CN102140513A - Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof - Google Patents
Primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and detection method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to primer sequences for detecting Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda, and a detection method and application thereof. In the method for polymerase chain reaction (PCR) detection through primers, primers for detecting pathogenic and nonpathogenic Edwardsiella and a method for single or multiplex amplification detection by applying the primers are further related; and the invention further relates to a kit comprising the primers and used for detecting the Edwardsiella tarda, Edwardsiella ictarda or pathogenic Edwardsiella tarda. By adopting multiplex PCR amplification, more than two pairs of primers are present in the same reaction system and can amplify more than two target genes, so that the multiplex PCR is reagent-saving, time-saving and labor-saving than single PCR; therefore, the cost is reduced and the efficiency is improved.
Description
Technical field
The present invention relates to biological technical field, relate to a kind of primer sequence and detection method and application that detects Edwardsiella tarda, catfish tarda or pathogenic Edwardsiella tarda particularly.
Background technology
Edwardsiella tarda (Edwardsiella tarda) is the hydrocoles common pathogenic bacteria, the host range of its infection is very extensive, batrachians (toad, the frog), reptiles (lizard, snake, green turtle, crocodile), fish (eel, catfish, flounder etc.), birds (penguin, American eagle, ostrich) is arranged and comprise people's Mammals (sea lion, cowfish, pig, dog, ox, monkey).Edwardsiella tarda can infect multiple wild and cultured fishes in seawater and fresh water environment existence, causes with the hueppe's disease to be the Edwardsiella disease (Edwardsiellosis) of cardinal symptom.Fast development along with China's aquaculture, the Wdwardsiella tarda disease not only influences the breed of multiple economic freshwater animal (common eel, soft-shelled turtle, bullfrog, crucian, grass carp etc.), also have a strong impact on the breed of multiple seawater economic fish (turbot, lefteye flounder, tongue sole, lithosporic, large yellow croaker etc.), cause enormous economic loss to aquaculture.Edwardsiella tarda be unique in the Edwardsiella can infected person, make the ill cause of disease of people, can be by approach infected person such as digestive tube and skin wounds, cause meningitis, cholecystitis, endocarditis, osteomyelitis, soft tissue infection, empyema, microbemia and septicemia, dysentery, gastroenteritis etc., harm humans health.The sick catalogue of planting of China's animal epidemic is classified Edwardsiella tarda as harmful organism, is the microorganism that China and other countries must quarantine in the fishery products foreign trade.Therefore, setting up the evaluation and the detection method of Edwardsiella tarda, the monitoring of setting up eqpidemic disease and pre-alarming system, is Comprehensive Control Edwardsiella disease, the needs that guarantee aquaculture healthy and sustainable development and aquatic product quality and public health security.
The evaluation of cause of disease and detection method have conventional Physiology and biochemistry method, immunological method and based on the molecular biology method of polymerase chain reaction (PCR).Conventional Physiology and biochemistry method is loaded down with trivial details time-consuming, and the immunological method detection sensitivity is low, specificity is not high, is difficult to satisfy disease epidemic situation complicated and changeable and handles needs.PCR method weak point detection time, high specificity, highly sensitive are the effective ways that detect cause of disease at present fast and accurately; Simultaneously, the multiplex PCR that grows up on the regular-PCR basis (multiplex PCR) can detect and analyze more target gene, has improved detection efficiency greatly, has reduced the detection cost, becomes the main means of medical science and veterinary science clinical diagnosis.
Tarda is the normal microflora in the water surrounding microecosystem, is present in settling, animals and plants surface and some animal digestive tracts of water surrounding, has pathogenic and two kinds of bions of non-virulent.When breeding environment discomfort or the reduction of susceptible animal immunizing power, the easy infection animal of pathogenic bacteria causes disease to take place.Differentiate that correctly pathogenic and nonpathogenic edwarsiella tarda is the important prerequisite that accurately diagnoses the illness, formulates the effective prophylactico-therapeutic measures of eqpidemic disease.Another of Edwardsiella kind of bacterium-catfish tarda (Edwardsiella ictaluri) also is the important pathogen of fish, and this cause of disease is survived in fresh water environment, can infect multiple freshwater fish.According to whole genome sequence comparative analysis result, the genome sequence similarity degree of Edwardsiella bacterium is very high, and the method for the PCR-based of report is used for detecting tarda and has following problem at present: (1) can not accurately differentiate the Edwardsiella bacterium from other microorganism on the level of planting; (2) can not accurately distinguish Edwardsiella tarda and catfish tarda on the bacterium of Edwardsiella; (3) can not accurately distinguish pathogenic and Edwardsiella bacterium non-virulent.At present increasing microorganism whole genome sequence obtains annotating, the difference of microorganism hereditary information and the biological function of microorganism can be in depth illustrated in research by comparative genomics and functional genomics comprehensively, also identify for microbial molecular and discriminating provides information accurately.Up to now, the whole genome sequence of an existing slow Edward of two strains and a strain catfish tarda obtains measuring, and the mechanism of causing a disease of tarda is obtaining illustrating progressively.Analysis by comparative genomics analysis and pathogenic functional gene, both there be the genetic information difference between planting in slow Edward and catfish tarda, also there is the identical gene information of function, differentiates the detection method of slow Edward and catfish tarda accurately for foundation and lay a good foundation.
