CN106399520A - Detection kit for aeromonas veronii and application method thereof - Google Patents
Detection kit for aeromonas veronii and application method thereof Download PDFInfo
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- CN106399520A CN106399520A CN201610880396.2A CN201610880396A CN106399520A CN 106399520 A CN106399520 A CN 106399520A CN 201610880396 A CN201610880396 A CN 201610880396A CN 106399520 A CN106399520 A CN 106399520A
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- 241000607574 Aeromonas veronii Species 0.000 title claims abstract description 111
- 238000001514 detection method Methods 0.000 title claims abstract description 41
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- 101150013736 gyrB gene Proteins 0.000 claims abstract description 46
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 238000012360 testing method Methods 0.000 claims description 30
- 238000011144 upstream manufacturing Methods 0.000 claims description 29
- 238000012408 PCR amplification Methods 0.000 claims description 28
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- 238000006062 fragmentation reaction Methods 0.000 claims description 24
- 238000003908 quality control method Methods 0.000 claims description 17
- 101150012629 parE gene Proteins 0.000 claims description 15
- 239000012488 sample solution Substances 0.000 claims description 14
- 241000376029 Tachysurus fulvidraco Species 0.000 claims description 12
- 239000011543 agarose gel Substances 0.000 claims description 10
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
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- 238000000137 annealing Methods 0.000 claims description 7
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- 108010054814 DNA Gyrase Proteins 0.000 claims description 6
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Abstract
The invention provides a detection kit for aeromonas veronii and an application method thereof. In the technical scheme, gyrB gene is found to be a single-copy house-keeping gene through experiments and has more remarkable advantages than conventional 16S rRNA in the distinguishing and identifying of aeromonas veronii and sibling species thereof; and meanwhile, Aha gene is found to be stably exist in the aeromonas veronii and related to the toxicity thereof. Based on the beneficial discoveries, the gyrB gene and Aha gene are adopted as target genes to develop a duplex PCR detection method for aeromonas veronii; in the method, specific primers are designed for the two target genes, and a PCR reaction system and reaction conditions are determined. In the invention, the sensitivity and specificity of aeromonas veronii detection are high, the pelteobagrus fulvidraco-source pathogenic aeromonas veronii can be quickly and accurately detected, and the wrong detection and missed detection are effectively avoided. Meanwhile, repeated culture and redundant series of biochemical reactions are prevented, and the time, labor and cost are saved.
Description
Technical field
The present invention relates to gene engineering technology field, further to the biological detecting method based on technique for gene engineering,
Specifically related to a kind of detection kit for Aeromonas veronii and its using method.
Background technology
Pelteobagrus fulvidraco (Pelteobagrusfulvidraco) is commonly called as youth's silk, beeswax fourth, river forehead, belongs to Silurformes
(Siluriformes), section (Bagridae), Pelteobagrus (Pelteobagrus), are the common one kind of China's rivers and lakes
Dwell small-sized economic fish in bottom, because of its fine and tender taste, delicious flavour, and favored by consumers.However, as easily ill Fish product
Kind, often there is disease to occur in the breeding process of Pelteobagrus fulvidraco, the common disease of report mainly has bacterial disease, mycosiss, posts
Infested, nutritional disease etc..Wherein again serious with bacterial disease harm, it is easily caused Pelteobagrus fulvidraco massive mortality, provisions
Grow family and cause heavy economic lossess.Pelteobagrus fulvidraco morbidity pathogen is led to include Aeromonas hydrophila, vibrio parahaemolytious, Shu Baite gas
Zymomonas mobiliss, Aeromonas veronii etc..Wherein by Aeromonas veronii diseases caused, it is often to go out in flying fish apoplexy due to endogenous wind in recent years
Existing fulminant bacterial diseases, Symptoms are that ill Pelteobagrus fulvidraco body colour is deepened, skin mucus increase, and fester in body surface many places, anus
Red and swollen bleeding, evagination, head lower jaw, the gill cover, fin ray base portion hyperemia are rubescent, abdominal distension.Dissecting in abdominal cavity has a large amount of pink
Ascites, liver enlargement turns white, and intestinal wall is rotten to the corn, gollbladder dilation.The ill initial stage only has a small amount of fish dead, and the later stage occurs dead on a large scale
Die, mortality rate reaches more than 90%, determine that this sick pathogenic bacterium is Aeromonas veronii by isolation identification.
