CN108384865A - Application of the Alpha fimbriae genes in grice diarrhoea enteropathogenic E. Coli monitors, differentiates - Google Patents

Abstract

The invention relates to the application of Alpha pili gene in the monitoring and identification of pathogenic Escherichia coli in piglets. The invention also provides a pair of specific primers and a kit for detecting pathogenic Escherichia coli of piglet diarrhea. The identification primers are used for the rapid detection of piglet diarrhea-causing E. coli in the clinical environment of pig farms; the bacteria isolated from feed, drinking water (or sewage), dust and feces are used as templates, and these primers are used for PCR amplification. For diarrhea-pathogenic Escherichia coli, the PCR amplification is expected to be positive; if it is other non-pathogenic Escherichia coli, such as Salmonella, Clostridium welchii and other genera, the result is negative. Based on the distribution difference of Alpha pili genes in Escherichia coli of different genus or different phenotypes, and the characteristics of the sequence conservation of Alpha pili cblB gene, the present invention provides a new method and selection for clinical rapid differentiation and identification of Escherichia coli causing piglet diarrhea .

Description

The role of Alpha pili gene in monitoring and identification of pathogenic Escherichia coli in piglets with diarrhea application

technical field

The present invention relates to the field of biotechnology. Since Alpha pili only exist in the genomes of Escherichia coli and some other pathogenic Escherichia coli, it can be used as a molecular marker, and a pair of specific primers can be designed using the conserved sequence in the gene fragment, and amplified by conventional PCR Characteristic gene fragments were detected.

Background technique

Piglet diarrhea is the most common disease in pig production, which can lead to reduced daily weight gain, dehydration and even death of piglets, bringing serious economic losses to the pig farming industry. In addition to piglet diarrhea caused by viruses such as porcine infectious gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (RDV), enterotoxigenic E. Bacteria such as Clostridioides are also the most common pathogens causing diarrhea in piglets. Among them, ETEC can be further classified into different serotypes such as K88+, F18+, 987P+, F41+, and K99+ according to the type of pilus expressed. At present, the clinical diagnosis of newly-occurring piglet diarrhea disease in pig farms is mainly: (Method 1) through the routine isolation and identification of different pathogens and further performing agglutination tests on glass plates or test tubes using single-factor antiserum to confirm positive results. Therefore, when determining diarrhea caused by bacterial pathogens, it is necessary to use single-factor antisera of different Escherichia coli K88, K99, F18, 987P, and F41 pili, different single-factor antisera of Salmonella, and the identification methods of Clostridium welchii. Detection, until a positive reaction result is obtained, the cost is relatively high; (Method 2) Routine isolation and identification of intestinal tract and feces samples through streaking, culture and other routine isolation and identification, after further biochemical tests to determine the genus of bacteria (usually culture takes one day) ), and then perform PCR with specific primers for different antigen serotypes to determine the specific pathogen serotype, which is cumbersome, time-consuming and labor-intensive. It is worth noting that many standard strains and most clinical isolates require screening of special media, and continuous laboratory culture and passage in a suitable environment (such as temperature, pH, etc.), which are used in serology The expression of the outer membrane antigen to be checked needs to be relatively sufficient. Since the adhesin of strains that have not been treated by the above method cannot reach a certain level of expression under in vitro culture conditions, when single-factor serum is used for glass plate or test tube agglutination test, often No visible agglutination reaction can be produced, which limits the applicability of glass plate or test tube agglutination tests based on pili detection to a great extent.

