CN105296617A - Gene chip for detecting vibrio harveyi colony and using method of gene chip - Google Patents
Gene chip for detecting vibrio harveyi colony and using method of gene chip Download PDFInfo
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- CN105296617A CN105296617A CN201510649243.2A CN201510649243A CN105296617A CN 105296617 A CN105296617 A CN 105296617A CN 201510649243 A CN201510649243 A CN 201510649243A CN 105296617 A CN105296617 A CN 105296617A
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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
- C12Q1/6876—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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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention discloses a gene chip for detecting a vibrio harveyi colony and a using method of the gene chip. The gene chip is characterized in that nucleotide probes for detecting vibrio alginolyticus, V. campbellii, V. harveyi, V. natriegens, V. parahaemolyticus and V. rotiferianus are immobilized on a chip carrier; the probes of various vibrios use a toxR gene as a target gene; the gene sequences of the various probes are as shown in SEQ ID NO: 11-14; and in a step of PCR amplification on a sample, the gene sequences of a PCR amplification forward primer designed for the toxR gene of various strains of the vibrio harveyi colony and a reverse primer with a digoxin marker are as shown in SEQ ID NO. 15-26. The gene chip disclosed by the invention has the advantage that the gene chip is capable of rapidly and effectively detecting 6 pathogenic vibrios of the pathogenic vibrio harveyi colony in a parallel mode, and the gene chip is high in accuracy and sensitivity and is strong in specificity.
Description
Technical field
The present invention relates to the gene chip detecting Marine Pathogenic Bacteria, especially relating to the gene chip for detecting Vibrio harveyi group and using method thereof.
Background technology
Vibrio harveyi group is the core monoid of Vibrio, is the former bacterium of most commonly encountered diseases causing Marine cultured fish and shrimp shellfish disease to occur, comprise vibrio alginolyticus (
vibrioalginolyticus), Vibrio campbellii (
v.campbellii), Vibrio harveyi (
v.harveyi), Vibrio natriegen (
v.natriegens), Vibrio parahaemolyticus (
v.parahaemolyticus) and wheel animalcule vibrios (
v.rotiferianus).Vibrio harveyi group not only can often cause breaking out of aquiculture disease, causes tremendous economic to lose to culture fishery; Simultaneously also by contact seawater, eat seafood raw or feed is caused human diseases by the food of this fungi pollution.Vibrio harveyi group has highly similar phenotype and genotype, cannot identify fast and accurately in kind of level.Therefore, in kind of level, Rapid identification vibrios pathogenic bacteria is for taking specific aim prophylactico-therapeutic measures early, alleviates disease and loses significant.Bacteria 16 S rRNA genes is often used to the Species estimation of bacterium, but in the various genome of Vibrio harveyi group, 16SrRNA gene is multiple copied, usually between 8-14; And the difference of the interior different copy 16SrRNA gene of genome can be greater than interspecific difference not, brings immense confusion to the qualification of planting.
Biochip technology ultimate principle is fixed nucleic acid on solid support, is detected the existence of sample target gene sequence by crossover process, and reaches semiquantitative object by detecting fluorescent signal.This technology has the features such as parallelism highly, diversity and specificity, is efficient, quick, the extensive important means obtaining relevant biological information.Utilize this technology can complete traditional biological method (comprising molecular biology method) several weeks, the several months even several years workload that just can complete in several minutes to several hours.