In the preventing and controlling of such disease, diagnosis early is the key link controlling epidemic situation, therefore except to symptom
Outside observation, the separation detection being directed to pathogenic microorganism is particularly important.Aeromonas veronii
(Aeromonasveronii) it is gram negative bacilli, the slightly curved song of thalline, belong to facultative anaerobe, thalline size is 0.3～0.7
1.2～2.5 μm of μ m.Well-grown when 25～30 DEG C, under 2～10 DEG C of cryogenic conditions and 42 DEG C of higher temperature conditions
Under also can growth and breeding.This bacterium grows vigorous, in 1.3mm more than colony diameter after culture 24h, in circle on ordinary nutrient agar
Shape, neat in edge, smooth surface, canescence, opaque, central slightly swell, well-grown on blood plate, can be formed significantly
Beta hemolysises.The detection method being directed to Aeromonas veronii in prior art is more extensive, the mode of commonly used separation and Culture, then
By being identified to the observation of colony characteristicses or thalline feature, its complex operation, take that longer and accuracy is not high.Existing skill
Researcher is had to attempt being based on as detection object realization to Aeromonas veronii identification inspection using specificity by PCR mode in art
Survey, but current stage correlation technique is generally using Aeromonas veronii 16S rRNA and nuclease gene as the target base of detection
Cause, practice finds that its specificity is not ideal enough, in addition design of primers and the factor such as PCR condition is not reasonable, leads to detect
Accuracy, speed of operation still have to be hoisted.
Content of the invention
It is contemplated that for the technological deficiency of prior art, providing a kind of detection kit for Aeromonas veronii
And its using method, with the relatively low skill of the Aeromonas veronii detection method accuracy that solves PCR-based technology in prior art
Art problem.
The invention solves the problems that another technical problem be PCR-based technology in prior art Aeromonas veronii detection side
Method is by the relatively low technical problem of selected detection unreasonable the led to accuracy of target gene.
The invention solves the problems that another technical problem be in prior art be directed to Aeromonas veronii part detection method
Complex operation, time-consuming longer.
The invention solves the problems that another technical problem be the Vickers gas with gyrB gene and Aha gene as target gene for the present invention
Zymomonas mobiliss PCR detection method, its primer performance has to be hoisted.
The invention solves the problems that another technical problem be the Vickers gas with gyrB gene and Aha gene as target gene for the present invention
Zymomonas mobiliss PCR detection method, its PCR expanding effect is not good.
For realizing above technical purpose, the present invention employs the following technical solutions：
A kind of detection kit for Aeromonas veronii, this test kit is included for Aeromonas veronii gyrB gene
Pair of primers and for Aeromonas veronii Aha gene pair of primers.
Preferably, the described pair of primers for Aeromonas veronii gyrB gene is sequence as shown in SEQ IDNO.1
Forward primer and the downstream primer as shown in SEQ ID NO.2.
Preferably, the described pair of primers for Aeromonas veronii Aha gene is sequence as shown in SEQ IDNO.3
Forward primer and the downstream primer as shown in SEQ ID NO.4.
Preferably, described test kit also includes 10 × PCR buffer, dNTP, MgCl2, rTaq archaeal dna polymerase, sun
Property quality-control product, negative quality-control product.
Preferably, described test kit is also included as the Aeromonas veronii bacterium solution of positive quality control product with as negative matter
The ddH of control product2O.
Meanwhile, present invention also offers a kind of application mentioned reagent box method of detecting Aeromonas veronii, include below
Step：
1) with testing sample solution as pcr template, with DNA fragmentation as shown in SEQ ID NO.1 for the sequence for Vickers gas list
Born of the same parents' bacterium gyrB upstream region of gene primer, with DNA fragmentation as shown in SEQ ID NO.2 for the sequence for Aeromonas veronii gyrB gene under
Trip primer, with DNA fragmentation as shown in SEQ ID NO.3 for the sequence for Aeromonas veronii Aha upstream region of gene primer, with sequence such as
DNA fragmentation shown in SEQ ID NO.4 is the execution double PCR amplification of Aeromonas veronii Aha downstream of gene primer, is expanded
Product；
2) take step 1) amplified production of gained execution agarose gel electrophoresiies, confirm at 745bp in electrophoresis result or
Whether there is band at 419bp.
