CN111676302A - Establishment and application of vibrio vulnificus RPA-LFS rapid detection method - Google Patents
Establishment and application of vibrio vulnificus RPA-LFS rapid detection method Download PDFInfo
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Abstract
The invention discloses establishment and application of a vibrio vulnificus RPA-LFS rapid detection method, which comprises the steps of primer design and screening, probe design and screening, RPA-LFS specificity detection, RPA-LFS reaction temperature optimization, RPA-LFS reaction time optimization and RPA-LFS minimum detection limit determination, wherein the length of a primer probe for screening is at least 46bp, the 5 ' tail end of the probe is labeled by FITC, the 3 ' tail end is introduced with 3C-spacer for tail end closure, THF (THF) is introduced in the middle of the probe, namely a purine-free and pyrimidine-free site is not included, at least 30bp of base is reserved in front of the AP site, at least 15bp of base is reserved behind the AP site, and biotin is labeled at the 5 ' tail end of a downstream primer. The invention has low requirement on the experimental operation of the detector, and can carry out detection without hiring professional technicians and farm workers during field detection without devices such as a gel imaging system and the like, thereby reducing the detection cost, shortening the detection practice and realizing visual detection.
Description
Technical Field
The invention relates to the field of marine product culture and food safety, in particular to establishment and application of a vibrio vulnificus RPA-LFS rapid detection method.
Background
At present, the common methods for detecting vibrio vulnificus include traditional culture methods, immunodiagnosis and nucleic acid diagnosis, wherein the nucleic acid diagnosis mainly comprises two methods, namely Polymerase Chain Reaction (PCR) and (LAMP), the traditional culture method is to culture a sample to be detected in a separated manner after enrichment, then to perform morphological and biochemical identification on a suspicious strain which is cultured in a separated manner, to perform comprehensive evaluation on an identification result, and to determine whether the suspicious strain is vibrio vulnificus. The method has the advantages of long detection period, large workload and high requirement on the experimental skill of workers, and the experimental operation can only be carried out in a laboratory and cannot be carried out on-site detection.
The detection period of the traditional culture method for detecting the vibrio vulnificus is long, the workload is large, the requirement on the experimental skill of workers is high, the experimental operation can only be carried out in a laboratory, and the field detection cannot be carried out. PCR is carried out in three temperature stages, and the experimental facility is high in requirement and long in time consumption, so that the PCR technology is limited to laboratory research and is not suitable for field detection (POCT detection). LAMP avoids the defects of PCR to a great extent, but has certain problems in itself: 1. LAMP primers are complex in design and need 2-3 pairs of primers; 2. LAMP is easy to generate false positive results, and the accuracy of the results is reduced; 4. the LAMP amplification time is generally 40min-60min, and the time cost is increased to a certain extent.
Immunodiagnosis is a visual detection of a highly efficient catalytic reaction combining antigen-antibody immunoreactions and enzymes. The amount of the detected antigen is judged by the shade of the color presented by the enzyme degradation substrate. The method has strong specificity, but has high selectivity to reagents, low sensitivity and easy occurrence of cross reaction.
The nucleic acid diagnosis technology is highly concerned by people due to strong specificity and high sensitivity, and the commonly used nucleic acid diagnosis technology mainly comprises two major types of conventional PCR amplification and LAMP. The PCR technology has been reported to be used for detecting more than 100 kinds of pathogens, but since three temperature stages are required, the experimental facility is high and time-consuming. Therefore, PCR technology is limited to laboratory studies and is not suitable for field in-situ detection (POCT detection). Compared with PCR, the isothermal amplification technology is simpler and faster, the specificity and the sensitivity of the isothermal amplification technology are equivalent to those of PCR, the technology overcomes the defect that a single-chain template is obtained by high-temperature denaturation of the traditional PCR technology, the process of temperature rise and drop is also avoided, temperature control equipment is not needed, and the detection cost is reduced to a great extent.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides the establishment and application of a vibrio vulnificus RPA-LFS rapid detection method.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly detecting vibrio vulnificus RPA-LFS comprises the following steps:
1) designing and screening primers:
an upstream primer: 5' -GAGATGGATTCTTTGTATAACATTGCGT
A downstream primer: 5' -ACGATGACGTTGGTTGTGTTACATTATC.
2) Designing and screening a probe:
and (3) probe:
FITC-GGTGAAGTTGGCTGGTGGTTATTTTCAGAA[THF]CATGGTTGTTGAGCTC-SpC3。
3) specific detection of RPA-LFS.
