CN108384865A - Application of the Alpha fimbriae genes in grice diarrhoea enteropathogenic E. Coli monitors, differentiates - Google Patents
Application of the Alpha fimbriae genes in grice diarrhoea enteropathogenic E. Coli monitors, differentiates Download PDFInfo
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Abstract
The present invention relates to application of the Alpha fimbriae genes in the monitoring of grice diarrhoea enteropathogenic E. Coli, discriminating.The present invention also provides a pair of specific primers and kit for detecting grice diarrhoea enteropathogenic E. Coli.Identify quick detection of the primer for causing grice diarrhoea Escherichia coli in the clinical setting of pig farm；To from feed, drinking-water (or sewage), airborne dust and excrement separation of bacterial be template, PCR amplification is carried out to primer using this, if it is grice diarrhoea enteropathogenic E. Coli is caused, then PCR amplification is expected positive；If it is other non-pathogenic escherichia colis, such as detection of Salmonella, clostridieum welchii other Pseudomonas, then result is feminine gender.The present invention is based on the characteristics of the distributional difference and Alpha pili cblB gene order conservatives of Alpha fimbriae genes, cause grice diarrhoea Escherichia coli to provide new method and selection for clinical quickly differentiation and identification in different Pseudomonas or different phenotype Escherichia coli.
Description
Technical field
The present invention relates to biotechnologies.In view of Alpha pili exist only in cause grice diarrhoea Escherichia coli and other
In some enteropathogenic E. Coli genomes, therefore molecular labeling is can be used as, is designed using the conserved sequence in its genetic fragment
The primer of a pair of of specificity, is detected by standard PCR amplification characteristic gene fragments.
Background technology
Grice diarrhoea is the disease most often sent out in live pig production, and piglet daily gain can be caused to reduce, and dehydration is even dead,
Serious economic loss is brought to pig-breeding industry.In addition to by pig infectious disease marcy agent (TGEV), Porcine Epidemic Diarrhea
Outside grice diarrhoea caused by the poison virus such as (PEDV) and porcine rotavirus (RDV), enterotoxigenic escherichia coli (ETEC), sramana
Bacterium, the bacteriums such as clostridieum welchii are also the most common pathogen for causing grice diarrhoea,.The pili class that wherein ETEC is expressed according to it
Type can be categorized further, as K88+, F18+, 987P+, the different serotypes such as F41+, K99+.Clinically pig farm is newly sent out at present
The diagnosis of grice diarrhoea disease is mainly：(method 1) is by the routine separation identification of different pathogens and further to utilizing list
Factor antiserum carry out separation bacterium glass plate or immunoprecipitation test to confirm the positive.Therefore, bacteria pathogeny initiation is being determined
Diarrhea when, need use different E.coli K88s, K99, F18,987P, the single-factor antiserum of F41 pili, different sramana
Bacterium single-factor antiserum and clostridieum welchii identification method are detected respectively, until obtain positive reaction as a result, cost compared with
It is high；(method 2) carries out the routinely separation identification such as scribing line separation, culture by laboratory to enteron aisle, fecal specimens, is further leading to
It crosses after biochemical test determines Pseudomonas and (is generally incubated needs one day), then by serum agglutination or for different antigen serotypes
Specific primer carries out PCR with the specific pathogen serotype of determination, and process is cumbersome, takes time and effort.It is worth noting that, many marks
Quasi- strain and most clinical separation strains are required for screening special culture medium, and (such as temperature, pH value under suitable environment
Deng) carry out laboratory continuous subculture, be used for Serological testing envelope antigen expression need it is more abundant, due to without
The expression that bacterial strain its adhesin of above method processing cannot reache a certain level under condition of culture in vitro, thus use Dan Yin
When sub- serum carries out glass plate or tube agglutination test detection, tend not to generate macroscopic agglutinating reaction, this results in base
It is significantly limited in the glass plate of pili detection or the application of tube agglutination test.
