CN106636394A - Isothermal amplication detection kit for nucleic acid of aeromonas and detection method of isothermal amplication detection kit - Google Patents

Isothermal amplication detection kit for nucleic acid of aeromonas and detection method of isothermal amplication detection kit Download PDF

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CN106636394A
CN106636394A CN201611181376.2A CN201611181376A CN106636394A CN 106636394 A CN106636394 A CN 106636394A CN 201611181376 A CN201611181376 A CN 201611181376A CN 106636394 A CN106636394 A CN 106636394A
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aeromonas
nucleic acid
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何琳
卜佳丽
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Zhejiang Wanli University
Zhejiang Wanli College
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Abstract

The invention provides an isothermal amplication detection kit for a nucleic acid of aeromonas. The isothermal amplication detection kit comprises a grinding liquid tube, a nucleic acid extract solution A tube, a nucleic acid extract solution B tube, a nucleic acid extract solution C tube, a TE buffer liquid tube, a UNG enzyme tube, an LAMP reaction liquid tube, a Bst DNA polymerase tube, a color developing agent tube, a positive control nucleic acid tube and a negative control tube. Common aeromonas bacterial can be effectively detected, and the on-site, programmed and standardized detection process is achieved. The invention further provides a detection method for the aeromonas. A target gene segment is optimized to design a LAMP specific primer, so that the aeromonas bacterial can be effectively detected by the primer, the detection specificity is good and the detection result is very easy to distinguish.

Description

A kind of virus nucleic acid isothermal amplification detection reagent kit and its detection method of Aeromonas
【Technical field】
The present invention relates to freshwater aquiculture animal pathogen detection technical, is specifically related to a kind of nucleic acid isothermal expansion of Aeromonas Increase detection kit and its detection method.
【Background technology】
Aeromonas is conditioned pathogen, in being widely distributed in fresh water culture environment of aquatic products.What is be reported so far is light Water cultivated animals cause of disease Aeromonas has many kinds.Wherein, Aeromonas hydrophila (Aeromonas hydrophila), cavy gas Monad (A.caviae), Aeromonas sobria (A.sobria), Aeromonas veronii (A.veronii), etc. various gas unit cells Bacterium is considered as the important pathogenic bacteria of fresh water aquatic livestock, can cause such as shell-fish (Sung, 2001；Soto-Rodrigu Deng 2010；Alagappan etc., 2010；Phumkhachom and Rattanachaikunsopon, 2010；Ji etc., 2011； Raissy etc., 2011), Bivalve (Tubiase etc., 1970；Beaz-Hidalgo etc., 2010), fish (Toranzo etc., 2005；Wo etc., 2008；Pal and Das, 2010；2011) etc. Frans etc., the Animal diseases for having important commercial value；Wherein thermophilic water Some species such as Aeromonas, Aeromonas sobria be also the mankind food borne pathogenses (Tracz etc., 2007).These bacteriums can With the economic loss for causing culture fishery huge, cause the complication that human body is serious.In addition, the aquatic products of China's export in recent years Also once there is the exceeded return of goods for causing of excessive cause pathogenicity Aeromonas, destruction, cancel register code name event in product, cause huge Big economic loss.Therefore the Fast Detection Technique of Pathogenic Aeromonas bacterium, to maintaining the healthy sustainable of freshwater aquaculture industry The aspects such as development, the supervision of aquatic products hygienic quality and protection public health all have being of great significance.But existing gas list Born of the same parents bacterium detection method is single for sample, and complex operation step, is difficult to realize live batch detection, it is difficult to meet fresh water support Grow the needs of animal diseases preventing and treating.Existing detection Pathogenic Aeromonas mainly have three kinds of methods：1. Physiology and biochemistry identification method, is somebody's turn to do Method complex operation step, wastes time and energy；2. EUSA (ELISA), though method detection is quick, sensitivity is high, can The features such as mass detection, but easily occur false positive issue when detecting to fresh sample, limit to a certain extent Application in production practices；3. PCR method (PCR), the method is quick, sensitive, but needs expensive nucleic acid to expand Increase instrument, equally limit the application in production practices.
Ring mediated isothermal amplification method (LAMP) developed in recent years, is a kind of new constant temperature nucleic acid amplification method, Can selectively efficient amplification target-gene sequence, product be the loop-stem structure that a series of target sequence of inverted repeats is constituted and The DNA fragmentation mixture of polycyclic cauliflower spline structure, shows the staged of different size zone composition on gel after electrophoresis Collection of illustrative plates.LAMP advantages compared with other Pathogenic Aeromonas detection methods are obvious：Although 1. reaction principle, design of primers compare Complexity, but expand under constant temperature, it is only necessary to and a thermostat (such as water-bath, thermos cup) can be carried out；② Efficient and sensible, template only needs 10 copies or less；3. high specificity, applies four primers for containing six sections by tight Case order is expanded；4. detection time is short, and amplification can be completed in 60min；5. product can be by directly in reaction tube Middle addition fluorescent dyeing, it is as a result visual in image.Find there are many places highly conserved by comparing common Aeromonas 16S RNA Sequence, according to these highly conserved sequence design specific primers, you can the presence of quick detection Aeromonas bacterium.Therefore The general detection that LAMP technology is applied to Pathogenic Aeromonas is capable of achieving into the high-volume quick detection of field samples.CN 102140512 B disclose the LAMP detection kit of pathogenic hydrophila gingivalis, are devised according to target-gene sequence pathogenic Aeromonas hydrophila detection primer sets, eight isolated areas on energy specific recognition target sequence, enhance pathogenic thermophilic aqueous vapor The specificity of monad detection, so the invention is only capable of for Aeromonas hydrophila this single Aeromonas to carry out specificity Detection.
