CN102154469A - General detection method for pathogenic vibrios and nucleic acid isothermal amplification detection kit used in same - Google Patents
General detection method for pathogenic vibrios and nucleic acid isothermal amplification detection kit used in same Download PDFInfo
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- CN102154469A CN102154469A CN 201110008044 CN201110008044A CN102154469A CN 102154469 A CN102154469 A CN 102154469A CN 201110008044 CN201110008044 CN 201110008044 CN 201110008044 A CN201110008044 A CN 201110008044A CN 102154469 A CN102154469 A CN 102154469A
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- 238000001514 detection method Methods 0.000 title claims abstract description 62
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- 238000011901 isothermal amplification Methods 0.000 title abstract description 4
- 239000007788 liquid Substances 0.000 claims abstract description 45
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- 239000013641 positive control Substances 0.000 claims abstract description 31
- 239000013642 negative control Substances 0.000 claims abstract description 27
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Abstract
The invention discloses a general nucleic acid isothermal amplification detection kit for pathogenic vibrios of mariculture animals. The detection kit comprises a grinding liquid tube into which grinding liquid is filled, a nucleic acid extracting solution tube A into which solution of sodium acetate is filled, a nucleic acid extracting solution tube B into which absolute ethanol is filled, a nucleic acid extracting solution tube C into which 70 mass percent ethanol solution is filled, a tris-hydrogen chloride ethylene diamine tetraacetic acid (TE) buffer solution tube into which TE buffer solution is filled, a uracil-DNA-glycosylase (UNG) tube into which uracil-DNA-glycosylase is filled, a loop-mediated isothermal amplification (LAMP) reaction liquid tube into which LAMP reaction liquid is filled, a bacillus stearothermophilus deoxyribonucleic acid (Bst DNA) polymerase tube into which Bst DNA polymerase is filled, a color-developing agent tube into which a nucleic acid dye SYBR Green I is filled, a positive control nucleic acid tube into which positive DNA of the vibrios is filled and a negative control tube into which sterilized double distilled water is filled. The invention also discloses a method for detecting the pathogenic vibrios of the mariculture animals by utilizing the detection kit.
Description
Technical field
The present invention relates to marine cultured animal cause of disease detection technique, the nucleic acid isothermal that specifically relates to the marine cultured animal kinds of pathogenic vibrio increase general detection kit and detection method.
Background technology
Kinds of pathogenic vibrio is one of common pathogenic bacteria monoid in the ocean environment, the vibriosis that causes is that a kind of popular scope is wide, endanger serious communicable disease, can endanger multiple marine cultured animals such as fish, crustaceans and shellfish, mariculture industry has been caused enormous economic loss.Flourish along with mariculture industry, vibriosis frequently takes place, the encountered pathogenic kind have Vibrio harveyi (
Vibrio harveyi), vibrio alginolyticus (
V. alginolyticus), Vibrio parahaemolyticus (
V. parahaemolyticus), Vibrio vulnificus (
V. vulnificus), the Fu Shi vibrios (
V. furnissii), vibrio fluvialis (
V. fluvialis), Vibrio mimicus (
V. mimicus), Vibrio anguillarum (
V. anguillarum) etc., the pathogenic bacterium that wherein most of kinds are still human.To infect the marine cultured animal disease that causes by the different sorts in the kinds of pathogenic vibrio, concrete prevention of adopting in breeding production or measure of control are similar basically, after marine cultured animal is ill, whether what plant (family) was concerned about is to culture kind to be infected by kinds of pathogenic vibrio, and not too can go to be concerned about to be that any kinds of pathogenic vibrio infects, universal test method and the detection kit of therefore setting up a kind of kinds of pathogenic vibrio are necessary.But existing vibrios detection method is single at sample, and complex operation step, is difficult for realizing on-the-spot batch detection, is difficult to satisfy the needs of marine cultured animal disease control.Existing detection kinds of pathogenic vibrio mainly contains three kinds of methods: 1. Physiology and biochemistry identification method, and this method complex operation step wastes time and energy; 2. enzyme linked immunosorbent assay (ELISA) though that this method detects is quick, highly sensitive, can be used for characteristics such as mass detection, is prone to the false positive problem when fresh sample is detected, limited the application in production practice to a certain extent; 3. polymerase chain reaction method (PCR), this method is quick, sensitive, but needs expensive nucleic acid amplification instrument, has limited the application in production practice equally.
The ring mediated isothermal amplification method (LAMP) that development in recent years is got up, it is a kind of novel constant temperature nucleic acid amplification method, can be selectively efficient amplified target gene order, product is loop-stem structure and the dna fragmentation mixture that encircles the Cauliflower spline structures that a series of reverse multiple target sequences constitute more, shows the staged collection of illustrative plates that different big sub-districts band is formed behind the electrophoresis on gel.It is obvious that LAMP and other kinds of pathogenic vibrio detection method are compared advantage: though 1. reaction principle, design of primers more complicated increase under constant temperature, only need thermostats (as water-bath, thermos cup etc.) just can carry out; 2. efficient and sensible, template only need 10 copies or still less; 3. high specificity is used four primers that comprised six sections and is increased in proper order by strictness; 4. lack detection time, amplification can be finished in 60min; 5. product can be by directly adding fluorescent dyeing in reaction tubes, and the result is visual in image.Although being used for vibrios, now existing LAMP method detects, as Vibrio parahaemolyticus (Chinese patent application number: 200710046637.4,200710030441.6,200710026389.7,200810030340.3; Wataru Y, 2009), vibrio cholerae (Chinese patent application number: 200710030446.9; 200710026391.4), Vibrio vulnificus (Chinese patent application number: 200810151271.1 Srisuk C, 2009), Vibrio mimicus (Chinese patent application number: 200710026390.X), vibrio fluvialis (Chinese patent application number:; Han F, 2008; Ren C H, 2008), but all are single kind at bacterium in the Vibrio.And cultivated animals faces the possibility that is infected by multiple morbid vibrio in sea farming is produced, what breeding production person was concerned about more in the practice is whether infective pathogen is vibrios, and needn't know that any vibrios infects, universal test method and the detection kit of therefore setting up a kind of kinds of pathogenic vibrio in the sea farming field are necessary.