Preferably, described testing sample solution is the microbial inoculum isolated from Pelteobagrus fulvidraco histoorgan.
Preferably, the reaction system of described double PCR amplification is as follows：Testing sample solution 1 μ L, 10 × PCR buffer
RTaq archaeal dna polymerase solution 0.25 the μ L, the MgCl of 250mmol/L of 2.5 μ L, 5U/ μ L2Solution 1.5 μ L, 20 μm of ol/L's
The gyrB downstream of gene primer 0.5 μ L of gyrB upstream region of gene primer 0.5 μ L, 20 μm of ol/L, the Aha upstream region of gene of 20 μm of ol/L draws
DNTP solution 0.5 the μ L, ddH of the Aha downstream of gene primer 0.6 μ L of thing 0.6 μ L, 20 μm of ol/L, 2.5mmol/L2O polishing is to 25
μL.
Preferably, the reaction condition of described double PCR amplification is as follows：94 DEG C of denaturations 4min；94 DEG C of degeneration 30s, 55
～65 DEG C of annealing 30s, 72 DEG C of extension 30s, 30～35 circulations of reaction；72 DEG C re-extend 10min.
Preferably, the temperature of described annealing is 61 DEG C, reaction cycle number of times is 30 times.
In above technical scheme, described positive quality control product is referred to as positive control with negative quality-control product again and feminine gender is right
According to, the two bacterium solution respectively clearly with purpose bacterium (Aeromonas veronii) and the clear and definite ddH without purpose bacterium2O；The two
Object of reference as PCR detection.In above technical scheme, the band coming across 745bp and 419bp position is respectively used to characterize
GyrB gene and the presence of Aha, in practical operation, can recognize in the case that above-mentioned two band simultaneously in one-time detection
It is set to Aeromonas veronii positive, only one band or there is not the situation of band then should to regard as Aeromonas veronii cloudy
Property.
The invention provides a kind of detection kit for Aeromonas veronii and its using method, this technical scheme with
The B subunit gene (gyrB) of gyrase and adhesin gene (Aha) devise two for target gene and cause a disease to for Pelteobagrus fulvidraco source
The high degree of specificity primer of property Aeromonas veronii.
GyrB gene is the house-keeping gene of single copy, and average base replacement rate is change 0.7%～0.8% in every 1,000,000 years,
The speed of the change 1% in every 50,000,000 years than 16S rDNA is fast, and not occurred level transfer, and it is prevalent in various thin
In bacterium.By laboratory facilities, the present invention finds that gyrB gene is being distinguished and identified Aeromonas veronii and its nearly edge than 16SrRNA
On kind, there is more significant advantage.Aha gene is found in 1961 earliest, is considered relevant with the synthesis of coli common pili,
Because the adhesive force of pili affects the colonization ability of many pathogenic bacterium, therefore Aha gene is considered the poison with part pathogenic bacterium
Power is relevant.The present inventor has cloned Aha gene in detached Aeromonas veronii from ill Pelteobagrus fulvidraco, and finds to have pathogenic
In the Aeromonas veronii of property, Aha gene is positive.Based on this beneficial discovery, the present invention selects it common with gyrB gene
Target gene as PCR amplification.
On this basis, the present invention devises the detection primer of two groups of high specifics, and around it establishes double PCR
Reaction system and reaction condition, define on this basis for Aeromonas veronii detection kit.The sensitivity of this test kit
Property is strong, specificity is high, can quickly and accurately detect the pathogenic Aeromonas veronii in Pelteobagrus fulvidraco source, be prevented effectively from false retrieval missing inspection.
Avoid culture, a series of tediously long biochemical reactions repeatedly simultaneously, save time, work capacity and cost.
Brief description
Fig. 1 is to be executed after double PCR amplification with various criterion thing for template in the embodiment of the present invention 1, and product is through 1.5% fine jade
The result figure of sepharose electrophoresis.In figure M：DL 2000DNA marker；1：Aha gene；2：Negative control；3：GyrB gene；
4：Negative control；5：GyrB gene-Aha gene compound；6：Negative control.