Summary of the invention
The object of the invention is to provide a kind of primer sequence and detection method thereof that detects Edwardsiella tarda, catfish tarda or pathogenic Edwardsiella tarda.
For achieving the above object, the technical solution used in the present invention is: the primer sequence that detects Edwardsiella tarda: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQID No.4, described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ IDNO.2:TCGTTCTGGGTGCAATCC; SEQ ID NO.3:CCTGGCGTTCCACCACTATGT; SEQID NO.4:GCCTTGATGACTATGCCGTTC.
Detect the primer sequence of catfish tarda: primer sequence is to SEQ ID No.1 and SEQ IDNo.2, SEQ ID No.3 and SEQ ID No.4; Described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ ID NO.3:CCTGGCGTTCCACCACTATGT; SEQ ID NO.4:GCCTTGATGACTATGCCGTTC.
Detect the primer sequence of pathogenic Edwardsiella tarda: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6; Described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ ID NO.3:CCTGGCGTTCCACCACTATGT; SEQ ID NO.4:GCCTTGATGACTATGCCGTTC; SEQ ID NO.5:GGTCAATAGCTGGCTACACAA; SEQ IDNO.6:GCGCCTCAGCGAGTATGCGAT.
Detect the primer sequence of pathogenic catfish tarda: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6; Described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ ID NO.3:CCTGGCGTTCCACCACTATGT; SEQ ID NO.4:GCCTTGATGACTATGCCGTTC; SEQ ID NO.5:GGTCAATAGCTGGCTACACAA; SEQ IDNO.6:GCGCCTCAGCGAGTATGCGAT.
Detect the detection method of the primer sequence of Edwardsiella tarda: with SEQ ID No.1 and SEQID No.2, SEQ ID No.3 and SEQ ID No.4 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 500bp appears in amplified production segment section, be the Edwardsiella tarda positive.
Detect the detection method of catfish tarda: with SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 500bp and 323bp appears in amplified production segment section simultaneously, be the catfish tarda positive.
Detect the detection method of pathogenic Edwardsiella tarda: with SEQ ID No.1 and SEQ IDNo.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 955bp and 500bp appears in amplified production segment simultaneously, be the pathogenic Edwardsiella tarda positive.
Detect the detection method of pathogenic catfish tarda: with SEQ ID No.1 and SEQ IDNo.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 955bp, 500bp, 323bp appears in amplified production segment simultaneously, be the pathogenic catfish tarda positive.
Detect the application of the primer sequence of Edwardsiella tarda: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of discriminating Edwardsiella tarda of animal, fishery products.
Detect the application of the primer sequence of catfish tarda: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of discriminating catfish tarda of animal, fishery products.
Detect the application of the primer sequence of pathogenic Edwardsiella tarda: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of the pathogenic Edwardsiella tarda of discriminating of animal, fishery products.
Detect the application of the primer sequence of pathogenic catfish tarda, it is characterized in that: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of the pathogenic catfish tarda of discriminating of animal, fishery products.The DNA band of the 500bp that amplifies by primer sequence SEQ ID No.1-4 of the present invention is Edwardsiella tarda and the conservative rpoS gene fragment of catfish tarda, and the DNA band of 323bp is the distinctive band of catfish tarda, in Edwardsiella tarda, do not have, the present invention utilizes the multiple PCR method of foundation, according to the appearance of above-mentioned two kinds of DNA specific bands, can detect and distinguish Edwardsiella tarda and catfish tarda.DNA band by primer sequence SEQ IDNo.5 of the present invention and 6 955bp that amplify is pathogenic Edwardsiella tarda and the conservative esaV gene fragment of pathogenic catfish tarda, the present invention utilizes the multiple PCR method of foundation, according to the appearance of above-mentioned three kinds of DNA bands, can detect and distinguish pathogenic Edwardsiella tarda and pathogenic catfish tarda.