Summary of the invention
General detection kit that the technical problem to be solved in the present invention provides that a kind of cost is low, detection sensitivity is high and the marine cultured animal kinds of pathogenic vibrio universal test method of easy scene operation in enormous quantities and used nucleic acid isothermal increase.
In order to solve the problems of the technologies described above, the invention provides a kind of marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal general detection kit that increases, this test kit comprises:
(1), the lapping liquid pipe: interior dress lapping liquid,
(2), nucleic acid extraction liquid A pipe: interior dress sodium acetate solution,
(3), nucleic acid extraction liquid B pipe: interior dress dehydrated alcohol,
(4), nucleic acid extraction liquid C pipe: the ethanolic soln of interior packing quality concentration 70%,
(5), TE damping fluid pipe: interior dress TE damping fluid;
(6), UNG enzyme pipe: interior dress uracil dna glycosylase;
(7), LAMP reaction solution pipe: interior dress LAMP reaction solution;
(8),
BstArchaeal dna polymerase pipe: interior dress
BstArchaeal dna polymerase;
(9), developer pipe: interior dress nucleic acid dye SYBR Green I;
(10), positive control nucleic acid pipe: the positive DNA of interior dress vibrios;
(11), negative control pipe: the distilled water of interior dress sterilization.
As the increase improvement of general detection kit of kinds of pathogenic vibrio nucleic acid isothermal of the present invention: lapping liquid is made up of the component with lower volume: 5 ~ 10 parts of the tris solutions of 1 ~ 2 M, 0.25 0.5 ~ 2.0 part of the disodium ethylene diamine tetra-acetic acid solution of ~ 1.0M, 5 ~ 10 parts of the sodium dodecyl sulfate solutions of mass concentration 5 ~ 10%, 0.01 ~ 1.0 part of mercaptoethanol, 0.01 ~ 0.02 part of oxine, 5 ~ 10 parts of 5 ~ 10 parts of balance phenols and chloroforms are with distilled water constant volume to 100 part.
As the increase further improvement of general detection kit of kinds of pathogenic vibrio nucleic acid isothermal of the present invention: the LAMP reaction solution is composed of the following components: each 1 ~ 4 μ M of LAMP primer VIBRIO-FIP and VIBRIO-BIP, each 0.1 ~ 0.5 μ M of LAMP primer VIBRIO-F3 and IVIBRIO-B3, each 0.5 ~ 2 mM of dATP, dGTP and dCTP, each 0.25 ~ 1 mM of dTTP, dUTP, Tris-HCl 10 ~ 40 mM, KCl 10 ~ 20 mM, (NH
4)
2SO
45 ~ 15 mM, MgSO
41 ~ 4 mM, Triton X-100 0.05%～1.0%, Betaine 0.8 ~ 1.2 M; All the other are distilled water.
As the increase further improvement of general detection kit of kinds of pathogenic vibrio nucleic acid isothermal of the present invention: the nucleotide sequence of LAMP primer is as follows:
VIBRIO-FIP（SEQ?ID?NO.1）：
5'-?CGGCTGCTGGCACGGAGTTTTTGCATTATTTGACGTTAGCGACAGAAG；
VIBRIO-BIP（SEQ?ID?NO.2）：
5'-?GAGCGTTAATCGGAATTACTGGGCTTTTCCGGGCTTTCACATCTGACTTAAC；
VIBRIO-F3（SEQ?ID?NO.3）:
5'-?CAGTCGTGAGGAAGGTGGTGT；
VIBRIO-B3（SEQ?ID?NO.4）:
5'-?CTAGTCTGCCAGTTTCAAATGCT。
The present invention also provides the detection method of utilizing the marine cultured animal kinds of pathogenic vibrio that detection kit carries out, comprises the following steps:
(1), getting testing sample organizes 0.2g to place centrifuge tube
In, add 0.5 ml lapping liquid, fully be milled to pulpous state with grinding rod;
(2), the pulpous state sample of above-mentioned grinding gained being boiled (100 ℃, l0 min) back gets supernatant liquor and places centrifuge tube with centrifugal 5 min of 10000 r/min
In;
(3), to centrifuge tube
The interior sodium acetate solution mixing that adds in the nucleic acid extraction liquid A pipe, the volume ratio of described sodium acetate solution and supernatant liquor is 1/10; The interior dehydrated alcohol of nucleic acid extraction liquid B pipe that adds-20 ℃ of precoolings again mixes, and leaves standstill 10 min, with the centrifugal 5min of 12000 r/min, abandons supernatant liquor, keeps throw out, and the volume ratio of described dehydrated alcohol and supernatant liquor is 2:1;
(4), with the throw out of ethanolic soln washing above-mentioned steps (3) gained in the nucleic acid extraction liquid C pipe, with centrifugal 5 min of 12000 r/min, abandon supernatant liquor then, keep throw out;
(5), with the throw out of ethanolic soln washing above-mentioned steps (4) gained in the nucleic acid extraction liquid C pipe, with centrifugal 5 min of 12000 r/min, abandon supernatant liquor then, keep throw out;
(6), the throw out of above-mentioned steps (5) gained is dried in room temperature, add 20 μ l TE damping fluid dissolution precipitation things, obtain sample nucleic acid;
(7), respectively with the positive control nucleic acid 2 μ l in the distilled water in above-mentioned sample nucleic acid, the negative control pipe, the positive control nucleic acid pipe, add in the 21 μ l LAMP reaction solutions, add 1 μ l UNG enzyme again, get detector tube, negative control pipe and positive control pipe respectively; Above-mentioned detector tube, negative control pipe and positive control Guan Jun are carried out enzymolysis in 37 ℃ of insulation 10 min;
(8), the reaction system behind above-mentioned detector tube, negative control pipe and the positive control pipe enzymolysis is carried out following operation respectively:, and then place rubble ice 5 min rapidly prior to 95 ℃ of insulation 5 min;
(9), in the reaction system of detector tube, negative control pipe and the positive control pipe of step (8) gained, add 1 μ l more respectively
BstArchaeal dna polymerase;
(10), the detector tube with above-mentioned steps (9) gained, negative control pipe and positive control pipe carry out following operation respectively: prior to 64 ℃ of insulation 45 min, finish the LAMP reaction in 90 ℃ of insulation 2 min then;
(11), after LAMP reaction finishes, add 1 μ l nucleic acid dye SYBR Green I, if the color of LAMP reaction product and the color of negative control tube reaction product all are orange in the detector tube, then product to be tested kinds of pathogenic vibrio detected result is negative; If the color of LAMP reaction product all is green with the color of positive control tube reaction product in the detector tube, then product to be tested kinds of pathogenic vibrio detected result is positive.
