CN105969907A - Kit for detecting ST251-type virulent aeromonas hydrophila and application - Google Patents
Kit for detecting ST251-type virulent aeromonas hydrophila and application Download PDFInfo
- Publication number
- CN105969907A CN105969907A CN201610604290.XA CN201610604290A CN105969907A CN 105969907 A CN105969907 A CN 105969907A CN 201610604290 A CN201610604290 A CN 201610604290A CN 105969907 A CN105969907 A CN 105969907A
- Authority
- CN
- China
- Prior art keywords
- dna
- aeromonas hydrophila
- pcr
- kit
- aeromonas
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a PCR kit for detecting ST251-type aeromonas hydrophila and application. The kit comprises a PCR Premix, a primer pair, a standard positive DNA template, a standard negative DNA template and sterilization double distilled water. An upstream primer sequence is 5'-GCGGCGTAAGCGGATTTGTAGC-3', a downstream primer sequence is 5'-CCGGTCGGATTGGTCAGGTTCAT-3', the annealing temperature is 63 DEG C, a correct amplification product is 826bp in size, the standard positive DNA template is genomic DNA extract of an ST251-type aeromonas hydrophila XS91-4-1 strain, and the DNA concentration is 500 nanograms per microliter; the negative DNA template is genomic extractive of an aeromonas veronii IB340 strain, and the DNA concentration is 500 nanograms per microliter. The kit is applicable to all kinds of gene amplification instruments, high in detection specificity and sensitivity, quick, accurate and good in stability. The kit can be used for quickly diagnosing freshwater fish outbreak diseases and blood poisoning caused by aeromonas hydrophila of other fishes and quickly detecting and identifying bacteria and then is widely applied to disease prediction and forecast and epidemiologic studies.
Description
Technical field
The invention belongs to biological technical field, be more particularly to a kind of quickly detection ST251 type pathogenic addicted to water
The test kit of Aeromonas, further relates to a kind of test kit detecting ST251 type pathogenic hydrophila gingivalis and uses
On the way, can be used for cultured freshwater fish disease outbreak and septicemia that other freshwater fish is caused by Aeromonas hydrophila
Rapid differential diagnosis and epidemiological study, it is also possible to provide technical support for related basic research.
Background technology
Aeromonas (aeromonads) is China's modal pathogenic bacteria of cultured freshwater fish apoplexy due to endogenous wind, almost
Various aquatic animal can be encroached on.Each Ecological niche that they are present in aquaculture system, is healthy
The main of aquatic animal intestinal often occupies one of bacterium.γ-deformation Gammaproteobacteria, Aeromonas section, gas unit cell is belonged in classification
Pseudomonas, the most approved kind is at least more than 25 kinds, and novel species is constantly being in the news.Aeromonas hydrophila
(Aeromonas hydrophila, Ah) is the representative species of this genus, is also the most often to be accredited as fish-pathogenic bacteria
Aeromonas.It is generally believed that most Aeromonas hydrophilas are conditioned pathogens.Aeromonas hydrophila causes Aquatic product
Animal diseases have acute, also have chronic, and symptom has different manifestations, as body surface fester, enteritis, rotten tail,
Rotten fin, erythrodermatitis, scale erection, Ascites Disease and septicemia etc., so between the different strains of Aeromonas hydrophila
Its virulence (pathogenic) is different.But, start so far from the end of the eighties in last century, occur in China
The sudden hemorrhage of fishes caused by Aeromonas hydrophila is but an exception, often causes in same water body many
Plant cultured fishes the most extensive fulminant death and (be also called motor type Aeromonas septicemia Motile
Aeromonas Septicemia, MAS), this Aeromonas hydrophila demonstrates powerful pathogenic.Become me
The fish bacterial disease that state's harm is maximum.These Aeromonas hydrophila bacterial strains are considered as pathogenic addicted to aqueous vapor list
Born of the same parents bacterium (virulent Aeromonas hydrophila).Owing to there is huge heterogeneity between Aeromonas hydrophila,
Different pathogenic bacterial strains is found to carry the combination of different virulence gene, and the no pathogenicity bacterial strain in environment is also
Often carry the most multiple virulence gene of more than one.
The classification of Aeromonas and identify extremely complex, but recently as the development of Protocols in Molecular Biology, right
Taxonomic identification and the typing method of Aeromonas achieve important breakthrough.The experimental results shows, these from
Its genotype of the bacterial strain of disease outbreak case broadly falls into ST251 type.These bacterial strains are composed at virulence gene, to Brachydanio rerio
LD50And the aspect such as serotype is the most consistent, it is believed that belong to same clone.From the beginning of 2009,
A kind of eruptive epidemic disease caused by Aeromonas hydrophila cultivates area generation at the channel catfish of the U.S., causes
Huge economic loss, is also ST251 type Aeromonas hydrophila through comparing its cause of disease of discovery.Obviously, ST251
Type Aeromonas hydrophila is different from other ST type Aeromonas hydrophilas.