Fig. 2 be in the embodiment of the present invention 1 with different strains solution for template execution double PCR amplification after, product is through 1.5%
The result figure of agarose gel electrophoresiies.In figure M：DL 2000DNA marker；1：Aeromonas veronii type strain ATCC35624；
2：The Aeromonas veronii being separated in nature；3：Wdwardsiella tarda；4：Vibrio parahaemolytious；5：Aeromonas hydrophila；6：
Salmonella enteritidis；7：Streptococcus agalactiae；8：Maxwell vibrio；9：Staphylococcus aureuses；10：Colon bacillus；11：Pneumonia
Klebsiella；12：Shu Baite bacterium；13：Negative control.
Fig. 3 is dual for template execution with the STb gene solution of Aeromonas veronii variable concentrations in the embodiment of the present invention 1
After PCR amplification, the result figure through 1.5% agarose gel electrophoresiies for the product.In figure M：DL 2000DNA marker；1：6.6ng/μ
L；2：6.6×10-1ng/μL；3：6.6×10-2ng/μL；4：6.6×10-3ng/μL；5：6.6×10-4ng/μL；6：6.6×10-5ng/μL；7：6.6×10-6ng/μL；8：Negative control.
Fig. 4 is with Aeromonas veronii variable concentrations bacterium solution for template execution double PCR amplification in the embodiment of the present invention 1
Afterwards, the result figure through 1.5% agarose gel electrophoresiies for the product.In figure：M：DL 2000DNA marker；1：3.2×106cfu/
mL；2：3.2×105cfu/mL；3：3.2×104cfu/mL；4：3.2×103cfu/mL；5：3.2×102cfu/mL；6：3×
101cfu/mL；7：3×100cfu/mL；8：Negative control.
Fig. 5 is to be executed after double PCR amplification with natural cases separator (pathological material of disease) for template in the embodiment of the present invention 1, produces
The result figure through 1.5% agarose gel electrophoresiies for the thing.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessively unnecessary details,
Will not be described in detail to belonging to known structure or function in following examples.
Approximating language used in following examples can be used for quantitative expression, shows in the feelings not changing basic function
Quantity can be allowed under condition to have certain variation.Therefore, it is not limited to this with the numerical value that the language such as " about ", " left and right " is revised accurate
Numerical value itself.In certain embodiments, " about " represent the numerical value allowing its correction in positive and negative 10 (10%) scope
Interior change, such as, what " about 100 " represented can be any numerical value between 90 to 110.Additionally, " the about first numerical value arrives
In the statement of second value ", at about revise the first and second numerical value two values.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, in following examples, technology used and scientific terminology have and art technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following examples, if no special instructions, is routine biochemistry reagent；Described experiment
Method, if no special instructions, is conventional method；Quantitative test in following examples, is respectively provided with three times and repeats to test, result
Average；% in following examples, if no special instructions, is weight/mass percentage composition.
Embodiment 1
1st, the design of primer
GyrB gene order (KX058388) according to Aeromonas veronii in GenBank and Aha gene order (log in
Number：KU877437) design and filter out two pairs of specific fragment primers.Each primer sequence and expected expanding fragment length such as table 1
Shown：
Table 1 gyrB gene, Aha gene-specific primer sequence and expected amplification
2nd, double PCR reaction system and reaction condition
Reaction system：Testing sample solution 1 μ L, 10 × PCR buffer 2.5 μ L, the rTaq archaeal dna polymerase of 5U/ μ L is molten
Liquid 0.25 μ L, the MgCl of 250mmol/L2The gyrB upstream region of gene primer 0.5 μ L of solution 1.5 μ L, 20 μm of ol/L, 20 μm of ol/L's
The Aha upstream region of gene primer 0.6 μ L of gyrB downstream of gene primer 0.5 μ L, 20 μm of ol/L, the Aha downstream of gene of 20 μm of ol/L draws
DNTP solution 0.5 the μ L, ddH of thing 0.6 μ L, 2.5mmol/L2O polishing is to 25 μ L.
Reaction condition：94 DEG C of denaturations 4min；94 DEG C of degeneration 30s, 61 DEG C of annealing 30s, 72 DEG C of extension 30s, react 30 times
Circulation；72 DEG C re-extend 10min.
3rd, double PCR expanding effect investigates experiment
Respectively using gyrB gene, Aha gene and the two mixture as template execution above-mentioned double PCR amplification, negative
Comparison is with ddH2O is template, and experimental result is as shown in Figure 1.It can be found that the double PCR reaction condition selected by the present embodiment can
Realize the accurate detection of gyrB gene, Aha gene and its mixture, band is consistent with desired location, for gyrB gene and Aha
Gene compound once expands and can assume two band simultaneously.