Alpha pili generally also known as 5 class pili claim class colonization factor antigen I (CFA/I-like) family.In people
On the ETEC of source, colonization factor (CFs) is very important adhesin, and is at least found that 25 kinds of different CF so far.Wherein
The antigenic determinant homology of CFA/I, CS4, CS14, CS1, CS17 and CS19 are very high.CFA/I be study CF the most thorough it
One：The pili is made of about 1000 major subunit CfaB, and the adhesin PROTEIN C faE of a copy is assembled on top；It is filled
With assisting to complete jointly by chaperone CfaA and leader protein CfaC.The composition of Alpha pili is similar therewith, by operon
Four subunits composition of cblABCD transcriptions：CblA and CblC is chaperone and leader protein respectively, and CblB is pili
Major subunit, CblD are top adhesins.Existing Alpha pili sequence data is all from full genome in GenBank
Group sequencing (such as accession number CP025036.1, which can be found by muca gene cblA or cblB or cblC or cblD,
Annotation is that full-length genome software annotates automatically).But the document about the description of Escherichia coli Alpha pili concrete functions does not have also
It delivers.For the statement of the pili be only capable of from about the homologous pili of onion Burkholderia article (Umadevi S.Sajjan,
Hong Xie,Matthew D.Lefebre,Miguel A.Valvanoand Janet
F.Forstner.Identification and molecular analysis of cable pilusbiosynthesis
Genes in Burkholderia cepacia.Microbiology (2003), 149,961-971) partial information is obtained in.
Invention content
The purpose of the present invention is to provide Alpha fimbriae genes in the monitoring of grice diarrhoea enteropathogenic E. Coli, differentiating
Application, and provide it is a kind of quickly, the specific detection agents box of simplicity, high specificity, suitable Clinical practice, it is anti-using PCR
Whether should quickly it differentiate in environmental samples comprising the enterotoxigenic escherichia coli or some other pathological form large intestine for causing grice diarrhoea
Bacillus is the enterotoxigenic escherichia coli or some other pathogenic for monitoring and effectively prevent to cause grice diarrhoea in the breeding process of pig farm
Type Escherichia coli occur to provide technical support.
Technical scheme of the present invention：Show Escherichia coli Alpha pili from known gene sequence information and comparative analysis
It is prevalent in the enterotoxigenic escherichia coli and other pathological form coli strains for causing grice diarrhoea, and the pili is grasped
It is relatively conservative to indulge sub- cbl sequences, is especially about the major subunit gene cblB sequences of 500bp.
In view of the features above of cbl operons, according in wherein Alpha pili major subunit gene cblB sequence designs
Downstream primer.DNA profiling, which is prepared, with measuring samples such as all feeds, drinking-water (or sewage), airborne dust and excrement carries out PCR detections,
When template can amplify the band for being expected to 500bp sizes, illustrate that the bacterial strain to be checked is to cause the production enterotoxin of grice diarrhoea big
Enterobacteria or other pathological form Escherichia coli；When template cannot expand respective strap, then the bacterial strain to be checked is not to cause grice diarrhoea
Enterotoxigenic escherichia coli or other pathological form Escherichia coli.
The invention discloses Alpha fimbriae genes as monitoring, differentiates the molecule mark of grice diarrhoea enteropathogenic E. Coli
The application of note.
The invention also discloses the specific primer for detecting grice diarrhoea enteropathogenic E. Coli, sequence is as follows：
AF-cblB-F:ATGAAAAAGGTATTTGCAAAATCTC(SEQ ID No.1)
AF-cblB-R:TTAGATATCCTGAGACAGAT(SEQ ID No.2)。
Further, the present invention provides a kind of reagents for detecting the pathogenic enterotoxigenic escherichia coli of grice diarrhoea
Box, including：
PCR buffer solutions, dNTPs, archaeal dna polymerase, positive control dna template and above-mentioned specific primer.
