CN111635951A - Establishment and application of AHPND pathogenic vibrio parahaemolyticus RPA-LFS rapid detection method - Google Patents
Establishment and application of AHPND pathogenic vibrio parahaemolyticus RPA-LFS rapid detection method Download PDFInfo
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Abstract
The invention discloses establishment and application of an AHPND pathogenic vibrio parahaemolyticus RPA-LFS rapid detection method, which comprises the steps of primer screening, primer inter-species specificity detection, probe design and screening, RPA-LFS inter-species specificity detection, RPA-LFS reaction temperature detection, RPA-LFS reaction time detection, RPA-LFS minimum detection limit determination, 46bp of the length of a primer probe screening probe, FITC for marking the 5 ' tail end of the probe, 3C-spacer for end closure by introducing the 3 ' tail end, THF (tetrahydrofuran) is introduced into the middle of the probe, namely a purine-free and pyrimidine-free site (AP site), at least 30bp of base is reserved in front of the AP site, at least 15bp of base is reserved behind the AP site, biotin is marked at the 5 ' tail end of a downstream primer, FITC is arranged at one end of amplification products of the probe and the downstream primer, and biotin is arranged at the other end of the amplification products of the probe. The invention solves the problem of equipment dependence, solves the problem of long detection period, and has simple requirement on experimental skills of operators.
Description
Technical Field
The invention relates to the field of marine product culture and food safety, in particular to establishment and application of a method for quickly detecting AHPND pathogenic vibrio parahaemolyticus RPA-LFS.
Background
Prawn hepatopancreatic necrosis syndrome (AHPND) is one of the current outbreaks of disease that poses a serious threat to prawn farming. AHPND initially outbreaks in china followed by successive outbreaks in countries such as vietnam, thailand, malaysia, mexico, philippines, etc. The disease has high lethality rate and high propagation speed, and once outbreak occurs, a large amount of prawns die, thereby causing serious economic loss in the prawn culture industry. There is a great need to take scientific and effective measures to monitor the pathogenic bacteria in real time and to take effective measures to prevent the outbreak and spread of pathogenic bacteria. Therefore, establishing a method capable of rapidly detecting AHPND on site is of great significance in preventing outbreak and spread of the disease.
Currently, the existing detection methods for detecting AHPND mainly include AP3, AP4 and LAMP detection methods. AP3 detection method: the AP3 detection method is a traditional PCR detection method, and a pair of primers with an amplification product of 336bp is designed by taking the PirA gene of AHPND as a target sequence for detection. The detection technology needs three temperature stages, amplification products of the detection technology need agarose gel electrophoresis detection, experimental equipment is high in requirement and long in time consumption, and therefore the detection technology is limited to laboratory research and is not suitable for field detection, and a rapid detection method for the pathogenic vibrio parahaemolyticus RPA-LFS of AHPND is provided for solving the problems.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides establishment and application of a rapid detection method for AHPND pathogenic vibrio parahaemolyticus RPA-LFS.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for rapidly detecting AHPND pathogenic vibrio parahaemolyticus RPA-LFS comprises the following steps:
1) designing and screening primers and probes:
an upstream primer: 5' -CATCTTTGACGGAATTTAACCCTAACAAT
A downstream primer: 5' -Biotin-TAACTAAACCTATGTAATGATCTTTTG
And (3) probe: 5' -FITC-ATGAGCCAGCTATTGATAATATTTGGGAAC [ THF ] ATTACGTGACTGAAT-SpC 3.
2) And (3) detecting specificity among RPA-LFS species.
3) The reaction temperature.
4) And (4) reaction time.
5) And (4) determining the lowest detection limit of RPA-LFS.
Preferably, the length of the probe for screening the primer and the probe is 46bp, the 5 ' end of the probe is labeled by FITC, the 3 ' end of the probe is introduced with 3C-spacer for end blocking, THF (purine-free pyrimidine-free site) (AP site) is introduced in the middle of the probe, at least 30bp of bases are reserved in front of the AP site, at least 15bp of bases are reserved behind the AP site, the 5 ' end of the downstream primer is labeled by biotin, one end of the amplification product of the probe and the downstream primer is provided with FITC, and the other end is provided with biotin.
An application of an RPA-LFS rapid detection method for AHPND pathogenic vibrio parahaemolyticus is disclosed, wherein RPA-LFS is primarily applied and detected, prawns are collected from a litopenaeus vannamei farm, 75 samples of aquaculture water are detected by the RPA-LFS detection method, an AP4 method is used as a calibration standard, 20 of 65 prawn samples are detected to be positive, 3 of 10 aquaculture water samples are detected to be positive, the result is consistent with the AP4 detection method, and the coincidence rate is 100%.