During existing gene chip and patented technology thereof are reported, as " a kind of can the gene chip of parallel detection Vibrio and monitoring method thereof " (patent of invention, CN103451310A, 2013) and " a kind of gene chip for detecting nine kinds of kinds of pathogenic vibrio in sea-food " (patent of invention, CN103820558A, 2014), biochip technology is utilized to detect vibrios although also report, but mostly probe designed in this kind of patent of invention is for 16SrRNA gene and hsp60 gene, and this kind of probe can not to be effective to phenotype all highly similar with genotype, and the Vibrio harveyi group that quick and precisely cannot identify in kind of level.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of 6 kinds of morbid vibrios rapidly and efficiently detecting cause of disease Vibrio harveyi group of energy parallelism, and accuracy and highly sensitive, high specificity gene chip and using method thereof for detecting the Sino-Kazakhstan arc maintenance flora of seawater.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of gene chip for detecting Vibrio harveyi group, comprise chip carrier, it is characterized in that: immobilized on described chip carrier have for detecting vibrio alginolyticus, Vibrio campbellii, Vibrio harveyi, Vibrio natriegen, the nucleotide probe of Vibrio parahaemolyticus and wheel animalcule vibrios, the described probe for detecting vibrio alginolyticus, the described probe for detecting Vibrio campbellii, the described probe for detecting Vibrio harveyi, the described probe for detecting Vibrio natriegen, described for detecting the probe of Vibrio parahaemolyticus and described all using for the probe detecting wheel animalcule vibrios
toxRgene is target gene, and wherein, vibrio alginolyticus has 4 probes to cover whole kind, covers kind of a cluster branch for interior 2 notable differences respectively with 2 probes.The gene order of each probe is as follows:
For detecting a using method for the gene chip of Vibrio harveyi group, comprise Vibrio harveyi group testing sample DNA extraction step to be measured; Sample P CR amplification step; PCR primer and chip hybridization step, the process after chip hybridization, the step of the scanner uni result interpretation of hybridization hybrid chip, is characterized in that: for Vibrio harveyi group different strain in described sample P CR amplification step
toxRthe gene order of the forward primer of the pcr amplification designed by gene and the reverse primer of band digoxigenin labeled is as follows:
In described sample P CR amplification step, amplification system is: sterilized water 5.3 μ L; 10 × PCR damping fluid 1.5 μ L; 2.5mMdNTP mixture 1 μ L; 10 μMs of each 0.5 μ L of forward primer; 10 μMs of each 0.5 μ L of reverse primer; Sample DNA 1 μ L; 5U/ μ LrTaq enzyme 0.2 μ L; Amplification program is: 94 DEG C of 4min; 94 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 30s; 35 circulations; 72 DEG C of 4min.
Compared with prior art, the invention has the advantages that: the present invention utilizes the Kazakhstan intracellular list of dimension group vibrios to copy conservative gene
toxRas target, by the probe of design for vibrio alginolyticus, Vibrio campbellii, Vibrio harveyi, Vibrio natriegen, Vibrio parahaemolyticus and wheel animalcule vibrios, make the gene chip that can be used for breathing out dimension flora Rapid identification, adopt multiple PCR technique to increase 6 vibrios simultaneously
toxRgene, parallel on gene chip, identify Vibrio harveyi group rapidly.The 6 kinds of morbid vibrios rapidly and efficiently detecting cause of disease Vibrio harveyi group of designed gene chip energy parallelism, and there is more special and sensitive feature.In addition, the probe design of gene chip is by Vibrio harveyi groups all in analytical database
toxRgene order, design Vibrio harveyi group Species specific probes, significantly improves the specificity of detection.The present invention has not only filled up the technological gap utilizing gene chip simultaneously to detect Vibrio harveyi group 6 sibling species, more improves the distinguishing ability of this sea farming pathogenic flora, greatly shortens the time of detection.
In sum, the present invention can reach the object detecting Vibrio harveyi group for the gene chip detecting Vibrio harveyi group, there is the advantages such as quick, accurate, easy and simple to handle and repeatability is strong, the application of achievement can instruct the quick application of the research of sea farming distress mechanism and corresponding means of prevention, reduces disease loss.
Accompanying drawing explanation
Fig. 1 is each probe location on gene chip;
Fig. 2 is vibrio alginolyticus ATCC17749
tspecific probe gene chip the result figure;
Fig. 3 is vibrio alginolyticus NBV00022 specific probe gene chip the result figure;
Fig. 4 is Vibrio campbellii MCCC1H0050
tauele Specific Primer gene chip the result figure;
Fig. 5 is Vibrio harveyi MCCC1H0031
tspecific probe gene chip the result figure;
Fig. 6 is for being Vibrio natriegen MCCC1H0025
tspecific probe gene chip the result figure;
Fig. 7 is Vibrio parahaemolyticus MCCC1A02609
tspecific probe gene chip the result figure;
Fig. 8 is wheel animalcule vibrios CAIM577
tspecific probe gene chip the result figure;
Fig. 9 is vibrio alginolyticus (ATCC17749
t) genechip detection result figure;
Figure 10 is the genechip detection result figure of vibrio alginolyticus (NBV00022);
Figure 11 is Vibrio campbellii (MCCC1H0050
t) genechip detection result figure;
Figure 12 is Vibrio harveyi (MCCC1H0031
t) genechip detection result figure;
Figure 13 is Vibrio natriegen (MCCC1H0025
t) genechip detection result figure;
Figure 14 is Vibrio parahaemolyticus (MCCC1A02609
t) genechip detection result figure;
Figure 15 is wheel animalcule vibrios (CAIM577
t) genechip detection result figure;
Figure 16 is vibrio alginolyticus (ATCC17749
t), vibrio alginolyticus (NBV00022), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t) and wheel animalcule vibrios (CAIM577
t) mixing masterplate genechip detection result figure;
Figure 17 is vibrio alginolyticus (ATCC17749
t), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t) and wheel animalcule vibrios (CAIM577
t) mixing masterplate detected result;
Figure 18 is vibrio alginolyticus (ATCC17749
t), vibrio alginolyticus (NBV00022), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025T) and wheel animalcule vibrios (CAIM577T) mix masterplate genechip detection result figure;
Figure 19 is the genechip detection result figure of pathogenic bacteria (XBF1001);
Figure 20 is the genechip detection result figure of pathogenic bacteria (XBF1002);
Figure 21 is the genechip detection result figure of pathogenic bacteria (XBF0906);
Figure 22 is the genechip detection result figure of pathogenic bacteria (XBF1004).