Heretofore described alpha fimbriae genes are as shown in SEQ ID No.3.Its major subunit gene cblB sequence
As shown in SEQ ID No.4.
The advantage of the invention is that：
1. causing grice diarrhoea enterotoxigenic escherichia coli or other pathological form large intestine bars in clinical sample for detecting, monitoring
Bacterium, by more plants of enteropathogenic E. Coli PCR detection verifications, the result which obtains is accurate, and does not have
The generation of false positive and the reaction that intersects.
2. the reagent and equipment cost needed for the detection method are relatively low, -20 DEG C of detection primer for designing synthesis can be long-term
It preserves, can be used for multiple times.
3. this method can be used for clinical monitoring and differentiate cause grice diarrhoea Escherichia coli enterotoxigenic escherichia coli or its
His pathological form Escherichia coli can directly be expanded using environmental samples such as feed, drinking-water (or sewage), airborne dust and excrement, be exempted from sample preparation
It goes to be separately cultured bacterium process, it is fast and convenient.
Description of the drawings
Fig. 1 causes the specificity of grice diarrhoea Escherichia coli quickly to detect electrophoretogram.Wherein：
M is Trans2K plus II DNA Marker molecular labelings, and swimming lane 1 is 987P wild strains 204, and swimming lane 2 is work
Journey bacterium DH5 α, swimming lane 3 are K99 type strain C83907, and swimming lane 4 is K99 wild strains 637, and swimming lane 5 is ox source ETEC wild strain B41,
Swimming lane 6 is F18ab type strain F107/86, and swimming lane 7 is F18ac type strain 2134P, and swimming lane 8 is F18ab wild strain SEC470, swimming
Road 9 is people source ETEC bacterial strain H10407, and swimming lane 10 is K88ab type strain C83901, and swimming lane 11 is K88ac type strain C83902,
Swimming lane 12 is K88ad type strain C83903, and swimming lane 13 is K88ac wild strain 2534-86, and swimming lane 14 is K88ac wild strain GT60,
Swimming lane 15 is K88ac wild strains Bd 1,107,/75 08, and swimming lane 16 is K88ac wild strain YZ20, and swimming lane 17 is K88ac wild strains
3030-2, swimming lane 18 are 987P wild strains 1592, and swimming lane 19 is Salmonella enteritidis 50336.
Specific implementation mode
Used biomaterial information is as follows in following embodiments：
C83901 plants, C83902 plants and C83903 plants are respectively that tri- kinds of serotypes of K88ab, K88ac and K88ad produce enterotoxin
Escherichia coli type strain is purchased from China Veterinary Drugs Supervisory Inst.'s Culture Collection Center；C83907 plants of K99 serotype Escherichia coli standards
Strain is purchased from China Veterinary Drugs Supervisory Inst.'s Culture Collection Center；F18ab serotype enterotoxigenic escherichia coli type strain F107/86,
K88ac serotypes field separation strains 3030-2, ox source F41 serotype type strain B41 reach Kodak state university David by American South
Professor Francis presents；Enteritis serotype detection of Salmonella 50336 is taught by Univ Pennsylvania USA Diter Schifferli
Present；Engineering bacteria DH5 α are purchased from Takara companies；Separation strains 2534-86 plants of K88ac serotypes field, GT60 plants, Bd
1107/75 08 plants and YZ20 plant, F18 serotypes field separation strains SEC470,204 plants of 987P serotypes field separation strains with
1592 plants, 637 plants of K99 serotypes field separation strains are detached and are preserved by Yangzhou University Zhu Guoqiang professors laboratory.
