CN117143718A - Vibrio harveyi detection kit - Google Patents
Vibrio harveyi detection kit Download PDFInfo
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- CN117143718A CN117143718A CN202311431527.5A CN202311431527A CN117143718A CN 117143718 A CN117143718 A CN 117143718A CN 202311431527 A CN202311431527 A CN 202311431527A CN 117143718 A CN117143718 A CN 117143718A
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- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 241000607618 Vibrio harveyi Species 0.000 title claims abstract description 31
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 77
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- 238000009835 boiling Methods 0.000 claims abstract description 22
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 239000008213 purified water Substances 0.000 claims abstract description 8
- 238000003752 polymerase chain reaction Methods 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 238000001962 electrophoresis Methods 0.000 claims description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 7
- 238000007400 DNA extraction Methods 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- FYFFGSSZFBZTAH-UHFFFAOYSA-N methylaminomethanetriol Chemical compound CNC(O)(O)O FYFFGSSZFBZTAH-UHFFFAOYSA-N 0.000 claims description 6
- 229920000936 Agarose Polymers 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000012154 double-distilled water Substances 0.000 claims description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 230000006037 cell lysis Effects 0.000 claims description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 claims description 4
- 229960005542 ethidium bromide Drugs 0.000 claims description 4
- 239000011535 reaction buffer Substances 0.000 claims description 4
- 102000016943 Muramidase Human genes 0.000 claims description 3
- 108010014251 Muramidase Proteins 0.000 claims description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 3
- JYNSMELUFHERAG-UHFFFAOYSA-N acetic acid;2-aminoethane-1,1,1-triol Chemical compound CC([O-])=O.[NH3+]CC(O)(O)O JYNSMELUFHERAG-UHFFFAOYSA-N 0.000 claims description 3
- 239000012916 chromogenic reagent Substances 0.000 claims description 3
- 229960000274 lysozyme Drugs 0.000 claims description 3
- 239000004325 lysozyme Substances 0.000 claims description 3
- 235000010335 lysozyme Nutrition 0.000 claims description 3
- 230000001681 protective effect Effects 0.000 claims description 3
- GFNANZIMVAIWHM-OBYCQNJPSA-N triamcinolone Chemical compound O=C1C=C[C@]2(C)[C@@]3(F)[C@@H](O)C[C@](C)([C@@]([C@H](O)C4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 GFNANZIMVAIWHM-OBYCQNJPSA-N 0.000 claims description 3
- 229960005294 triamcinolone Drugs 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 230000010354 integration Effects 0.000 abstract description 6
- 238000000034 method Methods 0.000 description 24
- 238000005516 engineering process Methods 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 230000001717 pathogenic effect Effects 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 230000035945 sensitivity Effects 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 241000607598 Vibrio Species 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000003298 DNA probe Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
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- 230000035755 proliferation Effects 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108010006464 Hemolysin Proteins Proteins 0.000 description 1
- 241001596950 Larimichthys crocea Species 0.000 description 1
- 241000238552 Penaeus monodon Species 0.000 description 1
- 241001417495 Serranidae Species 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
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- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
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- 239000003228 hemolysin Substances 0.000 description 1
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- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 101150012629 parE gene Proteins 0.000 description 1
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- 230000001376 precipitating effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1838—Means for temperature control using fluid heat transfer medium
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- 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
- C12R2001/63—Vibrio
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Abstract
The invention aims at solving the problem of low equipment integration level in the prior art, and provides a Vibrio harveyi detection kit which comprises a kit and a detection structure, wherein the detection structure comprises a base, a boiling water bath, a thermal circulation position, an observation position, an ultraviolet lamp and a lamp bracket; the base is provided with a boiling water bath, an observation position and a thermal circulation position, the boiling water bath is internally provided with a heating mechanism and purified water, the heating mechanism can keep the purified water in the boiling water bath in a boiling state, the observation position is internally provided with a fixing device capable of fixing a reagent bottle, the thermal circulation position is internally provided with the heating mechanism, the lamp holder is fixedly arranged on the base, the ultraviolet lamp is connected to the lamp holder and then arranged right above the observation position, and the light of the ultraviolet lamp faces the observation position. The kit equipment integration level is high, and during detection, the staff only needs to use the detection structure and the centrifugal instrument, and does not need to hold the reagent to walk back and forth among a plurality of equipment, so that the physical strength is saved, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the technical field of rapid detection of vibrio harveyi, in particular to a vibrio harveyi detection kit.