In test kit of the present invention, raw material can obtain in commercial mode, for example disodium ethylene diamine tetraacetate, Tutofusin tris, sodium laurylsulfonate, sodium-acetate, chloroform, balance phenol, mercaptoethanol, oxine, MgCl
2, dATP, dGTP, dCTP, dTTP be available from Shanghai biotechnology limited liability company, dUTP dodges brilliant molecular biosciences Science and Technology Ltd. available from Shanghai, uracil dna glycosylase (UNG enzyme),
BstThe dark grade of DNA polymerization is available from U.S. NEB company, and nucleic acid dye SYBR Green I is available from Beijing ancient cooking vessel state bio tech ltd, and Betaine, Triton X-100 are available from Sigma company.
LAMP primer VIBRIO-FIP, VIBRIO-BIP, VIBRIO-F3 and VIBRIO-B3 and PCR primer VIBRIO-Pf, VIBRIO-Pr can entrust U.S. invitrogen (Shanghai) English Weihe River Jie Ji Bioisystech Co., Ltd synthetic.
The acquisition mode of the positive DNA of kinds of pathogenic vibrio is: extract the Vibrio harveyi genomic dna, increase with PCR primer VIBRIO-Pf and VIBRIO-Pr, reaction system is: 25 μ l systems comprise 2.5 μ l, 10 * PCR reaction buffer, 1.0 μ l dNTPs(10mM), 1.0 μ l VIBRIO-Pf and VIBRIO-Pr(20 μ M), 0.5 μ l Taq archaeal dna polymerase (5 U/ μ l, Beijing ancient cooking vessel state bio tech ltd), all the other are distilled water.Amplification program is: 94 ℃ of pre-sex change 5 min, enter amplification cycles afterwards, and 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ of extension 60s finish an amplification cycles, and recirculation 30 is taken turns, last 72 ℃ of 10 min, 4 ℃ of preservations.Increase the band of 474bp, after reclaiming this fragment, be connected to T carrier (pMD-18 T-Vector by the requirement of T carrier specification sheets, available from the precious biotech firm in Dalian) on, and press the competent cell operational requirement with recombinant plasmid transformed to competent cell JM109(available from the precious biotech firm in Dalian) in, with blue hickie method screening positive bacteria, and carry out PCR with primer VIBRIO-Pf and VIBRIO-Pr and verify (program is the same).Extract the plasmid of positive bacteria at last with alkaline process, as the positive DNA of vibrios.
In the step of detection method of the present invention (7), eliminate the interference of template pollution to detected result by the UNG enzyme that in the sample nucleic acid that extracts, adds 1 unit.Also can be in step (9) add the nucleic acid dye that does not suppress the LAMP reaction in advance, uncapping after having avoided reaction to finish adds the dyestuff detected result, greatly reduces the possibility that template is polluted, and can avoid false-positive generation.
Compare with the LAMP technology of other reports, core of the present invention it-be at the conservative 16s rna gene of Vibrio, preferred goal gene section designs the LAMP Auele Specific Primer, make this primer can effectively detect the Vibrio bacterium, and detection specificity is good; Two of core is to carry out the processing of sample with custom-designed lapping liquid, makes that the treating processes of sample is extremely simple; Three of core is to adopt the uridylic glycosylase to eliminate the pollution of LAMP amplified production, has avoided the false positive results that causes greatly because of LAMP product amplification amount; Four of core is to have realized and can coveredly detect, and has avoided the generation of polluting from the source, has further avoided the appearance of false positive results; Five of core is technical parameters of having optimized the entire operation process, and with its stdn, supportingization, forms detection kit, is convenient to on-the-spot high volume applications.
Selected target sequence is the conservative 16S rna gene of Vibrio bacterium during design of primers in invention, so reaction can detect the interior bacterium (overwhelming majority is a kinds of pathogenic vibrio) of all Vibrios in principle; Instead would not expand, so can not detect the bacterium of non-Vibrio other bacterial genes.
The present invention is the conserved sequence region design Auele Specific Primer according to Vibrio bacterial 16 S rna gene, sets up the general detection technique of kinds of pathogenic vibrio LAMP, can realize the mass field rapid detection of the ill clinical sample of marine cultured animal.