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
The preparation of probe
1, target detect bacterial classification and target gene
The present invention selects encountered pathogenic bacteria important in marine cultured animal-Vibrio harveyi group as detected object, and comprise vibrio alginolyticus, Vibrio campbellii, Vibrio harveyi, Vibrio natriegen, Vibrio parahaemolyticus, wheel animalcule vibrios, each bacterial classification all uses
toxRgene is target gene.
2, probe design
The present invention adopts the oligonucleotide probe of 18-30bp.The basic demand of probe parameter is specificity and hybridization efficiently, probe T
mvalue keeps close, floats within the scope of upper and lower 10 DEG C; The continuous complementary base radix forming dimer and hairpin structure is less than 4 (value is less than 4.5kcal/mol); Probe is less than 7 bases with the continuous base number that mates of non-targeted gene order; The base mismatch number of probe and target gene is less than 4 bases.Probe after checking adds 16 T tails on 5 ' end, sterically hindered during to reduce DNA hybridization, improves hybridization efficiency.Carry out amido modified at probe 5 ' end, the cross-link with on slide, is fixed on probe on slide simultaneously.
The present invention is directed to different strain
toxr gene designs 14 probes altogether, has 16 poly-T and amido modified (being shown in Table 1) in 5 ' end band of probe.Each bacterium at least designs 2 different probes, and vibrio alginolyticus has 4 probes to detect two the different subspecies of vibrio alginolyticus on evolutionary tree respectively, and each subspecies have 2 different probes.
Table 1 detecting probe information table
Specific embodiment two
The probe preparation utilizing above-mentioned specific embodiment one to design is for detecting the gene chip of Vibrio harveyi group, and concrete steps are as follows:
1, the preparation of probe mother liquor
Probe lyophilized powder RNAase-free water (i.e. DEPC process water) is mixed with the mother liquor of 100 μm/L, after vibration mixing, in 4 DEG C, the centrifugal 1min of 5000rpm/min, obtains probe mother liquor ,-20 DEG C of preservations;
2, prepare before point sample
Get 0.2mL Eppendorf tube, add the spotting buffer (SSC) of the probe mother liquor of 10 μ L, 10 μ L successively, after vibration mixing, in 4 DEG C, the centrifugal 1min of 5000rpm/min, get supernatant and be loaded onto in 384 orifice plates;
3, point sample
Confirm the matrix wanting spot film, and control humidity (75%) and the temperature (23 DEG C) of point sample instrument, adopt high-accuracy mechanical hand deposition techniques, by probe points on aldehyde group modified sheet glass, every bar probe repeats 4 times;
4, point sample aftertreatment
Being placed on by the sheet glass put is equipped with in saturated nacl aqueous solution box, when placing 72 under the condition of temperature 30 DEG C, humidity 75%; After 3 days, distilled water put into by the sheet glass that point sample is good, boils 30s in 100 DEG C, namely obtains the gene chip for detecting Vibrio harveyi group, takes out after this gene chip dries naturally, puts into chip cartridges, 4 DEG C of preservations.On gene chip, the position of each group probe as shown in Figure 1, adopts high-accuracy mechanical hand deposition techniques, according to the design of probe in detecting array, is fixed on aldehyde radical substrate by corresponding probe.
