CN105132411A - Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri - Google Patents
Kit and detection method for simultaneously detecting vibrio bacteria and edwardsiella ictaluri Download PDFInfo
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- CN105132411A CN105132411A CN201510349562.1A CN201510349562A CN105132411A CN 105132411 A CN105132411 A CN 105132411A CN 201510349562 A CN201510349562 A CN 201510349562A CN 105132411 A CN105132411 A CN 105132411A
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Abstract
The invention discloses primer pairs shown in SEQ ID NO.1-2 and SEQ ID NO.3-4 and an application thereof, and further, discloses a kit and detection method for detecting vibrio bacteria and edwardsiella ictaluri. The kit and the method can amplify vibrio bacteria and edwardsiella ictaluri gene fragments respectively and simultaneously, achieve one-step detection of two pathogenic bacteria in a same reaction system, have the advantages of strong specificity, high sensitivity, short consuming time, simple steps, small workload, and low cost, are used for rapid detection of culture water bodies, can timely and effectively prevent happening of fish diseases, and have good application prospects.
Description
Technical field
The present invention relates to the detection technique of a kind of bacterium, particularly detect vibrio bacteria with the test kit of Channel-catfish tarda and detection method simultaneously.
Background technology
Pelteobagrus is in SILURIFORMES, and Chang section, Pelteobagrus, has another name called yellow peppery fourth, and its fine and tender taste, lipid content are low, protein content is high and deeply like by consumers in general.But along with the cultivation expansion of scale, the raising of cultivation density, breeding environment is constantly worsened, in breeding process, constantly breaks out disease, bring tremendous economic to lose to raiser.Wherein to have developed into the principal causative of Yellow catfish former for vibrio bacteria, Channel-catfish tarda.
Channel-catfish tarda, belongs to enterobacteriaceae Edwardsiella bacterium, " splits a disease " can cause Yellow catfish; In vibrio bacteria, as Vibrio anguillarum, Vibrio mimicus etc., the Yellow catfish vibriosis ， caused is similar to the symptom that Channel-catfish tarda causes.
At present, to vibrio bacteria with the detection of Channel-catfish tarda is all detect respectively, once can only detect a kind of pathogenic agent, be unfavorable for the detection of polyinfection, and need large batch of sample to detect, workload is comparatively large, and cost is also higher.
At present, have no and detect vibrio bacteria with the report of Channel-catfish tarda simultaneously.
Summary of the invention
In order to solve the problem, the invention provides a kind of detection vibrio bacteria simultaneously with the test kit of Channel-catfish tarda and detection method.
The present invention provide firstly the primer pair shown in SEQIDNO.1 ~ 2 Yu SEQIDNO.3 ~ 4.
Present invention also offers the primer pair shown in SEQIDNO.1 ~ 2 and SEQIDNO.3 ~ 4 preparation simultaneously detect vibrio bacteria He Channel-catfish tarda reagent in purposes.
The present invention detects vibrio bacteria simultaneously with the test kit of Channel-catfish tarda, and it comprises the primer pair shown in SEQIDNO.1 ~ 2 and SEQIDNO.3 ~ 4.
The present invention detects vibrio bacteria with the method for Channel-catfish tarda simultaneously, and it comprises the steps:
(1) extract sample DNA: get sample to be checked, extract DNA wherein;
(2) gene amplification: increase with the DNA that test kit according to claim 4 is treated in sample basis;
(3) result detects: detect DNA cloning result.
Wherein, described sample to be checked is aquaculture water.
Test kit provided by the invention and method can increase vibrio bacteria with the gene fragment of Channel-catfish tarda simultaneously respectively, achieve a step in same reaction system and detect two kinds of pathogenic bacterias, and can not influence each other therebetween, high specificity, highly sensitive, consuming time short, step is simple, under workload, with low cost, for rapid detection aquaculture water, the generation of fish disease can be prevented timely and effectively, have a good application prospect.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.The technology that all contents recorded based on claims of the present invention realize all belongs to scope of the present invention.