Therefore, positively effect of the present invention is: compiled vibrios and detect ten kinds of reagent and three kinds of equipment such as required lapping liquid, nucleic acid extraction liquid in test kit, making vibrios detect can carry out in an orderly manner, realized testing process scene, sequencing and stdn, make working specification, be difficult for makeing mistakes; Universal test method of the present invention is that a cover LAMP primer that designs according to the conservative 16s rna gene region sequence of Vibrio is that main body designs, thereby makes the vibrios detection have LAMP technology sensitivity, quick, easy, characteristic of accurate fully; The UNG enzyme can be eliminated the pollution of nucleic acid in the repeated detection process, has solved easy the to be contaminated interferential problem of restriction LAMP technology widespread use.Use test kit of the present invention and detection method just can finish the detection of kinds of pathogenic vibrio in 2 hours, testing process need not expensive plant and instrument such as PCR instrument and gel imaging system, only need have water-bath or metal bath and generic centrifuge to get final product; And detected result is very easy to differentiate; Compare with the existing detection technique of Vibrio bacterium, test kit of the present invention and corresponding method of detection have not only been realized the general detection of vibrios, and cost is low, highly sensitive, simple to operate, can realize field quick detection.In a word, method of the present invention has more in the past than sensitivity and the convenience that detection method was higher, can substitute related detecting method before the kinds of pathogenic vibrio such as Physiology and biochemistry method, PCR method, enzyme-linked immunosorbent assay etc.
Embodiment
The invention will be further described by the following examples.
Embodiment 1, a kind of marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal general detection kit (5 sample parts promptly can be used for the detection of 5 samples time) that increases, form by following content:
(1) lapping liquid pipe, 1 pipe, 3 ml.
Lapping liquid preparation: the tris solution of 1 M (Tris-HCl, pH8.0) 10 parts, the disodium ethylene diamine tetra-acetic acid solution (EDTANa of 0.25M
2) 2 parts, massfraction is 10 parts of the sodium dodecyl sulfate solutions (SDS) of 5 %, 0.5 part of mercaptoethanol, and 0.01 part of oxine, 7 parts of balance phenols, 8 parts of chloroforms part form with distilled water constant volume to 100.Above umber is volume parts.
(2) nucleic acid extraction liquid A pipe, 1 pipe, in adorn 400 μ l 3M sodium acetate solutions (pH5.2).
(3) nucleic acid extraction liquid B pipe, the l pipe, in adorn 10 ml dehydrated alcohols.
(4) nucleic acid extraction liquid C pipe, 1 pipe, in adorn the ethanolic soln of 10 ml mass concentrations 70%.
(5) TE damping fluid pipe, 1 pipe, in adorn 400 μ l TE damping fluids (contain 10 mM Tris-HCl and 1mM EDTA, pH8.0, all the other are distilled water).
(6) UNG enzyme pipe, 1 the pipe, in adorn 6 U uracil dna glycosylases (UNG enzyme),
(7) LAMP reaction solution pipe, 1 the pipe, in adorn 150 μ l LAMP reaction solutions,
The moiety of LAMP reaction solution is as follows: each 2 μ M of LAMP primer VIBRIO-FIP and VIBRIO-BIP, each 0.3 μ M of LAMP primer VIBRIO-F3 and VIBRIO-B3, each 1mM of dATP, dGTP and dCTP, each 0.5mM of dTTP, dUTP, Tris-HCl 20mM, KCl 15mM, (NH
4)
2SO
410mM, MgSO
42mM, Triton X-100 0.5%(volumetric concentration), Betaine 1M; All the other are distilled water.
LAMP primer described in the general detection kit of above-mentioned kinds of pathogenic vibrio gene is according to the conservative 16s rna gene sequences Design of Vibrio, the first-selected software of LAMP design of primers is PrimerExplore 4.0 (http://primerexplorer.jp/elamp4.0.0/index.html), also can use primer-design software commonly used (as Primer Primier 5.0 or Omiga 2.0) to require to design according to the LAMP primer design.Utilize among the present invention software PrimerExplore 4.0 online designs-dna sequence dna of cover LAMP primer is as follows:
VIBRIO-FIP：
5'-?CGGCTGCTGGCACGGAGTTTTTGCATTATTTGACGTTAGCGACAGAAG；
VIBRIO-BIP：
5'-?GAGCGTTAATCGGAATTACTGGGCTTTTCCGGGCTTTCACATCTGACTTAAC；
VIBRIO-F3：
5'-?CAGTCGTGAGGAAGGTGGTGT；
VIBRIO-B3：
5'-?CTAGTCTGCCAGTTTCAAATGCT。
(8)
BstThe archaeal dna polymerase pipe, 1 the pipe, in adorn 5 μ l
BstArchaeal dna polymerase.
(9) developer pipe, 1 pipe, in adorn the liquid dye of 5 μ l, these liquid dye are mixed according to the volume ratio of 1:100 by nucleic acid dye SYBR Green I and distilled water and get.
(10) positive control nucleic acid pipe, 1 pipe, in adorn the positive DNA of 10 μ l vibrios.
