogenes), abbreviated as listeria monocytogenes, is an important food-borne pathogenic bacterium whose tolerance and growth ability at low temperatures are one of the prominent features of the pathogen. The pathogen can form biological film on the surfaces of many objects, is not easy to clean, can grow in the environment of 4 ℃ and is the main pollution bacteria of refrigerated foods. Human listeriosis is caused by the consumption of foods contaminated with listeria monocytogenes. Intake of highly contaminated foods by most people can lead to moderate to severe gastroenteritis, symptoms including diarrhea, abdominal pain, fever, vomiting, mild and influenza-like illness.
At present, the detection of listeria monocytogenes in China comprises bacteriology, immunology and molecular biology. Bacteriology is a gold standard but is time-consuming and laborious, and immunological tests have low sensitivity to non-enriched food pathogen detection. The listeria monocytogenes has extremely low concentration in food, and has high requirement on the sensitivity of detection technology. With the development of molecular biology techniques, nucleic acid amplification methods including PCR, LAMP, RPA have been increasingly used for the identification of listeria monocytogenes. However, the existing molecular biology technology has high requirements on instruments or is easy to pollute in operation, complex in sample treatment and the like, and the amplified product can obtain a detection result only by agarose gel electrophoresis or fluorescent quantitative detection and other equipment, so that the field visualization can not be achieved.
In recent years, gene editing techniques based on CRISPR systems have been hot. CRISPR is essentially an adaptive immune system in bacteria that recognizes and activates the corresponding Cas protein through crRNA to break down the invader's DNA or RNA when re-invaded by a foreign microorganism. CRISPR/Cas12a is guided by single-stranded RNA (crRNA), and after specific binding to target DNA, structural changes in Cas12a result in its enzymatic activity, which can cleave not only target DNA in cis but also non-target DNA in trans. In 2018, the Jennifer Doudna team established a DETECTR ((DNA Endonuclease Targeted CRISPR Trans Reporter) by utilizing the properties of CRISPR/Cas12a in combination with isothermal amplification and reported in the paper that Human Papillomavirus (HPV) of patients was detected.
Disclosure of Invention
One of the purposes of the invention is to provide a site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application thereof.
The second object of the present invention is to provide a method for detecting listeria monocytogenes using the above kit.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention discloses a field visualization kit for detecting listeria monocytogenes (Listeria monocytogenes) based on CRISPR/Cas12a, which comprises the following components: an RPA amplification reagent and a CRISPR/Cas12a detection reagent;
the RPA amplification reagent comprises an RPA amplification primer pair, wherein the RPA amplification primer pair consists of an upstream primer shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No. 2;
the CRISPR/Cas12a detection reagent comprises LbCAs12a, crRNA, NEBuffer3.1 and a ssDNA fluorescent reporter, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 3.
Preferably, the RPA amplification reagent further comprises RPA Basic lyophilized powder, an RPA reaction buffer solution, mgOAc and DEPC water.
Furthermore, the invention also provides application of the field visualization kit in detection of listeria monocytogenes.
Wherein, preferably, the field visualization kit is used for detecting listeria monocytogenes existing in food or environment.
Furthermore, the invention also provides a method for detecting listeria monocytogenes existing in food or environment by using the field visualization kit, which comprises the following steps:
(1) Extracting genome DNA in listeria monocytogenes by adopting a water boiling method;
(2) Placing the RPA amplification reagent and the genomic DNA at the bottom of a centrifuge tube, and placing a CRISPR/Cas12a detection reagent on a centrifuge tube cover;
(3) RPA amplification was performed for 15 min;
(4) Centrifuging for 5 seconds, uniformly mixing all reagents in the centrifuge tube, and detecting CRISPR/Cas12 a;
(5) After 10 minutes, fluorescent signals were detected and observed.
Wherein, preferably, in the step (3), the reaction system for RPA amplification comprises: 0.4mg of RPA Basic lyophilized powder, 10. Mu.M of each of the upstream and downstream primers was 0.24. Mu.L, 2.95. Mu.L of RPA reaction buffer, 280mM MgOAc 0.25. Mu.L, 0.2. Mu.L of genomic DNA and DEPC water were made up to 5. Mu.L of the reaction system under the following conditions: amplified at 37℃for 15 minutes.