The advantage that the present invention had:
The regular-PCR amplification has a pair of primer in same reaction system, and a goal gene can only increase; The present invention adopts multiplex PCR amplification in same reaction system primer more than two pairs to be arranged, 2 the above goal gene that can increase, thereby multiplex PCR more saves reagent, time and workload than substance PCR, thus reduce cost, raise the efficiency.Primer sequence provided by the invention and multiplex PCR specific amplification are strong, highly sensitive, can be from the various bacteria microorganism rapid detection Edwardsiella tarda, and further distinguish pathogenic and non-pathogenic bacterial strains, in 3 hours, finish simultaneously Edwardsiella tarda kind, the discriminating of pathotype and non-pathogenic type detects, the detection sensitivity of each amplified reaction is 10～100 bacteriums.Detection method provided by the invention can be applicable to multiple analysis and the detection that contains the Edwardsiella tarda sample, water, settling, animal (fish, shrimp, shellfish, soft-shelled turtle, bullfrog, zooplankton etc.) and movement thereof, plant (plant plankton, algae etc.) as aquatic environment, import and export aquatic animals and plants, fishery products, the patient suspected's of hospital vomitus, intestines hydrops, movement etc.Detection kit provided by the invention is special, sensitive, quick and easy, can be used for aquaculture, commodity inspection, the hospital diagnosis to the tarda disease, in clinical diagnosis and disease controlling party mask very big using value is arranged.
Description of drawings
The electrophoresis colour developing figure of Edwardsiella tarda, wherein M:Trans2K Plus II DNA marker (Beijing Quanshijin Biotechnology Co., Ltd) are differentiated and detected to the multiplex PCR that Fig. 1 provides for the embodiment of the invention; C: blank (not adding any dna profiling); Swimming lane 1-36 is Edwardsiella tarda (E.tarda), 1-36 as shown in table 1; Swimming lane 37-66 is other kind bacterioid, 37-66 as shown in table 1; Swimming lane 67 is catfish tarda (E.ictaluri), as shown in table 1 67.
The substance PCR that Fig. 2 provides for the embodiment of the invention differentiates the electrophoresis colour developing figure of pathogenic Edwardsiella tarda, wherein M:Trans 2K Plus II DNA marker (Beijing Quanshijin Biotechnology Co., Ltd); C: blank (not adding any dna profiling); Swimming lane 1-9 is respectively pathogenic Edwardsiella tarda, 1-9 as shown in table 1; Swimming lane 10-21 is respectively nonpathogenic edwarsiella tarda, 19-30 as shown in table 1.
The multiplex PCR that Fig. 3 provides for the embodiment of the invention is differentiated the electrophoresis colour developing figure of pathogenic Edwardsiella tarda, wherein M:Trans2K Plus II DNA marker (Beijing Quanshijin Biotechnology Co., Ltd); C: blank (not adding any dna profiling); Swimming lane 1-9 is respectively pathogenic Edwardsiella tarda, 1-9 as shown in table 1; Swimming lane 10-12 is a nonpathogenic edwarsiella tarda, 19-21 as shown in table 1; Swimming lane 13 is pathogenic Edwardsiella tarda, as shown in table 1 10; Swimming lane 14-28 is non-Edwardsiella tarda, 22-36 as shown in table 1; Swimming lane 29-36 is pathogenic Edwardsiella tarda, 11-18 as shown in table 1; Swimming lane 37-44 is other kind bacterioid, 37-44 as shown in table 1; Swimming lane 45 is catfish tarda (E.ictaluri), as shown in table 1 67.
Whether the analysis lefteye flounder cultivating pool water sample that Fig. 4 provides for the embodiment of the invention has the electrophoresis colour developing figure of Edwardsiella tarda, wherein M:Trans 2K Plus II DNA marker (Beijing Quanshijin Biotechnology Co., Ltd); 1～12: water sample 1～12; 13: blank (not adding any dna profiling).