4th, the specificity verification experiment of double PCR amplification
Respectively to be separated to Aeromonas veronii, slow love moral in Aeromonas veronii type strain ATCC35624, nature
Magnificent bacterium, vibrio parahaemolytious, Aeromonas hydrophila, Salmonella enteritidis, streptococcus agalactiae, Maxwell vibrio, staphylococcus aureuses,
Colon bacillus, Klebsiella Pneumoniae, Shu Baite bacterium solution are template execution above-mentioned double PCR amplification, experimental result such as Fig. 2
Shown.It can be found that be separated to Aeromonas veronii in only Aeromonas veronii type strain ATCC35624 and nature existing
Band simultaneously in 745bp and 419bp, and the two pillar location is consistent；And all band in the amplification of remaining bacterial strain.Thus
It can be seen that the detection method of the present embodiment is good to Aeromonas veronii specificity.
5th, the susceptiveness confirmatory experiment of double PCR amplification
5.1 with the STb gene solution of Aeromonas veronii variable concentrations for sensitivity experiments during template
Extract Aeromonas veronii type strain ATCC35624 STb gene, prepare the STb gene solution of 6.6ng/ μ L, then carry out
Gradient dilution, respectively with each strength solution for template execution above-mentioned double PCR amplification, experimental result is as shown in Figure 3.It can be found that
When DNA concentration as little as 6.6 × 10-3Clear band is still can get it is believed that the method accuracy of detection reaches 10 during ng/ μ L-3ng/μ
The L DNA order of magnitude.
5.2 with Aeromonas veronii variable concentrations bacterium solution for sensitivity experiments during template
Prepare 3.2 × 10 with Aeromonas veronii type strain ATCC356246The bacterium solution of cfu/mL concentration, then carries out ladder
Degree dilution, respectively with the bacterium solution of each concentration for template execution above-mentioned double PCR amplification, experimental result is as shown in Figure 4.It can be found that
When bacterial concentration as little as 3.2 × 102Still bands visible during cfu/mL, and 3.2 × 103The bacterial concentration of cfu/mL can ensure band
Clearly, it can be considered that the method accuracy of detection reaches 3.2 × 102The cell concentration order of magnitude of cfu/mL.
6th, double PCR amplification method is to pathological material of disease Detection results confirmatory experiment
Collect sick fish, be separated to 21 parts of bacterial strain sample, using Aeromonas veronii type strain ATCC35624 as positive quality control
Product, aseptic ddH2O is as negative quality-control product.2 and 11 two parts of sample amplification go out specificity purpose band as a result.As shown in figure 3, its
Middle 1-21 is followed successively by 21 bacterial strains to be detected, and 22 is positive quality control product, and 23 is the amplification of negative quality-control product.By 21 parts of separation
Bacterial strain, after traditional microbial physiology biochemical identification technology combines the identification of 16S rDNA molecular biology method, verifies double
The accuracy of weight PCR detection method is 100%.
Embodiment 2
A kind of detection kit for Aeromonas veronii, this test kit is included for Aeromonas veronii gyrB gene
Pair of primers and for Aeromonas veronii Aha gene pair of primers.
On the basis of above technical scheme, meet following condition：
The described pair of primers for Aeromonas veronii gyrB gene is that upstream as shown in SEQ ID NO.1 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.2.
The described pair of primers for Aeromonas veronii Aha gene is that upstream as shown in SEQ ID NO.3 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.4.
Described test kit also includes 10 × PCR buffer, dNTP, MgCl2, rTaq archaeal dna polymerase, positive quality control product,
Negative quality-control product.
Described test kit is also included as the Aeromonas veronii bacterium solution of positive quality control product with as negative quality-control product
ddH2O.