Specific embodiment three
The using method of the gene chip for detecting Vibrio harveyi group that above-mentioned specific embodiment two prepares, concrete steps are as follows:
1, sample is chosen:
Adopt type strain: vibrio alginolyticus (ATCC17749
t), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t) and wheel animalcule vibrios (CAIM577
t); Environment bacterial strain: vibrio alginolyticus (NBV00022); The reference culture sample of seven kinds of bacteriums, utilizes the method for phenol chloroform isoamyl alcohol extracting to extract bacterial strain DNA.
2, the design of primer and synthesis
To adopt Primer5.0 for 6 kinds of vibrios
toxRgene conserved regions design primer.By each primer according to design of primers principles and requirements, by the parameter of Oligo7 software analysis primer, comprise secondary structure, get rid of inappropriate primer.Make the T of 10 pairs of primers as far as possible simultaneously
mclose, finally all primers are by BLAST comparison, and to ensure the specificity of primer, the gene order of the forward primer of the pcr amplification of Vibrio harveyi group target gene and the reverse primer of band digoxigenin labeled is as shown in table 2 below:
The amplimer of table 2 Vibrio harveyi group target gene
；
3, pcr amplification
The multiplexed PCR amplification system adopted is as shown in table 3 below:
Table 3 multiplexed PCR amplification system
Pcr amplification condition: 94 DEG C of 4min; 94 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 30s; 35 circulations; 72 DEG C of 4min; The PCR primer agarose gel electrophoresis of 2% (w/v) detects;
4, PCR primer and chip hybridization
Getting the pcr amplification product 2 μ L that above-mentioned steps 3 obtains joins in 0.2mL Eppendorf tube, then adds hybridization solution 10 μ L, fully mixes, 98 DEG C of sex change 5min; Be quickly transferred to after sex change in ice bath, leave standstill 5min, then fully mix, all solution is dropped in the point sample district of the gene chip that specific embodiment two prepares, covered, and get rid of all bubbles in point sample district; Gene chip is put in hybridizing box, after 37 DEG C of hybridization 30min, hybridizing box is placed in the thermostat container of 37 DEG C, and ensures in hybridizing box, there is certain humidity;
5, the process after gene chip hybridization
A, taking-up gene chip, carefully remove cover glass, immerse washing lotion I(0.3 × SSC, 0.2%SDS rapidly) in.On horizontal shaker, 90rpm/min swings and washes 10min.Then washing lotion II(0.06 × SSC is immersed) in, horizontal shaker 90r/min swings and washes 1min.Take out gene chip and remove the surplus liquid outside point sample district with thieving paper;
B, to be blockaded by 25 μ L liquid and 1 μ L antibody join in Eppendorf tube respectively, fully after mixing, all drip in point sample district, cover slide, be placed in hybridizing box, 37 DEG C of thermostat container 30min;
C, carefully remove cover glass, the surplus liquid outside removing point sample district, immerse in washing lotion II; Horizontal shaker, after 90rpm/min swings and washes 30s; Take out gene chip removing surplus liquid, drip balance liquid (100mMTris-HCl, 100mMNaCl, pH9.5) 50 μ L in point sample district, and leave standstill 1min, then remove surplus liquid;
D, nylon membrane put into nitrite ion (solution containing tetrazole indigo plant (NBT) and the bromo-4-of 5-chloro-3-indolol-phosphoric acid-4-toluene amine salt (BCIP)) and soak 10s, take out and carefully drain film nitrite ion unnecessary on the surface, then nylon membrane is covered in the point sample district (one-time-reach-place of gene chip, irremovable or pull nylon membrane), covered at once, compacting makes nylon membrane cover gene chip surface completely with exclusive segment air gently, be placed in hybridizing box, 37 DEG C, 20min(observes at any time in case develop the color excessively);
6, the scanner uni result interpretation of chip
Developed the color rear taking-up gene chip, and careful removing cover glass also uncovers nylon membrane, then swings gently with distilled water and washes 10 seconds, naturally dry, now can with the naked eye or magnifying glass observation hybridization signal (presenting purple round dot); By nylon membrane ordinary optical scanner (resolving power: dpi>1200) scanning, also can the kind of interpretation bacterium according to the position of signaling point.
Specific embodiment four
The gene chip for detecting Vibrio harveyi group that above-mentioned specific embodiment two prepares specific effect checking
The amplified production of experimental strain target gene 2% agarose gel electrophoresis is detected, detect qualified after carry out again hybridization colour developing, for gene chip specificity verification.The result as shown in Fig. 2 to Fig. 8, probe NBUZP001 and NBUZP002 can with vibrio alginolyticus (ATCC17749
t) specific binding, and there is no nonspecific hybridization signals.Simultaneously as shown in Figure 3, probe NBUZP003 and NBUZP004 also can specificity and vibrio alginolyticus (NBV00022) combine, and these four probes just in time cover the cluster branch of 2 notable differences of vibrio alginolyticus respectively.As Fig. 4 Vibrio campbellii (MCCC1H0050
t) specific probe; Fig. 5 Vibrio harveyi (MCCC1H0031
t) specific probe; Fig. 6 Vibrio natriegen (MCCC1H0025
t) specific probe; Fig. 7 Vibrio parahaemolyticus (MCCC1A02609
t) specific probe; Fig. 8 wheel animalcule vibrios (CAIM577
t) specific probe.Shown in detected result, 14 probes can the target gene of specific binding target pathogenic bacteria separately, comprises vibrio alginolyticus (ATCC17749
t), vibrio alginolyticus (NBV00022), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t), wheel animalcule vibrios (CAIM577
t).
Specific embodiment five
The embody rule effect of the gene chip for detecting Vibrio harveyi group that above-mentioned specific embodiment two prepares adopts type strain: vibrio alginolyticus (ATCC17749
t), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t) and wheel animalcule vibrios (CAIM577
t); Environment bacterial strain: vibrio alginolyticus (NBV00022); Unknown pathogenic bacteria: XBF0906, XBF1001, XBF1002 and XBF1004 detection validation.In addition, use this laboratory to be separated conservation 21 strain bacterium to verify, wherein vibrio alginolyticus 4 strain, Vibrio campbellii 4 strain, Vibrio harveyi 4 strain, Vibrio natriegen 2 strain, Vibrio parahaemolyticus 3 strain and wheel animalcule vibrios 4 strain, the amplified production of all bacterial strains is all through sequence verification, be target gene, and all mate completely with respective probe.Finally adopt 6 strain Vibrio harveyi mixing masterplate system detection validation and 2 kinds of multi-form 5 strain Vibrio harveyi group masterplate mixed system detection validation.Result is as shown in Fig. 9 to Figure 22, and the probe bacterium of all autonomous design can only the corresponding target gene of specific combination, without the signal of non-specific hybridization.Therefore, the probe of institute's autonomous design has the specificity of height.Fig. 9 is vibrio alginolyticus (ATCC17749
t) genechip detection result figure, only have the gene recombination signal of the type vibrios; Figure 10 is the genechip detection result figure of vibrio alginolyticus (NBV00022), and designed two kinds of probes for vibrio alginolyticus, can obviously distinguish NBV00022 type and ATCC17749
ttype vibrios; Figure 11 is Vibrio campbellii (MCCC1H0050
t) genechip detection result figure, the hybridization location result on chip is single, illustrates to have high specificity and sensitivity; Figure 12 is Vibrio harveyi (MCCC1H0031
t) genechip detection result figure; Figure 13 is Vibrio natriegen (MCCC1H0025
t) genechip detection result figure; Figure 14 is Vibrio parahaemolyticus (MCCC1A02609
t) genechip detection result figure; Figure 15 is wheel animalcule vibrios (CAIM577
t) genechip detection result figure; Figure 16 is vibrio alginolyticus (ATCC17749
t), vibrio alginolyticus (NBV00022), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t) and wheel animalcule vibrios (CAIM577
t) mixing masterplate genechip detection result figure, in hybrid template detects, all special being combined with each self-corresponding probe of target gene of all vibrios to be checked, shows the high specific that institute's independent research gene chip detects biased sample and sensitivity; Figure 17 is vibrio alginolyticus (ATCC17749
t), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025
t), Vibrio parahaemolyticus (MCCC1A02609
t) and wheel animalcule vibrios (CAIM577
t) mixing masterplate detected result; Figure 18 is vibrio alginolyticus (ATCC17749
t), vibrio alginolyticus (NBV00022), Vibrio campbellii (MCCC1H0050
t), Vibrio harveyi (MCCC1H0031
t), Vibrio natriegen (MCCC1H0025T) and wheel animalcule vibrios (CAIM577T) mix masterplate genechip detection result figure; Figure 19 is the genechip detection result of unknown pathogenic bacteria (XBF1001), through being accredited as Vibrio harveyi (MCCC1H0031
t); Figure 20 is the genechip detection result of unknown pathogenic bacteria (XBF1002), through being accredited as Vibrio harveyi (MCCC1H0031
t); Figure 21 is the genechip detection result of unknown pathogenic bacteria (XBF0906), through being accredited as Vibrio parahaemolyticus (MCCC1A02609
t); Figure 22 is the genechip detection result of pathogenic bacteria (XBF1004), through being accredited as Vibrio parahaemolyticus (MCCC1A02609
t).