The electrophoresis colour developing figure of the analysis batch production turbot aquaculture water water reservoir water sample that Fig. 5 provides for the embodiment of the invention, wherein M:Trans2K Plus II DNA marker (Beijing Quanshijin Biotechnology Co., Ltd); 1～7: the A～G of plant; 8: blank (not adding any dna profiling)
The electrophoresis colour developing figure of the analysis batch production turbot aquaculture water water reservoir bed mud that Fig. 6 provides for the embodiment of the invention, wherein M:Trans 2K Plus II DNA ma rke r (Beijing Quanshijin Biotechnology Co., Ltd); 1～7: the A～G of plant; 8: blank (not adding any dna profiling).
Fig. 7 provides the electrophoresis colour developing figure that analyzes the industrialized culture turbot, wherein M:Trans2K Plus II DNA marker (Beijing Quanshijin Biotechnology Co., Ltd) for the embodiment of the invention; 1～8: nephridial tissue 9: blank (not adding any template).
Embodiment
Differentiate fast and detect Edwardsiella tarda:
Present embodiment utilizes multiple PCR method, in order in a PCR reaction tubes, to react by a PCR, from the diagnosis sample that contains Edwardsiella tarda, differentiate fast and detect Edwardsiella tarda, differentiate fast in each bacterial strain that promptly from table 1, provides and detect Edwardsiella tarda, wherein said each bacterial strain all can obtain from the circulation of market referring to the report in the pertinent literature, provide preferred primer to SEQ ID No.1 and SEQ ID No.2 for this reason, SEQ ID No.3 and SEQ ID No.4, the preferred PCR reaction system that provides is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/L SEQ ID No.1,0.4 μ M/L SEQ ID No.2,0.2 μ M/L SEQ ID No.3,0.2 μ M/L SEQ ID No.4,1.0 μ l templates are added distilled water to 25 μ l.Provide preferred PCR reaction conditions to be: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds, 60.6～60.8 ℃ of annealing 45 seconds, 68 ℃～72 ℃ were extended 1.5～2.5 minutes; Carry out 30～35 circulations; 72 ℃ are extended 5～10min.Provide preferred qualitative observation procedure to be: amplified production to be carried out 1～1.5% agarose gel electrophoresis, show the amplified production size with ethidium bromide staining, observe amplified production segment size and have only the sample of 500bp to be judged to be the Edwardsiella tarda positive, as the swimming lane 1-36 of Fig. 1; That observes that amplified fragments has 500bp and 323bp simultaneously is judged to be the catfish tarda positive, as the swimming lane 67 of Fig. 1; The above-mentioned any segmental Edwardsiella tarda feminine gender that is judged as that do not increase is as the 37-66 swimming lane (referring to table 1) of Fig. 1.
Primer is to being respectively:
SEQ?ID?NO.1：GGATGAGGCATCGCGTAAG
SEQ?ID?NO.2：TCGTTCTGGGTGCAATCC
SEQ?ID?NO.3：CCTGGCGTTCCACCACTATGT
SEQ?ID?NO.4：GCCTTGATGACTATGCCGTTC。
Differentiate pathogenic bacterial strains from the Edwardsiella tarda bacterial strain, the Edwardsiella tarda that promptly filters out from embodiment 1 is positive to be template.
Present embodiment utilizes the substance PCR method, in order to differentiate pathogenic bacterial strains from the Edwardsiella tarda bacterial strain, the preferred sequence primer that for this reason provides is to SEQ ID No.5 and SEQ ID No.6, and the preferred PCR reaction system that provides is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/L SEQ ID No.5,0.4 μ M/L SEQ ID No.6,1.0 μ, 1 template is added distilled water to 25 μ l.Provide preferred PCR reaction conditions to be: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds; Annealed 45 seconds for 50～62 ℃; 72 ℃ were extended 50 seconds～1 minute and 30 seconds; Carry out 30～35 circulations; 72 ℃ are extended 5～10min.Provide preferred qualitative observation procedure to be: amplified production to be carried out 1～1.5% agarose gel electrophoresis, show the amplified production size with ethidium bromide staining, observe amplified production segment size and have only the sample of 955bp to be judged to be the Edwardsiella tarda positive, as the 1-9 swimming lane of Fig. 2; Any segmental Edwardsiella tarda feminine gender that is judged to be that do not increase is as the 10-21 swimming lane of Fig. 2.