Background
Vibrio harveyi (V.harveyi) is a light-emitting vibrio in seaborne river, bay and coastline areas and seafood that is widely distributed around the world. The bacterium is a conditional pathogenic bacterium, and can outbreak epidemic under certain conditions. In recent years, the reports of marine product morbidity or large-area death caused by Vibrio harveyi in coastal areas of China are increasing. At present, vibrio harveyi is known to infect various aquatic products such as weever, large yellow croaker, penaeus monodon, mo Ji prawn, grouper and the like, and brings great harm to the aquaculture industry. Bacterial diseases caused by Vibrio harveyi have the characteristics of rapid outbreak, wide spread, high death rate and the like, and the bacteria are extremely easy to generate drug resistance to antibiotics, so that the Vibrio harveyi disease is mainly prevented, and the rapid detection in early stage is mainly relied on. The existing vibrio detection technology mainly comprises traditional TCBS culture and further morphological and physiological biochemical identification methods, immunological methods (mainly comprising monoclonal antibody technology and enzyme-linked immunosorbent assay (ELISA)), immunoelectron microscope technology, molecular biological methods (mainly comprising molecular hybridization technology and Restriction Endonuclease Length Polymorphism (RELP)), and the like. Some of these detection methods have high requirements on technical conditions and long detection time. Some required materials are troublesome to prepare, the cost is high, and some methods are low in sensitivity and low in specificity, so that the technical difficulties mastered by vast medical staff and aquaculture owners are great, the popularization is difficult, and the application and development of the technologies are limited.
The early rapid detection of Vibrio harveyi is a main measure for preventing outbreaks of the vibriosis and an effective way for reducing losses in the aquatic economic animal breeding industry at present, so that the detection kit and the detection method thereof with the advantages of simplicity, rapidness, good specificity and high sensitivity are eagerly expected by vast medical workers and aquaculture workers.
The polymerase chain reaction (PCR technology for short) is a technology developed in the last 80 th century for in vitro amplification of specific DNA fragments, and is quickly applied to practice after being established due to the characteristics of rapidness, sensitivity, strong specificity and the like;
the Chinese patent with application number 200610124271.3 discloses a kit for rapidly detecting Vibrio harveyi (Vibrio harveyi) and a detection method. The kit and the detection method are designed by taking a pair of primers designed by the conserved region sequence of the heat-resistant direct hemolysin (gyrB) gene of Vibrio harveyi as a main body. The invention adopts Polymerase Chain Reaction (PCR) technology to qualitatively detect the specific DNA fragment of marine aquatic pathogenic bacteria-Vibrio harveyi, and has the advantages of simplicity, convenience, rapidness, good specificity and high sensitivity; the method can be used for tracking and detecting the Vibrio harveyi in the culture process of aquatic animals in each period, can also be used for environmental monitoring, avoids the spread and epidemic of germs, and has high practical value;
however, the device still has some defects in the actual use process: the kit equipment is not high in integration level, the steps of hot water bath, specific sheet growth and the like are involved in the use process, various equipment such as an alcohol lamp and a thermal cycler are required to be used, and when the kit is used for detection, workers need to hold reagents to walk back and forth between the equipment, so that the physical strength is very consumed, and the detection result is influenced due to the fact that the reagents are easy to scatter and leak when moved.
There is an urgent need for redesign to solve the above-described problems.