The invention has the following beneficial effects: the method solves the problem of equipment dependence, does not need expensive instruments and equipment, adopts the RPA-LFS technology as the normal-temperature isothermal amplification technology, has the optimal reaction temperature of 35-45 ℃, can carry out reaction only by a constant-temperature metal bath or a water bath kettle, and can carry out reaction at room temperature or by holding in hands in places with hard detection environmental conditions.
The detection method has the advantages that the detection period is long, the detection speed is high, the RPA-LFS technology is a constant-temperature amplification technology, double chains are opened to amplify depending on recombinase continuous displacement activity, and thermal cycle is not needed for denaturation, annealing and extension processes, so that rapid amplification can be carried out, and the result can be detected within 25 min. In addition, the amplification product of the technology does not need to be detected by agarose gel electrophoresis, and is detected by combining a lateral flow test strip technology, so that the detection time is reduced to a great extent.
The detection method has simple requirements on experimental skills of operators, and does not need strain culture, physiological and biochemical identification, use of complex instruments and the like. The method only needs to carry out simple sample adding according to requirements and then react at a proper temperature, and the whole detection process has no complex and high-difficulty experiment operation, so that the requirement on the experiment operation of a detector is not high, and a farm worker can carry out detection without engaging a professional technician when carrying out field detection.
Visual detection, the method utilizes the lateral flow test strip multiple amplification products to carry out visual detection, and can judge the detection result through color development. And devices such as a gel imaging system and the like are not needed, so that the detection cost is reduced, the detection practice is shortened, and the visual detection can be realized.
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FIG. 1 shows the detection of VP by RPA-LFSAHPNDSchematic diagram of specific detection among probe species;
FIG. 2 is a schematic diagram of the optimization of the RPA-LFS reaction conditions;
FIG. 3 is a schematic diagram of RPA-LFS sensitivity detection for different CFUs;
FIG. 4 is a schematic diagram of the detection of RPA-LFS sensitivity at different copy numbers.
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 AHPND pathogenic vibrio parahaemolyticus RPA-LFS comprises the following steps:
1) designing and screening primers and probes:
an upstream primer: 5' -CATCTTTGACGGAATTTAACCCTAACAAT
A downstream primer: 5' -Biotin-TAACTAAACCTATGTAATGATCTTTTG
And (3) probe: 5' -FITC-ATGAGCCAGCTATTGATAATATTTGGGAAC [ THF ] ATTACGTGACTGAAT-SpC 3;
six pairs of primers were designed with PirAB gene as the target sequence and VPAHPNDUsing genomic DNA as a template
Carrying out RPA reaction on the LiquidDNAamplification kit to amplify the PirAB gene, designing a probe according to a primer pair with the best amplification effect and better interspecies specificity, designing the probe in a primer5.0, and carrying out amplification reaction on the PirAB gene by using VPAHPNDAnd (3) amplifying the genome DNA serving as a template by using an RPAnfo kit, and detecting the amplification effect of the probe by combining a lateral flow test strip. And (3) selecting primers and probes which have good amplification effect and have no amplification of the NTC for the next step of interspecies specificity detection.
2) Specific detection among RPA-LFS species: respectively by VPAHPND，VPAHPND-freeThe primer probe combination which is used for detecting the interspecies specificity after mismatch with the common genome DNA of other vibrios is only used for detecting the VPAHPNDThe positive signal is shown when the genome DNA is used as a template, and the VP is usedAHPND-freeShows negative signals when taking genome DNA of other common vibrios as templates, and the results show that the primer probe combination VP after the introduction of mismatchAHPNDthe-1/mP 1 has good interspecific specificity.
3) Reaction temperature: in order to screen out the optimal reaction temperature, the reaction system is respectively incubated at 25 ℃, 30 ℃, 37 ℃, 42 ℃ and 45 ℃ for 30min, the result is shown in figure 2-a, when the reaction is carried out at 25 ℃, the color development of the detection line is weak, the color development of the detection line is deepened along with the increase of the temperature, the reaction temperature is in the range of 37 ℃ to 45 ℃, the color development of the detection line is consistent, namely, the RPA-LFS has better detection performance during the period of 37 ℃ to 45 ℃.
4) Reaction time: in order to screen out the optimal reaction time, the reaction system is respectively incubated for 5min, 10min, 15min, 20min, 25min, 30min, 35min and 40min at 37 ℃, the result is shown in figure 2-b, the detection line is not colored when the reaction is carried out for 5min, the detection line is provided with a weaker band when the reaction is carried out for 10min, the color development of the detection line is darker and darker along with the increase of the reaction time, namely, the amplification product is gradually increased, the color development of the detection line is not obviously changed when the reaction is carried out for 20min to 40min, and the detection can be finished when the reaction system is incubated for 20min, namely, the optimal reaction condition of the RPA-LFS system is 37 ℃ and.