4) The RPA-LFS reaction temperature is optimized.
5) RPA-LFS reaction time is optimized.
6) And (4) determining the lowest detection limit of RPA-LFS.
An application of a vibrio vulnificus RPA-LFS rapid detection method, wherein the length of a primer probe screening probe is at least 46 bp;
the 5 'end of the probe is labeled with FITC, the 3' end is introduced with 3C-spacer for end blocking, and THF (adenine-free pyrimidine-free site) (AP site) is introduced in the middle of the probe;
at least 30bp of base is reserved before the AP locus, at least 15bp of base is reserved after the AP locus, and biotin is marked at the 5' end of the downstream primer.
The application of a vibrio vulnificus RPA-LFS rapid detection method comprises the steps of initially applying and detecting RPA-LFS, detecting clinical samples by using an RPA-LFS detection system, comparing detection results with a traditional culture method and a qPCR method at the same time, detecting 7 positive results by the RPA-LFS in 55 prawn samples, detecting 5 positive results in 25 shellfish samples, and detecting the negative results of 6 fish samples and 2 crab samples;
the invention has the following beneficial effects:
1. the RPA-LFS technology is scientifically and effectively combined with the RPA technology and the LFS technology to establish a method for rapidly detecting AHPND, Vibrio parahaemolyticus and Vibrio vulnificus on site by the RPA-LFS;
2. the RPA-LFS detection method established by the scheme can finish detection in 20-25min, and compared with a detection method based on a PCR technology, the detection time is shortened to a great extent;
3. the RPA-LFS detection method established by the scheme overcomes the dependence on laboratory equipment, can carry out field detection in a farm, avoids the influence on a detection result caused by sample transportation, and shortens the detection period;
4. the RPA-LFS detection method established by the scheme is simple to operate, does not depend on high-quality technicians, enables farmers to carry out detection in a farm, simplifies the detection process and shortens the detection period;
5. the RPA-LFS established by the scheme realizes visual detection, and the detection result can be judged through color reaction, so that farmers can judge the result without any instrument or facility.
Drawings
FIG. 1 is a schematic diagram of the interspecies specific detection of Vibrio vulnificus probes by RPA-LFS;
FIG. 2 is a schematic diagram of the optimization of temperature variable RPA-LFS reaction conditions;
FIG. 3 is a schematic diagram of optimization of time variable RPA-LFS reaction conditions;
FIG. 4 is a schematic diagram of the RPA-LFS sensitivity detection.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
A method for rapidly detecting vibrio vulnificus RPA-LFS comprises the following steps:
1) designing and screening primers: five pairs of primers are designed in NCBIPrimerblast according to the sequence of virulence gene metalloprotease of vibrio vulnificus metalloprotease (VVP), RPA amplification is carried out by taking vibrio vulnificus standard strain ATCC27562 genome DNA as a template, amplification products are detected by 1.5 percent agarose gel electrophoresis, and a primer pair with the best amplification effect is screened out,
an upstream primer: 5' -GAGATGGATTCTTTGTATAACATTGCGT
A downstream primer: 5' -ACGATGACGTTGGTTGTGTTACATTATC.
2) Designing and screening a probe: designing a probe according to a primer pair with the best amplification effect and better interspecies specificity, designing the probe in a primer5.0, synthesizing the designed probe by a synthesis company, detecting, amplifying by using an RPAnfo kit by using vibrio vulnificus standard strain ATCC27562 genomic DNA as a template, detecting the amplification effect of the probe by combining a lateral flow test strip, and selecting the primer and the probe which have the good amplification effect and do not have amplification by NTC for next step of interspecies specificity detection
And (3) probe: FITC-GGTGAAGTTGGCTGGTGGTTATTTTCAGAA [ THF ] CATGGTTGTTGAGCTC-SpC 3.
3) Specific detection among RPA-LFS species: respectively using the genomic DNA of Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio fluvialis, Vibrio mediterranei, Vibrio shigae, Vibrio shigaviensis, Vibrio pisciformis, Vibrio mimicus, Vibrio canescens, Vibrio rotifer, Vibrio devil huidovorus, Vibrio shigaensis, Vibrio nigricans, Vibrio natriensis and the genomic DNA of common food-borne pathogenic bacteria Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus as templates to carry out RPA-LFS detection and obtain the data of figure 1, the detection result of the reaction system taking the vibrio vulnificus genome DNA as the template is positive, the result of the reaction system taking other genomes as the templates is negative, and the result shows that the interspecies specificity of the primer probe is better, so that the pair of primer probes is selected for optimizing the reaction condition.