Alpha pili are generally also known as type 5 pili or colonization factor antigen I (CFA/I-like) family. Colonization factors (CFs) are very important adhesins on human ETEC, and at least 25 different CFs have been discovered so far. The epitope homology of CFA/I, CS4, CS14, CS1, CS17 and CS19 is very high. CFA/I is one of the most well-studied CFs: the pilus is composed of about 1000 main subunits CfaB, and a copy of the adhesin protein CfaE is assembled at the top; its assembly is assisted by the chaperone protein CfaA and the leader protein CfaC. The composition of Alpha pili is similar, consisting of four subunits transcribed by the operon cblABCD: CblA and CblC are chaperone and leader proteins, respectively, CblB is the main subunit of pili, and CblD is apical adhesin. The existing Alpha pili sequence data in GenBank are all derived from whole genome sequencing (for example, the accession number CP025036.1, the sequence can be found by searching for the gene cblA or cblB or cblC or cblD, and the annotation is automatically annotated by the whole genome software). However, the literature on the specific function description of Escherichia coli Alpha pili has not yet been published. The expression of this pilus can only be obtained from the article (Umadevi S.Sajjan, Hong Xie, Matthew D.Lefebre, Miguel A.Valvano and JanetF.Forstner. Identification and molecular analysis of cable pilus biosynthesisgenes about Burkholderia cepacia homologous pilus in Burkholderia cepacia. Microbiology (2003), 149, 961–971).

Contents of the invention

The purpose of the present invention is to provide the application of Alpha pili gene in the monitoring and identification of piglet diarrhea pathogenic E. Quickly identify whether the environmental samples contain enterotoxigenic Escherichia coli or some other pathogenic Escherichia coli that cause piglet diarrhea. Escherichia coli to provide technical support.

Technical solution of the present invention: From known gene sequence information and comparative analysis, it is shown that Escherichia coli Alpha pili generally exist in enterotoxigenic Escherichia coli and other pathogenic Escherichia coli strains that cause diarrhea in piglets, and the pili operate The sub-cbl sequences are relatively conserved, especially the main subunit gene cblB sequence which is about 500bp long.

In view of the above characteristics of the cbl operon, the upstream and downstream primers were designed according to the cblB sequence of the main subunit gene of Alpha pili. Prepare DNA templates from various feed, drinking water (or sewage), fugitive dust, and feces waiting samples for PCR detection. When the template can amplify a band with an expected size of 500bp, it indicates that the strain to be tested is the product of diarrhea in piglets. Enterotoxigenic Escherichia coli or other pathogenic Escherichia coli; if the template cannot amplify the corresponding band, the strain to be tested is not enterotoxigenic Escherichia coli or other pathogenic Escherichia coli that causes diarrhea in piglets.

The invention discloses the application of the Alpha pili gene as a molecular marker for monitoring and identifying pathogenic Escherichia coli in piglets.

The invention also discloses specific primers for detecting pathogenic Escherichia coli of piglet diarrhea, the sequence of which 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 kit for detecting pathogenic enterotoxigenic Escherichia coli in piglet diarrhea, comprising:

PCR buffer, dNTPs, DNA polymerase, positive control DNA template and specific primers as above.

The alpha pili gene described in the present invention is shown in SEQ ID No.3. The main subunit gene cblB sequence is shown in SEQ ID No.4.

The advantages of the present invention are:

1. It is used to detect and monitor piglet diarrhea-causing enterotoxigenic E. coli or other pathogenic E. coli in clinical samples. It has been verified by PCR detection of multiple strains of pathogenic E. coli, and the results obtained by the PCR detection method are accurate. And there are no false positives and cross-reactions.

2. The cost of reagents and equipment required by this detection method is low, and the designed and synthesized detection primers can be stored at -20°C for a long time and can be used many times.

3. This method can be used for clinical monitoring and identification of piglet diarrhea-causing E. coli enterotoxigenic E. coli or other pathogenic E. coli, and environmental samples such as feed, drinking water (or sewage), dust and feces can be used for direct sample preparation and amplification , eliminating the need for isolation and culture of bacteria, fast and easy.

Description of drawings

Fig. 1 Electropherogram of specific rapid detection of Escherichia coli causing piglet diarrhea. in:

M is Trans2K plus II DNA Marker molecular marker, lane 1 is 987P wild strain 204, lane 2 is engineering strain DH5α, lane 3 is K99 standard strain C83907, lane 4 is K99 wild strain 637, lane 5 is bovine ETEC wild strain B41 , lane 6 is the F18ab standard strain F107/86, lane 7 is the F18ac standard strain 2134P, lane 8 is the F18ab wild strain SEC470, lane 9 is the human ETEC strain H10407, lane 10 is the K88ab standard strain C83901, and lane 11 is the K88ac standard strain C83902 , lane 12 is K88ad standard strain C83903, lane 13 is K88ac wild strain 2534-86, lane 14 is K88ac wild strain GT60, lane 15 is K88ac wild strain Bd 1107/75 08, lane 16 is K88ac wild strain YZ20, and lane 17 is K88ac wild strain 3030-2, lane 18 is 987P wild strain 1592, and lane 19 is Salmonella enteritidis 50336.