Disclosure of Invention
The invention aims to provide a Vibrio harveyi detection kit aiming at the problem of low equipment integration in the prior art.
The invention provides a Vibrio harveyi detection kit, which comprises a kit and a detection structure, wherein the detection structure comprises a base, a boiling water bath, a thermal circulation position, an observation position, an ultraviolet lamp and a lamp bracket; the base is provided with three blind grooves, the three blind grooves are respectively provided with a boiling water bath, an observation position and a thermal circulation position, the boiling water bath is internally provided with a heating mechanism and purified water, the heating mechanism can keep the purified water in the boiling water bath in a boiling state, the observation position is internally provided with a fixing device capable of fixing a reagent bottle, the thermal circulation position is internally provided with the heating mechanism and can keep constant temperature at any temperature between 60 ℃ and 100 ℃, the lamp holder is fixedly arranged on the base, the ultraviolet lamp is connected onto the lamp holder and is further arranged right above the observation position, and the light of the ultraviolet lamp faces the observation position.
Optionally, a chute is arranged on the lamp holder, and the ultraviolet lamp is connected with the lamp holder in a sliding way through the chute.
Optionally, the detecting structure further includes a sliding protection cover slidably connected to the base.
Optionally, the kit consists of the following reagents:
(a) DNA extraction reagent: comprises a buffer reagent A and a cell lysis reagent B,
reagent A: comprises NaCl0.1-0.3M, trihydroxymethyl aminomethane 1-100mM with pH8.0, ethylenediamine tetraacetic acid 0.1-10mM with pH8.0 and triamcinolone X-100 with V/V concentration of 0-20%;
reagent B: comprises lysozyme with a concentration of 1-10mg/ml and trihydroxymethyl aminomethane with a pH of 8.0 and a concentration of 1-100 mM;
(b) Polymerase chain reaction reagents: contains a reaction premixing reagent C and a reaction primer reagent D,
reagent C: contains 10 times polymerase chain reaction buffer solution, thermal polymerase and sterilized double distilled water, and the contents are respectively 2.5-5.0 μl, 0.5-2.5 units and 18-35.5 μl;
reagent D: contains specific primer pair VhF and VhR, deoxynucleotide and MgCl2, and the content is 0.2-1 mu M, 20-200 mu M and 0.5-10mM respectively;
(c) Electrophoresis and chromogenic reagent, comprising reagent E and reagent F,
reagent E: 50 times of electrophoresis buffer solution containing the trihydroxymethyl aminomethane-acetic acid and the ethylenediamine tetraacetic acid, and the concentration is 0.04M and 0.001M respectively;
reagent F: agarose with W/V of 0.8-2% and ethidium bromide with W/V of 0.5-20 mug/ml;
specific primer pairs VhF and VhR in the reagent D are specific primers and essential components for performing PCR reaction, and VhF is particularly 5' -TCTAACTATCCACCGCGG; vhR in particular 5' -AGCAATGCCATCTTCACGTTC.
The beneficial effects of the invention are as follows: the kit equipment has high integration level, omits a plurality of equipment such as an alcohol lamp, a thermal cycler and the like which are needed to be used in the detection process, and when in detection, a worker only needs to use a detection structure and a centrifugal instrument, and does not need to hold reagents to walk back and forth among the plurality of equipment, so that the physical strength is saved, the detection efficiency is improved, the detection technology based on nucleic acid comprises a DNA probe and a PCR technology (polymerase chain reaction technology), and compared with other vibrio pathogen detection technologies and other pathogen detection technologies, the method has several remarkable advantages: firstly, the method is very rapid in detection, the detection result can be obtained in 3-4 hours, and other methods at least need 3 days or more than one week; secondly, the method has extremely high detection sensitivity and low detection lower limit of about 10 bacteria, and other methods can detect bacteria after the pure culture proliferation reaches 105 in a certain time. The invention can be used for detecting the infection of the marine aquatic animals by the Vibrio harveyi in the culture process of the marine aquatic animals in each period, can also detect pathogenic Vibrio harveyi in the marine water environment, and can also detect whether various aquatic products are polluted by the Vibrio harveyi.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic perspective view of a detection structure.