4) RPA-LFS reaction temperature optimization: using the vibrio vulnificus standard strain ATCC27562 genome DNA as a template, amplifying by using the screened optimal primers and probes, and setting different temperature gradients: 25 ℃,30 ℃,35 ℃,37 ℃ and 42 ℃. And respectively putting the prepared reaction systems at corresponding temperatures for reaction. Where NTC was placed at 37 ℃. LFS detection is carried out after 20min of reaction. As shown in FIG. 2, the detection effect was the best when the reaction temperature was between 35 ℃ and 42 ℃. At a reaction temperature of 50 ℃, the recombinase and the polymerase in the reaction system are inactivated due to an excessively high temperature, and thus amplification cannot be performed.
5) RPA-LFS reaction time optimization: and (3) amplifying by using the screened optimal primer and probe by using the vibrio vulnificus standard strain ATCC27562 genome DNA as a template. And (3) putting the prepared reaction system into a 37 ℃ reaction system, and carrying out LFS detection after 5min, 10min, 15min, 20min, 25mmin, 30min, 35min and 40min of reaction, wherein the NTC reaction time is 40min, the result is shown in figure 3, the detection system can detect the result after 10min of reaction, but the detection line is weak in color, and a clear detection line can be detected after 20min of reaction.
6) Determination of RPA-LFS minimum detection limit: using 1mL of 107CFU/mL～101The CFU/mL vibrio vulnificus pure culture solution is subjected to boiling method to obtain genome DNA, 1 mu L of the genome DNA is taken as a template to evaluate the lowest detection lower limit of RPA-LFS, and 1mL of the genome DNA is used for polluting the vibrio vulnificus pure culture solution with the final concentration of 107CFU/mL～101The prawn hepatopancreas tissue homogenate of the CFU/mL vibrio vulnificus liquid is boiled to obtain genome DNA, and 1 mu L of the genome DNA is taken asThe template was used to assess whether shrimp homogenate affected the detection sensitivity of RPA-LFS.
The application of the method for quickly detecting the vibrio vulnificus RPA-LFS is characterized in that the RPA-LFS is primarily applied for detection, a RPA-LFS detection system is used for detecting clinical samples, meanwhile, compared with a traditional culture method and a qPCR method, the RPA-LFS detects 7 positive results in 55 prawn samples, 5 positive results are detected in 25 shellfish samples, the detection results of 6 fish samples and 2 crab samples are negative, the results are consistent with the detection results of the traditional culture method and the qPCR method, the coincidence rate reaches 100%, and the RPA-LFS detection system can be well applied to actual detection.
Designing a primer: in the research, virulence gene metalloprotease of vibrio vulnificus metalloprotease (VVP) is used as a target sequence, NCBI primer-blast software is used for designing a primer, the designed primer is sent to a synthesis company for synthesis, and the primer design principle is as follows: 1. the length of the primer is 30-36 bp; 2. the GC content of the primer is 20-80 percent; 3. the Tm value of the primer is 50 to 100.
Primer screening: taking vibrio vulnificus standard strain ATCC27562 genome DNA as a template, amplifying by using an RPA kit, detecting the amplification effect of primers, wherein the total volume of an amplification Reaction system is 50 mu L, sequentially adding 2.1ul of an upstream primer (10uM), 2.1ul of a downstream primer (10uM), 25ul of a 2x Reaction buffer, 10x Basic E-mix5ul, 2.5ul of a 20 x core mix and 2.5uM of dNTP (10uM) into a 200 mu L EP tube, fully whirling, uniformly mixing and centrifuging, to ensure the synchronization of all the reaction systems, 1. mu.L of template DNA and 2.5. mu.L of 280mM magnesium acetate were added to the lid, and the template and magnesium ions were simultaneously added to the reaction system during the flash centrifugation, finally, after fully whirling and mixing evenly and instantaneously centrifuging, the sample is put in a constant temperature metal bath for reaction at 37 ℃ for 30min, and purifying the amplification product by using a PCR cleaning kit, and carrying out electrophoresis detection on the cleaned sample by using 1.5% agarose gel.