5) The lowest detection limit of RPA-LFS is determined, in order to determine the lowest detection limit of RPA-LFS, VPAHPND genomic DNA of different CFUs is used as a template to detect the sensitivity, the result is shown in figure 3, the RPA-LFS reaction system can detect 101CFU/mL at the lowest, and simultaneously the plasmid is diluted to 107-100The individual copies/. mu.L were used as templates to evaluate the sensitivity of RPA-LFS, and the results are shown in FIG. 4 when the amount of the template is 102The test line showed positive with copies/. mu.L, and the color of the test line gradually deepened with the increase of the amount of the template. Indicating a sensitivity of 10 for RPA-LFS2copies/μL。
A method for quickly detecting AHPND pathogenic vibrio parahaemolyticus RPA-LFS comprises the steps of screening a probe with a length of 46bp by a primer probe, labeling the 5 ' tail end of the probe with FITC, introducing 3C-spacer into the 3 ' tail end for carrying out tail end closure, introducing THF (tetrahydrofuran) in the middle of the probe, namely a purine-free and pyrimidine-free site (AP site), reserving at least 30bp of base before the AP site, at least 15bp of base after the AP site, labeling biotin at the 5 ' tail end of a downstream primer, and carrying FITC at one end and biotin at the other end of an amplification product of the probe and the downstream primer.
An application of an RPA-LFS rapid detection method for AHPND pathogenic vibrio parahaemolyticus is disclosed, wherein RPA-LFS is primarily applied and detected, prawn is taken from a litopenaeus vannamei farm, 75 samples of aquaculture water are detected by the RPA-LFS detection method, an AP4 method is used as a calibration standard, 20 of 65 prawn samples are detected to be positive, and 3 of 10 aquaculture water samples are detected to be positive. The result is consistent with the AP4 detection method, and the coincidence rate is 100%.
Clinical sample detection of RPA-LFS
Designing a primer and a probe: in the research, virulence genes PirAB for expressing toxA and ToxB toxins are taken as target sequences, primers are designed by NCBIprimer-blast software, and the designed primers are sent to a synthesis company for synthesis, wherein the design principle of the primers 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. 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.
And (3) probe screening: screening a primer probe by adopting a TwintAmpnfo kit, wherein the total volume of an amplification reaction is 50 mu L, and using VPAHPNDGenomic DNA as template, 29.5. mu.L of rehydration buffer, 2.1. mu.L of upstream and downstream primers (10. mu.L), 12.2. mu.L of ddH2O were added to dry powder tubes containing various enzyme components, and to ensure that all reactions were performed simultaneously, 1. mu.L of template DNA (VP)AHPNDGenomic DNA) and 2.5. mu.L of 280mM magnesium acetate were added to the tube cap, centrifuged instantaneously and vortexed well, followed by centrifugation instantaneously, and finally the reaction system was placed at 37 ℃ 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: the TwintAmpnfo kit is adopted to detect the screened probes and primers, the total volume of the amplification reaction is 50 mu L, and Vibrio Parahaemolyticus (VP) is respectively usedAHPND) Vibrio Parahaemolyticus (VP)AHPND-free) The genome DNA of Vibrio vulnificus, Vibrio alginolyticus, Vibrio cholerae, Vibrio harveyi, Vibrio mediterranei, Vibrio shiluo, Vibrio splendidus, Vibrio mimicus, and Vibrio pisciformis is as a template, and packaged in a containerTo a dry powder tube containing various enzyme components, 29.5. mu.L of rehydration buffer, 2.1. mu.L of upstream and downstream primers (10. mu.L), 2.2. mu.L of ddH2O were added, and to ensure that all reactions were performed simultaneously, 1. mu.L of template DNA (VP)AHPNDGenomic DNA) and 2.5. mu.L of 280mM magnesium acetate were added to the tube cap, centrifuged instantaneously and vortexed well, followed by centrifugation instantaneously, and finally the reaction system was placed at 37 ℃ 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.
RPA-LFS reaction temperature optimization: by VPAHPNDGenome 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 ℃, 37 ℃, 42 ℃ and 45 ℃. Taking 10 dry powder tubes containing recombinase, polymerase and other components, adding 29.5. mu.L rehydration buffer, 2.1. mu.L upstream and downstream primers (10. mu.L), 12.2. mu.L ddH2O, and adding 1. mu.L template DNA (VP) to ensure the synchronous operation of all reaction systemsAHPNDGenomic DNA) and 2.5 mu L of 280mM magnesium acetate are added on a tube cover, NTC is added with 1 mu L ddH2O to complement the volume, the mixture is instantaneously centrifuged, and is instantaneously centrifuged after being fully vortexed and mixed evenly, and finally the reaction system is reacted for 20min at the temperature corresponding to the reaction system. 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.