Accompanying drawing explanation
Fig. 1 double PCR primer test electrophorogram.1:MarkerI; 2: blank; 3: tarda; 4: vibrios; 5: tarda+vibrios; 6: tarda+vibrios
Fig. 2 double PCR specificity test electrophorogram.1:MarkerI; 2: streptococcus agalactiae; 3: Streptococcus iniae; 4: Pseudomonas fluorescens; 5: Aeromonas hydrophila; 6: flavobacterium columnare; 7: standard Aeromonas caviae; 8: flavobacterium columnare; 9: Salmonellas; 10: genus bacillus; 11: Aeromonas sobria; 12: germ oligotrophy unit cell; 13: Vibrio parahaemolyticus; 14: positive.
Fig. 3 double PCR sensitivity test electrophorogram.1：100；2：10
-1；3：10
-2；4：10
-3；5：10
-4；6：10
-5；7：10
-6；8：10
-7；9：MarkerI。
Fig. 4 actual sample detects electrophorogram.1: negative control; 2-4 is No. 1 fish tissues: body surface muscle, body surface muscle, abdominal muscles; 5-7 is No. 3 fish tissues: body surface muscle, body surface muscle, abdominal muscles; 8-10 is No. 2 fish tissues: body surface muscle, body surface muscle, abdominal muscles; 11:MarkerI.
Embodiment
One, experiment material and instrument
1.1 bacterial isolates
(Tong Wei Instituut Voor Veehouderij En Diergezondheid (Id-Dlo) is separated streptococcus agalactiae, preserve), (Sichuan Agricultural University is separated Streptococcus iniae, Tong Wei Instituut Voor Veehouderij En Diergezondheid (Id-Dlo) preserves), Pseudomonas fluorescens (aquatic institute of the Chinese Academy of Sciences, 56-12-10), (Sichuan Agricultural University is separated Aeromonas hydrophila, Tong Wei Instituut Voor Veehouderij En Diergezondheid (Id-Dlo) preserves), flavobacterium columnare (aquatic institute of Chinese Academy of Sciences G4), standard Aeromonas caviae (aquatic institute of the Chinese Academy of Sciences, DMA1-A), Salmonellas (ATCC13311), genus bacillus (CICC1.126), Aeromonas sobria (ATCC43979), (Sichuan Agricultural University is separated germ oligotrophy unit cell, Tong Wei Instituut Voor Veehouderij En Diergezondheid (Id-Dlo) preserves), tarda (ATCC33202), Vibrio parahaemolyticus (CICC10552).
1.2 key instruments and reagent
Regular-PCR instrument (Mycycler, Bio-RAD), gel imaging system (universalHOOD-SN, Beijing 6 1), high speed desktop refrigerated centrifuge (KDC-160HR, University of Science and Technology innovate), electrophoresis apparatus (PowerPAC3000, Bio-RAD), electric heating constant temperature shaking table (SKY-211B, Shanghai Su Kun), microsyringe (10 μ L, 100 μ L, 200 μ L, 1000 μ L, Eppendorf), Bechtop (SW-CJ-2FD, Suzhou purify), electro-heating standing-temperature cultivator (SPX-150B, PVG Rong Feng).
Bacterial genomes DNA extraction kit (Tian Gen biochemical technology company limited); 2 × PCRMIX (Tian Gen biochemical technology company limited), Greenview (BioBRK), agarose (Sigma).
Embodiment 1 design of primers
One, experimental technique
1, PCR primer Design and synthesis
According to the total rpoA gene order Yi of vibrio bacteria Ji Channel-catfish tarda Phosphoserine aminotransferase gene order, design two pairs of primers (table 1) respectively.Primer is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Table 1PCR primer data
2, Template preparation
Activation tarda, Vibrio parahaemolyticus, and the bacterial genomes DNA extraction kit using Tian Gen biochemical technology company limited to produce is extracted.
3, pcr amplification
Using the DNA of step 2 extraction as template, the template of negative control is ddH
2o, the two pairs of primers got in step 1 increase respectively according to following system and program:
PCR reaction system (20 μ l):
PCR response procedures:
After mixing PCR reaction system, be placed in PCR instrument, after 95 DEG C of denaturation 4min, circulate as follows:
94℃30sec
58℃30sec
72℃1min
35 circulations, last 72 DEG C extend 5min.
4, result detects
Adopt the sepharose of 1.5%, electrophoresis 60min under constant voltage 100V condition, observes pcr amplification result under being placed in gel imaging system.