In the practice such as Defect inspection, Food Safety Analysis in aquaculture, the very first time determines whether Aeromonas Infection is bigger than knowing the meaning specifically by which kind of Aeromonas infection.And in prior art due to different Aeromonas (such as Aeromonas hydrophila, Aeromonas sobria, Aeromonas veronii etc.) specific detection one by one is carried out, waste time and energy, it is impossible to make Aeromonas infection is determined whether with one-time detection.
【The content of the invention】
The technical problem to be solved is, for the shortcoming of above prior art, there is provided a kind of Aeromonas Virus nucleic acid isothermal amplification detection reagent kit and its detection method, can one-time detection determine whether Aeromonas.
The technical scheme is that：A kind of virus nucleic acid isothermal amplification detection reagent kit of Aeromonas, it includes：
1) liquid pipe is ground：Built-in lapping liquid,
2) nucleic acid extraction liquid A pipes：Built-in sodium acetate solution；
3) nucleic acid extraction liquid B pipes：Built-in absolute ethyl alcohol；
4) nucleic acid extraction liquid C pipes：The ethanol solution of built-in mass concentration 70%；
5) TE bufferings liquid pipe：Built-in TE buffer solutions；
6) UNG enzymes pipe：Built-in uracil dna glycosylase；
7) LAMP reactions liquid pipe：Built-in LAMP reactant liquors；
8) Bst archaeal dna polymerases pipe：Built-in Bst archaeal dna polymerases；
9) developer pipe：Built-in nucleic acid dye SYBR Green I；
10) positive control nucleic acid pipe：Built-in Aeromonas positive DNA；
11) negative control pipe：The distilled water of built-in sterilizing.
Preferably, the lapping liquid is made up of the component of volumes below percentage：Trishydroxymethylaminomethane 5～10%, Disodium ethylene diamine tetraacetate 0.5～2.0%, dodecyl sodium sulfate 5～10%, mercaptoethanol 0.01～1.0%, 8- hydroxyl quinolines Quinoline 0.01～0.02%, balance phenol 5～10%, chloroform 5～10%, balance of distilled water；The trishydroxymethylaminomethane Concentration is 1～2M；The concentration of the disodium ethylene diamine tetraacetate is 0.25～1.0M；The quality of the dodecyl sodium sulfate point Number is 5～10%.
Preferably, the LAMP reactant liquors are made up of the component of following concentration：LAMP primer Aeromonas-FIP and Aeromonas-BIP is each 1～4 μM, and LAMP primer Aeromonas-F3 and Aeromonas-B3 are each 0.1～0.5 μM, dATP, Each 0.5～the 2mM of dGTP and dCTP, each 0.25～1mM of dTTP, dUTP, 10～40mM of Tris-HCl, 10～20mM of KCl, (NH4)2SO45～15mM, MgSO40.8～1.2M of 1～4mM, Triton X-100 0.05%～1.0%, Betaine, it is molten Agent is distilled water.
It is further preferred that the nucleotide sequence of LAMP primer is as follows：
Aeromonas-FIP[SEQ ID NO.1]：
GCAATATTCCCCACTGCTGCCTAGGATCAGCCACACTGGAACTG；
Aeromonas-BIP[SEQ ID NO.2]：
CATGCCGCGTGTGTGAAGAAGTTTGCTACCAACCTTTCCTCCTC；
Aeromonas-F3[SEQ ID NO.3]：
CCCTAGCTGGTCTGAGAGG；
Aeromonas-B3[SEQ ID NO.4]：
TCTTCTGCGAGTAACGTCAC。
Preferably, the preparation method of Aeromonas positive DNA is as follows：Drawn with the PCR positioned at LAMP target fragment upstream and downstream Thing (Aeromonas-Pf [SEQ ID NO.5]:5'-GAGGAAAGGTTGGTAGCGAA；Aeromonas-Pr[SEQ ID NO.6]:5'-ACCCCCCTCTACAAGACTC), Aeromonas hydrophila genomic DNA is expanded, amplification condition is：92～ 96 DEG C of 5～10min of denaturation, 92～96 DEG C of 20～40s of denaturation, 56～60 DEG C annealing 20～40s, 70～74 DEG C extend 50～ 70s completes an amplification cycles, and repetitive cycling 35～35 is taken turns, and 8～12min of last 70～75 DEG C of extensions, 2～6 DEG C of preservations are obtained The band of 474bp, obtains positive DNA fragmentation (sequence of the positive DNA fragmentation is SEQ ID NO.7)；Reclaim positive DNA pieces Section, is connected to carrier pMD-18T-Vector, and converts competent escherichia coli cell JM109, Jing after PCR checkings, carries Plasmid is taken, Aeromonas positive DNA is obtained, as Aeromonas positive control after dilution.During the Aeromonas hydrophila derives from State's General Microbiological Culture preservation administrative center bacterial classification, bacterium numbering：1.1027.The sequence of the positive DNA fragmentation is： GAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGA TTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGATTTGGAGGCTGTGTCCTTGAGACGTGGCTTCCGGAGCTA ACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATGTCTGGAATCCCTAAGAGATT GGGGAGTGCCTTCGGGAATCAGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGT CCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCAGCACGTAATGGTGGGAACTCAAGGGAGACTGCCGGTGATAAA CCGGAGGAAGGT.In this kit, this section of sequence is connected on JM109 plasmids, together positive as Aeromonas DNA。
In the kit of the present invention, raw material is obtained in commercial mode, wherein disodium ethylene diamine tetraacetate, trihydroxy methyl Aminomethane, dodecyl sodium sulfate, sodium acetate, chloroform, balance phenol, mercaptoethanol, 8-hydroxyquinoline, Tris-HCl, KCl, (NH4)2SO4、MgSO4, dATP (deoxyadenosine triphosphate), dGTP (triphosphoric acid guaninesDeoxynucleotide), dCTP (triphosphoric acids Deoxycytidylic acid), dTTP (triphosphoric acidsThymidylic acid) Shanghai bioengineering Co., Ltd is purchased from, DUTP is purchased from Shanghai Shan Jing molecular biosciences Science and Technology Ltd., and uracil dna glycosylase (UNG enzymes), Bst DNA are polymerized dark etc. Purchased from NEB companies of the U.S., nucleic acid dye SYBR Green I are purchased from Beijing Ding Guo bio tech ltd, Betaine, Triton X-100 are purchased from Sigma companies.