Protocols in Molecular Biology has proven to maximally effective Bacteria Detection, identification technology, have simplicity, quickly,
Inexpensively, the sensitive and feature of high specificity.In conjunction with traditional bacteria distribution, culture technique, it has also become bacteriology
The basic fundamental of research.
About detection and the qualification of pathogenic hydrophila gingivalis, many documents and patented technology is had all to report phase
The PCR detection technique closed.But, these technology are both for certain or some virulence gene of this bacterium and design,
Such as hemolysin gene, aerolysin gene, enterotoxin genes etc..As previously mentioned, Aeromonas hydrophila has
The biggest heterogeneity, although these genes generally exist ST251 type Aeromonas hydrophila, but in many non-causes
Characteristic of disease bacterial strain also has distribution, even exists too in the Aeromonas of other kind.So, such
Detection technique cannot be distinguished by the Aeromonas hydrophila of ST251 type Aeromonas hydrophila and other monoid.
There is obvious technology inferior position in traditional Bacteria Identification based on phenotype and detection technique, as time-consuming, laborious
Unstable with accuracy.The PCR detection scheme that a lot of document reports or patented technology are introduced, causes a disease screening
Property Aeromonas hydrophila aspect be blank, be not the most specifically designed for ST251 type pathogenic addicted to aqueous vapor list
The PCR detection technique of born of the same parents bacterium.And the Aeromonas hydrophila of this monoid is science of fish disease field pay close attention to problem it
One, detection and qualification to it are the necessary technology means such as relevant disease diagnosis and epidemiological study.
Summary of the invention
It is an object of the invention to there are provided a kind of reagent detecting ST251 type pathogenic hydrophila gingivalis
Box, the Aeromonas hydrophila of this monoid all has the most pathogenic, is freshwater fish disease outbreak (bacillary deteriorated blood
Disease) pathogen.Very harmful on Aquatic product of this pathogenic bacterium, the kind of infringement Fish is many, is most important
Modal fish pathogens antibacterial.
Further object is that and provide a kind of PCR reagent detecting ST251 type Aeromonas hydrophila
Box application in preparation treatment or prevention cultured freshwater fish disease outbreak medicine.Without special in implementation process
Reagent, and provide positive control and negative control DNA profiling, be possible to prevent occur that false positive and vacation are cloudy
Property extension.Be applicable to the most all types of gene-amplificative instrament, detection high specificity, highly sensitive,
Quickly and accurately, good stability.Can be used for freshwater fish disease outbreak and other Fish in aquaculture process (as
Channel catfish, tilapia, Siniperca chuatsi etc.) caused by Aeromonas hydrophila the quick diagnosis of septicemia, antibacterial
Quickly detection and qualification, and be then used widely in prediction, forecast and the epidemiological study of disease.
To achieve these goals, the present invention uses techniques below measure:
The technology design of the present invention is: by many strains ST251 type Aeromonas hydrophila is carried out gene order-checking,
From Genbank data, additionally download multiple Aeromonas hydrophila whole genome sequence, pass through comparative genomics
Research, it has been found that a kind of dehydrogenase coding genes be ST251 type pathogenic hydrophila gingivalis exclusive,
Non-pathogenic and other types pathogenic hydrophila gingivalis do not exist.To this end, we are for this base
Because of design specific PCR amplimer, carry out test and constantly optimize.By the different bacterium to 15 strain stocks
Strain and some clinic case bacterial strains be repeatedly detected it was confirmed this to primer in detection, identify that ST251 type causes a disease
High degree of specificity in property Aeromonas hydrophila.Basis at this, we establish corresponding detection kit, use
In freshwater fish bacterial septicemia and the quick diagnosis of channel catfish septicemia, the quickly detection of cause of disease and mirror
Fixed.
Being found by comparative genomics research, the so-called virulence gene which kind of does not has report at present is certain monoid
Institute is exclusive, and the genome of environment bacterial strain also contains virulence gene.Relevant document analysis also demonstrate that this point.
Therefore, it is understood that false positive easily occurs in the testing result for virulence gene, and present invention overcomes this
Individual shortcoming, and given confirming through substantial amounts of experiment.
The detection method that the present invention provides, can not only be applied to the qualification of the most isolated and purified bacteria samples,
Can also be applied in disease fish blood sample, tissue sample, water sample, sediment of pond sample and Feed Sample cause
The detection of characteristic of disease Aeromonas hydrophila.