Wherein, preferably, in step (4), the CRISPR/Cas12a detection system comprises: 1 μM LbCAs12 a2 μL,1 μM crRNA2 μL, NEBuffer3.13 μL,5 μM ssDNA fluorescent reporter 2 μL and DEPC water 16 μL, at 37℃for 10 minutes.
Preferably, in the step (5), fluorescence generated by the reaction system is observed under a blue light transmittance meter with a wavelength of 470-520 nm.
Wherein, preferably, the minimum detection limit of the listeria monocytogenes concentration is 4.4CFU/g.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12 a; a method for detecting Listeria monocytogenes using the in situ visualization kit is also provided. According to the invention, the CRISPR/Cas12a detection reagent and the RPA amplification reagent are completely separated in the same centrifuge tube, so that the two reactions are not affected, the RPA reaction is carried out for 15 minutes and then the centrifugation is carried out, and the detection sensitivity of 4.4CFU/g is realized in 25 minutes. The invention does not need expensive equipment and greatly reduces the cost and time of diagnosis for field detection. The primer group has the advantages of strong specificity and good sensitivity, the kit provided by the invention has the advantages of greatly reducing the loss of an RPA system, completely separating an amplification system from a detection system, greatly shortening the reaction time, visualizing the result and the like. The CRISPR/Cas12a method established by the invention can be used for field visual detection of listeria monocytogenes, thereby being beneficial to popularization in basic level and field work and reducing infection risk of consumers.
Drawings
FIG. 1 is a schematic diagram of CRISPR/Cas12a detection of Listeria monocytogenes provided by the present invention;
FIG. 2 is a diagram showing the result of screening specific PCR primers against Listeria monocytogenes provided by the present invention (P represents positive control, N represents negative control);
FIG. 3 shows the result of screening specific RPA primers against Listeria monocytogenes provided by the present invention (P represents positive control, N represents negative control);
FIG. 4 is a graph showing the results of RPA volume optimization provided by the present invention;
FIG. 5 is a graph of crRNA screening results provided by the present invention;
FIG. 6 is a bar chart of endpoint fluorescence and a visual result chart of specific detection of different food-borne pathogens provided by the invention; in the bar graph, from left to right, listeria monocytogenes (Listeria monocytogenes), pathogenic enterocolitis yersinia (pathogenic Yersinia enterocolitica), klebsiella pneumoniae (Klebsiella Pneumoniae) shigella flexneri (Shigella flexneri), and negative control (NTC), respectively;
FIG. 7 is a bar graph of endpoint fluorescence and visualization results of sensitivity detection by different Listeria monocytogenes genome concentrations provided by the present invention;
FIG. 8 is a bar graph of endpoint fluorescence and visualization results of sensitivity detection of different Listeria monocytogenes bacteria concentrations in labeled pork provided by the present invention;
FIG. 9 is a standard curve of concentration versus fluorescence intensity for different Listeria monocytogenes bacteria solutions in a labeled pork provided by the present invention.
Detailed Description
The experimental procedures, without specific conditions noted in the examples below, were selected according to methods and conditions conventional in the art, or according to the commercial specifications. Reagents and starting materials were commercially available, for which specific components were not identified in the examples below.
The basic principle of the method for detecting listeria monocytogenes DNA based on CRISPR/Cas12a provided by the invention is as shown in figure 1: the three target genes of the listeria monocytogenes firstly amplify specific DNA fragments through trace RPA, then specifically combine through a complex of Cas12a/crRNA, finally cut ssDNA fluorescent reporter by utilizing DNA nonspecific enzyme digestion activity, and detect the listeria monocytogenes through fluorescent signals.