The preparation method is as follows: with the PCR primer (VIBRIO-Pf [SEQ ID NO.5]: 5'-AGACACGGTCCAGACTCCTAC that is positioned at LAMP target fragment upstream and downstream; VIBRIO-Pr [SEQ ID NO.6]: 5'-AGGGTATCTAATCCTGTTTGCT), the Vibrio harveyi genomic dna is increased, amplification condition is: 94 ℃ of pre-sex change 5 min, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ of extension 60s finish an amplification cycles, and recirculation 30 is taken turns, last 72 ℃ of 10min, 4 ℃ of preservations, obtain the band of 474bp, reclaim this fragment after, be connected to carrier pMD-18 T-Vector, and transformed into escherichia coli competent cell JM109, after PCR checking, extract plasmid, be diluted to behind the 100 copy/μ l as the vibrios positive control.This Vibrio harveyi for example can be available from Chinese common micro-organisms culture presevation administrative center, as is CGMCC 1.1600.
(11) negative control pipe, 1 pipe, in adorn 10 μ l sterilization distilled water.
(12) grinding rod, 5.
(13) packing box (78 * 78 * 50mm).
(14) cystoses, foam plate hight 22 mm have three row holes, four holes of first row, aperture 5.5 mm, three skies of secondary series, aperture 10 mm, three skies of the 3rd row, aperture 15.5mm.Each tubule of above-mentioned 1 ~ 11 is positioned in these apertures, and this cystose is identical with the bottom surface size of above-mentioned packing box, is loaded in the packing box.
(15) test kit working instructions-part.
Embodiment 2, a kind of marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal general detection kit (5 sample part) that increases, only following content is different from embodiment 1, and all the other are all with embodiment 1.
Lapping liquid preparation: the tris solution of 2 M (Tris-HCl, pH8.0) 5 parts, the disodium ethylene diamine tetra-acetic acid solution (EDTANa of 0.5M
2) 1 part, 7 parts of the sodium dodecyl sulfate solutions (SDS) of massfraction 7 %, 0.01 part of mercaptoethanol, 0.02 part of oxine, 5 parts of balance phenols, 10 parts of chloroforms part form with distilled water constant volume to 100.Above umber is volume parts.
The moiety of LAMP reaction solution is as follows: each 4 μ M of LAMP primer VIBRIO-FIP and VIBRIO-BIP, each 0.5 μ M of LAMP primer VIBRIO-F3 and VIBRIO-B3, each 2mM of dATP, dGTP and dCTP, each 1.0mM of dTTP and dUTP, Tris-HCl 40mM, KCl 20mM, (NH
4)
2SO
415mM, MgSO
44mM, Triton X-100 1.0%, Betaine 0.8M; All the other are distilled water.
Embodiment 3, a kind of marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal general detection kit (5 sample part) that increases, only following content is different from embodiment 1, and all the other are all with embodiment 1.
Lapping liquid preparation: the tris solution of 1.5 M (Tris-HCl, pH8.0) 7 parts, the disodium ethylene diamine tetra-acetic acid solution (EDTANa of 1.0M
2) 0.5 part, 5 parts of the sodium dodecyl sulfate solutions of massfraction 10% (SDS), 0.01 part of mercaptoethanol, 0.015 part of oxine, 10 parts of balance phenols, 10 parts of chloroforms part form with distilled water constant volume to 100.Above umber is parts by volume.
The moiety of LAMP reaction solution is as follows: each 1 μ M of LAMP primer VIBRIO-FIP and VIBRIO-BIP, each 0.25 μ M of LAMP primer VIBRIO-F3 and VIBRIO-B3, each 0.5mM of dATP, dGTP and dCTP, each 0.5mM of dTTP, dUTP, Tris-HCl 40mM, KCl 10mM, (NH
4)
2SO
45mM, MgSO
41mM, Triton X-100 0.05%, Betaine 0.8M; All the other are distilled water.
The universal test method of embodiment 4, a kind of marine cultured animal kinds of pathogenic vibrio utilizes any one detection kit of embodiment 1 ~ embodiment 3, carries out following steps successively:
(1), gets ill green hata liver organization 0.2 g to be measured and place centrifuge tube
In, add the 0.5ml lapping liquid in the lapping liquid pipe, fully be milled to pulpous state with grinding rod;
(2), the pulpous state sample of above-mentioned grinding gained being boiled (100 ℃, l0 min) back gets supernatant liquor and places centrifuge tube with centrifugal 5 min of 10000 r/min
In;
(3), to centrifuge tube
The interior sodium acetate solution mixing that adds in the nucleic acid extraction liquid A pipe, the volume ratio of sodium acetate solution and supernatant liquor is 1/10; The interior dehydrated alcohol of nucleic acid extraction liquid B pipe that adds-20 ℃ of precoolings again mixes, and leaves standstill 10min, with the centrifugal 5min of 12000 r/min, abandons supernatant liquor, keeps throw out, and the volume ratio of dehydrated alcohol and supernatant liquor is 2:1;
(4), with the throw out of 1 ml ethanolic soln washing above-mentioned steps (3) gained in the nucleic acid extraction liquid C pipe, with centrifugal 5 min of 12000 r/min, abandon supernatant liquor then, keep throw out;
(5), with the throw out of 1ml ethanolic soln washing above-mentioned steps (4) gained in the nucleic acid extraction liquid C pipe, with centrifugal 5 min of 12000 r/min, abandon supernatant liquor then, keep throw out;
(6), the throw out with above-mentioned steps (5) gained (is positioned at centrifuge tube
The bottom) dry in room temperature, add 20 μ l TE damping fluid dissolution precipitation things in the TE damping fluid pipe, obtain sample nucleic acid;
(7), respectively with the positive control nucleic acid 2 μ l in the distilled water in above-mentioned sample nucleic acid, the negative control pipe, the positive control nucleic acid pipe, add in the 21 μ l LAMP reaction solutions, add 1 μ l UNG enzyme again, thereby get detector tube, negative control pipe and positive control pipe respectively; Above-mentioned detector tube, negative control pipe and positive control Guan Jun are carried out enzymolysis in 37 ℃ of insulation 10 min, and purpose is to eliminate the pollution template that wherein may exist with enzyme solution;
(8), the reaction system behind above-mentioned detector tube, negative control pipe and the positive control pipe enzymolysis is carried out following operation respectively:, and then place rubble ice 5 min rapidly prior to 95 ℃ of insulation 5 min;
(9), in the reaction system of detector tube, negative control pipe and the positive control pipe of step (8) gained, add 1 μ l more respectively
BstArchaeal dna polymerase;
(10), the detector tube with above-mentioned steps (9) gained, negative control pipe and positive control pipe carry out following operation respectively: prior to 64 ℃ of insulation 45 min, finish the LAMP reaction in 90 ℃ of insulation 2 min then;
(11), after LAMP reaction finishes, add 1 μ l nucleic acid dye SYBR Green I, if detector tube and positive control pipe all show green, the demonstration of negative control pipe is orange, represents that the vibrios detected result of this sample is positive; If it is orange that detector tube and negative control pipe all show, the positive control pipe shows green, represents that the vibrios detected result of this sample is negative; If it is orange that the positive control pipe shows, represent that this detection reagent lost efficacy, need to detect again after the replacing detection reagent; If the negative control pipe shows green, represent that this detection reagent is contaminated, need to detect again after replacing detection reagent and the detection site.