LAMP primer Aeromonas-FIP, Aeromonas-BIP, Aeromonas-F3 and Aeromonas-B3 and PCR Primer Aeromonas-Pf, Aeromonas-Pr are designs of the invention, raw work bioengineering (Shanghai) share in commission Co., Ltd synthesizes.The primer of the present invention can detect common Pathogenic Aeromonas, as long as detection Aeromonas then illustrates There is the presence of Aeromonas in animal or breeding environment, you can take appropriate measures.Because the therapy apparatus of Aeromonas As Pathogen is pre- Anti- means are similar, in production and need not know the species of specific Aeromonas the very first time.
The virus nucleic acid isothermal amplification detection reagent kit of any one Aeromonas causes a disease in a kind of utilization claim 1～3 The detection method of property Aeromonas, comprises the following steps：
1) take testing sample to be organized in centrifuge tube I, add lapping liquid, with grinding rod pulpous state is fully milled to,
2) 5～10min is centrifuged with 8000～12000r/min after is boiled the slurried sample obtained by above-mentioned grinding, supernatant is taken Liquid is placed in centrifuge tube II；
3) sodium acetate solution in nucleic acid extraction liquid A pipes is added to mix into the centrifuge tube II equipped with supernatant, add- Absolute ethyl alcohol mixing in the nucleic acid extraction liquid B pipes of 20 DEG C of precoolings, stands 5～20min, and with 10000～14000r/min 5 are centrifuged ～10min, abandons supernatant, retains sediment；Sodium acetate solution is 1 with the volume ratio of supernatant:12～1:8；Absolute ethyl alcohol with The volume ratio of sediment is 4:1～4:3；
4) above-mentioned steps 3 are washed with the ethanol solution in nucleic acid extraction liquid C pipes) obtained by sediment, then 10000～ 14000r/min is centrifuged 5～10min, abandons supernatant, retains sediment；
5) above-mentioned steps 4 are washed with the ethanol solution in nucleic acid extraction liquid C pipes) obtained by sediment, then 10000～ 14000r/min is centrifuged 5～10min, abandons supernatant, retains sediment；
6) by above-mentioned steps 5) obtained by sediment dry in room temperature, add TE buffer solution sediments, obtain sample Nucleic acid；
7) respectively by the positive control in the distilled water in above-mentioned sample nucleic, negative control pipe, positive control nucleic acid pipe Nucleic acid, in adding to LAMP reactant liquors, adds UNG enzymes, and detection pipe, negative control pipe and positive control pipe are obtained respectively；To above-mentioned Detection pipe, negative control pipe and positive control Guan Jun are incubated 5～20min and are digested in 36～38 DEG C；
8) by the reaction system after above-mentioned detection pipe, negative control pipe and positive control pipe enzymolysis prior to 90～100 DEG C of guarantors 5～10min of temperature, is then immediately placed in again in rubble ice；
9) to step 8) obtained by detection pipe, negative control pipe and positive control pipe reaction system in be separately added into again Bst archaeal dna polymerases；
10) by above-mentioned steps 9) obtained by detection pipe, negative control pipe and positive control pipe proceed as follows respectively：First 40～50min is incubated in 63～65 DEG C, then 2～5min is incubated in 85～95 DEG C and is completed LAMP reactions；
11) after LAMP reactions terminate, nucleic acid dye SYBR Green I are added, observes LAMP product in detection pipe Color.If the color of LAMP product and the color of negative control tube reaction product are to be measured in orange in detection pipe Product Pathogenic Aeromonas testing result is feminine gender；If the color of LAMP product is with positive control tube reaction in detection pipe In green, then product to be tested Pathogenic Aeromonas testing result is positive to the color of product.