Example 1 specific target Gene screening and construction of RPA amplification method
1.1 target Gene screening and primer design
The invention designs 3 pairs of primers for the prfA and hly genes of Listeria monocytogenes respectively, and a primer Lm8 corresponding to a certain assumed protein gene is derived from the literature (Tao T, chen Q, bie X, et al, mining of novel patterns-specific primers for PCR detection of Listeria monocytogenes based on genomic approach [ J)].World Journal of Microbiology&Biotechnology,2015,31 (12): 1955-1966), the three genes are highly conserved and exist in the genome of all pathogenic serotypes of Listeria monocytogenes. According to
Primer design rules of the basic kit, prfA (FR 733646.1), hly (FR 733646.1) in GenBank, specific primers were designed by Primer premier 5.0. The nucleotide sequences of the primers are shown in Table 1:
RPA primers described in Table 1
First, the primers are initially screened by ordinary PCR, and then re-screened by RPA. PCR amplification System and procedure: the total system was 20. Mu.L, including 2 XM 5 HiPer plus Taq HiFi PCR mix. Mu.L, 1. Mu.L (10 mM) each of the upstream primer F and the downstream primer R, 2. Mu.L of the genomic template, and the rest of DEPC water were made up. Pre-denaturation at 95℃for 2min, denaturation at 95℃for 30s, annealing for 30s, extension at 72℃for 30s, total of 35 cycles, extension at 72℃for 2min. RPA amplification system and procedure: 4mg RPABasic lyophilized powder, 10. Mu.M upstream and downstream primer F/R each 2.4. Mu.L, genome template 2. Mu.L, RPA reaction buffer 29.5. Mu.L, 280mM MgOAc 2.5. Mu.L, and DEPC water 11.2. Mu.L at 37℃for 15min. The amplified products and DL500 DNA markers for molecular marking are electrophoresed for 30min in 2% agarose gel at 120V, and the result of PCR amplification reaction is shown in figure 2 by using a gel imaging system to observe the result, and the prfA-2F/R, hly-1F/R and Lm-8F/R have better effects according to the thickness of the amplified product strip corresponding to each target point. The result of RPA amplification screening is shown in FIG. 3, and the amplification effect of the primer pair prfA-2F/R, hly-3F/R and Lm-8F/R is better according to the thickness of the amplified product.
1.2 RPA amplification
1.2.1 cultivation of bacteria: culturing at 37 deg.c and 150rpm for 12 hr to obtain bacterial liquid, 10 times gradient dilution with physiological saline to prepare live bacterial solutions in different concentration, and coating and counting with proper 3 serial gradient dilutions;
1.2.2 extraction of genomic DNA: extracting genome DNA of bacteria by water boiling method. The bacterial culture was centrifuged at 12000r/min for 2min and the supernatant was discarded. The pellet was suspended in DEPC water, boiled for 20 minutes, and immediately frozen at-20 ℃. The solution was then centrifuged at 12000r/min for 2 minutes. The supernatant was used as a template for the reaction.
1.2.3 RPA reaction system optimization and establishment
The optimal reaction volume for RPA of the present invention was determined to be 5 μl by setting a volume gradient of 15 μl,10 μl,5 μl,2 μl, considering that the volume of RPA reaction may affect subsequent CRISPR/Cas12a detection. The results are shown in FIG. 4 (P is the positive experimental group, N is the negative control group). The 5 μl reagent system is as in table 2:
TABLE 2 RPA reaction System
| Reaction components | Dosage of |
| Upstream primer F (10. Mu.M) | 0.24μL |
| Downstream primer R (10. Mu.M) | 0.24μL |
| RPA Basic freeze-dried powder | 0.4mg |
| RPA reaction buffer | 2.95μL |
| DNA template | 0.2μL |
| DEPC water | 1.12μL |
| 280mM MgOAc | 0.25μL |
| Sum up | 5μL |
The reagents were mixed well and removed and added to the 0.2. Mu.L genomic DNA to make up a 5. Mu.L reaction system. The reaction conditions were metal bath or water bath for 15 minutes at 37 ℃.
Example 2 crRNA screening and CRISPR/cas12a detection System establishment
2.1 CrRNA sequence design
3 crRNA sequences for prfA, hly, some putative protein genes were designed and the specific sequences are shown in Table 3:
TABLE 3 crRNA sequence
2.2 selection of optimal primers and crRNA
As a result of fluorescence kinetics screening of the three crrnas, it was found that the fluorescence-time profile and fluorescence intensity of the reaction system containing crRNA-hly and its primer pair were significantly better than those of the reaction system containing crRNA-prfA and its primer pair and crRNA-Lm8 and its primer pair (fig. 5), and thus crRNA-hly and its primer pair were selected for subsequent reaction system studies. Therefore hly-3F was chosen as the optimal upstream primer and hly-3R as the optimal downstream primer for the RPA amplification reaction.
The optimal primers and corresponding crRNA sequences were determined as described above, in particular as shown in table 4.
TABLE 4 optimal primers and crRNA
2.3 Optimization and establishment of CRISPR/Cas12a detection system
The present invention performs CRISPR/Cas12a detection with the system described in table 5:
table 5 CRISPR/Cas12a detection system
Wherein the ssDNA reporter has the sequence of 6-FAM-/TTTTTT/BHQ-1.