A kind of method that application mentioned reagent box detects Aeromonas veronii, comprises the following steps：
1) with testing sample solution as pcr template, with DNA fragmentation as shown in SEQ ID NO.1 for the sequence for Vickers gas list
Born of the same parents' bacterium gyrB upstream region of gene primer, with DNA fragmentation as shown in SEQ ID NO.2 for the sequence for Aeromonas veronii gyrB gene under
Trip primer, with DNA fragmentation as shown in SEQ ID NO.3 for the sequence for Aeromonas veronii Aha upstream region of gene primer, with sequence such as
DNA fragmentation shown in SEQ ID NO.4 is the execution double PCR amplification of Aeromonas veronii Aha downstream of gene primer, is expanded
Product；
2) take step 1) amplified production of gained execution agarose gel electrophoresiies, confirm at 745bp in electrophoresis result or
Whether there is band at 419bp.
On the basis of above technical scheme, meet following condition：
Described testing sample solution is the microbial inoculum isolated from Pelteobagrus fulvidraco histoorgan.
The reaction system of described double PCR amplification is as follows：Testing sample solution 1 μ L, 10 × PCR buffer 2.5 μ L,
RTaq archaeal dna polymerase solution 0.25 the μ L, the MgCl of 250mmol/L of 5U/ μ L2Solution 1.5 μ L, the gyrB gene of 20 μm of ol/L
The Aha upstream region of gene primer 0.6 μ L of the gyrB downstream of gene primer 0.5 μ L of forward primer 0.5 μ L, 20 μm of ol/L, 20 μm of ol/L,
DNTP solution 0.5 the μ L, ddH of the Aha downstream of gene primer 0.6 μ L of 20 μm of ol/L, 2.5mmol/L2O polishing is to 25 μ L.
The reaction condition of described double PCR amplification is as follows：94 DEG C of denaturations 4min；94 DEG C of degeneration 30s, 55 DEG C of annealing 30s,
72 DEG C of extension 30s, 35 circulations of reaction；72 DEG C re-extend 10min.
Embodiment 3
A kind of detection kit for Aeromonas veronii, this test kit is included for Aeromonas veronii gyrB gene
Pair of primers and for Aeromonas veronii Aha gene pair of primers.
On the basis of above technical scheme, meet following condition：
The described pair of primers for Aeromonas veronii gyrB gene is that upstream as shown in SEQ ID NO.1 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.2.
The described pair of primers for Aeromonas veronii Aha gene is that upstream as shown in SEQ ID NO.3 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.4.
A kind of method that application mentioned reagent box detects Aeromonas veronii, comprises the following steps：
1) with testing sample solution as pcr template, with DNA fragmentation as shown in SEQ ID NO.1 for the sequence for Vickers gas list
Born of the same parents' bacterium gyrB upstream region of gene primer, with DNA fragmentation as shown in SEQ ID NO.2 for the sequence for Aeromonas veronii gyrB gene under
Trip primer, with DNA fragmentation as shown in SEQ ID NO.3 for the sequence for Aeromonas veronii Aha upstream region of gene primer, with sequence such as
DNA fragmentation shown in SEQ ID NO.4 is the execution double PCR amplification of Aeromonas veronii Aha downstream of gene primer, is expanded
Product；
2) take step 1) amplified production of gained execution agarose gel electrophoresiies, confirm at 745bp in electrophoresis result or
Whether there is band at 419bp.
On the basis of above technical scheme, meet following condition：
The reaction system of described double PCR amplification is as follows：Testing sample solution 1 μ L, 10 × PCR buffer 2.5 μ L,
RTaq archaeal dna polymerase solution 0.25 the μ L, the MgCl of 250mmol/L of 5U/ μ L2Solution 1.5 μ L, the gyrB gene of 20 μm of ol/L
The Aha upstream region of gene primer 0.6 μ L of the gyrB downstream of gene primer 0.5 μ L of forward primer 0.5 μ L, 20 μm of ol/L, 20 μm of ol/L,
DNTP solution 0.5 the μ L, ddH of the Aha downstream of gene primer 0.6 μ L of 20 μm of ol/L, 2.5mmol/L2O polishing is to 25 μ L.
The reaction condition of described double PCR amplification is as follows：94 DEG C of denaturations 4min；94 DEG C of degeneration 30s, 65 DEG C of annealing 30s,
72 DEG C of extension 30s, 35 circulations of reaction；72 DEG C re-extend 10min.
Embodiment 4
A kind of detection kit for Aeromonas veronii, this test kit is included for Aeromonas veronii gyrB gene
Pair of primers and for Aeromonas veronii Aha gene pair of primers.