The present invention has following beneficial technique effect：
1) ten kinds of reagents such as Aeromonas detection required lapping liquid, nucleic acid extraction liquid and three kinds are summarized in kit Equipment so that Aeromonas detection can be carried out in an orderly manner, realize detection process scene, sequencing and standardization so that behaviour Make specification, not error-prone, can the various common Aeromonas bacteriums of effective detection, i.e. one-time detection determines whether gas list Born of the same parents bacterium；
2) process of sample is carried out with the lapping liquid of special design so that the processing procedure of sample is extremely simple；
3) UNG enzymes can eliminate the pollution of nucleic acid during repeated detection, eliminate LAMP using uracil dna glycosylase and expand The pollution of volume increase thing, it is to avoid because of the false positive results that LAMP product amplification amounts are caused greatly, solves restriction LAMP technology extensive Using easily contaminated interference problem；
4) realize and covered can detect, the generation of pollution is avoided from source, further avoid false positive results Appearance；
5) the 16s rna genes area that detection method is guarded according to Aeromonas, preferred genes of interest section is designing LAMP specific primers so that the common Aeromonas bacterium of the primer energy effective detection, and Aeromonas detection is complete Be provided with LAMP technology it is sensitive, quick, easy, it is accurate the characteristics of, detection specificity it is good, testing result be very easy to differentiate；
6) optimize the technical parameter of whole operation process, and standardized, supporting, form detection kit, just In live high volume applications；
7) detection of Pathogenic Aeromonas can be completed in 2 hours using the kit and detection method of the present invention, Detection process is without the need for expensive instrument and equipment such as PCR instrument and gel imaging system, it is only necessary to have water-bath or metal bath and commonly from Scheming；
8) compared with the existing detection technique of Aeromonas bacterium, the kit and corresponding method of detection of the present invention are not only Realize the general detection of Aeromonas, and low cost, sensitivity are high, simple to operate, are capable of achieving field quick detection；
9) with the higher sensitivity of detection method than ever and convenience, can substitute before Pathogenic Aeromonas Related detecting method such as Physiology and biochemistry method, PCR methods, enzyme linked immunosorbent assay etc..
【Specific embodiment】
Below in conjunction with specific embodiment, the present invention is described further.
Embodiment provided below and being not used to limits the scope that the present invention is covered, described step nor with To limit its execution sequence.Those skilled in the art do conspicuously improved with reference to existing common knowledge to the present invention, also fall Enter within the protection domain of application claims.
Embodiment one
A kind of Aeromonas virus nucleic acid isothermal amplification detection reagent kit (5 sample parts, you can for the detection of 5 samples time), by following Content is constituted：
1) liquid pipe, 1 pipe, 3ml are ground；Lapping liquid is prepared：1M tris solution (Tris-HCl, PH8.0) 10 parts, the disodium ethylene diamine tetra-acetic acid solution (EDTANa of 0.25M2) 2 parts, mass fraction is 5% dodecyl sulphur 10 parts of acid sodium solution (SDS), 0.5 part of mercaptoethanol, 0.01 part of 8-hydroxyquinoline, 7 parts of balance phenol, 8 parts of chloroform is fixed with distilled water Hold 100 parts to form.Above number is volume parts；
2) nucleic acid extraction liquid A pipes, 1 pipe, built-in 400 μ l 3M sodium acetate solutions (pH5.2)；
3) nucleic acid extraction liquid B pipes, l pipes, built-in 10ml absolute ethyl alcohols；
4) nucleic acid extraction liquid C pipes, 1 pipe, the ethanol solution of built-in 10ml mass concentrations 70%；
5) TE bufferings liquid pipe, 1 pipe, built-in 400 μ l TE buffer solutions (Tris-HCl containing 10mM and 1mM EDTA, pH8.0, Remaining is distilled water)；
6) UNG enzymes pipe, 1 pipe, built-in 6U uracil dna glycosylases (UNG enzymes)；
7) LAMP reactions liquid pipe, 1 pipe, built-in 150 μ l LAMP reactant liquors,
The constituent of LAMP reactant liquors is as follows：LAMP primer Aeromonas-FIP and Aeromonas-BIP are each 2 μM, LAMP primer Aeromonas-F3 and Aeromonas-B3 are each 0.3 μM, each 1mM of dATP, dGTP and dCTP, and dTTP, dUTP are each 0.5mM,Tris-HCl 20mM,KCl 15mM,(NH4)2SO4 10mM,MgSO42mM, Triton X-100 0.5%, Betaine 1M；Solvent is distilled water；
The 16s rna gene sequences Designs that above-mentioned LAMP primer is guarded according to Aeromonas, LAMP primer design First-selected software is the (http of PrimerExplore 4.0://primerexplorer.jp/elamp4.0.0/index.html), Conventional primer-design software (such as Primer Primier 5.0 or Omiga 2.0) setting according to LAMP primer can also be used Meter requirement is designed.In the present invention using the Photographing On-lines of software PrimerExplore 4.0-set LAMP primer DNA sequences Row are as follows：
Aeromonas-FIP[SEQ ID NO.1]：
GCAATATTCCCCACTGCTGCCTAGGATCAGCCACACTGGAACTG；
Aeromonas-BIP[SEQ ID NO.2]：
CATGCCGCGTGTGTGAAGAAGTTTGCTACCAACCTTTCCTCCTC；
Aeromonas-F3[SEQ ID NO.3]：
CCCTAGCTGGTCTGAGAGG；
Aeromonas-B3[SEQ ID NO.4]：
TCTTCTGCGAGTAACGTCAC。