Placing the CRISPR/Cas12a detection reagent on a centrifuge tube cover, centrifuging for 5 seconds after the RPA amplification reaction is finished, uniformly mixing an RPA amplification product and the CRISPR/Cas12a detection reagent, and continuing to react for 10 minutes at 37 ℃.
2.4 specific assay
The genomic DNA from the specific assays was tested using the genome extracted in example 1, including Listeria monocytogenes (Listeria monotygenes), yarrowia enterocolitica (Yersinia enterocolitica), klebsiella pneumoniae (Klebsiella pneumoniae), shigella flexneri (Shigella flexneri), with DEPC water added as a negative control. The specificity of the methods of the invention was verified according to the CRISPR/Cas12a detection system described above. The detection results in FIG. 6 show that (corresponding to Listeria monocytogenes, yersinia enterocolitica, klebsiella pneumoniae and Shigella flexneri from left to right), CRISPR/Cas12a has very high specificity to the fluorescence intensity containing the Listeria monocytogenes gene, other bacteria and negative control have no fluorescence reaction, the method has no cross reaction in the detection process, the specificity is good, and the visualization can be realized.
2.5 genome sensitivity detection
Quantification and gradient dilution are carried out on a listeria monocytogenes genome template for sensitivity detection, and the prepared genome concentrations are respectively 4.7x10 -5 ng/μL、4.7×10 -7 ng/μL、4.7×10 -9 ng/μL、4.7×10 -10 ng/. Mu.L, while DEPC water was used as a negative control. Genome extraction by the method of example 1DNA, as described above, is added to the bottom of the tube with RPA amplification reagents and varying concentrations of Listeria monocytogenes genomic DNA, the RPA amplification reagents comprising: 0.4mg of RPA Basic lyophilized powder, 10. Mu.M of each of the upstream and downstream primers, 0.24. Mu.L of RPA reaction buffer, 2.95. Mu.L, 280mM MgOAc 0.25. Mu.L and DEPC water 1.12. Mu.L. After reacting for 15min at 37 ℃, centrifuging the CRISPR/Cas12a detection reagent at the tube cover for 5 seconds, uniformly mixing at the bottom, and continuing to react for 10min at 37 ℃, wherein the CRISPR/Cas12a detection reagent comprises: 1 μM LbCAs12 a2 μL,1 μM crRNA2 μL,3 μL NEBuffer3.1,5 μM ssDNA fluorescent reporter 2 μL and 16 μL DEPC water. The results of the blue light projector and the microplate reader were observed, and the fluorescence intensities were statistically different from those of the negative control, and as a result, as shown in FIG. 7, the lowest detection limit of the genome was determined to be 4.7X10 -9 ng/μL。
2.6 detection of bacterial liquid sensitivity
Colony counting the cultured bacterial liquid and gradient diluting the bacterial liquid to 4.4X10 concentration 8 CFU/mL，4.4×10 6 CFU/mL，4.4×10 4 CFU/mL，4.4×10 2 CFU/mL，4.4×10 0 CFU/mL. Pork samples were taken, washed with sterile PBS, then the samples were divided into 5 small portions (1 g) and subjected to uv irradiation to ensure sterility. 1 hour of the sample is randomly selected as a negative control, and the other 4 hours of the sample are respectively inoculated with 1mL of the diluted bacterial liquid and homogenized to obtain the labeled pork sample. Genomic DNA was extracted from a labeled pork sample by the method of example 1, and RPA amplification reagents and Listeria monocytogenes genomic DNA at different concentrations were added to the bottom of the tube as described above, wherein the RPA amplification reagents contained: 0.4mg of RPA Basic lyophilized powder, 10. Mu.M of each of the upstream and downstream primers, 0.24. Mu.L of RPA reaction buffer, 2.95. Mu.L, 280mM MgOAc 0.25. Mu.L and DEPC water 1.12. Mu.L. After reacting for 15min at 37 ℃, centrifuging the CRISPR/Cas12a detection reagent at the tube cover for 5 seconds, uniformly mixing at the bottom, and continuing to react for 10min at 37 ℃, wherein the CRISPR/Cas12a detection reagent comprises: 1 μM LbCAs12 a2 μL,1 μM crRNA2 μL,3 μL NEBuffer3.1,5 μM ssDNA fluorescent reporter 2 μL and 16 μL DEPC water. The results observed by the blue light projector and the enzyme label instrument are observed by the blue light projector and the enzyme label instrument, and compared with the negative control, the results are fluorescentThe light intensities are all statistically different, and the result is shown in FIG. 8, and the minimum detection limit of the bacterial liquid of the labeled sample is determined to be 4.4X10 0 CFU/g。
2.5.3 drawing a standard curve with the concentration of Listeria monocytogenes bacteria liquid as the abscissa and the fluorescence intensity at the concentration as the ordinate. The regression equation of the fitted standard curve is y=579.6x+3069 (R 2 = 0.9620), wherein Y represents fluorescence intensity, X represents bacterial liquid concentration, and the standard curve is shown in fig. 9.