On the basis of above technical scheme, meet following condition：
The described pair of primers for Aeromonas veronii gyrB gene is that upstream as shown in SEQ ID NO.1 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.2.
The described pair of primers for Aeromonas veronii Aha gene is that upstream as shown in SEQ ID NO.3 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.4.
A kind of method that application mentioned reagent box detects Aeromonas veronii, comprises the following steps：
1) with testing sample solution as pcr template, with DNA fragmentation as shown in SEQ ID NO.1 for the sequence for Vickers gas list
Born of the same parents' bacterium gyrB upstream region of gene primer, with DNA fragmentation as shown in SEQ ID NO.2 for the sequence for Aeromonas veronii gyrB gene under
Trip primer, with DNA fragmentation as shown in SEQ ID NO.3 for the sequence for Aeromonas veronii Aha upstream region of gene primer, with sequence such as
DNA fragmentation shown in SEQ ID NO.4 is the execution double PCR amplification of Aeromonas veronii Aha downstream of gene primer, is expanded
Product；
2) take step 1) amplified production of gained execution agarose gel electrophoresiies, confirm at 745bp in electrophoresis result or
Whether there is band at 419bp.
On the basis of above technical scheme, meet following condition：
The reaction system of described double PCR amplification is as follows：Testing sample solution 1 μ L, 10 × PCR buffer 2.5 μ L,
RTaq archaeal dna polymerase solution 0.25 the μ L, the MgCl of 250mmol/L of 5U/ μ L2Solution 1.5 μ L, the gyrB gene of 20 μm of ol/L
The Aha upstream region of gene primer 0.6 μ L of the gyrB downstream of gene primer 0.5 μ L of forward primer 0.5 μ L, 20 μm of ol/L, 20 μm of ol/L,
DNTP solution 0.5 the μ L, ddH of the Aha downstream of gene primer 0.6 μ L of 20 μm of ol/L, 2.5mmol/L2O polishing is to 25 μ L.
Embodiment 5
A kind of detection kit for Aeromonas veronii, this test kit is included for Aeromonas veronii gyrB gene
Pair of primers and for Aeromonas veronii Aha gene pair of primers.
On the basis of above technical scheme, meet following condition：
The described pair of primers for Aeromonas veronii gyrB gene is that upstream as shown in SEQ ID NO.1 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.2.
The described pair of primers for Aeromonas veronii Aha gene is that upstream as shown in SEQ ID NO.3 for the sequence is drawn
Thing and the downstream primer as shown in SEQ ID NO.4.
A kind of method that application mentioned reagent box detects Aeromonas veronii, comprises the following steps：
1) with testing sample solution as pcr template, with DNA fragmentation as shown in SEQ ID NO.1 for the sequence for Vickers gas list
Born of the same parents' bacterium gyrB upstream region of gene primer, with DNA fragmentation as shown in SEQ ID NO.2 for the sequence for Aeromonas veronii gyrB gene under
Trip primer, with DNA fragmentation as shown in SEQ ID NO.3 for the sequence for Aeromonas veronii Aha upstream region of gene primer, with sequence such as
DNA fragmentation shown in SEQ ID NO.4 is the execution double PCR amplification of Aeromonas veronii Aha downstream of gene primer, is expanded
Product；
2) take step 1) amplified production of gained execution agarose gel electrophoresiies, confirm at 745bp in electrophoresis result or
Whether there is band at 419bp.
Embodiment 6
A kind of detection kit for Aeromonas veronii, this test kit is included for Aeromonas veronii gyrB gene
Pair of primers and for Aeromonas veronii Aha gene pair of primers.
Above embodiments of the invention are described in detail, but described content have been only presently preferred embodiments of the present invention,
Not in order to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should
It is included within protection scope of the present invention.
Claims (10)
1. a kind of detection kit for Aeromonas veronii is it is characterised in that this test kit is included for Aeromonas veronii
The pair of primers of gyrB gene and the pair of primers for Aeromonas veronii Aha gene.
2. a kind of detection kit for Aeromonas veronii according to claim 1 is it is characterised in that described be directed to
The pair of primers of Aeromonas veronii gyrB gene is forward primer as shown in SEQ ID NO.1 for the sequence and such as SEQ ID
Downstream primer shown in NO.2.