Reference numerals indicate 1, boiling water bath; 2. a thermal cycling station; 3. observing the position; 4. an ultraviolet lamp; 5. a lamp holder; 501. a chute; 6. the protective cover is slid.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. It should be further noted that, for convenience of description, only some, but not all of the structures related to the present invention are shown in the drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the invention. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
A Vibrio harveyi detection kit, as shown in FIG. 1, comprises: the kit and the detection structure comprise a base, a boiling water bath 1, a thermal circulation position 2, an observation position 3, an ultraviolet lamp 4 and a lamp bracket 5; three blind grooves are formed in the base, a boiling water bath 1, an observation position 3 and a thermal circulation position 2 are respectively arranged in the three blind grooves from left to right, a heating mechanism and purified water are arranged in the boiling water bath 1, the heating mechanism can keep the purified water in the boiling water bath 1 in a boiling state, a fixing device capable of fixing a reagent bottle is arranged in the observation position 3, the heating mechanism is arranged in the thermal circulation position 2 and can keep constant temperature at any temperature between 60 ℃ and 100 ℃, a lamp bracket 5 is fixedly arranged on the base, an ultraviolet 4 lamp is connected to the lamp bracket 5 and is further arranged right above the observation position 3, and the light of the ultraviolet lamp 4 faces the observation position 3; the kit equipment integration level is high, has saved multiple equipment such as alcohol burner, thermal cycler that need use in the inspection process, and during the detection, the staff only need use detection structure and centrifugal instrument, and no longer need hold reagent and walk back and forth between a plurality of equipment, has saved physical power, has improved detection efficiency.
Further, a chute 501 is arranged on the lamp holder, and the ultraviolet lamp 4 is connected with the lamp holder 5 in a sliding way through the chute 501; when in observation, the illumination position of the ultraviolet lamp can be adjusted according to the requirement, so that the observation is convenient.
Further, the detecting structure further includes a sliding protection cover 6, and the sliding protection cover 6 is slidably connected to the base.
Wherein, the kit consists of the following reagents:
(a) DNA extraction reagent: comprises a buffer reagent A and a cell lysis reagent B,
reagent A: comprises NaCl0.1-0.3M, trihydroxymethyl aminomethane 1-100mM with pH8.0, ethylenediamine tetraacetic acid 0.1-10mM with pH8.0 and triamcinolone X-100 with V/V concentration of 0-20%;
reagent B: comprises lysozyme with a concentration of 1-10mg/ml and trihydroxymethyl aminomethane with a pH of 8.0 and a concentration of 1-100 mM;
(b) Polymerase chain reaction reagents: contains a reaction premixing reagent C and a reaction primer reagent D,
reagent C: contains 10 times polymerase chain reaction buffer solution, thermal polymerase and sterilized double distilled water, and the contents are respectively 2.5-5.0 μl, 0.5-2.5 units and 18-35.5 μl;
reagent D: contains specific primer pair VhF and VhR, deoxynucleotide and MgCl2, and the content is 0.2-1 mu M, 20-200 mu M and 0.5-10mM respectively;
(c) Electrophoresis and chromogenic reagent, comprising reagent E and reagent F,
reagent E: 50 times of electrophoresis buffer solution containing the trihydroxymethyl aminomethane-acetic acid and the ethylenediamine tetraacetic acid, and the concentration is 0.04M and 0.001M respectively;
reagent F: agarose with W/V of 0.8-2% and ethidium bromide with W/V of 0.5-20 mug/ml;
specific primer pairs VhF and VhR in the reagent D are specific primers and essential components for performing PCR reaction, and VhF is particularly 5' -TCTAACTATCCACCGCGG; vhR in particular 5' -AGCAATGCCATCTTCACGTTC.