8) Bst archaeal dna polymerases pipe, 1 pipe, built-in 5 μ l Bst archaeal dna polymerases；
9) developer pipe, 1 pipe, the liquid dyes of built-in 5 μ l, the liquid dyes are by nucleic acid dye SYBR Green I and double Water is steamed according to 1:100 volume ratio is obtained by mixing；
10) positive control nucleic acid pipe, 1 pipe, built-in 10 μ l Aeromonas positive DNA.Aeromonas positive DNA preparation methods It is as follows：Aeromonas hydrophila genomic DNA is extracted, with the PCR primer (Aeromonas-Pf positioned at LAMP target fragment upstream and downstream [SEQ ID NO.5]:5'-GAGGAAAGGTTGGTAGCGAA；Aeromonas-Pr[SEQ ID NO.6]:5'- ACCCCCCTCTACAAGACTC), Aeromonas hydrophila genomic DNA is expanded.Reaction system is：25 μ l systems include 2.5 μ l 10 × PCR reaction buffers, 1.0 μ l dNTPs (10mM), 1.0 μ l Aeromonas-Pf and Aeromonas-Pr (20 μM), 0.5 μ l Taq archaeal dna polymerases (5U/ μ l, Beijing Ding Guo bio tech ltd), remaining is distilled water.Amplification condition For：94 DEG C of denaturations 5min, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 60s and complete an amplification cycles, and repetition is followed Ring 30 is taken turns, last 72 DEG C of 10min, 4 DEG C of preservations, obtains the band of 474bp, after reclaiming the fragment, is connected by the requirement of carrier T specification It is connected in carrier T (pMD-18T-Vector, purchased from Dalian treasured biotech firm), and will recombinates matter by competent cell operation requirement Grain is converted to competent cell JM109 (purchased from Dalian treasured biotech firm), and with blue hickie method positive bacteria is screened, and uses primer VIBRIO-Pf and VIBRIO-Pr enter performing PCR checking (program is ibid).Finally with the plasmid of alkalinity extraction positive bacteria, gas list is obtained Born of the same parents bacterium positive DNA, is diluted to after 100 copies/μ l as Aeromonas positive control.Above-mentioned Aeromonas hydrophila is from China General Microbiological Culture preservation administrative center bacterial classification, bacterium numbering：1.1027；
11) negative control pipe, 1 pipe, built-in 10 μ l sterilizings distilled water；
12) grinding rod, 5；
13) packing box (78 × 78 × 50mm)；
14) one block of cystosepiment, foam plate hight 22mm, there is three row, totally 11 holes, four holes of first row, aperture 5.5mm, the Two holes of row three, aperture 10mm, the 3rd hole of row three, aperture 15.5mm.Above-mentioned 1～11 each tubule is positioned over these apertures In, the cystosepiment is identical with the bottom surface size of above-mentioned packing box, loaded in packing box；
15) kit operation instructions-part.
Embodiment two
A kind of Aeromonas virus nucleic acid isothermal amplification detection reagent kit (5 sample part), only herein below are different from embodiment one, its Yu Jun is with embodiment one.
Lapping liquid is prepared：5 parts of the tris solution (Tris-HCl, pH8.0) of 2M, the ethylenediamine tetraacetic of 0.5M Sodium solution (the EDTANa of acetic acid two2) 1 part, 7 parts of the sodium dodecyl sulfate solution (SDS) of mass fraction 7%, mercaptoethanol 0.01 Part, 0.02 part of 8-hydroxyquinoline, 5 parts of balance phenol, 10 parts of chloroform is formed with distilled water constant volume to 100 parts.Above number is body Product number.
The constituent of LAMP reactant liquors is as follows：LAMP primer Aeromonas-FIP and Aeromonas-BIP are each 4 μM, LAMP primer Aeromonas-F3 and Aeromonas-B3 are each 0.5 μM, and each 2mM of dATP, dGTP and dCTP, dTTP and dUTP is each 1.0mM,Tris-HCl 40mM,KCl 20mM,(NH4)2SO4 15mM,MgSO44mM, Triton X-100 1.0%, Betaine 0.8M；Solvent is distilled water.
Aeromonas positive DNA preparation methods are as follows：With the PCR primer positioned at LAMP target fragment upstream and downstream (Aeromonas-Pf[SEQ ID NO.5]:5'-GAGGAAAGGTTGGTAGCGAA；Aeromonas-Pr[SEQ ID NO.6]: 5'-ACCCCCCTCTACAAGACTC), Aeromonas hydrophila genomic DNA is expanded, amplification condition is：92 DEG C of denaturations 10min, 92 DEG C of denaturation 40s, 56 DEG C of annealing 40s, 70 DEG C extend 70s and complete an amplification cycles, and repetitive cycling 25 is taken turns, and last 70 DEG C extend 8min, 2 DEG C preservation, obtain the band of 474bp, after reclaiming the fragment, be connected to carrier pMD-18T-Vector, And competent escherichia coli cell JM109 is converted, and Jing after PCR checkings, plasmid is extracted, Aeromonas positive DNA is obtained, it is diluted to As Aeromonas positive control after 100 copy/μ l.
Embodiment three
A kind of Aeromonas virus nucleic acid isothermal amplification detection reagent kit (5 sample part), only herein below are different from embodiment one, its Yu Jun is with embodiment one.
Lapping liquid is prepared：7 parts of the tris solution (Tris-HCl, pH8.0) of 1.5M, the ethylenediamine of 1.0M Tetraacethyl disodium solution (EDTANa2) 0.5 part, 5 parts of the sodium dodecyl sulfate solution (SDS) of mass fraction 10%, sulfydryl second 0.01 part of alcohol, 0.015 part of 8-hydroxyquinoline, 10 parts of balance phenol, 10 parts of chloroform is formed with distilled water constant volume to 100 parts.Above part Number is parts by volume.
The constituent of LAMP reactant liquors is as follows：LAMP primer Aeromonas-FIP and Aeromonas-BIP are each 1 μM, LAMP primer Aeromonas-F3 and Aeromonas-B3 are each 0.25 μM, each 0.5mM of dATP, dGTP and dCTP, and dTTP, dUTP are each 0.5mM,Tris-HCl 40mM,KCl 10mM,(NH4)2SO4 5mM,MgSO41mM, Triton X-100 0.05%, Betaine 0.8M；Solvent is distilled water.