Detailed ways

The biological material information used in the following examples is as follows:

C83901 strains, C83902 strains and C83903 strains are K88ab, K88ac and K88ad three serotypes of enterotoxigenic Escherichia coli standard strains were purchased from China Veterinary Drug Control Institute Culture Collection Center; C83907 K99 serotype Escherichia coli standard strains were purchased from China Veterinary Drug Control Institute Bacteria Collection Center; F18ab serotype enterotoxigenic Escherichia coli standard strain F107/86, K88ac serotype field isolate 3030-2, and bovine source F41 serotype standard strain B41 by Professor David Francis of South Dakota State University, USA Gift; Salmonella enteritidis serotype 50336 was donated by Professor Diter Schifferli of the University of Pennsylvania; engineering bacteria DH5α was purchased from Takara Company; K88ac serotype field isolates 2534-86 strains, GT60 strains, Bd1107/75 08 strains and YZ20 strains, F18 Serotype SEC470, 204 and 1592 serotype 987P field isolates, and 637 K99 serotype field isolates were all isolated and preserved by the laboratory of Professor Zhu Guoqiang of Yangzhou University.

F18ab serotype enterotoxigenic Escherichia coli standard strain 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):H1 associated with enterotoxaemiain pigs.Veterinary Microbiology.25,267–281

K88ac serotype field isolate 3030-2: Francis, D.H. and J.A. Willgohs. 1991. Evaluation of a live avirulent Escherichia coli vaccine for K881, LT1 enterotoxigenic colibacillosis inweaned pigs. American Journal of Veterinary Research. 52:1051–1055.

Bovine F41 serotype standard strain B41: J.A.MORRIS,*C.THORNS,A.C.SCOTT,W.J.SOJKA,ANDG.A.WELLS.,1982.Adhesion In Vitro and In Vivo Associated with an AdhesiveAntigen(F41)Produced by a K99Mutant of the ReferenceStrainEscherichia coli B41.Infection and Immunity.36(3),1146-1153

Salmonella enteritidis serovar 50336; Xia Meng, Xianchen Meng, Chunhong Zhu, Heng Wang, Jinqiu Wang, Jiajia Nie, Philip R. Hardwidge4&Guoqiang Zhu., 2013. The RNA chaperone Hfq regulates expression of fimbrial-related genes and virulence of Salmonella enteritidis. Enter FEMS microbiology letters. 346(2):90-6

F18 serotype field isolate SEC470: H Liu, L Song, Y Cai, Y Wang, L Yu., 2016. Draft Genome Sequence of Escherichia coliStrain SEC470, Isolated from a PigletExperiencing Diarrhea. Genome Announcements.4(2):e00088-16

K88ac serotype field isolates GT60, YZ20 strains, 204 strains and 1592 strains 987P serotype field isolates, Zhang Xinjun, Hu Huijie, He Zhangke, Zhou Mingxu, Xu Mian, Zhu Guoqiang*., 2015. Escherichia coli type I fimA Gene cloning and sequence comparison analysis. Journal of Yangzhou University (Agriculture and Life Science Edition). 36(4):13-17

K99 serotype field isolates 637 strains: CM Ferreiros, MT Criado., 1983. Purification and partial characterization of a K99-antigen associated adhesin in Escherichia coli (637 strain). Revista De Fisiología. 39(1):45-50

K88ac serotype field isolate 2534-86: NM Clark, EM Berberov, M Wang, RAMoxley., 2006. Anti-capsular antibodies activate killing of Escherichia coli O8: K87 by the alternate complement pathway in porcine serum. Veterinary Immunology&Immunopathology.114( -2):185-191

K88ac serotype field isolate Bd 1107/75 08 strain: L.Blomberg&P.L.Conway.,2009.AnIn vitro Study of Ileal Colonization Resistance to Escherichia coli Strain Bd1107/75 08(K88) in Relation to Indigenous Squamous Gastric Colonization in Piglets of Varying Ages. Microbial Ecology in Health & Disease. 2(4):285-291.