Two, result
Result as shown in Figure 1, blank is without amplified band, during with tarda DNA for template, the fragment of 124bp size can be amplified, with Vibrio parahaemolyticus DNA for the fragment of 524bp size can be amplified during template, during with the mixture of tarda and Vibrio parahaemolyticus for template, the fragment of storage 524bp, 124bp size that can simultaneously increase, consistent with expected results (as Fig. 1).
Experiment proves, primer pair shown in SEQIDNO.1 ~ 2 of the present invention and SEQIDNO.3 ~ 4 can distinguish the vibrio bacteria that simultaneously accurately increases with the gene of Channel-catfish tarda, and does not mutually disturb between two pairs of primers.
Embodiment 2 primer specificity is tested
One, test method
1, PCR primer
The shown two pairs of primers of table 1 in Example 1.
2, Template preparation:
Get the genomic DNA as template of streptococcus agalactiae, Streptococcus iniae, Pseudomonas fluorescens, Aeromonas hydrophila, flavobacterium columnare, standard Aeromonas caviae, flavobacterium columnare, Salmonellas, genus bacillus, Aeromonas sobria respectively, the bacterial genomes DNA extraction kit using Tian Gen biochemical technology company limited to produce is extracted.
3, pcr amplification
Get the genomic DNA as template that step 2 obtains respectively, the template of negative control is ddH
2o, using the mixture of 2.5 μ l tarda DNA and 2.5 μ l Vibrio parahaemolyticus DNA as positive template, increases according to amplification program in embodiment 1.
4, result detects
Adopt the sepharose of 1.5%, electrophoresis 60min under constant voltage 100V condition, observes pcr amplification result under being placed in gel imaging system.
Two, result
Result as shown in Figure 2, except positive (tarda and vibrios) amplifies except the target band of 524bp, 124bp size, all the other test strain streptococcus agalactiaes, Streptococcus iniae, Pseudomonas fluorescens, Aeromonas hydrophila, flavobacterium columnare, standard Aeromonas caviae, flavobacterium columnare, Salmonellas, genus bacillus, Aeromonas sobria, germ oligotrophy unit cell are all without band.
Experiment proves, detection kit provided by the invention and method high specificity, can increase vibrio bacteria exactly with the gene of Channel-catfish tarda simultaneously, and other bacteriums of can not increasing.
Embodiment 3 primer sensitivity test
One, test method
1, PCR primer
The shown two pairs of primers of table 1 in Example 1.
2, Template preparation:
Get positive DNA (mixtures of 2.5 μ l tarda DNA and 2.5 μ l Vibrio parahaemolyticus DNA), carry out concentration determination, and according to 10 of original content
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7,10
-8, 10
-9doubly carry out doubling dilution.
3, pcr amplification
Get the genomic DNA as template that step 2 obtains respectively, the template of negative control is ddH
2o, increases according to amplification program in embodiment 1.
4, result detects
Adopt the sepharose of 1.5%, electrophoresis 60min under constant voltage 100V condition, observes pcr amplification result under being placed in gel imaging system.
Two, result
As shown in Figure 3, according to original content 10
-3when carrying out doubling dilution, still can effectively detect.That is, adopt the method set up to carry out pcr amplification, can detect that the minimum template concentrations of the nucleic acid of tarda and Vibrio parahaemolyticus is respectively 8.333ng/ μ l and 20.825ng/ μ l.
Experiment prove, detection kit provided by the invention and method highly sensitive, can detect that the minimum template concentrations of the nucleic acid of tarda and Vibrio parahaemolyticus is respectively 8.333ng/ μ l and 20.825ng/ μ l.The detection of embodiment 4 actual sample
One, experimental technique
Sample to be checked: the channel catfish (picking up from Leshan) choosing three censorships, wherein two is disease fish, and they have manifest symptom (body surface has the stove that festers, and belly is congested), are numbered 1,2; Another be healthy with, non-evident sympton, is numbered 3.Get muscle 3 place (body surface fester 2 places, congested 1 place of belly) of 1, No. 2 fish, get muscle 3 place (body surface muscle 2 place, abdominal muscles 1 place) of No. 3 fishes.
1, PCR detects
(1) PCR primer
The shown two pairs of primers of table 1 in Example 1.
(2) Template preparation
Utilize chelex-100 to complete the extraction of got tissue DNA, and carry out PCR detection as template.