Aeromonas positive DNA preparation methods are as follows：With the PCR primer positioned at LAMP target fragment upstream and downstream (Aeromonas-Pf[SEQ ID NO.5]：5'-GAGGAAAGGTTGGTAGCGAA；Aeromonas-Pr[SEQ ID NO.6]： 5'-ACCCCCCTCTACAAGACTC), Aeromonas hydrophila genomic DNA is expanded, amplification condition is：96 DEG C of denaturations 5min, 96 DEG C of denaturation 20s, 60 DEG C of annealing 20s, 74 DEG C extend 50s and complete an amplification cycles, and repetitive cycling 25 is taken turns, and last 75 DEG C extend 8min, 6 DEG C preservation, obtain the band of 474bp, after reclaiming the fragment, be connected to carrier pMD-18 T- Vector, and competent escherichia coli cell JM109 is converted, Jing after PCR checkings, plasmid is extracted, obtain Aeromonas positive DNA, is diluted to after 100 copies/μ l as Aeromonas positive control.
Example IV
A kind of detection method of Aeromonas, using any one detection kit of one～embodiment of embodiment 3, successively Carry out following steps：
1) take ill Carassius auratus liver tissue 0.2g to be measured to be placed in centrifuge tube I, add the 0.5ml grindings in grinding liquid pipe Liquid, with grinding rod pulpous state is fully milled to,
2) slurried sample obtained by above-mentioned grinding is boiled and 5min is centrifuged with 10000r/min after (100 DEG C, l0min), taken Supernatant is placed in centrifuge tube II；
3) the sodium acetate solution mixing in nucleic acid extraction liquid A pipes, sodium acetate solution and supernatant are added into centrifuge tube II Volume ratio be 1：10；The absolute ethyl alcohol mixing in the nucleic acid extraction liquid B pipes of -20 DEG C of precoolings is added, 10min is stood, with 12000r/min is centrifuged 5min, abandons supernatant, retains sediment, and absolute ethyl alcohol is 2 with the volume ratio of sediment:1；
4) above-mentioned steps 3 are washed with the ethanol solution in nucleic acid extraction liquid C pipes) obtained by sediment, then 12000r/ Min is centrifuged 5min, abandons supernatant, retains sediment；
5) above-mentioned steps 4 are washed with the ethanol solution in nucleic acid extraction liquid C pipes) obtained by sediment, then 12000r/ Min is centrifuged 5min, abandons supernatant, retains sediment；
6) by above-mentioned steps 5) obtained by the sediment bottom of centrifuge tube II (be located at) dry in room temperature, add TE buffer solutions 20 μ l TE buffer solution sediments in pipe, obtain sample nucleic；
7) respectively by the positive control in the distilled water in above-mentioned sample nucleic, negative control pipe, positive control nucleic acid pipe The μ l of nucleic acid 2, in adding to 21 μ l LAMP reactant liquors, add 1 μ l UNG enzymes, so as to obtain detection pipe, negative control pipe and sun respectively Property control tube；10min is incubated to above-mentioned detection pipe, negative control pipe and positive control Guan Jun in 37 DEG C to digest, it is therefore an objective to Pollution template wherein that may be present is eliminated with enzyme solution；
8) by the reaction system after above-mentioned detection pipe, negative control pipe and positive control pipe enzymolysis prior to 95 DEG C of insulations 5min, is then immediately placed in again 5min in rubble ice；
9) to step 8) obtained by detection pipe, negative control pipe and positive control pipe reaction system in be separately added into 1 μ again L Bst archaeal dna polymerases；
10) by above-mentioned steps 9) obtained by detection pipe, negative control pipe and positive control pipe proceed as follows respectively：First 45min is incubated in 64 DEG C, then 2min is incubated in 90 DEG C and is completed LAMP reactions；
11) after LAMP reactions terminate, 1 μ l nucleic acid dye SYBR Green I are added, LAMP reactions in observation detection pipe are produced The color of thing, if detection pipe shows green with positive control pipe, negative control pipe shows orange, represents the gas list of the sample Born of the same parents bacterium testing result is the positive；If detection pipe shows orange with negative control pipe, positive control pipe shows green, and representing should The Aeromonas testing result of sample is feminine gender；If positive control pipe shows orange, represent that the detection reagent fails, need more Change after detection reagent and detect again；If negative control pipe shows green, represent that the detection reagent is contaminated, detection need to be changed Detect again after reagent and detection site.
Embodiment five
A kind of detection method of Aeromonas, using any one detection kit of embodiment one, two, three, to sick sample Carried out with nutrient broth after increasing bacterium, Pathogenic Aeromonas are detected according to following steps：
1) take ill crucian fish liver organization 2g to be measured and be placed in homogenate in aseptic homogenizer, homogenate is added into nutrient broth (5ml) 25 DEG C～28 DEG C shaking table culture 5h～6h in.Nutrient broth formula is：Albumen freezes 10g, beef extract 3g, sodium chloride 5g, steams Distilled water 1000ml, pH7.4.By mentioned component mixing, dissolving post-equalization pH, 121 DEG C of autoclaving 15min；
2) 2ml bacterium solutions are taken, 10000r/m centrifugation 1min abandon supernatant, add 50 μ l distilled waters, water-bath to boil 10min；
3) 10000r/m centrifugations 1min, takes supernatant；
4) using any one detection kit of one～embodiment of embodiment 3, according to step 7 in embodiment 4)～step 11) methods described is detected.
The Aeromonas testing result that the sample is represented if detection pipe shows green is the positive, if detection pipe shows orange Color then represents that the Aeromonas testing result of the sample is feminine gender.