A kind of PCR kit detecting ST251 type Aeromonas hydrophila, the content of this test kit includes:
The PCR premix of a.PCR premix: a kind of lyophilizing, comprise high-fidelity DNA polymerase,
DNTPs, MgCl2, reaction buffer, PCR increased response agent and stabilizer；
B. primer pair；
C. standard positive DNA profiling；
D. standard female DNA profiling；
E. sterilizing distilled water.
It is characterized in that: forward primer sequence is 5 '-GCGGCGTAAGCGGATTTGTAGC-3 ',
Downstream primer sequence is 5 '-CCGGTCGGATTGGTCAGGTTCAT-3 ', and annealing temperature is 63 DEG C,
Correct amplified production size is 826bp.Standard positive DNA profiling is ST251 type Aeromonas hydrophila
The extracting genome DNA thing of XS91-4-1 strain, DNA concentration is 500ng/ μ l；Negative DNA profiling is dimension
The genome extract of family name Aeromonas (Aeromonas veronii IB340 strain), DNA concentration is 500ng/ μ l.
The positive is the bacterial strain that applicant place laboratory oneself separates, identifies and preserves with negative control bacterial strain.Described
Primer the design of primers plate PimerSelect being made by DNAstar software completed, Cong Zhongxuan
Select 10 and optimal primer carried out Blast comparison analysis again, screen and obtain the primer pair that following specificity is the highest:
Forward primer sequence is 5 '-GCGGCGTAAGCGGATTTGTAGC-3 '；Downstream primer sequence is
5’-CCGGTCGGATTGGTCAGGTTCAT-3’。
A kind of PCR kit of ST251 type Aeromonas hydrophila that detects is in preparation treatment or prevention freshwater aquiculture
Application in Fish disease outbreak medicine.Its use step is:
1. need measuring samples is carried out pre-treatment, to extract STb gene therein for using the present invention to provide
Test kit detect.The detection kit that the present invention provides, can not only be applied to the most isolated and purified
Bacteria samples, it is also possible to be applied to disease fish blood sample, tissue sample, water sample, sediment of pond sample and raise
The detection of pathogenic hydrophila gingivalis in material sample.In order to extract the high-quality DNA examination for the present invention
Agent box detects, and can select commercialization DNA extraction kit ripe in the market according to sample classification
(such as DNeasy Blood&Tissue Kit and the QIAamp DNA Stool Mini Kit of QIAGEN company).
1) for water sample: taking 1 liter of water sample, sterile working, under the conditions of 4 DEG C, 12800 × g is centrifuged 20 points
Clock, collects precipitate and is used for follow-up DNA extraction.
2) for solid or semi-solid sample such as sediment of pond, feedstuff or animal intestinals: first in aseptic condition
Fully smash to pieces down, then take appropriate sterilized water carry out 1:2～5 dilution, shake 2min, then with 400 × g from
Heart 1min, collects supernatant, by step 1) it is centrifuged and collects its precipitate for follow-up DNA extraction.
3) for step 1) and step 2) centrifugal sediment that obtains, use the QIAamp of QIAGEN company
DNA Stool Mini Kit extracts STb gene therein.
4) for bacterial clump sample, Promega company can be usedGenomic DNA
Purification Kit extracts its genomic DNA, detects for follow-up PCR.For bacterium colony sample, sometimes
Can also be placed directly within PCR reaction system with the minimal amount of antibacterial of sterilizing toothpick picking, and without carrying in advance
Take its DNA.But, in this case need to be adjusted the denaturation time in PCR amplification procedure by 2min
Whole for 5min.
The foundation of 2.PCR reaction system.In each Premix pipe, add 2 μ l forward primer, 2 μ l downstreams
Primer, 1 μ lDNA template, and 45 μ l sterilizing distilled waters, be about 50 μ l to cumulative volume.After slight mixing,
Dodge from 2s, make all liq all concentrate at the bottom of pipe.
3. it is placed in the PCR amplification instrument enterprising performing PCR amplification of routine.The program of PCR amplification is by following reaction ginseng
Number is carried out: denaturation 2min at 95 DEG C, the cyclic process enter degeneration, annealing and extending: hold at 94 DEG C
There is degenerative process in continuous 30s, continues 30s and complete annealing process at 63 DEG C, continues 60s and prolong at 72 DEG C
Extend through journey.Above-mentioned degeneration, anneal and the cyclic process that extends carries out 32 circulations, finally again prolong at 72 DEG C
Stretch 10min.
4. amplified production gel electrophoresis analysis.The agarose using 1.5% concentration carries out gel electrophoresis, bromination second
After ingot dyeing, put and observe under uviol lamp and take pictures.According to DNA molecular amount labelling, it is judged that amplified production DNA
Clip size.