Primer is to being respectively:
SEQ?ID?NO.5：GGTCAATAGCTGGCTACACAA
SEQ?ID?NO.6：GCGCCTCAGCGAGTATGCGAT。
Simultaneously also can utilize multiple PCR method, in order in a PCR reaction tubes, to react by a PCR, differentiate and detect pathogenic Edwardsiella tarda from other microorganism fast, the preferred primer that for this reason provides is SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.The preferred multi-PRC reaction system that provides is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/L SEQ ID No.1,0.4 μ M/L SEQ ID No.2,0.2 μ M/L SEQ ID No.3,0.2 μ M/L SEQ ID No.4,0.4 μ M/L SEQ ID No.5,0.4 μ M/L SEQ ID No.6,1.0 μ l templates are added distilled water to 25 μ l.Provide preferred PCR reaction conditions to be: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds, 60.6～60.8 ℃ of annealing 45 seconds, 68 ℃～72 ℃ were extended 1.5～2.5 minutes; Carry out 30～35 circulations; 72 ℃ are extended 5～10mi n.Provide preferred qualitative observation procedure to be: amplified production to be carried out 1～1.5% agarose gel electrophoresis, show the amplified production size with ethidium bromide staining, observe the big or small sample of amplified production segment simultaneously and be judged to be the pathogenic Edwardsiella tarda positive, as swimming lane 1-9,13, the 29-36 of Fig. 3 for 955bp and 500bp; Only observe amplified production segment size and be judged to be the nonpathogenic edwarsiella tarda positive, as swimming lane 10-12, the 14-28 of Fig. 3 for the sample of 500bp; Observe the big or small sample of amplified production segment simultaneously and be judged to be Edwardsiella tarda feminine gender, the pathogenic catfish tarda positive, as the swimming lane 45 of Fig. 3 for 955bp, 500bp, 323bp; The above-mentioned any segmental Edwardsiella tarda feminine gender that is judged as that do not increase is as the swimming lane 37-44 (referring to table 1) of Fig. 3.
Primer is to being respectively:
SEQ?ID?NO.1：GGATGAGGCATCGCGTAAG
SEQ?ID?NO.2：TCGTTCTGGGTGCAATCC
SEQ?ID?NO.3：CCTGGCGTTCCACCACTATGT
SEQ?ID?NO.4：GCCTTGATGACTATGCCGTTC
SEQ?ID?NO.5：GGTCAATAGCTGGCTACACAA
SEQ?ID?NO.6：GCGCCTCAGCGAGTATGCGAT
Bacterium source and PCR detected result thereof that table 1 embodiment uses
ATCC: U.S. representative microbial preservation center
AVA: Singapore agricultural food product veterinary administration office
AU: fishery system of U.S. Auburn university
TU: aquatic living things science system of Tokyo Univ Japan
MOA: the national animal exotic disease diagnositc center (Qingdao) of Chinese Animal Quarantine institute
CGMCC: Chinese microbial preservation center (Beijing)
IOH: Inst. of Hydrobiology, Chinese Academy of Sciences
Reference
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Yang Chun will Wang Xiu Hua Huangjian.2008。Culture the turbot Edwardsiella tarda and divide luxuriant evaluation and Phylogenetic Analysis.Shanghai Aquatic Products Univ. 9CN)'s journal, 17 (3): 280-284
Ling，S.H.，Wang，X.H.，Xie，L.，Lim，T.M.and?Leung，K.Y.(2000)Use?of?green?fluorescent?protein(GFP)to?study?the?invasion?pathways?of?Edwardsiella?tarda?in?in?vivo?and?in?vitro?fish?models.Microbiology?146，7-19.
Mo，Z.L.，Xiao，P.，Mao，Y.X.，Zou，Y.X.，Wang，B.，Li，J.，Xu，Y.L.and?Zhang，P.J.(2007)Construction?and?characterization?of?a?live，attenuated?esrB?mutant?of?Edwardsiella?tarda?and?its?potential?as?a vaccine?against?the?haemorrhagic?septicaemia?in?turbot，Scophthamus?maximus(L.).Fish?Shellfish?Immunol?23，521-530.
Tan，Y.P.，Lin，Q.，Wang，X.H.，Joshi，S.，Hew，C.L.and?Leung，K.Y.(2002)Comparative?proteomic?analysis?of?extracellular?proteins?of?Edwardsiella?tarda.Infect?Immun?70，6475-6480.