From the results of the above examples, it can be seen that: the method can be used for rapidly, sensitively and accurately detecting the listeria monocytogenes, and avoids cross contamination of samples or environment. The method has stronger specificity and sensitivity, can exclude the interference of other pathogenic bacteria, and can detect target bacteria as low as 4.4CFU/g. By combining CRISPR/Cas12a as a signal recognition element and ssDNA reporter as a signal amplification element, the amplification reaction and the detection reaction are not mutually interfered, and the RPA system has low loss, so that the experimental steps are simplified, the detection cost is reduced, and the rapid and on-site visual detection of listeria monocytogenes in food samples with complex components can be realized.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A site-visualized kit for detecting listeria monocytogenes (Listeria monocytogenes) based on CRISPR/Cas12a, characterized in that the site-visualized kit comprises the following components: an RPA amplification reagent and a CRISPR/Cas12a detection reagent;
the RPA amplification reagent comprises an RPA amplification primer pair, wherein the RPA amplification primer pair consists of an upstream primer shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No. 2;
the CRISPR/Cas12a detection reagent comprises LbCAs12a, crRNA, NEBuffer3.1 and a ssDNA fluorescent reporter, and the nucleotide sequence of the crRNA is shown as SEQ ID NO. 3.
2. The field visualization kit of claim 1, wherein the RPA amplification reagents further comprise RPABasic lyophilized powder, RPA reaction buffer, mgOAc, and DEPC water.
3. Use of the field visualization kit of claim 1 or 2 for detecting listeria monocytogenes.
4. The use of claim 3, wherein said field visualization kit is for detecting listeria monocytogenes present in a food product or environment.
5. A method of detecting listeria monocytogenes present in a food product or environment using the site visualization kit of claim 1 or 2, comprising the steps of:
(1) Extracting genome DNA in listeria monocytogenes by adopting a water boiling method;
(2) Placing the RPA amplification reagent and the genomic DNA at the bottom of a centrifuge tube, and placing a CRISPR/Cas12a detection reagent on a centrifuge tube cover;
(3) RPA amplification was performed for 15 min;
(4) Centrifuging for 5 seconds, uniformly mixing all reagents in the centrifuge tube, and detecting CRISPR/Cas12 a;
(5) After 10 minutes, fluorescent signals were detected and observed.
6. The method of claim 5, wherein in step (3), the reaction system for RPA amplification comprises: 0.4mg of RPABasic freeze-dried powder, 10. Mu.M of each of the upstream and downstream primers, 2.95. Mu.L of RPA reaction buffer, 280mM MgOAc, 0.25. Mu.L, 0.2. Mu.L of genomic DNA and DEPC water were made up to 5. Mu.L of the reaction system under the following reaction conditions: amplified at 37℃for 15 minutes.
7. The method of claim 5, wherein in step (4), the CRISPR/Cas12a detection system comprises: 1 μM LbCAs12 a2 μL,1 μM crRNA2 μL, NEBuffer3.13 μL,5 μM ssDNA fluorescent reporter 2 μL and DEPC water 16 μL, at 37℃for 10 minutes.
8. The method according to claim 5, wherein the fluorescence generated by the reaction system is observed in step (5) under a blue light transmittance meter having a wavelength of 470 to 520 nm.