5. result judges.If the DNA stripe size of amplification is about 826bp, i.e. think that amplification is sun
Property, say, that sample DNA exists the DNA of ST251 type Aeromonas hydrophila.If gone out without amplified band
Now, or stripe size is not substantially inconsistent with 826bp, then amplification is judged to feminine gender, illustrates in sample DNA
There is not the DNA of ST251 type Aeromonas hydrophila.
6. quality control: only amplify correct band in positive control sample, and negative control sample is without bar
The result of the amplification that band occurs just can be recognized.If positive control sample does not amplify band, or band is big
Little with anticipated is not inconsistent, or negative control sample amplifies band, and that explanation reaction system or experimental implementation are deposited
In certain problem, need to reset reaction system and expand.
One preferred scheme is, the DNA fragmentation (826bp) similar to expection size that will obtain in step 5,
Use standard method to carry out reclaiming purification to it, then clone and check order.The result of order-checking is SEQ ID
Nucleotide sequence shown in NO:1.This sequence similarity should be more than 95%.
The present invention compared with prior art, has the following advantages and effect:
1., compared with traditional detection and authentication method, the detection method of the PCR that the present invention provides has highly sensitive
Property, high specific and the feature of high accuracy.
2. the detection kit that the present invention provides be directed to Aeromonas hydrophila that genotype is ST251 this
Specific bacteria monoid, the most with strong points.Very harmful on Aquatic product of this pathogenic bacterium, the kind of infringement Fish
Class is many, is most important modal fish pathogens antibacterial, is also the important object of academia research.Imply that this
The range of application of invention will be widely.In view of the significant damage of this monoid Aeromonas hydrophila, and
The detection always do not set up for this specific monoid or identification technology, it should say that the present invention has significantly wound
The property made.
3. the present invention is on the basis of comparative genomics is studied, it was found that this monoid Aeromonas hydrophila
Specific enzyme, and design specific primer based on this enzyme.Than conventional needle to Aeromonas virulence gene
The primer of design, its reliability is higher.Do not have false-positive testing result, say, that will not be to any
The raw positive amplification reaction of non-ST251 type Aeromonas hydrophila.This illustrates that its accuracy detected is than existing phase
Close detection technique and there is obvious advantage and advance.
4. with strong points due to the detection object of the present invention, therefore its using value will be the highest.Can be used for water
Produce freshwater fish disease outbreak and other Fish (such as channel catfish, tilapia, Siniperca chuatsi etc.) in breeding process thin
In the quick diagnosis of bacterium property septicemia, non-biological specimen (such as feedstuff, bed mud, water body example etc.) this monoid
The quickly detection of antibacterial and qualification, and then obtain wide in prediction, forecast and the epidemiological study of disease
General application.
5. the present invention may be used for various types of sample detection, applied widely.There is provided for different samples
Different operation scheme.
6. the primer that the present invention provides carries out nucleotide Blast search, and result is kissed with forward primer height
Close (100%query cover) bacterial groups be mainly Aeromonas hydrophila and enterobacteriaceae antibacterial (see
Fig. 1)；The bacterial groups being identical with downstream primer is Aeromonas hydrophila (see Fig. 2), and is ST251
Type bacterial strain.So, use upstream and downstream primer that DNA of bacteria is expanded, use the present invention to recommend simultaneously
High annealing temperature (63 DEG C), then only ST251 type Aeromonas hydrophila can be amplified out.This fully demonstrate,proves
The detection method that the bright present invention provides, has specificity highly to the antibacterial of this monoid.
7., during daily fish diseases diagnosis and treatment, the detection kit that the present invention provides obtains and well detects knot
Really (it is shown in Table 1).Table 1 testing result shows, only silver carp, flathead, Megalobrama amblycephala, Carassius auratus and channel catfish
In fulminant hueppe's disease disease fish, testing result is positive, and this testing result and the knot of bacteria distribution
Fruit is identical.Even if the sick fish of other kind has septicemia symptom, do not detect this monoid addicted to water
Aeromonas, illustrates that its cause of disease is not ST251 type Aeromonas hydrophila, and its result is same and bacteria distribution
Result is highly consistent.Water, sediment of pond and feedstuff the most also can detect positive findings, it was demonstrated that this
The susceptiveness of detection technique.Some disease (such as enteritis and the tail-rot disease of Ctenopharyngodon idellus) although the separated qualification of its cause of disease
Confirm as Aeromonas hydrophila, but this Aeromonas hydrophila is not belonging to ST251 type, so testing result is still
For feminine gender.
The test kit that table 1 present invention provides is the inspection of ST251 type Aeromonas hydrophila in daily sick fish sample
Survey result (2013-2016)
+ represent in sample containing ST251 type Aeromonas hydrophila；Without ST251 type addicted to aqueous vapor list in-expression sample
Born of the same parents bacterium
Accompanying drawing explanation
The upstream amplification primer that Fig. 1 provides for the present invention, through high coverage (100%) sequence that Blast comparison obtains
Bacteria name corresponding to row, to show the specificity of primer.