Yamada，Y.and?Wakabayashi，H.(1999)Identification?of?fish?pathogenic?strains?belonging?to?the?genus?Edwardsiella?by?sequence?analysis?of?sodB.Fish?Pathology?34，145-150.
Application examples 1
Sample: lefteye flounder cultivating pool water sample
Purpose: analyze and whether contain Edwardsiella tarda
1.DNA template preparation
Sample is the cultivating pool 1-12 of a Shandong Province lefteye flounder plant water sample.Each sample is got 10～100ml breeding seawater, centrifugal 3～the 5min of 5000 * g, get the cellulose nitrate membrane filtration of supernatant, filter membrane is shredded buffered soln (100mM Tris-HCl, the 10mMEDTA that is suspended in 500 μ l～1ml by 0.22 μ m, 5～10%Chelex100, pH7.6), place boiling water bath 5～10min, the centrifugal 2～5min of 12000 * g, go precipitation, get supernatant liquor and do template.
2. multiplex PCR amplification
The PCR reaction system is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/L SEQ ID No.1,0.4 μ M/L SEQ ID No.2,0.2 μ M/L SEQ ID No.3,0.2 μ M/L SEQ ID No.4,1.0 μ l templates are added distilled water to 25 μ l; The PCR reaction conditions is: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds, 60.6～60.8 ℃ of annealing 45 seconds, 68 ℃～72 ℃ were extended 1.5～2.5 minutes; Carry out 30～35 circulations; 72 ℃ are extended 5～10min.
3. electrophoretic analysis
Get 10 μ l pcr amplification products electrophoresis on 1～1.5% sepharose, observe electrophoresis result with the dyeing of bromination ingot back.According to Fig. 4 result, water sample 1-4 (corresponding to the swimming lane 1-4) size that only increases is judged to be and contains Edwardsiella tarda for the band of the 500bp size that do not increase is the band of 323bp; Water sample 5-12 (corresponding to swimming lane 5-12) any band that do not increase is judged to be and does not contain Edwardsiella tarda.
Application examples 2
Sample: Shandong Province batch production turbot plant water reservoir water sample
Purpose: analyze and whether contain pathogenic Edwardsiella tarda
1.DNA template preparation
Sample is the water reservoir water sampling of the coastal batch production turbot A of plant in Shandong Province, B, C, D, E, F, G.Each sample is got 10～100ml breeding seawater, centrifugal 3～the 5min of 5000 * g, get the cellulose nitrate membrane filtration of supernatant, filter membrane is shredded buffered soln (100mM Tris-HCl, the 10mM EDTA that is suspended in 500 μ l～1ml by 0.22 μ m, 5～10%Chelex100, pH7.6), place boiling water bath 5～10min, the centrifugal 2～5min of 12000 * g, go precipitation, get supernatant liquor and do template.
2. multiplex PCR amplification
The PCR reaction system is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/L SEQ ID No.1,0.4 μ M/L SEQ ID No.2,0.2 μ M/L SEQ ID No.3,0.2 μ M/L SEQ ID No.4,0.4 μ M/L SEQ ID No.5,0.4 μ M/L SEQ ID No.6,1.0 μ l templates are added distilled water to 25 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds, 60.6～60.8 ℃ of annealing 45 seconds, 68 ℃～72 ℃ were extended 1.5～2.5 minutes; Carry out 30～35 circulations; 72 ℃ are extended 5～10min.
3. electrophoretic analysis
Get 10 μ l pcr amplification products electrophoresis on 1～1.5% sepharose, observe electrophoresis result with the dyeing of bromination ingot back.According to Fig. 5 result, A of plant and B (the corresponding swimming lane 1-2 respectively) size that only increases is the 500bp band, is judged to be and contains the non-virulent tarda; The C of plant, D, E and F (the corresponding respectively to swimming lane 3-6) size that increases simultaneously is the band of 500bp and 955bp, is judged to be and contains pathogenic Edwardsiella tarda; The G of plant (swimming lane 7) the above-mentioned fragment that do not increase is judged to be and does not contain Edwardsiella tarda.
Application examples 3
Sample: batch production turbot plant water reservoir bed mud
Purpose: analyze and whether contain pathogenic Edwardsiella tarda
1.DNA template preparation
Sample is the water reservoir bed mud of the coastal batch production turbot A of plant in Shandong Province, B, C, D, E, F, G.Each sample takes by weighing 1g, adds 10ml TSB liquid nutrient medium shaking table and cultivates 4～8 hours, and get an amount of bacterium liquid and put boiling water bath 5～10 minutes, centrifugal 2～5 minutes of 12000 * g, getting supernatant is template.