F18ab serotype enterotoxigenic escherichia coli type strains F107/86：Bertschinger,H.U.,Bachman,
M.,Mettler,C.,Pospischil,A.,Schraner,E.M.,Stamm,M.,Sydler,T.,Wild,P.,
1990.Adhesive fimbriae produced invivo by Escherichia coli 0139:K12(B):
H1associated with enterotoxaemiain pigs.Veterinary Microbiology.25,267–281
K88ac serotypes field separation strains 3030-2：Francis,D.H.and
J.A.Willgohs.1991.Evaluation of a live avirulentEscherichia coli vaccine for
K881,LT1enterotoxigenic colibacillosis inweaned pigs.American Journal of
Veterinary Research.52:1051–1055.
Ox source F41 serotype type strains B41：J.A.MORRIS,*C.THORNS,A.C.SCOTT,W.J.SOJKA,AND
G.A.WELLS.,1982.Adhesion In Vitro and In Vivo Associated with an
AdhesiveAntigen(F41)Produced by a K99Mutant of the ReferenceStrain
Escherichia coli B41.Infection and Immunity.36(3),1146-1153
Enteritis serotype detection of Salmonella 50336；Xia Meng,Xianchen Meng,Chunhong Zhu,Heng Wang,
Jinqiu Wang,Jiajia Nie,Philip R.Hardwidge4&Guoqiang Zhu.,2013.The
RNAchaperone Hfq regulates expression of fimbrial-relatedgenes and virulence
of Salmonella enterica serovar Enteritidis.,FEMS microbiology letters.346(2):
90-6
F18 serotypes field separation strains SEC470:H Liu, L Song, Y Cai, Y Wang, L Yu., 2016.Draft
Genome Sequence ofEscherichia coliStrain SEC470,Isolated from a Piglet
Experiencing Diarrhea.Genome Announcements.4(2):e00088-16
K88ac serotypes field separation strains GT60, YZ20 plants, 204 plants and 1592 plants of 987P serotypes field separation strains,
Believe army, Hu Huijie, what Zhang Ke, Zhou Mingxu, perhaps continuous, Zhu Guoqiang *, the clone of 2015. escherichia coli I type pili fimA genes
With sequence comparing analysis Yangzhou Universitys journal (agricultural and life science version) .36 (4):13-17
637 plants of K99 serotypes field separation strains:CM Ferreiros, MT Criado., 1983.Purification
and partial characterization of a K99-antigen associated adhesin in
Escherichia coli(637strain).Revista De Fisiología.39(1):45-50
Separation strains 2534-86 plants of K88ac serotypes field:NM Clark, EM Berberov, M Wang, RAMoxley.,
2006.Anti-capsular antibodies activate killing of Escherichia coli O8:K87by
the alternate complement pathway in porcine serum.Veterinary Immunology&
Immunopathology.114(1-2):185-191
1,107,/75 08 plants of K88ac serotypes field separation strains Bd:L.Blomberg&P.L.Conway.,2009.An
In vitro Study of Ileal Colonisation Resistance to Escherichia coli Strain Bd
1107/75 08(K88)in Relation to Indigenous Squamous Gastric Colonisation in
Piglets of Varying Ages.Microbial Ecology in Health&Disease.2(4):285-291。
Embodiment 1
The nucleotide sequence of the PCR detection method the primer of the present invention is as follows：
AF-cblB-F:ATGAAAAAGGTATTTGCAAAATCTC(SEQ ID No.1)
AF-cblB-R:TTAGATATCCTGAGACAGAT(SEQ ID No.2)
Detection method process is as follows：
1. environmental sample is collected：(1) feed：Take feed 0.1g, grind into powder spare；(2) (or sewage) is drunk water：
1.5mL centrifuge tube water samplings 1mL, 10000rpm centrifugation 5 minutes, removes supernatant, and precipitation is spare；(3) airborne dust：In pig farm difference
Position is placed sterilized template one and is opened, and for 24 hours afterwards collects the dust fallen on template, takes 0.1g spare；(4) excrement
Just：Sterilized cotton swab takes swine excrement 0.1g spare.