(3) pcr amplification
Get above-mentioned DNA respectively as template, the template of negative control is ddH
2o, increases according to amplification program in embodiment 1.
(4) result detects
Adopt the sepharose of 1.5%, electrophoresis 60min under constant voltage 100V condition, observes pcr amplification result under being placed in gel imaging system.
Two, result
As shown in Figure 3, result shows, and No. 1,2, the channel catfish of censorship all detects vibrios, Channel-catfish tarda, and No. 3 are not detected target cause of disease, consistent with practical situation.
Experimental result illustrates, test kit of the present invention and method accurately can detect vibrio bacteria with Channel-catfish tarda.
To sum up, test kit of the present invention and method can increase vibrio bacteria with the gene fragment of Channel-catfish tarda simultaneously respectively, achieve a step in same reaction system and detect two kinds of pathogenic bacterias, high specificity, highly sensitive, consuming time short, step is simple, under workload, with low cost, for rapid detection aquaculture water, the generation of fish disease can be prevented timely and effectively, have a good application prospect.
Claims (5)
1.SEQIDNO.1 the primer pair shown in ~ 2 Yu SEQIDNO.3 ~ 4.
Primer pair shown in 2.SEQIDNO.1 ~ 2 and SEQIDNO.3 ~ 4 preparation simultaneously detect vibrio bacteria He Channel-catfish tarda reagent in purposes.
3. detect a test kit for vibrio bacteria and Channel-catfish tarda simultaneously, it is characterized in that: it comprises the primer pair shown in SEQIDNO.1 ~ 2 and SEQIDNO.3 ~ 4.
4. detect a method for vibrio bacteria and Channel-catfish tarda simultaneously, it is characterized in that: it comprises the steps:
(1) extract sample DNA: get sample to be checked, extract DNA wherein;
(2) gene amplification: increase with the DNA that test kit according to claim 4 is treated in sample basis;
(3) result detects: detect DNA cloning result.
5. method according to claim 4, is characterized in that: described sample to be checked is aquaculture water.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111273007A (en) * | 2020-03-13 | 2020-06-12 | 内江师范学院 | Kit and detection method for rapidly detecting fish Edwardsiella ictaluri |
Citations (5)
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| CN102382881A (en) * | 2011-11-03 | 2012-03-21 | 通威股份有限公司 | Kit and method for detecting fish pathogenic bacteria |
| CN103060186A (en) * | 2012-12-24 | 2013-04-24 | 山东省海水养殖研究所 | Aquatic product Edwardsiella tarda rapid drug allergy detection kit |
| CN103333946A (en) * | 2013-03-29 | 2013-10-02 | 广西壮族自治区水产研究所 | Rapid detection method for vibrio vulnificus and vibrio harveyi |
| CN103388029A (en) * | 2013-08-08 | 2013-11-13 | 通威股份有限公司 | Detection kit and detection method for vibrio bacteria |
| CN104017873A (en) * | 2014-05-30 | 2014-09-03 | 浙江万里学院 | A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof |
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- 2015-06-23 CN CN201510349562.1A patent/CN105132411A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102382881A (en) * | 2011-11-03 | 2012-03-21 | 通威股份有限公司 | Kit and method for detecting fish pathogenic bacteria |
| CN103060186A (en) * | 2012-12-24 | 2013-04-24 | 山东省海水养殖研究所 | Aquatic product Edwardsiella tarda rapid drug allergy detection kit |
| CN103333946A (en) * | 2013-03-29 | 2013-10-02 | 广西壮族自治区水产研究所 | Rapid detection method for vibrio vulnificus and vibrio harveyi |
| CN103388029A (en) * | 2013-08-08 | 2013-11-13 | 通威股份有限公司 | Detection kit and detection method for vibrio bacteria |
| CN104017873A (en) * | 2014-05-30 | 2014-09-03 | 浙江万里学院 | A multiple PCR primer used for simultaneously detecting five pathogenic bacteria and three virulence genes in marine, and a designing method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111273007A (en) * | 2020-03-13 | 2020-06-12 | 内江师范学院 | Kit and detection method for rapidly detecting fish Edwardsiella ictaluri |
| CN111273007B (en) * | 2020-03-13 | 2023-08-01 | 内江师范学院 | Kit for rapidly detecting Edwardsiella ictaluri and detection method |
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Application publication date: 20151209 |