9. The method of claim 5, wherein the listeria monocytogenes has a minimum detection limit of 4.4CFU/g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211090936.9A CN116121408A (en) | 2022-09-07 | 2022-09-07 | Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211090936.9A CN116121408A (en) | 2022-09-07 | 2022-09-07 | Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116121408A true CN116121408A (en) | 2023-05-16 |
Family
ID=86296155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211090936.9A Pending CN116121408A (en) | 2022-09-07 | 2022-09-07 | Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116121408A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568514A (en) * | 2023-11-29 | 2024-02-20 | 南京微测生物科技有限公司 | Method and kit for detecting listeria monocytogenes based on inferior PAM one-pot method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113462795A (en) * | 2021-05-31 | 2021-10-01 | 浙江大学 | Combined detection method for rapidly detecting Listeria monocytogenes, system and application thereof |
| CN114075607A (en) * | 2020-08-21 | 2022-02-22 | 吉林大学 | On-site visualization kit for detecting listeria monocytogenes based on SHERLOCK and application |
-
2022
- 2022-09-07 CN CN202211090936.9A patent/CN116121408A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114075607A (en) * | 2020-08-21 | 2022-02-22 | 吉林大学 | On-site visualization kit for detecting listeria monocytogenes based on SHERLOCK and application |
| CN113462795A (en) * | 2021-05-31 | 2021-10-01 | 浙江大学 | Combined detection method for rapidly detecting Listeria monocytogenes, system and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| TIAN YACHEN等: "An ultrasensitive and contamination-free on-site nucleic acid detection platform for Listeria monocytogenes based on the CRISPR-Cas12a system combined with recombinase polymerase amplification", 《LWT》, vol. 152, 4 August 2021 (2021-08-04), pages 2, XP086805414, DOI: 10.1016/j.lwt.2021.112166 * |
| WANG BEI等: "Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection", 《ANALYTICAL CHEMISTRY》, 28 August 2019 (2019-08-28) * |
| 王明华主编: "《生物检测技术在食品检验中的应用研究》", 31 July 2022, 中华工商联合出版社, pages: 144 - 145 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117568514A (en) * | 2023-11-29 | 2024-02-20 | 南京微测生物科技有限公司 | Method and kit for detecting listeria monocytogenes based on inferior PAM one-pot method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111020008B (en) | Rapid isothermal detection method and kit for nucleic acid of vibrio cholerae O1 group | |
| CN113249499B (en) | Salmonella typhi detection kit, and preparation method and application thereof | |
| CN112831580B (en) | Reaction system for detecting vibrio parahaemolyticus DNA, kit and application thereof | |
| CN106661606A (en) | Method for detecting and characterising a microorganism | |
| CN112159854B (en) | Primer composition for detecting CRISPR/Cas12a of escherichia coli O157-H7 and detection method | |
| CN107746879A (en) | Detect RPA primers, probe, kit and the detection method of staphylococcus aureus | |
| CN114075607B (en) | On-site visualization kit for detecting listeria monocytogenes based on SHERLOCK and application | |
| US20220098645A1 (en) | Fast and portable microfluidic detection system as an alternative to salmonella's classical culture method | |
| CN107022632A (en) | A kind of Salmeterol fluticasone propionate primer sets, kit and application | |
| CN111378774A (en) | Primer group, kit and method for rapidly detecting Listeria monocytogenes | |
| CN102676664B (en) | Fluorescent quantitative polymerase chain reaction (PCR) primers and probes for detecting pathogenic bacteria of multiple aquatic products simultaneously and detection method | |
| CN115747353A (en) | Primer group, reagent, kit and detection method for detecting Listeria monocytogenes | |
| CN114196766B (en) | Molecular marker, primer pair, kit and method for specifically identifying rice ralstonia solanacearum Xoo | |
| CN116121408A (en) | Site visualization kit for detecting listeria monocytogenes based on CRISPR/Cas12a and application | |
| CN107236812B (en) | Detection kit for bacillus cereus, detection method and application | |
| CN105567802A (en) | Fluorescence PCR (polymerase chain reaction) detection kit for Chlamydia pneumoniae | |
| CN113512601B (en) | Molecular targets for screening for Proteus and quantitative detection methods | |
| CN109971873A (en) | Identify the method for Listeria Monocytogenes, Yi Shi listeria spp and listeria innocua | |
| JP2013048611A (en) | Method for detecting clostridium perfringens and kit for detecting clostridium perfringens | |
| CN103874766B (en) | Molecular Detection is determined | |
| CN112359133A (en) | RPA primer group, kit and rapid detection method for detecting candida auricula | |
| TWI692528B (en) | Methods for detecting E. coli and molecular markers used | |
| CN114196768B (en) | Specific molecular target for identifying pseudomonas aeruginosa serogroup and rapid detection method thereof | |
| CN114196767B (en) | Specific molecular target and method for detecting staphylococcus aureus ST type by using same | |
| CN111172305B (en) | Method and kit for detecting escherichia coli |
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 |