2. preparing the chromosomal DNA templates of measuring samples：Take the 50- after previous step measuring samples addition sterilization treatment
The ultra-pure water of 100 μ l is suspended in 1.5mL centrifuge tubes, after mixing well, is placed in water-bath 5 minutes in 100 DEG C of boiling water, is cooled to room
Wen Hou, quiescent setting 10 minutes (having ready conditions can be centrifuged 1 minute with 10000rpm) draw 2-3 μ l supernatants and do pcr template.
3.PCR is expanded and product detection：50 μ l amplification systems contain 1 × PCR buffer solutions, 0.2mM dNTP, upstream and downstream
Each 1mM of primer, 5 μ l measuring samples pcr templates and 0.5 μ lDNA polymerases.Amplification cycles parameter is 94 DEG C of pre-degeneration 4min；
94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 25 cycles；72 DEG C of extension 10min.Take 5 μ l PCR products in 1.2% agarose gel
In with 90V constant voltage electrophoresis 40min, ethidium bromide EB dyeing, with Trans2K plus II DNA Marker, (full formula gold is public
Department) it is standard molecular weight analysis result, sample to be tested is 500bp or so band as positive control can amplify expected size
PCR product, then sample, which contains, causes grice diarrhoea enterotoxigenic escherichia coli or other pathological form Escherichia coli.
Cause the specificity quickly detection of grice diarrhoea Escherichia coli
With AF-cblB-F/AF-cblB-R a pair of upstream and downstream primers, pig enterotoxigenic escherichia coli C83901, C83902,
C83903,3030-2, F107/86,2143P, SEC470,2534-86, GT60, Bd 1,107,/75 08, YZ20, C83907,
637,1592 and 204 can amplify about 500bp size strips, and enterotoxin is produced in other serotype bacillus coli DH 5 alphas, ox source
Escherichia coli B41, people source enterotoxigenic escherichia coli H10407 and Salmonella enteritidis 50336 are negative right without any band
According to as shown in Figure 1.
SEQUENCE LISTING
<110>Yangzhou University
<120>Application of the Alpha fimbriae genes in grice diarrhoea enteropathogenic E. Coli monitors, differentiates
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
atgaaaaagg tatttgcaaa atctc 25
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ttagatatcc tgagacagat 20
<210> 3
<211> 5015
<212> DNA
<213>Escherichia coli
<400> 3
atggctgttt ccattaatag tcagggtgaa ggcaacgttc gcgtaatatc taaaagtaat 60
gaagttcagt acatcaaggc gacagtattc cgtatcgata atccatcgac gcctcaggaa 120
aatgaagttg agattaaatc cggtgatgca aatcatctgg ttgttatgcc acctaaattc 180
gctttaccag ccggtagtag caaaaccgta cgttttgttg cgatggaacc agagcaaaaa 240
gagaaaaatt atcgcgttaa atttgaagcg gttcccagta ttgatgacgt tgccacagat 300
aaaaaagatc tctctatgca gttaacagtt aacttaattt gggggattgt tgttagtgtt 360
ccacctcagc aacctattgc taagttagaa gtaaatgctg cccaaaaatt agttaatgca 420
gggaatcaac gcttaaagat tttaacgatc gcttattgta aaaataatag caaagaaaat 480