The optimal composition of each tube of reactant is: reagent C:5.0 μl of 10xPCRBuffer,0.5 μl TaqE (concentration 5U/. Mu.l), 35.5 μl dH2O, reagent D:4.0 mu ldNTP (2.5 mMeach), 3.0 mu l MgCl2 (25 mM), 10pMVhF,10pMVhR.
The reagent A is a DNA extraction buffer solution, provides a proper buffer environment for DNA extraction and inhibits DNA degradation; reagent B is a cell lysis reagent, which lyses bacteria and releases DNA; reagent C is a PCR reaction premixing reagent, is one of the main components of the PCR reaction reagent, and reagent D is a PCR primer reagent, is also the main component of the PCR reaction, and comprises specific primers VhF and VhR which are the most critical of the kit. Reagent E is 50 times TAE (electrophoresis buffer solution), and provides a buffer system with proper pH value, ionic strength and the like for sample electrophoresis; reagent F is agarose as electrophoresis medium and ethidium bromide as color developing agent.
The using method of the kit sequentially comprises the following steps:
(a) Sample treatment: taking 0.1-0.5 g of sample, suspending the sample with 100-400 mu l of reagent A, and fully and uniformly mixing the sample; centrifuging 10000-12000g for 2-5 min, and discarding supernatant;
(b) DNA extraction: suspending and precipitating again with 100-300 μl of reagent A, adding 20-100 μl of reagent B, mixing, standing at room temperature for more than 2 minutes, and keeping the temperature in boiling water bath for 1-10 minutes to completely rupture cells and release DNA; centrifuging 10000-12000g for 8-10 min, collecting supernatant, and adding absolute ethanol with volume 2 times of that of the supernatant, namely 240-800 μl, to precipitate DNA; standing at room temperature for 5-10 min, and centrifuging 10000-12000g for 3-8 min; removing the supernatant, washing with 75% ethanol once, volatilizing ethanol thoroughly, and dissolving the precipitate in 10-50 μl sterilized double distilled water to obtain DNA extractive solution;
(c) And (3) PCR amplification: taking 0.5-1 μl of extracted DNA as a template for polymerase chain reaction amplification; mixing 21-42 μl of reagent C with 4-8 μl of reagent D; placing the reagent on a thermal circulation position to amplify the extracted DNA template, and melting the template for 2-5 minutes at 94-98 ℃; then denaturation is carried out for 30 seconds to 1 minute at 93 to 95 ℃, renaturation is carried out for 30 seconds to 1 minute at 60 to 64 ℃, extension is carried out for 30 seconds to 1.5 minutes at 72 ℃, the cycle is carried out for 30 to 35 times, and finally, the temperature is kept for 8 to 10 minutes at 72 ℃, so that the specific fragment of the Vibrio harveyi is increased to more than 108 mol;
(d) Electrophoresis and chromogenic detection: taking 5-10 mu l of amplified sample, mixing with 2-6 mu l of bromophenol blue with the V/V of 0.25%, carrying out agarose electrophoresis and color development with the W/V' of 0.8-1.2%, spinning the reagent at an observation position, detecting whether a 363 nucleotide pair strip exists or not under ultraviolet light, and if a 363 nucleotide pair strip exists, indicating that the sample has Vibrio harveyi infection or pollution, otherwise, not.
The invention has the advantages and positive effects that:
nucleic acid-based detection techniques, including DNA probes and PCR techniques (polymerase chain reaction techniques), have several significant advantages over other vibrio pathogen detection techniques and other pathogen detection techniques: firstly, the method is very rapid in detection, the detection result can be obtained in 3-4 hours, and other methods at least need 3 days or more than one week; secondly, the method has extremely high detection sensitivity and low detection lower limit of about 10 bacteria, and other methods can detect bacteria after the pure culture proliferation reaches 105 in a certain time. The invention can be used for detecting the infection of the marine aquatic animals by the Vibrio harveyi in the culture process of the marine aquatic animals in each period, can also detect pathogenic Vibrio harveyi in the marine water environment, and can also detect whether various aquatic products are polluted by the Vibrio harveyi.