Example 1

The nucleotide sequence of primer used in PCR detection method of the present invention is as follows:

AF-cblB-F:ATGAAAAAGGTATTTGCAAAATCTC (SEQ ID No. 1)

AF-cblB-R:TTAGATATCCTGAGACAGAT (SEQ ID No. 2)

The detection method process of the present invention is as follows:

1. Environmental sample collection: (1) Feed: Take 0.1g of feed, grind it into powder, and set aside; (2) Drinking water (or sewage): Take 1mL of water sample in a 1.5mL centrifuge tube, centrifuge at 10000rpm for 5 minutes, remove the supernatant, Precipitation for standby; (3) Dust: Place a piece of sterilized sulfuric acid paper in different positions of the pig farm, collect the dust falling on the sulfuric acid paper after 24 hours, and take 0.1g for standby; (4) Feces: Sterilized cotton Take 0.1g of pig feces for swab.

2. Prepare the chromosomal DNA template of the sample to be tested: take the sample to be tested in the previous step and add 50-100 μl of sterilized ultrapure water to suspend in a 1.5mL centrifuge tube, mix well, and place in boiling water at 100°C Bath in water for 5 minutes, cool to room temperature, and settle for 10 minutes (centrifuge at 10,000 rpm for 1 minute if possible). Take 2-3 μl of supernatant as PCR template.

3. PCR amplification and product detection: 50 μl amplification system includes 1×PCR buffer, 0.2 mM dNTP, 1 mM upstream and downstream primers, 5 μl PCR template of the sample to be tested and 0.5 μl DNA polymerase. The amplification cycle parameters were pre-denaturation at 94°C for 4 min; 25 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and extension at 72°C for 10 min. Take 5 μl of the PCR product and electrophoresis at a constant voltage of 90V for 40min in 1.2% agarose gel, stain with ethidium bromide EB, and use Trans2K plus II DNA Marker (Quanshijin Company) as the standard molecular weight analysis result. The sample to be tested can be amplified like a positive control. If a PCR product with an expected band size of about 500bp is added, the tested sample contains enterotoxigenic Escherichia coli or other pathogenic Escherichia coli that cause diarrhea in piglets.

Specific and rapid detection of Escherichia coli causing piglet diarrhea

Using a pair of upstream and downstream primers AF-cblB-F/AF-cblB-R, porcine enterotoxigenic Escherichia coli C83901, C83902, C83903, 3030-2, F107/86, 2143P, SEC470, 2534-86, GT60, Bd 1107 /75 08, YZ20, C83907, 637, 1592 and 204 can all amplify bands with a size of about 500bp, while other serotypes E. coli DH5α, bovine enterotoxigenic E. coli B41, human enterotoxigenic E. coli H10407 and Salmonella enteritidis 50336 does not have any bands, which is a negative control, as shown in Figure 1.

SEQUENCE LISTING

<110> Yangzhou University

<120> Application of Alpha Pili Gene in Monitoring and Identification of Pathogenic Escherichia coli in Piglet Diarrhea

<130>

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 25

<212>DNA

<213> Artificial sequence

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atgaaaaagg tatttgcaaa atctc 25

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ttagatatcc tgagacagat 20

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<211> 5015

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<213> Escherichia coli

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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 tcagggacaag 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

tattaaacc 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 aaagggggt tgcaccatga gaaatcgatt gattgcggcg atattggggct 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 tcaggggaggg 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. The application of Alpha pili gene as a molecular marker for monitoring and identifying pathogenic Escherichia coli in piglets. 2. be used for detecting the specificity primer of piglet diarrhea pathogenic escherichia coli, it is characterized in that sequence is as follows: AF-cblB-F:ATGAAAAAGGTATTTGCAAAATCTC AF-cblB-R:TTAGATATCCTGAGACAGAT. 3. A kit for detecting pathogenic Escherichia coli of piglet diarrhea, comprising PCR buffer, dNTPs, DNA polymerase, positive control DNA template, characterized in that, also comprising the specific primers claimed in claim 2.