tgtaagatac agaccgtaaa taaaaacatc ttccctggtc aggaaagaaa tcttgaaagc 540
atatctggct acgacaaaat cgttgtcaaa tataataact ggatcaccaa agataatggc 600
gagtttgaac tggcggtcca ttaattatca ttgtaataaa cgaaggaata gtaatgaaaa 660
aggtatttgc aaaatctctt ctggtcgcag cgatgttttc tgttgcaggc tccgcattgg 720
cagtacaaaa ggatattact gtaacggcca acgttgatgc cgctctggat atgacacaga 780
ccgataacac cgcgttgccg aaagcagtag aaatgcaata tctgccgggg cagggtctgc 840
aatcttacca gttgatgacc aaaatctggt ctaacgacgt taccaaagat gtaaaaatgc 900
agttagtgtc cccagcgcaa ctggttcaaa gcctggatgc tagcaagatt gttccgctga 960
cagtaacctg gggcggtgaa gaaattaaag ctgatgcggc aaccactttt accgcaacca 1020
aaatctttgc ttccgatgcg ttaactaacg gttctctggc taaaaacctg atgtttgctc 1080
aaaccaccaa aggtgttctg gagacaggca tctaccgtgg tgtagtaagc atttatctgt 1140
ctcaggatat ctaatccaga taacagaatt aacacgtaac aaagggaggg gtagccctcc 1200
cttattttaa ctgttttagt atttgttatg gatttcagga ttgccatgga taaaaaatta 1260
ctggctcttt tgatcctggc gagtctcagc ccggcagagg cgacattaac caaaattccc 1320
gcagggtttg aggttattgc tcagggacag caggagtata tcgaggttta tttttcaggg 1380
aaaagtctcg gtaaatatta tgcaatggtt aatcttgata ccgtaacatt tcttgatcca 1440
gcaagtctat ataacaagct ggagctggat gtagacgatc agaaaatcgc gcatatagtg 1500
aaagaaaaat tatcgcagcc gctagctcgc cacggtgaat tggcttgcgg ttatgtacgt 1560
actgactcag ggtgtggttt tctgaatacc gatacgctgg aaataatcta taatgatgaa 1620
gaaagttcgg caacgttgtt tattaatccg caatggaatt cagctttcga tgcgaagtca 1680
ttatatttaa atccagacaa aaatactgtt aatgctttta tacatcagca agacatcaat 1740
gttctggcac aggatgatta ccaatcgttg tctattcagg gaaacggtgc gctgggaata 1800
acagaaaata gctatattgg tgcacactgg aatttcaacg gttatgatgc agatgatgtc 1860
agtgacagta atgctgacgt cagcgatctc tattatcgtt atgatttttt acgtcgttat 1920
tatgtgcagg cgggtcgcat ggacaaccgc acactattta atgcacaagg cgggaacttt 1980
acctttaact ttctgccact cggtgcaatt gatgggatgc gtatcgggtc gacactgagc 2040
tatttaaacc aggcgcaaag ccagcaagga accccagtaa tggttctgct ttcgcgcaat 2100
tctcgtgttg acgcttatcg taatgagcaa cttttgggat cgttttatct caatagtggt 2160
tcgcaattta ttgataccag ttcctttccg cctggcagct acagcgtggc attaaaagtc 2220
tacgaaaata accaactcac ccgcaccgag cttgtgccgt ttaccaaaac cggcggtctg 2280
actgacggaa atgcgcaatg gttcttacag gcaggtaaaa ctacatcaca ggtttctgat 2340
gatgaaagtt cagcttatca gctaggagta cgcctgccat tacatccgca atatgagctc 2400
tacgcagggc tggcgaatgc cgatgatgtg agtgctttcg agttaggtaa taactggacg 2460
gcagatttag gcggggtggg gaatcttgca atcagcgcca gcgtgttccg taacgatgac 2520
ggcggcaaag gtgatatgca acaggccaac tggagtaatc cgggatggcc gacgttgggc 2580
ttttatcgga ccaactctga cggcgatgct tgtacaaccg acagcagaga gagctataac 2640
gccttaagct gttatgaaag tatttccgcg acggtttcac agaattttgt cggctggaat 2700
atgatgctgg gttatacccg cacacaaaat aacactgatg atagtttgcg ttgggataaa 2760
cagcagagct ttgaaaataa ctatcttcgc cagacaacag cgcaaagtat ctctgaaact 2820
gtacaactta gcgcttcccg cgcttttgtg atgcgtgact ggattttgag tacgtcagtc 2880