High coverage (100%) sequence that the downstream amplification primer that Fig. 2 provides for the present invention obtains through Blast comparison
Corresponding bacteria name, to show the specificity of primer.
Fig. 3 is that a kind of test kit is for different bacterium, the testing result of different Aeromonas.
To prove the specificity of this context of detection.1st swimming lane is molecular weight standard；It is Aeromonas the most successively
Hydrophila IB101, Megalobrama amblycephala blood separation strain (ST251 type)；Aeromonas hydrophila LNB101,
Silver carp blood separation strain (ST251 type)；Aeromonas veronii IH317, for Megalobrama amblycephala intestinal separation strain；
Aeromonas media 4pW15, for pond water separation strain；Aeromonas hydrophila XS91-4-1, for
Silver carp blood separation strain (ST251 type)；Aeromonas sobria CR79-1-1, for pathogenic strains；Aeromonas
Media 4LNC204, for silver carp intestinal separation strain；Streptococcus agalactiae 0718flz, for tilapia liver
Dirty separation strain；Streptococcus agalactiae 0722XY, for liver of hybrid tilapia separation strain；Citrobacter
Freudii HD00103, for Acipenser Sinensis pathogen；Aermonas schubertii HYL2, for Ophicephalus argus pathogen；Yersinia
Ruckeri SC90-2-4, for silver carp flathead pathogen；Vibrio anguillarum E-3-11, for seawater fish pathogen；
Aeromonas veronii IB340, for Megalobrama amblycephala blood separation strain；Pseudomonas fluoresces 0704CCl,
For channel catfish blood separation strain.
Fig. 4 is a kind of detection kit testing result of ST251 type Aeromonas hydrophila in sick fish sample.
1st swimming lane is molecular weight standard, and the 2nd road is pathogen Aeromonas hydrophila XS91-4-1
The amplification of (ST251 type) DNA；3rd and the 4th road is the sick fish liver specimens that 2 tails infect XS91-4-1；
5th road and the 6th road are the sick fish liver specimens infecting Aeromonas veronii IB340.
Fig. 5 be a kind of detection kit in feedstuff, water and sediment of pond sample ST251 type addicted to aqueous vapor list
The result of the detection of born of the same parents bacterium.
M: molecular weight standard, the 1st and the 2nd swimming lane is Feed Sample, and the 3rd and the 4th swimming lane respectively with the addition of disease
The Feed Sample of former bacterium Aeromonas hydrophila Aeromonas hydrophila XS91-4-1 (ST251 type) bacterium solution；
5,6th swimming lane is conventional pond water sample；7th and 8 swimming lanes are conventional sediment of pond sample.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be appreciated that these embodiments are only used for
The bright present invention rather than restriction the scope of protection of present invention, unreceipted specific experiment bar in the following example
Part and method, generally according to normal condition such as: J. Pehanorm Brooker etc. are edited, Science Press, 2002, point
Sub-cloning experimentation guide (third edition)；Commercial kit is operated by its subsidiary operation instruction.All
Relate to DNA extraction and the operating procedure of PCR reaction system configuration, aseptic technique must be used.
In the description of the invention, term " test specimen " refers to suspect and includes all right of antibacterial to be detected
As thing.Test specimen without particular limitation of, as long as the nucleic acid amplification specific region to chromosomal DNA can be passed through
Amplification detects the existence of antibacterial.Preferably example includes the water in breeding water body, the end in breeding water body
Mud or other solid adhesions, bait and biological sample etc..Biological sample is primarily referred to as bacteria samples, Aquatic product is supported
Grow the blood of animal, mucus, histoorgan or intestinal contents etc..
Bacterial genomes DNA extraction kit in description of the invention be not limited only to QIAGEN company and
The product of Promega company, the similar test kit of any other companies satisfactory.
In addition to the PCR Premix that PCR reaction system provides except using the present invention, it is also possible to select business
The like product of product, DNA profiling and the primer that only need to add ormal weight in Premix can carry out PCR
Amplification.Its premise is that positive control sample must amplify correct band, and negative control sample goes out without amplified band
Existing.
Embodiment 1:
A kind of test kit detecting pathogenic hydrophila gingivalis, this test kit contains:
A.PCR premix: a kind of PCR premix lyophilized powder, comprise the archaeal dna polymerase of high-fidelity, dNTPs,
MgCl2, reaction buffer, PCR increased response agent and stabilizer；
B. primer pair；
C. standard positive DNA profiling；
D. standard female DNA profiling；
E. sterilizing distilled water.
The forward primer of described primer pair is 5 '-GCGGCGTAAGCGGATTTGTAGC-3 '；
The downstream primer of described primer pair is 5 '-CCGGTCGGATTGGTCAGGTTCAT-3 '.