Certainly, above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned citing.The change that those skilled in the art make in essential scope of the present invention, remodeling, interpolation or replacement, also should belong to scope.
Claims (3)
1. one kind for detecting the gene chip of Vibrio harveyi group, comprise chip carrier, it is characterized in that: immobilized on described chip carrier have for detecting vibrio alginolyticus, Vibrio campbellii, Vibrio harveyi, Vibrio natriegen, the nucleotide probe of Vibrio parahaemolyticus and wheel animalcule vibrios, the described probe for detecting vibrio alginolyticus, the described probe for detecting Vibrio campbellii, the described probe for detecting Vibrio harveyi, the described probe for detecting Vibrio natriegen, described for detecting the probe of Vibrio parahaemolyticus and described all using for the probe detecting wheel animalcule vibrios
toxRgene is target gene, and the gene order of each probe is as follows:
。
2. a using method for the gene chip for detecting Vibrio harveyi group according to claim 1, comprises Vibrio harveyi group testing sample DNA extraction step to be measured; Sample P CR amplification step; PCR primer and chip hybridization step, the step of the scanner uni result interpretation of the process after chip hybridization and its chip, is characterized in that: for Vibrio harveyi group different strain in described sample P CR amplification step
toxRthe gene order of the forward primer of the pcr amplification designed by gene and the reverse primer of band digoxigenin labeled is as follows:
。
3. the using method of the gene chip for detecting Vibrio harveyi group according to claim 2, is characterized in that: in described sample P CR amplification step, amplification system is: sterilized water 5.3 μ L; 10 × PCR damping fluid 1.5 μ L; 2.5mMdNTP mixture 1 μ L; 10 μMs of each 0.5 μ L of forward primer; 10 μMs of each 0.5 μ L of reverse primer; Sample DNA 1 μ L; 5U/ μ LrTaq enzyme 0.2 μ L; Amplification program is: 94 DEG C of 4min; 94 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 30s; 35 circulations; 72 DEG C of 4min.
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| CN106939347A (en) * | 2017-04-26 | 2017-07-11 | 淮海工学院 | Portunus trituberculatus Miers cause of disease Vibrio natriegen double PCR quick detection kit and method |
| CN107273711A (en) * | 2017-06-22 | 2017-10-20 | 宁波大学 | A kind of shrimp disease quantitative forecasting technique based on enteron aisle bacterial indicator |
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| CN106636073A (en) * | 2017-01-20 | 2017-05-10 | 青岛农业大学 | Preparation method of pathogenic vibrio harveyi DNA (Deoxyribonucleic Acid) template in prawn culture |
| CN106939347A (en) * | 2017-04-26 | 2017-07-11 | 淮海工学院 | Portunus trituberculatus Miers cause of disease Vibrio natriegen double PCR quick detection kit and method |
| CN106939347B (en) * | 2017-04-26 | 2020-08-04 | 淮海工学院 | Dual PCR (polymerase chain reaction) rapid detection kit and method for pathogenic vibrio natriegens of portunus trituberculatus |
| CN107273711A (en) * | 2017-06-22 | 2017-10-20 | 宁波大学 | A kind of shrimp disease quantitative forecasting technique based on enteron aisle bacterial indicator |
| CN107273711B (en) * | 2017-06-22 | 2021-03-23 | 宁波大学 | Screening method of prawn health condition indicating flora |
| CN108546743A (en) * | 2018-05-09 | 2018-09-18 | 宁波海洋研究院 | A kind of Vibrio harveyi rapid detection method |
| CN110699473A (en) * | 2019-11-27 | 2020-01-17 | 中国水产科学研究院黄海水产研究所 | Method for rapidly detecting vibrio rotifer in culture site |
| CN111304347A (en) * | 2019-11-27 | 2020-06-19 | 中国水产科学研究院黄海水产研究所 | PCR reaction system for rapidly detecting vibrio rotifer in culture site |
| CN111518928A (en) * | 2019-12-05 | 2020-08-11 | 广东美格基因科技有限公司 | TaqMan probe quantitative detection method for detecting vibrio harveyi and corresponding kit |
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