The universal test method of embodiment 5, a kind of marine cultured animal kinds of pathogenic vibrio, sick sample increased bacterium with nutrient broth after, according to following steps kinds of pathogenic vibrio is detected:
(1), get ill green hata liver organization 2 g to be measured and place aseptic homogenizer homogenate, homogenate is added in the nutrient broth (5ml) 25 ℃ ~ 28 ℃ shaking tables cultivates 5h ~ 6h.The nutrient broth prescription is: albumen freezes 10g, extractum carnis 3g, sodium-chlor 5g, distilled water 1000ml, pH7.4.Mentioned component is mixed, dissolves post-equalization pH, 121 ℃ of autoclaving 15min.
(2), get 2ml bacterium liquid, the centrifugal 1min of 10000r/m abandons supernatant, adds 50 μ l distilled waters, 10min is boiled in water-bath.
(3), the centrifugal 1min of 10000r/m, get supernatant.
(4), use any one detection kit of embodiment 1 ~ embodiment 3, detect according to step (7) among the embodiment 4 ~ described method of step (11).
If detector tube shows green then represents that the vibrios detected result of this sample is positive, represents that then the vibrios detected result of this sample is negative if the detector tube demonstration is orange.
Can be by this embodiment 5 explanation the present invention through comparatively complicated dna profiling preparation, if tissue infection bacterium and need boil after amplification, centrifugally can prepare the DNA detection template, this method is simple and practical, need not nucleic acid extraction liquid.
The universal test method of embodiment 6, a kind of marine cultured animal kinds of pathogenic vibrio as detected object, is verified the specificity of detection method with bacterium not of the same race.According to following steps bacterium is detected:
(1), vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, vibrio fluvialis, Aeromonas hydrophila, Aeromonas caviae, intestinal bacteria, nitrobacteria are enriched to OD respectively at cultivating in the suitable culture medium
600=0.6.Carry out following step then respectively:
(2), get 1ml bacterium liquid, the centrifugal 1min of 10000r/m abandons supernatant, adds 50 μ l distilled waters, 10min is boiled in water-bath.
(3), the centrifugal 1min of 10000r/m, get supernatant, promptly get DNA of bacteria and detect template.Above-mentioned template can be carried out 10 with the TE damping fluid
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Times serial dilution, thus respectively 10
-1-10
-7Template.
(4), use any one detection kit of embodiment 1 ~ embodiment 3, according to step (7) among the embodiment 4 ~ described method of step (11), respectively to 10
-1-10
-7Template detects.
The result shows, the LAMP reaction can detect kinds of pathogenic vibrio such as vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, vibrio fluvialis, and the detected result of common bacteria in the water surroundings such as Aeromonas hydrophila, Aeromonas caviae, intestinal bacteria, nitrobacteria is presented feminine gender.
The The above results explanation: LAMP detection kit of the present invention has excellent specificity to kinds of pathogenic vibrio.
The contrast experiment 1: with same infected animal sample (having confirmed to infect Vibrio harveyi) as testing sample, utilize the test kit among the embodiment 1, prepare the detection template of ill green hata (being sample nucleic acid) by the method for step (1) among the embodiment 4 ~ step (6).Above-mentioned template is carried out 10 with the TE damping fluid
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Times serial dilution detects template as LAMP and PCR respectively.
1), according to step (7) among the embodiment 4 ~ described method of step (11), respectively to 10
-1-10
-7Template is checked.
2), carrying out PCR with same template detects use primer VIBRIO-Pf and VIBRIO-Pr.
The result shows that the LAMP reaction can detect 10
-6The template of dilution doubly, and PCR can only detect to 10
-3The template of dilution doubly, the remolding sensitivity PCR that LAMP detects is high 1000 times, and the time spent still less, need not complex apparatus.
The contrast experiment 2: as testing sample, utilize the test kit among the embodiment 2 with same ill green hata, experiment method is with contrast experiment 1.
The result shows: the LAMP reaction can detect 10
-6The template of dilution doubly, and PCR can only detect to 10
-3The template of dilution doubly.
The contrast experiment 3: as testing sample, utilize the test kit among the embodiment 3 with same ill green hata, experiment method is with contrast experiment 1.