Specific operation process is as follows:
1) genome DNA extraction of 15 strain antibacterials: use bacterial genomes to extract test kit, such as Promega
CompanyGenomic DNA Purification Kit, by specification requires operation.
2) PCR reaction system uses PCR Master Mix, is subsequently adding following ingredients
After the mixing of the most each component, dodge from 2s, make liquid concentrate at the bottom of pipe.Amplification condition is: 94 DEG C of 2min,
Then 32 circulations: 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 60s, last 72 DEG C extend 10min.
3) analysis of the agarose gel electrophoresis of amplified production；
PCR primer uses ethidium bromide staining after carrying out electrophoresis with 1.5% (W/V) agarose gel.Electrophoresis is tied
Fruit is observed under uv analyzer and takes pictures.
4) result:
Some swimming lanes have the appearance of DNA band, and its size is judged as 862bp according to molecular weight standard, and this says
Bright bacterial cultures is ST251 type Pathogenic Aeromonas.Bacterial strain corresponding to the sample of band does not occurs not
It it is ST251 type Pathogenic Aeromonas.The amplified band that positive appearance is correct, and negative control sample
In amplified band (Fig. 3) does not occurs.Being without false positive and false negative result occur, testing result can
Letter.Additionally, repeat for 3 times to test all to obtain identical result, the favorable reproducibility of illustration method.
Embodiment 2:
The test kit of a kind of quick detection pathogenic hydrophila gingivalis, is used for detecting aquatic animal tissue sample
Target bacteria that may be present in product.For realizing this purpose it may first have to sample to be carried out pre-treatment, extract
STb gene in sample, the test kit then using the present invention to provide carries out PCR Amplification Analysis.
A kind of PCR kit of ST251 type Aeromonas hydrophila that detects is in preparation treatment or prevention freshwater aquiculture
Application in Fish disease outbreak medicine.Its operating procedure is:
1) sterile working, the viscera tissue such as Liver and kidney spleen taking animal, or the blood of animal.
2) the DNeasy Blood&Tissue Kit using QIAGEN company extracts tissue and includes antibacterial base
Because group DNA requires operation at interior all DNA, by specification.
3) the PCR Premix during PCR reaction system uses the test kit of the present invention, is subsequently adding following
Composition
After the mixing of the most each component, dodge from 2s, make liquid concentrate at the bottom of pipe.Amplification condition is: 94 DEG C of 2min,
Then 32 circulations: 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 90s, last 72 DEG C extend 10min.
4) analysis and the result of the agarose gel electrophoresis of amplified production judges.
PCR primer uses ethidium bromide staining after carrying out electrophoresis with 1.5% (W/V) agarose gel.Electrophoresis is tied
Fruit is observed under uv analyzer and takes pictures.
5) result: have DNA band to occur in the swimming lane corresponding to some measuring samples, its size is according to dividing
Sub-amount standard is judged as 862bp.This illustrates have ST251 type pathogenic in the tissue sample corresponding to these swimming lanes
Aeromonas exists.Positive control and negative control sample all present expected results (Fig. 4).That is
False positive and false negative result do not occur, testing result is credible.Additionally, repeat to test and all obtain phase for 3 times
Same result, the favorable reproducibility of illustration method.
Embodiment 3:
A kind of test kit of quick detection pathogenic hydrophila gingivalis, be used for detecting non-biological specimen (water sample,
Bed mud sample, feedstuff sample) in target bacteria that may be present.For realizing this purpose it may first have to sample is entered
Row pre-treatment, extracts the STb gene in sample, and the test kit then using the present invention to provide carries out PCR amplification point
Analysis.
A kind of PCR kit of ST251 type Aeromonas hydrophila that detects is at preparation treatment or prevention cultured freshwater fish
Application in class disease outbreak medicine.Its operating procedure is:
1) for water sample: taking 1 liter of water sample, sterile working, under the conditions of 4 DEG C, 12800 × g is centrifuged 20
Minute, precipitate is collected and is used for DNA extraction.
2) for solid or semi-solid sample such as sediment of pond, feedstuff or animal intestinals: first in aseptic condition
Fully smash to pieces down, then take appropriate sterilized water carry out 1:2～5 dilution, shake 2min, then with 400 × g from
Heart 1min, collects supernatant, by step 1) it is centrifuged and collects its precipitate for follow-up DNA extraction.
3) for step 1) and step 2) centrifugal sediment that obtains, use the QIAamp of QIAGEN company
DNA Stool Mini Kit extracts STb gene therein, and by specification requires operation, for following PCR
Amplification.