3. a kind of detection kit for Aeromonas veronii according to claim 2 is it is characterised in that described be directed to
The pair of primers of Aeromonas veronii Aha gene is forward primer as shown in SEQ ID NO.3 for the sequence and such as SEQ ID NO.4
Shown downstream primer.
4. a kind of detection kit for Aeromonas veronii according to claim 3 is it is characterised in that described reagent
Box also includes 10 × PCR buffer, dNTP, MgCl2, rTaq archaeal dna polymerase, positive quality control product, negative quality-control product.
5. a kind of detection kit for Aeromonas veronii according to claim 4 is it is characterised in that described reagent
Box also includes the Aeromonas veronii bacterium solution as positive quality control product and the ddH as negative quality-control product2O.
6. test kit described in a kind of application claim 4 detects the method for Aeromonas veronii it is characterised in that including following walking
Suddenly：
1) with testing sample solution as pcr template, with DNA fragmentation as shown in SEQ ID NO.1 for the sequence as Aeromonas veronii
GyrB upstream region of gene primer, is drawn with DNA fragmentation as shown in SEQ ID NO.2 for the sequence for Aeromonas veronii gyrB downstream of gene
Thing, with DNA fragmentation as shown in SEQ ID NO.3 for the sequence for Aeromonas veronii Aha upstream region of gene primer, with sequence such as SEQ
DNA fragmentation shown in ID NO.4 is the execution double PCR amplification of Aeromonas veronii Aha downstream of gene primer, obtains amplification and produces
Thing；
2) take step 1) amplified production of gained execution agarose gel electrophoresiies, confirm at 745bp in electrophoresis result or at 419bp
Whether there is band.
7. method according to claim 6 is it is characterised in that described testing sample solution is from Pelteobagrus fulvidraco histoorgan
The microbial inoculum isolated.
8. method according to claim 6 is it is characterised in that the reaction system of described double PCR amplification is as follows：Treat test sample
The rTaq archaeal dna polymerase solution 0.25 μ L of product solution 1 μ L, 10 × PCR buffer 2.5 μ L, 5U/ μ L, 250mmol/L's
MgCl2GyrB downstream of gene primer 0.5 μ of the gyrB upstream region of gene primer 0.5 μ L of solution 1.5 μ L, 20 μm of ol/L, 20 μm of ol/L
The Aha downstream of gene primer 0.6 μ L of the Aha upstream region of gene primer 0.6 μ L of L, 20 μm of ol/L, 20 μm of ol/L, 2.5mmol/L's
DNTP solution 0.5 μ L, ddH2O polishing is to 25 μ L.
9. method according to claim 8 is it is characterised in that the reaction condition of described double PCR amplification is as follows：94 DEG C pre-
Degeneration 4min；94 DEG C of degeneration 30s, 55～65 DEG C of annealing 30s, 72 DEG C of extension 30s, 30～35 circulations of reaction；72 DEG C re-extend
10min.
10. it is characterised in that the temperature of described annealing is 61 DEG C, reaction cycle number of times is method according to claim 9
30 times.
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| CN109554449A (en) * | 2019-01-18 | 2019-04-02 | 集美大学 | A kind of multiple PCR method that can detect 7 virulence genes of Aeromonas simultaneously |
| CN110106268A (en) * | 2019-05-28 | 2019-08-09 | 电子科技大学中山学院 | A kind of the LAMP detection primer composition and kit of Aeromonas veronii |
| CN115976237A (en) * | 2022-09-28 | 2023-04-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Specific new molecular target for identifying aeromonas and rapid detection method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109554449A (en) * | 2019-01-18 | 2019-04-02 | 集美大学 | A kind of multiple PCR method that can detect 7 virulence genes of Aeromonas simultaneously |
| CN109554449B (en) * | 2019-01-18 | 2022-02-22 | 集美大学 | Multiplex PCR method capable of simultaneously detecting 7 virulence genes of aeromonas |
| CN110106268A (en) * | 2019-05-28 | 2019-08-09 | 电子科技大学中山学院 | A kind of the LAMP detection primer composition and kit of Aeromonas veronii |
| CN115976237A (en) * | 2022-09-28 | 2023-04-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Specific new molecular target for identifying aeromonas and rapid detection method thereof |
| CN115976237B (en) * | 2022-09-28 | 2023-08-22 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Specific new molecular target for identifying aeromonas and rapid detection method thereof |
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