This embodiment explanation can require no considerably complicated DNA profiling and prepare, and only need after as a result bacterium amplification Boiling can prepare DNA detection templates, illustrate that the method is easy.
Embodiment six
A kind of detection method of Aeromonas, using bacterium not of the same race as detection object, verifies the specificity of detection method, Bacterium is detected according to following steps：
1) by Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Aeromonas veronii, bacillus licheniformis, Vibrio parahaemolytious, Escherichia coli, nitrobacteria cultivate enrichment to OD600=0.6 in suitable culture medium.
2) 1ml bacterium solutions are taken, 10000r/m centrifugation 1min abandon supernatant, add 50 μ l distilled waters, water-bath to boil 10min；
3) 10000r/m centrifugations 1min, takes supernatant, obtains final product DNA of bacteria detection template；
4) using any one detection kit of one～embodiment of embodiment 3, according to step 7 in embodiment 4)～step 11) methods described, respectively to 10-1-10-7Template is detected.
As a result show, LAMP reactions can detect Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria, Vickers The Pathogenic Aeromonas such as Aeromonas, and to water rings such as bacillus licheniformis, vibrio parahaemolytious, Escherichia coli, nitrobacterias The testing result of common bacteria presents negative in border.
The above results explanation：The LAMP detection kit of the present invention has specificity well to Pathogenic Aeromonas.
Contrast experiment 1：, as testing sample, utilized using same infected animal sample (having confirmed that infection Aeromonas hydrophila) Kit in embodiment one, by step 1 in embodiment 4)～step 6) method prepare detection template (the i.e. sample of ill crucian Product nucleic acid).Above-mentioned template is carried out into 10 with TE buffer solutions-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7It is serially diluted again, respectively As LAMP and PCR detection templates.
1) according to step 7 in embodiment 4)～step 11) methods described, respectively to 10-1-10-7Template is checked.
2) performing PCR detection is entered with same template, using primer Aeromonas-Pf and Aeromonas-Pr.
As a result show, LAMP reactions can detect 10-6The template for diluting again, and PCR can only be detected to 10-3Dilute again Template, the remolding sensitivity PCR of LAMP detections is high 1000 times, and the used time is less, without the need for complex device.
Contrast experiment 2：Using same ill crucian as testing sample, using the kit in embodiment two, experiment side Formula is with contrast experiment 1.
As a result show：LAMP reactions can detect 10-6The template for diluting again, and PCR can only be detected to 10-3Dilute again Template.
Contrast experiment 3：Using same ill crucian as testing sample, using the kit in embodiment three, experiment side Formula is with contrast experiment 1.
As a result show：LAMP reactions can detect 10-6The template for diluting again, and PCR can only be detected to 10-3Dilute again Template.
SEQUENCE LISTING
<110>Wanli College, Zhejiang
<120>A kind of virus nucleic acid isothermal amplification detection reagent kit and its detection method of Aeromonas
<130> 2016
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 44
<212> DNA
<213>Artificial sequence
<400> 1
gcaatattcc ccactgctgc ctaggatcag ccacactgga actg 44
<210> 2
<211> 44
<212> DNA
<213>Artificial sequence
<400> 2
catgccgcgt gtgtgaagaa gtttgctacc aacctttcct cctc 44
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
ccctagctgg tctgagagg 19
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
tcttctgcga gtaacgtcac 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
gaggaaaggt tggtagcgaa 20
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<400> 6
acccccctct acaagactc 19
<210> 7
<211> 474
<212> DNA
<213>Artificial sequence
<400> 7
gaggaatacc ggtggcgaag gcggccccct ggacaaagac tgacgctcag gtgcgaaagc 60
gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgatttgg 120
aggctgtgtc cttgagacgt ggcttccgga gctaacgcgt taaatcgacc gcctggggag 180
tacggccgca aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat 240
gtggtttaat tcgatgcaac gcgaagaacc ttacctggcc ttgacatgtc tggaatccct 300
aagagattgg ggagtgcctt cgggaatcag aacacaggtg ctgcatggct gtcgtcagct 360
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccctgtcc tttgttgcca 420
gcacgtaatg gtgggaactc aagggagact gccggtgata aaccggagga aggt 474

Claims (10)

1. a kind of virus nucleic acid isothermal amplification detection reagent kit of Aeromonas, it is characterised in that it includes：
1) liquid pipe is ground：Built-in lapping liquid,
2) nucleic acid extraction liquid A pipes：Built-in sodium acetate solution；
3) nucleic acid extraction liquid B pipes：Built-in absolute ethyl alcohol；
4) nucleic acid extraction liquid C pipes：The ethanol solution of built-in mass concentration 65～75%；
5) TE bufferings liquid pipe：Built-in TE buffer solutions；
6) UNG enzymes pipe：Built-in uracil dna glycosylase；
7) LAMP reactions liquid pipe：Built-in LAMP reactant liquors；
8) Bst archaeal dna polymerases pipe：Built-in Bst archaeal dna polymerases；
9) developer pipe：Built-in nucleic acid dye SYBR Green I；
10) positive control nucleic acid pipe：Built-in Aeromonas positive DNA；
11) negative control pipe：The distilled water of built-in sterilizing.
2. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 1, it is characterised in that the grinding Liquid is made up of the component of volumes below percentage：Trishydroxymethylaminomethane 5～10%, disodium ethylene diamine tetraacetate 0.5～ 2.0%th, dodecyl sodium sulfate 5～10%, mercaptoethanol 0.01～1.0%, 8-hydroxyquinoline 0.01～0.02%, balance phenol 5～10%, chloroform 5～10%, balance of distilled water；The concentration of the trishydroxymethylaminomethane is 1～2M；The ethylenediamine The concentration of tetraacethyl disodium is 0.25～1.0M；The mass fraction of the dodecyl sodium sulfate is 5～10%.
3. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 1, it is characterised in that the LAMP Reactant liquor is made up of the component of following concentration：LAMP primer Aeromonas-FIP and Aeromonas-BIP are each 1～4 μM, LAMP Primer Aeromonas-F3 and Aeromonas-B3 is each 0.1～0.5 μM, each 0.5～2mM of dATP, dGTP and dCTP, dTTP, Each 0.25～the 1mM of dUTP, 10～40mM of Tris-HCl, 10～20mM of KCl, (NH4)2SO45～15mM, MgSO41～4mM, Triton X-100 0.05%～1.0%, Betaine0.8～1.2M, solvent is distilled water.
4. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 3, it is characterised in that the LAMP The nucleotide sequence of primer Aeromonas-FIP is SEQ ID NO.1.
5. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 3, it is characterised in that the LAMP The nucleotide sequence of primer Aeromonas-BIP is SEQ ID NO.2.
6. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 3, it is characterised in that the LAMP The nucleotide sequence of primer Aeromonas-F3 is SEQ ID NO.3.
7. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 3, it is characterised in that the LAMP The nucleotide sequence of primer Aeromonas-B3 is SEQ ID NO.4.
8. the virus nucleic acid isothermal amplification detection reagent kit of Aeromonas according to claim 1, it is characterised in that the gas list Born of the same parents bacterium positive DNA is prepared by following steps：Aeromonas hydrophila genomic DNA is expanded, i.e., 92～96 DEG C denaturations 5 ～10min, 92～96 DEG C of 20～40s of denaturation, 56～60 DEG C of 20～40s of annealing, 70～74 DEG C extend 50～70s and complete an expansion Increase circulation, repetitive cycling 25～35 is taken turns, 8～12min of last 70～75 DEG C of extensions, 2～6 DEG C of preservations obtain the band of 474bp, Obtain positive DNA fragmentation；Positive DNA fragmentation is reclaimed, carrier pMD-18T-Vector is connected to, and converts Escherichia coli sense By state cell JM109, Jing after PCR checkings, plasmid is extracted, obtain Aeromonas positive DNA；The sequence of the positive DNA fragmentation For SEQ ID NO.7.
9. in a kind of utilization claim 1～8 virus nucleic acid isothermal amplification detection reagent kit of any one Aeromonas gas unit cell The detection method of bacterium, it is characterised in that it comprises the following steps：
1) take testing sample to be organized in centrifuge tube I, add lapping liquid, with grinding rod pulpous state is fully milled to,
2) 5～10min is centrifuged with 8000～12000r/min after is boiled the slurried sample obtained by above-mentioned grinding, supernatant is taken and is put In centrifuge tube II；
3) add the sodium acetate solution in nucleic acid extraction liquid A pipes to mix into the centrifuge tube II equipped with supernatant, add precooling Nucleic acid extraction liquid B pipes in absolute ethyl alcohol mixing, stand 5～20min, with 10000～14000r/min be centrifuged 5～10min, Supernatant is abandoned, retains sediment；
4) above-mentioned steps 3 are washed with the ethanol solution in nucleic acid extraction liquid C pipes) obtained by sediment, then 10000～ 14000r/min is centrifuged 5～10min, abandons supernatant, retains sediment；
5) above-mentioned steps 4 are washed with the ethanol solution in nucleic acid extraction liquid C pipes) obtained by sediment, then 10000～ 14000r/min is centrifuged 5～10min, abandons supernatant, retains sediment；
6) by above-mentioned steps 5) obtained by sediment dry in room temperature, add TE buffer solution sediments, obtain sample nucleic；
7) respectively by the positive control nucleic acid in the distilled water in above-mentioned sample nucleic, negative control pipe, positive control nucleic acid pipe, In adding to LAMP reactant liquors, UNG enzymes are added, detection pipe, negative control pipe and positive control pipe are obtained respectively；To above-mentioned detection Pipe, negative control pipe and positive control Guan Jun are incubated 5～20min and are digested in 36～38 DEG C；
8) by above-mentioned detection pipe, negative control pipe and positive control pipe enzymolysis after reaction system prior to 90～100 DEG C insulation 5～ 10min, is then immediately placed in again in rubble ice；
9) to step 8) obtained by detection pipe, negative control pipe and positive control pipe reaction system in be separately added into Bst again Archaeal dna polymerase；
10) by above-mentioned steps 9) obtained by detection pipe, negative control pipe and positive control pipe proceed as follows respectively：Prior to 63 ～65 DEG C of 40～50min of insulation, are then incubated 2～5min in 85～95 DEG C and complete LAMP reactions；
11) after LAMP reactions terminate, nucleic acid dye SYBR Green I are added, observes the face of LAMP product in detection pipe Color.
10. the detection method of Aeromonas according to claim 9, it is characterised in that the step 3) in sodium acetate it is molten Liquid is 1 with the volume ratio of supernatant:12～1:8；The step 3) in the volume ratio of absolute ethyl alcohol and sediment be 4:1～4:3.
CN201611181376.2A 2016-12-20 2016-12-20 Isothermal amplication detection kit for nucleic acid of aeromonas and detection method of isothermal amplication detection kit Pending CN106636394A (en)

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Application publication date: 20170510