4) the PCR Premix during PCR reaction system uses the test kit of the present invention, is subsequently adding following ingredients
After the mixing of the most each component, dodge from 2s, make liquid concentrate at the bottom of pipe.Amplification condition is: 94 DEG C of 2min,
Then 32 circulations: 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 60s, last 72 DEG C extend 10min.
5) analysis and the result of the agarose gel electrophoresis of amplified production judges.
PCR primer uses ethidium bromide staining after carrying out electrophoresis with 1.5% (W/V) agarose gel.Electrophoresis is tied
Fruit is observed under uv analyzer, has DNA band to occur in the swimming lane corresponding to sample being found to have.It is big
Little it is judged as 862bp according to molecular weight standard, the sample corresponding to explanation has ST251 type pathogenic gas unit cell
Bacterium exists.Positive and negative control sample all presents expected result (Fig. 5).It is without appearance
False positive and false negative result, testing result is credible.Additionally, repeat for 3 times to test all to obtain identical result,
The favorable reproducibility of illustration method.
SEQUENCE LISTING
<110>
Inst. of Hydrobiology, Chinese Academy of Sciences
<120>
A kind of test kit detecting ST251 type pathogenic hydrophila gingivalis and application
<130>
A kind of test kit detecting ST251 type pathogenic hydrophila gingivalis and application
<160>
1
<170>
PatentIn version 3.1
<210>
1
<211>
826
<212>
DNA
<213>
ST251 type pathogenic hydrophila gingivalis
<400>
1
gcggcgtaag cggatttgta gcgttgcagg
aagaagtgtt ccggcagcgc gctggtgcag 60
ccctgttttc cgtagtgctg cacctgattt
tcaagaatat tgcccgcgca gagcagccct 120
tcggagccat gtagctccag acgctggtcg
tagccgtaag aggagcggcg gctgttaacg 180
atggtcgcca ttgcgccgga ggcatatctc
agaacaataa aagcggtgtc gatgtctccc 240
gcctcgccaa ttgccggatc caccaggttg
ctgccctggg catacaccga caccggctct 300
tcacccatga tgaagcgcgc catatcaaag
tcgtgaatgg tcatatcgcg gaacatgccg 360
ccagagacgc ggacatactc cgccggtggt
ggagacggat cgcgggagat gatcagcagc 420
gattccggtt tgccgataca cccggcctgg
gcgtcggttt tcacgcggcg gaactgtggg 480
tcaaagcggc ggttgaaacc aacaaagaga
ggaacgttgc aagctgctac cgtcgcaagg 540
caatcacgga cccgagctaa atccagatgc
accggttttt cgcaaaagat tgtttttcca 600
gcacgtgccg catgttcaat gagatcggca
tgggtatccg tcgccgaggc aatcagcacc 660
ccatgaatct ggggatcaac cattgcttct
tcacaacttt gtaccttggc accgtgcttg 720
gcggccaaag tcaaagcccc ctcctgatga
gggtcgatga cagaataaag acgagtttca 780
ttgtgatccg cgatgttgac cgcatgaacc
tgaccaatcc gaccgg
826
Claims (2)
1. the PCR kit detecting ST251 type Aeromonas hydrophila, this test kit contains: a) PCR premix: the PCR premix of a kind of lyophilizing, comprises high-fidelity DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR increased response agent and stabilizer；B) primer pair；C) standard positive DNA template；D) standard female DNA template；E) sterilizing distilled water；It is characterized in that: forward primer sequence is 5 '-GCGGCGTAAGCGGATTTGTAGC-3 ', downstream primer sequence is 5 '-CCGGTCGGATTGGTCAGGTTCAT-3 ', annealing temperature is 63 DEG C, amplified production size is 826bp, standard positive DNA template is the extracting genome DNA thing of ST251 type Aeromonas hydrophila XS91-4-1 strain, and DNA concentration is 500ng/ μ l；Negative DNA profiling is the genome extract of Aeromonas veronii IB340 strain, and DNA concentration is 500ng/ μ l.