The foregoing description is only of embodiments of the present invention, and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes using the descriptions and the drawings of the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.
Claims (5)
1. A vibrio harveyi detection kit, comprising: the kit and the detection structure comprise a base, a boiling water bath (1), a thermal circulation position (2), an observation position (3), an ultraviolet lamp (4) and a lamp bracket (5); three blind grooves are formed in the base, a boiling water bath (1), an observation position (3) and a thermal circulation position (2) are respectively arranged in the three blind grooves, a heating mechanism and purified water are arranged in the boiling water bath (1), the heating mechanism can keep the purified water in the boiling water bath (1) in a boiling state, a fixing device capable of fixing a reagent bottle is arranged in the observation position (3), the heating mechanism is arranged in the thermal circulation position (2), a lamp holder (5) is fixedly arranged on the base, an ultraviolet lamp (4) is connected onto the lamp holder (5) and is arranged right above the observation position (3), and the light of the ultraviolet lamp (4) faces the observation position.
2. The vibrio harveyi detection kit according to claim 1, wherein the lamp holder is provided with a chute (501), and the ultraviolet lamp (4) is slidably connected to the lamp holder (5) through the chute (501).
3. The kit according to claim 1, wherein the detecting structure further comprises a sliding protective cover (6), and the sliding protective cover (6) is slidably connected to the base.
4. The kit for detecting vibrio harveyi according to claim 1, wherein the kit is composed of the following reagents:
(a) DNA extraction reagent: comprises a buffer reagent A and a cell lysis reagent B,
reagent A: comprises NaCl0.1-0.3M, trihydroxymethyl aminomethane 1-100mM with pH8.0, ethylenediamine tetraacetic acid 0.1-10mM with pH8.0 and triamcinolone X-100 with V/V concentration of 0-20%;
reagent B: comprises lysozyme with a concentration of 1-10mg/ml and trihydroxymethyl aminomethane with a pH of 8.0 and a concentration of 1-100 mM;
(b) Polymerase chain reaction reagents: contains a reaction premixing reagent C and a reaction primer reagent D,
reagent C: contains 10 times polymerase chain reaction buffer solution, thermal polymerase and sterilized double distilled water, and the contents are respectively 2.5-5.0 μl, 0.5-2.5 units and 18-35.5 μl;
reagent D: contains specific primer pair VhF and VhR, deoxynucleotide and MgCl2, and the content is 0.2-1 mu M, 20-200 mu M and 0.5-10mM respectively;
(c) Electrophoresis and chromogenic reagent, comprising reagent E and reagent F,
reagent E: 50 times of electrophoresis buffer solution containing the trihydroxymethyl aminomethane-acetic acid and the ethylenediamine tetraacetic acid, and the concentration is 0.04M and 0.001M respectively;
reagent F: agarose with W/V of 0.8-2% and ethidium bromide with W/V of 0.5-20 mug/ml;
specific primer pairs VhF and VhR in the reagent D are specific primers and essential components for performing PCR reaction, and VhF is particularly 5' -TCTAACTATCCACCGCGG; vhR in particular 5' -AGCAATGCCATCTTCACGTTC.
5. The kit for detecting Vibrio harveyi according to claim 4, wherein each tube of the reaction reagent has a formulation of 5.0. Mu.l of 10-fold polymerase chain reaction buffer, 2.5 units of thermal polymerase, 35.5. Mu.l of sterilized double distilled water 4.0. Mu.l of 2.5mM dNTP, 3.0. Mu.l of 25mM MgCl2, 10pMVhF,10pMVhR.
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| CN118240909A (en) * | 2024-04-28 | 2024-06-25 | 中华人民共和国日照海关 | A rapid detection method for Vibrio harveyi |
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