The result shows: the LAMP reaction can detect 10
-6The template of dilution doubly, and PCR can only detect to 10
-3The template of dilution doubly.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
SEQ?ID?NO.1：
cggctgctgg?cacggagttt?ttgcattatt?tgacgttagc?gacagaag 48
SEQ?ID?NO.2：
gagcgttaat?cggaattact?gggcttttcc?gggctttcac?atctgactta?ac 52
SEQ?ID?NO.3：
cagtcgtgag?gaaggtggtg?t 21
SEQ?ID?NO.4：
ctagtctgcc?agtttcaaat?gct 23
SEQ?ID?NO.5：
agacacggtc?cagactccta?c 21
SEQ?ID?NO.6：
agggtatcta?atcctgtttg?ct 22
Claims (5)
1. a marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal general detection kit that increases is characterized in that comprising:
(1), the lapping liquid pipe: interior dress lapping liquid,
(2), nucleic acid extraction liquid A pipe: interior dress sodium acetate solution,
(3), nucleic acid extraction liquid B pipe: interior dress dehydrated alcohol,
(4), nucleic acid extraction liquid C pipe: the ethanolic soln of interior packing quality concentration 70%,
(5), TE damping fluid pipe: interior dress TE damping fluid;
(6), UNG enzyme pipe: interior dress uracil dna glycosylase;
(7), LAMP reaction solution pipe: interior dress LAMP reaction solution;
(8),
BstArchaeal dna polymerase pipe: interior dress
BstArchaeal dna polymerase;
(9), developer pipe: interior dress nucleic acid dye SYBR Green I;
(10), positive control nucleic acid pipe: the positive DNA of interior dress vibrios;
(11), negative control pipe: the distilled water of interior dress sterilization.
2. the marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal according to claim 1 general detection kit that increases, it is characterized in that: described lapping liquid is made up of the component with lower volume: 5 ~ 10 parts of the tris solutions of 1 ~ 2 M, 0.25 0.5 ~ 2.0 part of the disodium ethylene diamine tetra-acetic acid solution of ~ 1.0M, 5 ~ 10 parts of the sodium dodecyl sulfate solutions of mass concentration 5 ~ 10%, 0.01 ~ 1.0 part of mercaptoethanol, 0.01 ~ 0.02 part of oxine, 5 ~ 10 parts of 5 ~ 10 parts of balance phenols and chloroforms are with distilled water constant volume to 100 part.
3. the marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal according to claim 2 general detection kit that increases, it is characterized in that: described LAMP reaction solution is composed of the following components: each 1 ~ 4 μ M of LAMP primer VIBRIO-FIP and VIBRIO-BIP, each 0.1 ~ 0.5 μ M of LAMP primer VIBRIO-F3 and IVIBRIO-B3, each 0.5 ~ 2 mM of dATP, dGTP and dCTP, each 0.25 ~ 1 mM of dTTP, dUTP, Tris-HCl 10 ~ 40 mM, KCl 10 ~ 20 mM, (NH
4)
2SO
45 ~ 15 mM, MgSO
41 ~ 4 mM, Triton X-100 0.05%～1.0%, Betaine 0.8 ~ 1.2 M; All the other are distilled water.
4. the marine cultured animal kinds of pathogenic vibrio nucleic acid isothermal according to claim 3 general detection kit that increases, it is characterized in that: the nucleotide sequence of described LAMP primer is as follows:
VIBRIO-FIP：
5'-?CGGCTGCTGGCACGGAGTTTTTGCATTATTTGACGTTAGCGACAGAAG；
VIBRIO-BIP：
5'-?GAGCGTTAATCGGAATTACTGGGCTTTTCCGGGCTTTCACATCTGACTTAAC；
VIBRIO-F3:
5'-?CAGTCGTGAGGAAGGTGGTGT；
VIBRIO-B3:
5'-?CTAGTCTGCCAGTTTCAAATGCT。
5. utilize the detection method of carrying out the marine cultured animal kinds of pathogenic vibrio as any one detection kit in the claim 1 ~ 4, comprise the following steps:
(1), getting testing sample organizes 0.2g to place centrifuge tube
In, add 0.5 ml lapping liquid, fully be milled to pulpous state with grinding rod;
(2), the pulpous state sample of above-mentioned grinding gained being boiled (100 ℃, l0 min) back gets supernatant liquor and places centrifuge tube with centrifugal 5 min of 10000 r/min
In;
(3), to centrifuge tube
The interior sodium acetate solution mixing that adds in the nucleic acid extraction liquid A pipe, the volume ratio of described sodium acetate solution and supernatant liquor is 1/10; The interior dehydrated alcohol of nucleic acid extraction liquid B pipe that adds-20 ℃ of precoolings again mixes, and leaves standstill 10 min, with the centrifugal 5min of 12000 r/min, abandons supernatant liquor, keeps throw out, and the volume ratio of described dehydrated alcohol and supernatant liquor is 2:1;
(4), with the throw out of ethanolic soln washing above-mentioned steps (3) gained in the nucleic acid extraction liquid C pipe, with centrifugal 5 min of 12000 r/min, abandon supernatant liquor then, keep throw out;
(5), with the throw out of ethanolic soln washing above-mentioned steps (4) gained in the nucleic acid extraction liquid C pipe, with centrifugal 5 min of 12000 r/min, abandon supernatant liquor then, keep throw out;
(6), the throw out of above-mentioned steps (5) gained is dried in room temperature, add 20 μ l TE damping fluid dissolution precipitation things, obtain sample nucleic acid;
(7), respectively with the positive control nucleic acid 2 μ l in the distilled water in above-mentioned sample nucleic acid, the negative control pipe, the positive control nucleic acid pipe, add in the 21 μ l LAMP reaction solutions, add 1 μ l UNG enzyme again, get detector tube, negative control pipe and positive control pipe respectively; Above-mentioned detector tube, negative control pipe and positive control Guan Jun are carried out enzymolysis in 37 ℃ of insulation 10 min;
(8), the reaction system behind above-mentioned detector tube, negative control pipe and the positive control pipe enzymolysis is carried out following operation respectively:, and then place rubble ice 5 min rapidly prior to 95 ℃ of insulation 5 min;
(9), in the reaction system of detector tube, negative control pipe and the positive control pipe of step (8) gained, add 1 μ l more respectively
BstArchaeal dna polymerase;
(10), the detector tube with above-mentioned steps (9) gained, negative control pipe and positive control pipe carry out following operation respectively: prior to 64 ℃ of insulation 45 min, finish the LAMP reaction in 90 ℃ of insulation 2 min then;
(11), after LAMP reaction finishes, add 1 μ l nucleic acid dye SYBR Green I, if the color of LAMP reaction product and the color of negative control tube reaction product all are orange in the detector tube, then product to be tested kinds of pathogenic vibrio detected result is negative; If the color of LAMP reaction product all is green with the color of positive control tube reaction product in the detector tube, then product to be tested kinds of pathogenic vibrio detected result is positive.