ggtgttttcc atcgtaatga caacggtggc gataacgacg acaacggctt gtacttatcg 2940
ttttcgttat ctgacacgcc aacgatggat agcaataaca acagccattc aacaaatgtg 3000
tctacggatt atcgttatag cgaacaggat ggcgatcaaa cgtcatggca gttatcgcat 3060
accttttata acgattcatt cagccataaa gaacttggcg taaccgtcgg aggcctgaac 3120
accgatacca taaacagcgc ggttaacggg cgttgggatg ggcaatacgg aaatgtctac 3180
gctaccgtat ctgatagtta tgaccgtaag aatcatgatc atctttcggc ctttacgggc 3240
acttatagct ctacgctggc tgtcagtcgc tatggcgtta atttgggagc cagtggtaca 3300
gacgatttgc tgggtgcggt actggtggat gtgaaaggct tctctgaaca ggatgaagag 3360
agtcaggatc tgcaactcga agcgcgggtg gcaggcagcc gaactttgca gcttggtcaa 3420
agtgacagtg tgttgttccc ttatcctgga tttcagtctg gttttgttga ggttaacgac 3480
agtagccagg gcaatcagca ggggacaaca aacatcatta acggtgcagg gaatcgtgaa 3540
ttaatgttgt tgcctggcaa gctgcgctat cgcgaagtgt ctgccagctt taactacaac 3600
tatatcggtc gtttattact gccggcagcg gtgaaaaaat tcccgattgt ggggctgaac 3660
agcgccatgt tactggtagc tgaagatggc ggatttacac ttgaaattaa cggcagcgaa 3720
aaagagctct atctgctttc cgggcagcaa ttccttaagt gtccactaag tgttgtaaag 3780
aaacgcgcca gcattcgtta cagcggagat gttacttgta gtgtggtgac ttattcacaa 3840
ttaccggagt cgattcaggt tcaggcacag ttgaaacagc ctaaattacg tggaaacgtt 3900
cagacggcgc aaagggaggt tgcaccatga gaaatcgatt gattgcggcg atattgggct 3960
tgtttggcac gctcactggc gttcaggcag ctcctgacgt gaccagtgaa attacgtatg 4020
atttggcatc tggtagagcg gattattact tctggaagga tgaagcgtcg gcaggaaata 4080
atggatatat gtggtatgaa tgttcgtatc ctgacctcca acaaacctgt acagctaatg 4140
gaaatatatc gacagtacaa atctatttaa ctgaacaacg cagtgggatg cgttggccgg 4200
taaaactcaa aggatttaaa acagccattg taagtagcga tgaagcgccg ccaggatgca 4260
aggggggcaa agggcttcag acgaatctta aggattctaa tagatcttca tgtacagaag 4320
atggtcaaca ttattatata tacgatacaa agtttcttac gctctacctt gagcagacag 4380
agatgaagaa tttgccgatt ggtggcgtct ggaaggggaa agttaaatta cattcgaaca 4440
gcccggccca ggactatttc gcaaatatta ccctgaatac gctcgacccc aaccatattg 4500
acgtgttctt cccggagttc gcccacgcca cgccaagggt gcagttagac ttgcatccaa 4560
caggaagcgt taacggcagc aactacgcgc aagatctgac catgttggac atgtgcctgt 4620
acgatggttt taacggtaat gccatcagtt atgaaatcat gctcaaagat gaagggcgac 4680
cagctgcagg gcgcagagac ggttacttct ctatctatcg tcagggaggg accaccaccg 4740
acgagggaga acgcattgat taccgggtca aaatgtacaa cccggaaacc ggtgggcaaa 4800
ttgatgtgcg caataatgaa aatatggtct ggaacagcat taacctgaaa cgtgtgcgtc 4860
cggtggtact gcccggtatt cgctatgccg tgatgtgtgt gccgacaccc ttgactctgg 4920
cagtcgataa attcagcgtg atggataaac aggccgggta ctacatgggc aaattgtcag 4980
taatctttac gccttccttg ccaaccatca attaa 5015
<210> 4
<211> 500
<212> DNA
<213>Escherichia coli
<400> 4
atgaaaaagg tatttgcaaa atctcttctg gtcgcagcga tgttttctgt tgcaggctcc 60
gcattggcag tacaaaagga tattactgta acggccaacg ttgatgccgc tctggatatg 120
acacagaccg ataacaccgc gttgccgaaa gcagtagaaa tgcaatatct gccggggcag 180
ggtctgcaat cttaccagtt gatgaccaaa atctggtcta acgacgttac caaagatgta 240