And (3) introducing species specific detection: the interspecific specificity of the primer with the best amplification effect is detected and screened by respectively using Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio fluvialis, Vibrio mediterranei, Vibrio shigella, Vibrio shigawa, Vibrio enterobacter pisiformis, Vibrio mimicus, Vibrio campylobacter, Vibrio rotifer, Vibrio devil, Vibrio labrum, Vibrio lautus, Vibrio chaugensis, Vibrio nigripes, Vibrio naturoides, genomic DNA of Vibrio natriensis and common food-borne pathogenic bacterium Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus and genomic DNA of Staphylococcus aureus as templates, the total volume of an amplification Reaction system is 50 mu L, and 2.1ul of an upstream primer (10uM), 2.1ul of a downstream primer (10uM), 2.2 x Reaction buffer25ul,10x Basic E-mix5ul,20 x mix 2.5 and dNTP (10uM, after fully vortexing, mixing and centrifuging, in order to ensure the synchronous operation of all reaction systems, 1 mu L of template DNA and 2.5 mu L of 280mM magnesium acetate are added on a cover, the template and magnesium ions are simultaneously added into the reaction systems during instantaneous centrifugation, and finally, after fully vortexing, mixing and instantaneous centrifugation, the sample is placed in a constant temperature metal bath for reaction at 37 ℃ for 30 min. And purifying the amplification product by using a PCR cleaning kit, and carrying out electrophoresis detection on the cleaned sample by using 1.5% agarose gel.
Designing a probe: designing probes according to the screened optimal primers, and designing the probes in primer5.0 software, wherein the probes are positioned between the upstream primer and the downstream primer. The design principle of the probe is as follows: 1. the length of the primer probe screening probe is at least 46 bp; 2. the 5 'end of the probe is labeled with FITC, the 3' end is introduced with 3C-spacer for end blocking, and THF (adenine-free pyrimidine-free site) (AP site) is introduced in the middle of the probe; 3. at least 30bp of base is reserved before the AP locus, at least 15bp of base is reserved after the AP locus, and biotin is labeled at the 5' end of the downstream primer, so that the probe and the amplification product of the downstream primer are provided with FITC at one end and biotin at the other end, and thus detection can be realized by LFS.
And (3) probe screening: screening a primer probe by adopting a Twint Amp nfo kit, wherein the total volume of amplification reaction is 50 mu L, taking vibrio vulnificus standard strain ATCC27562 genome DNA as a template, adding 29.5 mu L of rehydration buffer, 2.1 mu L of upstream and downstream primers (10 mu LM) and 12.2 mu L of ddH2O into a dry powder tube filled with various enzyme components, adding 1 mu L of template DNA (vibrio vulnificus genome DNA) and 2.5 mu L of 280mM magnesium acetate onto a tube cover, performing instant centrifugation, fully vortexing and uniformly mixing, then performing instant centrifugation, and finally placing the reaction system into 37 ℃ for reaction for 20 min. After the reaction is finished, taking 2mL of EP tube, numbering and adding 95 mu L of dilution buffer into the EP tube, then adding 5 mu L of amplification product into the EP tube, fully mixing uniformly, centrifuging, inserting the test strip into the EP tube according to the correct direction, and judging the result after 2 min.
Specific detection among RPA-LFS species: detecting the screened probe and primer by adopting a Twint Amp nfo kit, wherein the total volume of amplification reaction is 50 muL, taking the genome DNA of common vibrio bacteria, the genome DNA of vibrio vulnificus and common food-borne pathogenic bacteria from different sources as templates, respectively, adding 29.5 muL of rehydration buffer, 2.1 muL of upstream and downstream primers (10 muLM) and 2.2 muL of ddH2O into a dry powder tube filled with various enzyme components, in order to ensure the synchronous progress of all reaction systems, adding 1 muL of template DNA (vibrio vulnificus genome DNA) and 2.5 muL of 280mM magnesium acetate onto a tube cover, instantly centrifuging, fully whirling and uniformly mixing, then instantly centrifuging, finally putting the reaction system into a reaction system at 37 ℃ for 20min, taking a 2mL EP tube after the reaction is finished, numbering, adding 95 muL of dilution buffer into the EP tube, then adding 5 muL of amplification product into the EP tube, fully uniformly mixing, centrifuging, inserting the EP tube into an EP test strip according to the correct direction, the result can be judged after 2 min.