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| CN103614455A (en) * | 2013-07-03 | 2014-03-05 | 淮海工学院 | Kit for rapid detection of Vibrio aestuarianus through loop-mediated isothermal amplification and detection method |
| CN106636394A (en) * | 2016-12-20 | 2017-05-10 | 浙江万里学院 | Isothermal amplication detection kit for nucleic acid of aeromonas and detection method of isothermal amplication detection kit |
| CN107012227A (en) * | 2017-04-21 | 2017-08-04 | 丹东出入境检验检疫局综合技术服务中心 | The universal primer pair detected for kinds of pathogenic vibrio and its application |
| CN107227378A (en) * | 2017-08-10 | 2017-10-03 | 广东出入境检验检疫局检验检疫技术中心 | Detect the RPA IAC primers and method of vibrio mimicus |
| CN111534620A (en) * | 2020-05-11 | 2020-08-14 | 福建省闽东水产研究所 | Universal pathogenic vibrio kit and rapid detection method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852992A (en) * | 2003-10-08 | 2006-10-25 | 札幌啤酒株式会社 | Primer for detecting alicyclobacillus |
| CN101008037A (en) * | 2007-01-18 | 2007-08-01 | 中国科学院南海海洋研究所 | Kit and method for detecting Bibrio Parahemolyticus using loop-mediated equal-temperature amplification technology |
| CN101008038A (en) * | 2007-01-18 | 2007-08-01 | 中国科学院南海海洋研究所 | Kit and method for detecting V.mimicus using loop-mediated equal-temperature amplification technology |
| CN101475986A (en) * | 2009-01-20 | 2009-07-08 | 中国水产科学研究院黄海水产研究所 | Chip for gene detection of multiple vibrios at the same time, and detection and use thereof |
| CN101792817A (en) * | 2009-11-30 | 2010-08-04 | 浙江大学 | Detection method of prawn IHHNV (infectious hypodermal and hematopoietic necrosis virus) and used nucleic acid isothermal amplification detection kit |
-
2011
- 2011-01-14 CN CN 201110008044 patent/CN102154469A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1852992A (en) * | 2003-10-08 | 2006-10-25 | 札幌啤酒株式会社 | Primer for detecting alicyclobacillus |
| CN101008037A (en) * | 2007-01-18 | 2007-08-01 | 中国科学院南海海洋研究所 | Kit and method for detecting Bibrio Parahemolyticus using loop-mediated equal-temperature amplification technology |
| CN101008038A (en) * | 2007-01-18 | 2007-08-01 | 中国科学院南海海洋研究所 | Kit and method for detecting V.mimicus using loop-mediated equal-temperature amplification technology |
| CN101475986A (en) * | 2009-01-20 | 2009-07-08 | 中国水产科学研究院黄海水产研究所 | Chip for gene detection of multiple vibrios at the same time, and detection and use thereof |
| CN101792817A (en) * | 2009-11-30 | 2010-08-04 | 浙江大学 | Detection method of prawn IHHNV (infectious hypodermal and hematopoietic necrosis virus) and used nucleic acid isothermal amplification detection kit |
Non-Patent Citations (1)
| Title |
|---|
| 《中国海洋大学学报》 20101031 孙颖等 弧菌16S rRNA 基因特异引物设计及海洋弧菌菌群结构分析 122-130 1－4 , * |
Cited By (6)
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| CN103614455A (en) * | 2013-07-03 | 2014-03-05 | 淮海工学院 | Kit for rapid detection of Vibrio aestuarianus through loop-mediated isothermal amplification and detection method |
| CN106636394A (en) * | 2016-12-20 | 2017-05-10 | 浙江万里学院 | Isothermal amplication detection kit for nucleic acid of aeromonas and detection method of isothermal amplication detection kit |
| CN107012227A (en) * | 2017-04-21 | 2017-08-04 | 丹东出入境检验检疫局综合技术服务中心 | The universal primer pair detected for kinds of pathogenic vibrio and its application |
| CN107227378A (en) * | 2017-08-10 | 2017-10-03 | 广东出入境检验检疫局检验检疫技术中心 | Detect the RPA IAC primers and method of vibrio mimicus |
| CN107227378B (en) * | 2017-08-10 | 2020-11-13 | 广东出入境检验检疫局检验检疫技术中心 | RPA-IAC primer and method for detecting vibrio mimicus |
| CN111534620A (en) * | 2020-05-11 | 2020-08-14 | 福建省闽东水产研究所 | Universal pathogenic vibrio kit and rapid detection method thereof |
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