2. multiplex PCR amplification
The PCR reaction system is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/LSEQ ID No.1,0.4 μ M/L SEQ ID No.2,0.2 μ M/L SEQ ID No.3,0.2 μ M/L SEQ ID No.4,0.4 μ M/L SEQ ID No.5,0.4 μ M/L SEQ ID No.6,1.0 μ l templates are added distilled water to 25 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds, 60.6～60.8 ℃ of annealing 45 seconds, 68 ℃～72 ℃ were extended 1.5～2.5 minutes; Carry out 30～35 circulations; 72 ℃ are extended 5～10min.
3. electrophoretic analysis
Get 10 μ l pcr amplification products electrophoresis on 1～1.5% sepharose, observe electrophoresis result with the dyeing of bromination ingot back.According to Fig. 6 result, the A of plant, B, C, D, E (the corresponding respectively to swimming lane 1-5) size that increases simultaneously are the band of 500bp and 955bp, are judged to be and contain pathogenic Edwardsiella tarda; F of plant and G (the corresponding respectively to swimming lane 6-7) size that only increases is the 500bp band, is judged to be and contains the non-virulent tarda.
Application examples 4
Sample: culture turbot
Purpose: analyze and whether infect pathogenic Edwardsiella tarda
1.DNA template preparation
Sample is the nephridial tissue of the ill turbot of certain plant's 8 tail.Aseptic technique takes by weighing nephridial tissue 1g, fully after the homogenate, add 100 μ l N,O-Diacetylmuramidase (50mg/L) solution, 37 ℃ act on 10～30 minutes, add TE buffered soln (100mM Tris-HCl, 10mM EDTA, pH7.6) 500 μ l mix, add the saturated phenol of equal-volume (pH 8.0), concuss, centrifugal 3～5 minutes of 12000 * g draws supernatant and repeats phenol extracting 1 time.The gained supernatant adds the sodium acetate (2M/L) of 0.1 times of volume, and mixing adds equal-volume ice ethanol, and stand at low temperature is 15～30 minutes behind the mixing, and centrifugal 3～5 minutes of 12000 * g abandons supernatant, and precipitation is dissolved in 50 μ l TE buffered soln as template.
2. multiplex PCR amplification
The PCR reaction system is: 1 * PCR buffer, 200 μ M/L dNTP, 50mM/L MgCl
2, 1.0U Taq enzyme, 0.4 μ M/L SEQ ID No.1,0.4 μ M/L SEQ ID No.2,0.2 μ M/L SEQ ID No.3,0.2 μ M/L SEQ ID No.4,0.4 μ M/L SEQ ID No.5,0.4 μ M/L SEQ ID No.6,1.0 μ l templates are added distilled water to 25 μ l.The PCR reaction conditions is: 94 ℃ of pre-sex change 3～5min; 94 ℃ of sex change 45 seconds, 60.6～60.8 ℃ of annealing 45 seconds, 68 ℃～72 ℃ were extended 1.5～2.5 minutes; Carry out 30～35 circulations; 72 ℃ are extended 5～10min.
3. electrophoretic analysis
Get 10 μ l pcr amplification products electrophoresis on 1～1.5% sepharose, observe electrophoresis result with the dyeing of bromination ingot back.According to Fig. 7 result, sample 1-6 (the corresponding respectively to swimming lane 1-6) size that increases simultaneously is the band of 500bp and 955bp, is judged to be and contains pathogenic Edwardsiella tarda; Sample 7-8 (corresponding respectively to swimming lane 7-8) any band that do not increase is judged to be and does not contain Edwardsiella tarda.
Claims (12)
1. primer sequence that detects Edwardsiella tarda, it is characterized in that: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, and described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ ID NO.3:CCTGGCGTTCCACCACTATGT; SEQIDNO.4:GCCTTGATGACTATGCCGTTC.
2. primer sequence that detects the catfish tarda, it is characterized in that: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4; Described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ ID NO.3:CCTGGCGTTCCACCACTATGT; SEQ IDNO.4:GCCTTGATGACTATGCCGTTC.