RPA-LFS reaction temperature optimization: the vibrio vulnificus standard strain ATCC27562 genome DNA is used as a template, a group of screened primer probes is selected for carrying out reaction temperature optimization, and the reaction temperature gradient is set to be 25 ℃,30 ℃,35 ℃,37 ℃ and 40 ℃. Taking 10 dry powder tubes filled with components such as recombinase, polymerase and the like, adding 29.5 mu L of rehydration buffer, 2.1 mu L of upstream and downstream primers (10 mu LM) and 12.2 mu L of ddH2O into the tubes, adding 1 mu L of template DNA (vibrio vulnificus genome DNA) and 2.5 mu L of 280mM magnesium acetate onto tube covers, adding 1 mu L of LddH2O into NTC to complement the volume, performing instantaneous centrifugation, performing vortex mixing, performing instantaneous centrifugation, performing reaction at the temperature corresponding to each reaction system for 20min, taking 2mL of EP tubes after the reaction is finished, adding 95 mu L of dilution buffer into the EP tubes, adding 5 mu L of amplification products into the EP tubes, performing full mixing, performing centrifugation, inserting test paper strips into the EP tubes in the correct direction, and determining the result after 2 min.
RPA-LFS reaction time optimization: and (3) selecting a group of screened primer probes to optimize the reaction time by using the genomic DNA of the vibrio vulnificus standard strain ATCC27562 as a template. Taking 9 dry powder tubes filled with recombinase, polymerase and other components, adding 29.5 mu L rehydration buffer, 2.1 mu L upstream and downstream primers (10 mu LM), 12.2 mu L ddH2O into the tubes, adding 1 mu L template DNA (Vibrio vulnificus genome DNA) and 2.5 mu L280 mM magnesium acetate onto the tube covers, adding 1 mu L LddH2O into NTC to complement the volume, performing instant centrifugation, sufficiently whirling and mixing uniformly, then performing instant centrifugation, finally reacting the reaction systems at 37 ℃ for 5min, 10min, 15min, 20min, 25mM, 30min, 35min and 40min respectively, performing LFS detection, taking 2mL EP tubes after the reaction is finished, adding 95 mu L dilution buffer into the EP tubes in serial number, adding 5 mu L amplification products into the EP tubes, sufficiently mixing uniformly, inserting the EP tubes in the correct direction, and judging the result after 2 min.
Determination of RPA-LFS detection limit: using 1mL of 107CFU/mL～101The CFU/mL vibrio vulnificus pure culture solution is subjected to boiling method to obtain genome DNA, 1 mu L of the genome DNA is taken as a template to evaluate the lowest detection lower limit of RPA-LFS, and 1mL of the genome DNA is used for polluting the vibrio vulnificus pure culture solution with the final concentration of 107CFU/mL～101The genomic DNA of the prawn hepatopancreas homogenate of the CFU/mL vibrio vulnificus bacterial liquid is obtained by a boiling method, and 1 mu L of the prawn hepatopancreas homogenate is taken as a template to evaluate whether the prawn hepatopancreas homogenate influences the detection sensitivity of RPA-LFS. Taking a dry powder tube filled with components such as recombinase, polymerase and the like, adding 29.5 mu L of rehydration buffer, 2.1 mu L of upstream and downstream primers (10 mu LM) and 12.2 mu L of ddH2O into the tube, adding 1 mu L of template DNA (vibrio vulnificus genome DNA) and 2.5 mu L of 280mM magnesium acetate onto a tube cover for ensuring the synchronous operation of all reaction systems, performing instantaneous centrifugation, sufficiently whirling and uniformly mixing, then performing instantaneous centrifugation, adding 1 mu L of ddH2O into NTC for complementing volume, finally reacting the reaction systems at 37 ℃ for 30min respectively, performing LFS detection, taking 2mL of EP tube after the reaction is finished, numbering the EP tube into the EP tube, and performing LFS detection on the EP tubeAdding 95 mu L of dilution buffer, then adding 5 mu L of amplification product into an EP tube, fully mixing uniformly, centrifuging, inserting the test strip into the EP tube according to the correct direction, and judging the result after 2 min.
RPA-LFS preliminary application: 88 samples are collected from the aquaculture base in Nantong city, the samples are dissected in an ultra-clean workbench to obtain corresponding tissues and the tissues are ground into homogenate, a proper amount of the homogenate is taken, a genome is extracted by using a tissue genome DNA extraction kit to carry out RPA-LFS detection and qPCR experiment, meanwhile, the identification is carried out by applying a national standard culture method, the identification of the culture method refers to the working manual for detecting food pollution and harmful factor risks in 2013, and the qPCR experiment primer adopts a qPCR detection method of vibrio vulnificus reported by Caroline D' Souza and the like.