RPA-LFS reaction time optimization: by VPAHPNDAnd (3) taking the genome DNA as a template, and selecting a group of screened primer probes to optimize the reaction time. A dry powder tube containing recombinase, polymerase and the like is taken, 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 are added into the tube, and 1 mu L of template DNA (VP) is added to ensure that all reaction systems are synchronously performedAHPNDGenomic DNA) and 2.5. mu.L of 280mM magnesium acetate were added to the vial cap, NTC was added to 1. mu.L ddH2O to make up the volume, and the reaction was centrifuged instantaneously and vortexed thoroughly before centrifuging it again, and finally the reaction was runThe system is reacted for 5min, 10min, 15min, 20min, 25mmin, 30min, 35min and 40min respectively at 37 ℃, and then LFS detection is carried out. 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.
Determination of RPA-LFS detection limit: using 1mL of 107CFU/mL～101VP of CFU/mLAHPNDThe pure culture solution is boiled to obtain genome DNA, 1 microliter is taken as a template to evaluate the lowest detection lower limit of RPA-LFS, and the concentration is 107-100The plasmid pVA of copies/. mu.L was used as a template, and 1. mu.L was used as a template to evaluate 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 and 2.5 mu L of 280mM magnesium acetate onto a tube cover for ensuring the synchronous operation of all reaction systems, carrying out instantaneous centrifugation, fully whirling, mixing uniformly, then carrying out instantaneous centrifugation, adding 1 mu L of LddH2O to NTC for volume supplementation, and finally carrying out LFS detection after respectively reacting the reaction systems at 37 ℃ for 30 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.
RPA-LFS preliminary application: 65 litopenaeus vannamei samples and 10 culture environment water samples are purchased from Taobao merchants, corresponding tissues are dissected and taken from the samples in a super clean workbench and are ground into homogenate, and after genome DNA is extracted, RPA-LFS and AP4 detection are respectively carried out.
The invention establishes a portable and rapid VP based on a recombinant polymerase-free isothermal amplification technology (RPA) and a lateral flow test strip technology (LFS)AHPNDThe nucleic acid detection method provides a reference basis for aquatic product culture. The invention establishes RPA-LFS to rapidly detect VP by taking AHPND pathogenic vibrio parahaemolyticus PirA and PirB virulence genes as target sequencesAHPNDThe method of (1). The intervarietal specificity of detecting AHPND by RPA-LFS is verified by taking the common vibrio genome DNA as a template,and the RPA-LFS reaction time and temperature are optimized to 107CFU/mL-101Bacterial liquid of CFU/mL and 107-100The plasmid of copies/reaction is used as a template to determine the sensitivity of the RPA-LFS detection, and the established method is used for detecting low-concentration samples and clinical samples. The result shows that the method has good specificity and sensitivity, and the lowest detection limit is 101CFU/mL or 102In the clinical sample detection, the method detects 23 positive samples from 75 clinical samples, the detection result is consistent with that of the AP4 method, and the coincidence rate is 100%. The invention creates a rapid detection VPAHPNDThe method is an efficient, rapid and portable VP detection methodAHPNDThe method has important significance for outbreak and spread of diseases in the aquiculture process. Provides important technical support for the detection of aquatic product culture sites.
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 AHPND pathogenic vibrio parahaemolyticus RPA-LFS is characterized by comprising the following steps:
1) designing and screening primers and probes:
an upstream primer: 5' -CATCTTTGACGGAATTTAACCCTAACAAT
A downstream primer: 5' -Biotin-TAACTAAACCTATGTAATGATCTTTTG
And (3) probe: 5' -FITC-ATGAGCCAGCTATTGATAATATTTGGGAAC [ THF ] ATTACGTGACTGAAT-SpC 3.
2) And (3) detecting specificity among RPA-LFS species.
3) The reaction temperature.
4) And (4) reaction time.
5) And (4) determining the lowest detection limit of RPA-LFS.
2. The use of the method of claim 1 for rapid detection of AHPND pathogenic Vibrio parahaemolyticus RPA-LFS, wherein:
the length of the primer probe screening probe is 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 needs to be reserved before the AP locus, and at least 15bp of base needs to be reserved after the AP locus;
the 5' end of the downstream primer is labeled with biotin, and one end of the amplification product of the probe and the downstream primer is provided with FITC, and the other end of the amplification product of the probe and the downstream primer is provided with biotin.
3. The method of claim 1, wherein the RPA-LFS is used for preliminary detection, wherein 75 samples of the aquaculture water of litopenaeus vannamei farms are detected by the RPA-LFS detection method, the AP4 method is used as a calibration standard, 20 of 65 prawn samples are detected as positive samples, 3 of 10 aquaculture water samples are detected as positive samples, and the result is consistent with the AP4 detection method, and the coincidence rate is 100%.
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Application publication date: 20200908 |