aaaatgcagt tagtgtcccc agcgcaactg gttcaaagcc tggatgctag caagattgtt 300
ccctgacagt aacctggggc ggtgaagaaa ttaaagctga tgcggcaacc acttttaccg 360
caaccaaaat ctttgcttcc gatgcgttaa ctaacggttc tctggctaaa aacctgatgt 420
ttgctcaaac caccaaaggt gttctggaga caggcatcta ccgtggtgta gtaagcattt 480
atctgtctca ggatatctaa 500
Claims (3)
1.Alpha fimbriae genes are as monitoring, the application for the molecular labeling for differentiating grice diarrhoea enteropathogenic E. Coli.
2. the specific primer for detecting grice diarrhoea enteropathogenic E. Coli, it is characterised in that sequence is as follows：
AF-cblB-F:ATGAAAAAGGTATTTGCAAAATCTC
AF-cblB-R:TTAGATATCCTGAGACAGAT。
3. a kind of kit for detecting grice diarrhoea enteropathogenic E. Coli, including PCR buffer solutions, dNTPs, DNA polymerization
Enzyme, positive control dna template, which is characterized in that also include the specific primer described in claim 2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234360A (en) * | 2018-11-15 | 2019-01-18 | 江苏农牧科技职业学院 | A kind of newborn piglet source Escherichia coli LAMP detection method based on fimbriae gene |
| CN111454872A (en) * | 2020-03-16 | 2020-07-28 | 扬州大学 | Avian pathogenic escherichia coli VI type secretion system clpV gene deletion strain and construction method and application thereof |
| CN114107108A (en) * | 2021-11-29 | 2022-03-01 | 中牧实业股份有限公司 | Enterotoxigenic escherichia coli for piglets and application thereof |
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|---|---|---|---|---|
| CN1314953A (en) * | 1997-05-20 | 2001-09-26 | 生物技术研究及发展公司 | Method and compositions to identify swine genetically resistant to F18 E. coli associated diseases |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314953A (en) * | 1997-05-20 | 2001-09-26 | 生物技术研究及发展公司 | Method and compositions to identify swine genetically resistant to F18 E. coli associated diseases |
Non-Patent Citations (2)
| Title |
|---|
| TIMOTHY J. JOHNSON ET AL.: "Pathogenomics of the Virulence Plasmids of Escherichia coli", 《MICROBIOL MOL BIOL REV》 * |
| 周明旭: "K88ac+产肠毒素大肠杆菌相关新毒力因子的研究", 《中国博士学位论文全文数据库（农业科技辑）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109234360A (en) * | 2018-11-15 | 2019-01-18 | 江苏农牧科技职业学院 | A kind of newborn piglet source Escherichia coli LAMP detection method based on fimbriae gene |
| CN111454872A (en) * | 2020-03-16 | 2020-07-28 | 扬州大学 | Avian pathogenic escherichia coli VI type secretion system clpV gene deletion strain and construction method and application thereof |
| CN114107108A (en) * | 2021-11-29 | 2022-03-01 | 中牧实业股份有限公司 | Enterotoxigenic escherichia coli for piglets and application thereof |
| CN114107108B (en) * | 2021-11-29 | 2023-08-08 | 中牧实业股份有限公司 | Colibacillus for producing enterotoxin of piglets and application thereof |
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