3. primer sequence that detects pathogenic Edwardsiella tarda, it is characterized in that: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6; Described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ IDNO.3:CCTGGCGTTCCACCACTATGT; SEQ ID NO.4:GCCTTGATGACTATGCCGTTC; SEQ ID NO.5:GGTCAATAGCTGGCTACACAA; SEQ ID NO.6:GCGCCTCAGCGAGTATGCGAT.
4. primer sequence that detects pathogenic catfish tarda, it is characterized in that: primer sequence is to SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6; Described primer is respectively, SEQ ID NO.1:GGATGAGGCATCGCGTAAG; SEQ ID NO.2:TCGTTCTGGGTGCAATCC; SEQ IDNO.3:CCTGGCGTTCCACCACTATGT; SEQ ID NO.4:GCCTTGATGACTATGCCGTTC; SEQ ID NO.5:GGTCAATAGCTGGCTACACAA; SEQ ID NO.6:GCGCCTCAGCGAGTATGCGAT.
5. the detection method of the primer sequence of the described detection Edwardsiella tarda of claim 1, it is characterized in that: with SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 500bp appears in amplified production segment section, be the Edwardsiella tarda positive.
6. the detection method of the described detection of a claim 2 catfish tarda, it is characterized in that: with SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ IDNo.4 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 500bp and 323bp appears in amplified production segment section simultaneously, be the catfish tarda positive.
7. the detection method of the pathogenic Edwardsiella tarda of the described detection of claim 3, it is characterized in that: with SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 955bp and 500bp appears in amplified production segment simultaneously, be the pathogenic Edwardsiella tarda positive.
8. the detection method of the pathogenic catfish Edwardsiella of the described detection of claim 4, it is characterized in that: with SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQID No.4, SEQ ID No.5 and SEQ ID No.6 is that primer sequence is right, carry out the multiplex PCR amplification, amplified production carries out qualitative analysis with gel electrophoresis, if the band of 955bp, 500bp, 323bp appears in amplified production segment simultaneously, be the pathogenic catfish tarda positive.
9. the application of the primer sequence of the described detection Edwardsiella tarda of claim 1 is characterized in that: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of discriminating Edwardsiella tarda of animal, fishery products.
10. the application of the primer sequence of the described detection of a claim 2 catfish tarda is characterized in that: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of discriminating catfish tarda of animal, fishery products.
11. the application of the primer sequence of the pathogenic Edwardsiella tarda of the described detection of claim 3 is characterized in that: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of the pathogenic Edwardsiella tarda of discriminating of animal, fishery products.
12. the application of the primer sequence of the pathogenic catfish tarda of the described detection of claim 4 is characterized in that: described primer sequence can be applicable to water sample, mud sample, animal and movement thereof, animal feed, the plant to culture environment of aquatic products or imports and exports in the detection kit of the pathogenic catfish tarda of discriminating of animal, fishery products.
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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 |
| CN103146827A (en) * | 2013-03-06 | 2013-06-12 | 厦门市农产品质量安全检验测试中心 | Multiplex PCR (Polymerase Chain Reaction) primer for simultaneously detecting salmonella, citrobacter, proteus and Edwardsiellas and design method thereof |
| CN104164514A (en) * | 2014-09-01 | 2014-11-26 | 厦门出入境检验检疫局检验检疫技术中心 | Fluorescent probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri |
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Cited By (6)
| 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 |
| CN102382881B (en) * | 2011-11-03 | 2013-07-03 | 通威股份有限公司 | Kit and method for detecting fish pathogenic bacteria |
| CN103146827A (en) * | 2013-03-06 | 2013-06-12 | 厦门市农产品质量安全检验测试中心 | Multiplex PCR (Polymerase Chain Reaction) primer for simultaneously detecting salmonella, citrobacter, proteus and Edwardsiellas and design method thereof |
| CN103146827B (en) * | 2013-03-06 | 2014-04-16 | 厦门市农产品质量安全检验测试中心 | Multiplex PCR (Polymerase Chain Reaction) primer for simultaneously detecting salmonella, citrobacter, proteus and Edwardsiellas and design method thereof |
| CN104164514A (en) * | 2014-09-01 | 2014-11-26 | 厦门出入境检验检疫局检验检疫技术中心 | Fluorescent probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri |
| CN104164514B (en) * | 2014-09-01 | 2016-03-16 | 厦门出入境检验检疫局检验检疫技术中心 | Detect Edwardsiella tarda with the fluorescent probe PCR method of Channel-catfish tarda simultaneously |
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