The invention relates to a portable and rapid vibrio vulnificus nucleic acid detection method based on a recombinant polymerase-free isothermal amplification technology (RPA) and a lateral flow test strip technology (LFS), which provides a reference basis for aquatic product culture, wherein a virulence gene metalloprotease of vibrio vulnificus metalloprotease (VVP) is used as a target sequence design primer, and a pair of optimal primers and probes are screened out to establish the rapid detection method of the RPA-LFS; and evaluating the detection time, temperature, specificity and sensitivity of the method; the specificity, sensitivity and initial application of samples collected on site were performed simultaneously using RPA-LFS, qPCR techniques and culture methods. The result shows that the method can obtain the detection result within 25min at the temperature of 35-42 ℃. When the genomic DNAs of the vibrio vulnificus environmental isolation strains from different sources are taken as templates, the detection results are positive, and the detection specificity reaches 100 percent without cross reaction with other common vibrios and common food-borne pathogenic bacteria; the detection offline of the RPA-LFS for detecting the vibrio vulnificus can reach 10 CFU per reaction, and is not influenced by tissue components; when detecting low concentration samples, 100，10-1，10-2CFU/mL bacterial solution is polluted into homogenate of tissues of prawns, oysters and pomfret, and results are detected after enrichment for 2 hours or 4 hours respectively. The clinical samples are detected by the RPA-LFS detection method, and the traditional culture method and the qPCR detection method are compared, and the result shows that 13 clinical samples are detected to be positive in 88 clinical samples, and the result shows thatConsistent with the culture and qPCR methods. The method for rapidly detecting the vibrio vulnificus is an efficient, rapid and portable method for detecting the vibrio vulnificus, and provides important technical support for field detection of aquatic product culture.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (3)
1. A method for rapidly detecting vibrio vulnificus RPA-LFS is characterized by comprising the following steps:
1) designing and screening primers:
an upstream primer: 5' -GAGATGGATTCTTTGTATAACATTGCGT
A downstream primer: 5' -ACGATGACGTTGGTTGTGTTACATTATC
2) Designing and screening a probe:
and (3) probe:
FITC-GGTGAAGTTGGCTGGTGGTTATTTTCAGAA[THF]CATGGTTGTTGAGCTC-SpC3。
3) specific detection of RPA-LFS.
4) The RPA-LFS reaction temperature is optimized.
5) RPA-LFS reaction time is optimized.
6) And (4) determining the lowest detection limit of RPA-LFS.
2. The use of the method for rapidly detecting the vibrio vulnificus RPA-LFS according to claim 1, wherein the method comprises the following steps:
the length of the primer probe screening probe is at least 46 bp;
the 5 'end of the probe is labeled with FITC, the 3' end is introduced with 3C-spacer for end blocking, and THF (adenine-free pyrimidine-free site) (AP site) is introduced in the middle of the probe;
at least 30bp of base is reserved before the AP locus, at least 15bp of base is reserved after the AP locus, and biotin is marked at the 5' end of the downstream primer.
3. The method for rapidly detecting the vibrio vulnificus RPA-LFS according to claim 1, wherein the RPA-LFS is primarily used for detection, a clinical sample is detected by using an RPA-LFS detection system, the detection result is compared with that of a traditional culture method and a qPCR method, 7 positive results are detected by the RPA-LFS in 55 prawn samples, 5 positive results are detected by 25 shellfish samples, and the detection results of 6 fish samples and 2 crab samples are negative.
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| CN112646908A (en) * | 2020-12-31 | 2021-04-13 | 广州赛哲生物科技股份有限公司 | Vibrio vulnificus isothermal amplification primer, probe, kit and detection method |
| CN113249500A (en) * | 2021-03-03 | 2021-08-13 | 中国科学院大学宁波华美医院 | Method for rapidly detecting vibrio vulnificus in clinical blood |
| CN116267722A (en) * | 2023-01-31 | 2023-06-23 | 江苏海洋大学 | Simple and easy shellfish offspring seed even seeder |
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| CN112646908A (en) * | 2020-12-31 | 2021-04-13 | 广州赛哲生物科技股份有限公司 | Vibrio vulnificus isothermal amplification primer, probe, kit and detection method |
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| CN116267722A (en) * | 2023-01-31 | 2023-06-23 | 江苏海洋大学 | Simple and easy shellfish offspring seed even seeder |
| CN116267722B (en) * | 2023-01-31 | 2024-02-27 | 江苏海洋大学 | Simple and easy shellfish offspring seed even seeder |
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Application publication date: 20200918 |