2. a kind of PCR kit detecting ST251 type Aeromonas hydrophila described in claim 1 in preparation treatment or prevents the application in cultured freshwater fish disease outbreak medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610604290.XA CN105969907B (en) | 2016-07-27 | 2016-07-27 | Kit for detecting ST251 type pathogenic aeromonas hydrophila and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610604290.XA CN105969907B (en) | 2016-07-27 | 2016-07-27 | Kit for detecting ST251 type pathogenic aeromonas hydrophila and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105969907A true CN105969907A (en) | 2016-09-28 |
| CN105969907B CN105969907B (en) | 2020-02-07 |
Family
ID=56951035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610604290.XA Active CN105969907B (en) | 2016-07-27 | 2016-07-27 | Kit for detecting ST251 type pathogenic aeromonas hydrophila and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105969907B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119384A (en) * | 2016-08-04 | 2016-11-16 | 广州市刑事科学技术研究所 | A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects |
| CN108707679A (en) * | 2018-04-18 | 2018-10-26 | 海南大学 | Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420373A (en) * | 2015-12-22 | 2016-03-23 | 于辉 | Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas |
-
2016
- 2016-07-27 CN CN201610604290.XA patent/CN105969907B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420373A (en) * | 2015-12-22 | 2016-03-23 | 于辉 | Multiple PCR primer set and probes and detecting method for simultaneously detecting three kinds of aeromonas |
Non-Patent Citations (3)
| Title |
|---|
| MAODA PANG ET AL: "Novel insights into the pathogenicity of epidemic Aeromonas hydrophila ST251 clones from comparative genomics", 《SCIENTIFIC REPORTS》 * |
| 刘礼辉: "斑点叉尾鮰败血症３种病原多重ＰＣＲ", 《西北农林科技大学学报》 * |
| 朱大玲: "嗜水气单胞菌毒力基因及工程疫苗", 《中国优秀博硕士学位论文全文数据库（博士）农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119384A (en) * | 2016-08-04 | 2016-11-16 | 广州市刑事科学技术研究所 | A kind of Aeromonas hydrophila method for nucleic acid analysis and the application in legal medical expert detects |
| CN106119384B (en) * | 2016-08-04 | 2019-12-20 | 广州市刑事科学技术研究所 | Aeromonas hydrophila nucleic acid analysis method and application thereof in forensic detection |
| CN108707679A (en) * | 2018-04-18 | 2018-10-26 | 海南大学 | Aeromonas veronii detection primer, kit, detection method and its development approach based on specific sequence |
| CN108707679B (en) * | 2018-04-18 | 2021-08-03 | 海南大学 | Aeromonas veronii detection primer, kit, detection method and development method thereof based on specific sequence |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105969907B (en) | 2020-02-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2010524443A (en) | Analysis of microbial populations | |
| CN106811541A (en) | Primer, probe and kit for field quick detection pasteurella multocida | |
| CN107385111A (en) | The real-time fluorescence quantitative PCR detection primer and its kit of a kind of goose astrovirus | |
| CN109554449B (en) | Multiplex PCR method capable of simultaneously detecting 7 virulence genes of aeromonas | |
| Mandakovic et al. | Genomic-based restriction enzyme selection for specific detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP | |
| CN103898227A (en) | Nucleotide specific to cronobacter O antigen and application of nucleotide | |
| Ding et al. | A simple PCR method for the detection of pathogenic spiroplasmas in crustaceans and environmental samples | |
| CN104232783B (en) | Quick detection method for cow brucella attenuated vaccine strain A19 | |
| Liu et al. | Simultaneous identification of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari with smartcycler-based multiplex quantitative polymerase chain reaction | |
| CN103276103A (en) | Kit with RT-LAMP nucleic acid test strips for detecting porcine epidemic diarrhea virus and applications of kit | |
| CN104745689A (en) | Primers, probe and kit used for detecting bordetella pertussis | |
| CN103993090B (en) | To Providence O31, O41, O42, the nucleotides that O43 and O50 are special and application thereof | |
| CN105969907A (en) | Kit for detecting ST251-type virulent aeromonas hydrophila and application | |
| CN101381767A (en) | Universal real time fluorescent PCR detection method of trichinella | |
| CN103451305B (en) | Detect the primer of disperse adhesivity colon bacillus, probe and method and test kit | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| CN102329858B (en) | Sugarcane smut bacteria nest type polymerase chain reaction (PCR) quick detection method | |
| CN107475411A (en) | A kind of Taqman real-time fluorescence PCR detection methods for detecting Borrelia burgdoyferi nucleic acid | |
| CN102417930B (en) | Loop-mediated isothermal amplification-based nucleic acid test method for Riemerella anatipestifer | |
| CN105200045B (en) | The nucleotides special to vibrio fluvialis O11, O14, O16 and O17 and its application | |
| CN105200044B (en) | The nucleotides special to vibrio fluvialis O1, O6, O7, O8 and O9 and its application | |
| CN107236827A (en) | Transmissible gastro-enteritis virus detection kit and detection method | |
| CN110358850B (en) | Primer group and kit for detecting serotype of streptococcus Parauberis | |
| CN104561343B (en) | Distinguish the primer and kit of detection low pathogenicity and highly pathogenic babesia motasi | |
| CN112176080A (en) | Nested PCR primer group, kit and detection method for specifically detecting purple sisal leaf roll disease phytoplasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |