AU2020210232B2 - Inert vector Escherichia coli and potential use thereof - Google Patents
Inert vector Escherichia coli and potential use thereofInfo
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- AU2020210232B2 AU2020210232B2 AU2020210232A AU2020210232A AU2020210232B2 AU 2020210232 B2 AU2020210232 B2 AU 2020210232B2 AU 2020210232 A AU2020210232 A AU 2020210232A AU 2020210232 A AU2020210232 A AU 2020210232A AU 2020210232 B2 AU2020210232 B2 AU 2020210232B2
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Abstract
Disclosed are an inert carrier Escherichia coli and the use thereof, wherein the inert carrier Escherichia coli has been deposited in the China General Microbiological Culture Collection Center (CGMCC), with a deposit address of Beijing, China, a deposit number of CGMCC No. 17339, a deposit date of March 18, 2019, a classification name of Escherichia coli, and a strain code of SE1. The Escherichia coli does not cause any macroscopic agglutination reaction with various chicken sera of different genetic backgrounds, i.e., does not generate a non-specific agglutination reaction with different kinds of chicken sera, has the properties of being expressed and displayed on the surface a specific antigen protein, and is used as an inert carrier in an indirect agglutination test for detecting antigens or antibodies.
Description
Inert Vector Inert Vector Escherichia Escherichia Coli Coli and Potential Use and Potential Use Thereof Thereof
TechnicalField Technical Field The present invention belongs to the field of biological detection, and relates to an inert The present invention belongs to the field of biological detection, and relates to an inert
vector Escherichia vector Escherichiacoli, coli,andand isolation, isolation, identification identification and and use thereof. use thereof. In ofview In view the of the characteristics that characteristics thethebacteria that number bacteria numberofof the the vector vector Escherichia Escherichia coli coli atat aa working working
concentration has concentration hasnono non-specific non-specific agglutination agglutination reactions reactions with with various various chickenchicken sera of sera of different genetic different genetic backgrounds, backgrounds, asaswell wellasasititprovides providesanan inertvector inert vector andand hashas potential potential use use
prospects in prospects in the the development development of of an an indirect indirect agglutination agglutination testmethod test method for for simple simple and and rapidrapid
detection of the target antigens or infected antibodies. detection of the target antigens or infected antibodies.
Backgroud Backgroud
Thedevelopment The developmentof of immunology immunology has provided has provided many technical many technical methods methods for the for the simple simple and rapid and rapid clinical clinical detection detection of ofpathogenic pathogenic bacteria bacteria and and infection infection antibodies. antibodies.Among them,the Among them, the agglutination test agglutination test is is aa traditional traditionaland and classic classic rapid rapid diagnosis diagnosis method forepidemic method for epidemic diseases diseases
whichisis widely which widelyused used in in medicine medicine and and veterinary veterinary clinics, clinics, and and its principle its principle is that is that after after thethe
insoluble particle antigens such as bacteria and red blood cells are bound to the corresponding insoluble particle antigens such as bacteria and red blood cells are bound to the corresponding
antibodies, when antibodies, anelectrolyte when an electrolyte is is present present and optimal, the and optimal, the antigen antigen particles particles will will agglomerate agglomerate
and aggregate and aggregatewith witheach eachother, other,forming formingsmall smallaggregates aggregates or or particlesthat particles that are are visible visible to to naked naked
eyes. The eyes. antigeninvolved The antigen involvedininthe thereaction reactionisis called called agglutinogen, agglutinogen,and andthe theantibody antibodyisiscalled called
lectin. Plate lectin. Plateagglutination agglutinationtest is is test a amore more commonly usedqualitative commonly used qualitative method methodininthethe agglutination reaction. agglutination reaction.AA drop of aa diagnostic drop of diagnostic serum serumcontaining containinga known a known antibody antibody
(appropriately diluted) (appropriately diluted) and and aa drop dropofofthe thebacteria bacteriasuspension suspensiontotobebetested testedarearedropped dropped on on a a clean transparent clean transparent glass glass plate, plate, and and gently gently mixed mixedin inequal equal volume. volume. Waiting Waiting for 2for 2 minutes minutes at at roomtemperature, room temperature,ififthere thereisisgranular granularagglutination agglutinationvisible visibletotonaked naked eyes, eyes, it is it is a positive a positive
reaction, which reaction, is often which is often used usedfor foridentification identification of of bacteria, bacteria, serotyping andthe serotyping and thelike. like. On Onthe the contrary, known contrary, diagnosticantigens known diagnostic antigenscan canalso alsobebeused usedtotodetect detectthe thepresence presenceofofcorresponding corresponding antibodies in antibodies in the the serum orwhole serum or wholeblood blood to to be be detected, detected, andand areare commonly commonly used used in theinglass the glass plate agglutination plate agglutination reaction reaction for for the thediagnosis diagnosis of of Brucella Brucella infection infectionand and the the whole blood plate whole blood plate agglutination test agglutination test for for Salmonella pullorum/typhimurium Salmonella pullorum/typhimurium and and the the like. like. It It is is worth worth noting noting that that
the indirect the indirect agglutination agglutination test test reaction reaction developed developedonon thethe basis basis of of the the direct direct agglutination agglutination
-1- -1-1 - reaction with inert vectors such as latex particles has expanded the detection application range reaction with inert vectors such as latex particles has expanded the detection application range and increased the sensitivity of the detection reaction. and increased the sensitivity of the detection reaction.
The agglutination test is suitable for the rapid diagnosis of some pathogenic infections. It The agglutination test is suitable for the rapid diagnosis of some pathogenic infections. It
has many has manyadvantages advantagessuch suchasassimple simpleandand fast,nonoadditional fast, additionalequipment, equipment,nonoexpensive expensive
equipment,low equipment, low cost cost andand can can be detected be detected on but on site, site,inbut in practice, practice, some drawbacks some drawbacks and and technical bottlenecks technical bottlenecks have haveappeared, appeared,forforexample, example, taking taking the the detection detection of the of the whole whole bloodblood
plate agglutination plate agglutination antigens infected with antigens infected Salmonellapullorum/typhimurium with Salmonella pullorum/typhimuriumas anasexample, an example, the agglutination antigen detection has certain limitations in practice applications. It has been the agglutination antigen detection has certain limitations in practice applications. It has been
reported that there are a variety of obvious cross-reactions, inconsistent detection results of reported that there are a variety of obvious cross-reactions, inconsistent detection results of
each batch, each batch, poor poor repeatability repeatability and and stability, stability, missed missed detections detections due due to to weak positive reactions weak positive reactions and other factors that affect the detection results, and the method is only relatively sensitive to and other factors that affect the detection results, and the method is only relatively sensitive to
the detection the detection effect effect of of adult adult flocks flocks rather rather than thanyounger younger chickens, chickens, andand there there may may be large be large
missed detection missed detection errors errors for for chicks. chicks. For For the the preliminary preliminary study, study, data data from fromthethesame same laboratorylocated, the laboratorylocated, thesame same technical technicalperson personused used the the most widely used most widely used commercially commercially
available Salmonella available pullorum/typhimurium Salmonella pullorum/typhimurium stained stained agglutination agglutination antigen antigen to detect to detect the the samesame
batch of batch of 200 serumsamples 200 serum samplesfrom from a chicken a chicken farm farm twice twice at at differenttimes, different times,itit was wasfound foundthat that the the total coincidence total rate of coincidence rate of the the detection detection results resultswas was only only 81%. When 81%. When comparing comparing the detection the detection
results with results with the the Salmonella Salmonella DDgroup groupELISA ELISA kit kit of BioChek of BioChek in Netherlands, in the the Netherlands, it found it was was found that the that the total total coincidence coincidencerate rateof of the the detection detection results results was 79.5%, was only only the 79.5%, the positive positive
coincidencerate coincidence rate (detection (detection rate rate or or sensitivity) sensitivity)was was 75.2-79.4%, and the 75.2-79.4%, and the negative negativecoincidence coincidence rate was rate 79.5-85.5%.The was 79.5-85.5%. Theabove above detection detection resultsand results andcomparative comparative analysis analysis showed showed thatthat whenwhen the commercial the commercial agglutination agglutination antigen antigen was wasusedused to detect to detect the Salmonella the Salmonella
pullorum/typhimurium pullorum/typhimurium serum serum antibody, antibody, the the sensitivity,specificity sensitivity, specificityand andaccuracy accuracy of of resultsdid results did not reach not reach aa level level to to be be desired, desired, and and the the detection detectionresults results of of each each batch batchwere werenotnotstable stableandand
consistent, and consistent, and the therepeatability repeatabilitywas was poor. poor. WhenWhen using using theagglutination the same same agglutination antigen antigen prepared from prepared fromSalmonella Salmonella gallinarum gallinarum isolatedfrom isolated from thethe same same farm farm to detect to detect thethe same same 200 200 sera, sera,
it was it foundthat was found that the thecoincidence coincidencerate ratebetween between thethe detection detection results results andand the the Salmonella Salmonella D D group ELISA group ELISAkit kitincreased increased from from 79.5% 79.5%toto88%, 88%, of of which which thethe positivecoincidence positive coincidencerate rate (detection rate or sensitivity) also increased to 97.5%. This result also proved that there was a (detection rate or sensitivity) also increased to 97.5%. This result also proved that there was a
certain degree certain degree or or more obviousfalse more obvious false positive positive misdetection and false misdetection and false negative negative missed detection missed detection in the results of the current commercial agglutination antigens. in the results of the current commercial agglutination antigens.
In view In viewofofthe thebottleneck bottlenecktechnology technologyof of thethe accuracy, accuracy, stabilityand stability andrepeatability repeatabilityofofthe the aboveagglutination above agglutinationantigen antigendetection detection results results that that need need to to be be improved andperfected, improved and perfected,the the root root cause is cause is that that the the agglutination agglutination antigens antigenscurrently currentlyused usedin inthetheagglutination agglutination testareare test whole whole
bacterial antigens, bacterial antigens, and and their their components arenot components are notsingle singlesomatic somaticantigen antigenofofandand non-bacterial non-bacterial
antigens O1, antigens O1,O9, 01, O9, 09, andand O12 O12 012 single-factor single-factor components, components, but bacterial but bacterial particleparticle antigensantigens that that compound compound multiple multiple antigen antigen components. components. In theory, In theory, there there areare non-specific non-specific cross-reactions cross-reactions with with
the same the sameororhomologous homologous components components of bacteria of bacteria of same of the the same family, family, same genera same genera and and other other different genera different (especially in genera (especially in Enterobacteriaceae), Enterobacteriaceae), inin view viewofofthethefact factthat thatthe thenumber number of of
bacteria in bacteria in the theworking working concentration concentration of agglutination of agglutination antigenantigen is generally is generally large, large, the the disadvantagesofofnon-specific disadvantages non-specificcross-reactions cross-reactions will will inevitably inevitably affect affect or even or even significantly significantly
interfere with the detection and diagnosis results, including use of the agglutination test as an interfere with the detection and diagnosis results, including use of the agglutination test as an
epidemicpurification epidemic purification technology technologyininanimal animalclinics clinicsininscreening screeningpullorum pullorum positiveflocks positive flocksandand eliminating the eliminating the breeding breedingpoultry, poultry,thereby therebyaffecting affectingthe theepidemic epidemic purificationeffect purification effectandand thethe
advancement of epidemic purification work. Therefore, if there is an inert vector bacteria, and advancement of epidemic purification work. Therefore, if there is an inert vector bacteria, and
at the same time a specific inert vector bacteria expressing a single target antigen is used to at the same time a specific inert vector bacteria expressing a single target antigen is used to
replace the replace the multi-component multi-component whole whole bacterial bacterial antigen antigen as theasagglutination the agglutination antigen, antigen, while while preserving the preserving the advantages advantagesofofintuitive intuitiveagglutination agglutinationreaction reactionresults, results, easy easy operation operationand andthe the like, itit can like, accurately and can accurately andperfectly perfectlyimprove improve the the specificity specificity of the of the agglutination agglutination antigen antigen
reaction, improve and perfect the repeatability and stability of the agglutination reaction, and reaction, improve and perfect the repeatability and stability of the agglutination reaction, and
has great has great application application prospects for the prospects for the improvement andperfection improvement and perfectionofofon-site on-siterapid rapiddiagnosis diagnosis methodsfor methods forsome somepathogen pathogen infections. infections.
Summary Summary Summary Objective ofofthe Objective theinvention: invention:ininview view of the of the urgent urgent need need to improve to improve and the and perfect perfect the
agglutination antigens agglutination antigenswith withspecific specific sensitivity,accurate sensitivity, accurate andand reliable reliable testtest results, results, goodgood
repeatability and stability in the current medical and veterinary clinics for the rapid diagnosis repeatability and stability in the current medical and veterinary clinics for the rapid diagnosis
of pathogenic of infections and pathogenic infections andepidemics, epidemics,ininthe thepresent presentinvention inventionwewe isolatesandand isolates identifiesa a identifies
strain inert strain inert vector vector Escherichia coli, which Escherichia coli, hasnononon-specific which has non-specific agglutination agglutination reaction reaction with with
different kinds different of chicken kinds of chickensera. sera.Based Basedon on thethe inert inert vector vector bacteria bacteria and and its its vector vector bacteria bacteria
should have should haveexpression expressioncharacteristics, characteristics, it it is is expected to provide expected to an inert provide an inert vector vector bacteria bacteria and and
potential use thereof in the development of a simple and rapid indirect agglutination detection potential use thereof in the development of a simple and rapid indirect agglutination detection method method .
Technical solution: in order to solve the problems in the existing technology, the present Technical solution: in order to solve the problems in the existing technology, the present
invention adopts invention adoptsthe thefollowing following technical technical solutions: solutions: an an inert inert vector vector Escherichia Escherichia coli coli SE1, SE1, whichhas which hasbeen beendeposited deposited in in thethe China China General General Microbiological Microbiological Culture Culture Collection Collection Center Center
(CGMCC) (CGMCC) on on March March 18, 18, 20192019 withwith the the accession accession number number of CGMCC of CGMCC No. No.17339, 17339, No.17339, is and andis and is classified as Escherichia coli with a strain code of SE1. classified as Escherichia coli with a strain code of SE1.
Wherein the vector bacteria (i.e., the inert vector Escherichia coli) can be cultured in LB Wherein the vector bacteria (i.e., the inert vector Escherichia coli) can be cultured in LB
and eosin-methylene and eosin-methyleneblue blue agar agar medium, medium, and cultivation and the the cultivation method method is as is as follows: follows: picking picking a a small amount small amountofofthe thestored storedstrains strains and and marking markingononLBLB or or eosin-methylene eosin-methylene blueblue agaragar medium, medium,
the cultivation the cultivation temperature is 37°C, temperature is 37℃, wherein whereingray-white gray-white round round colonies colonies can can be formed be formed afterafter
incubation at incubation at 37°C 37°CininthetheLBLB agar agar medium; medium; black black colonies colonies withoutwithout metallic metallic luster luster can be can be formedafter formed after incubation incubation at at 37°C in the 37°C in the eosin-methylene blueagar eosin-methylene blue agarplate. plate. PCRprimers PCR primers targeting targeting thethe species-specific species-specific uidA uidA genegene of Escherichia of Escherichia coli used coli are are used to to identify the identify the SE1 vectorbacteria SE1 vector bacteriaSE1, SE1,andand thethe resultof of result PCRPCR amplification amplification of uidA of uidA gene gene is is
consistent with consistent with the the size sizeofofamplified amplifiedbands bands of K12 of K12 Escherichia Escherichia coli engineering coli engineering bacteria bacteria
standard strain standard strain DH5α DH5a and and DH5 and Escherichiacoli Escherichia Escherichia coliEDL933 coli EDL933 EDL933 (O157: (0157: (0157: H7 Escherichia H7 type H7 type type Escherichia Escherichia coli coli coli weakenedstrain weakened strain(lack (lack of of eae eae and and stx stx genes)) genes)) . Accordingtotothe According thenational nationalstandard standard method method (GB4789.38-2012), (GB4789.38-2012), indole indole (indole, (indole, I) I) test, test, methyl red (MR) test, VP test, citrate (C) utilization test and five sugar fermentation tests are methyl red (MR) test, VP test, citrate (C) utilization test and five sugar fermentation tests are
used to used to conduct conductbiochemical biochemical testdetection test detectionandand identificationononthetheabove identification above SE1SE1 Escherichia Escherichia
coli vector coli vector bacteria, bacteria, and and the the results results meet meet the the biochemical characteristics of biochemical characteristics of Escherichia Escherichia coli coli described in described in the the national national standard standard method. method.
Theinert The inert vector vectorEscherichia Escherichiacoli colidescribed described in in thethe present present invention invention is isolated is isolated fromfrom
healthy chicken healthy chicken flocks. flocks.
Theglass The glassplate plate agglutination agglutinationtest test is is used used toto verify verify that that the the vector vector bacteria bacteria suspension suspension does not does not have haveself-coagulation self-coagulationphenomenon, phenomenon,and and has has no non-specific no non-specific agglutination agglutination reactions reactions
with various with various chicken chickensera sera of of different different genetic genetic backgrounds. Thechicken backgrounds. The chicken serainclude, sera include,but butare are not limited not limited to, to,SPF SPF chicken serum, healthy chicken serum, healthychicken chickenserum, serum,chicken chickenbacterial bacterialinfection infection positive positive serum(e.g., serum (e.g., avian avian Escherichia Escherichia coli coli positive positiveserum, serum, Salmonella Salmonella positive positive serum, serum, Staphylococcus Staphylococcus
positive serum, positive serum, Pasteurella Pasteurella positive positive serum serumand andthethelike), like),chicken chickenparasite parasiteinfection infectionpositive positive
- 44 - serum(e.g., serum (e.g., chicken chickencoccidiosis coccidiosispositive positive serum, serum, taeniasis taeniasis positive positive serumserum and and the the like), like), chicken viral chicken viral infection infection positive positive serum (e.g., Chicken serum (e.g., Chicken Newcastle diseasepositive Newcastle disease positiveserum, serum,avian avian influenza positive influenza positive serum, serum, chicken chickenMarek's Marek's disease disease positive positive serum, serum, chicken chicken infectious infectious bursal bursal disease positive disease positive serum, chickenegg serum, chicken eggdrop dropsyndrome syndrome positive positive serum, serum, avian avian encephalomyelitis encephalomyelitis virus infection virus infection positive positiveserum serum and the like) and the like) and and various various immunized chicken immunized chicken seraofofwhich sera which thethe detected agglutination results are negative. detected agglutination results are negative.
Thebacteria The bacterianumber numberof of thethe inert inert vector vector Escherichia Escherichia coli coli of of thethe present present invention invention at aat a working concentrationhashasno no working concentration non-specific non-specific agglutination agglutination reactions reactions with with SPF SPF chicken chicken serum serum
infected with infected with O1 avian Escherichia 01 avian Escherichia coli, coli, SPF SPF chicken chickenserum seruminfected infectedwith withO2 O2 avian avian
Escherichia coli Escherichia coli and and SPF SPFchicken chicken serum serum infected infected withwith 078 O78 avianavian Escherichia Escherichia coli; coli; and and K12 K12 Escherichia coli Escherichia coli engineering bacteria standard engineering bacteria standard strain strain DH5α, DH5a, Escherichia Escherichia DH5, Escherichia coli coli coli EDL933 EDL933 EDL933 (O157: (0157: (O157:
H7type H7 typeEscherichia Escherichiacoli) coli)and andother otherEscherichia Escherichia coli coli cancan react react with with thethe SPFSPF chicken chicken serumserum
infected by the above Escherichia coli. infected by the above Escherichia coli.
The content of the present invention also includes use of the inert vector Escherichia coli The content of the present invention also includes use of the inert vector Escherichia coli
as an as an the the inert inert vector vector in in an an indirect indirect agglutination agglutination test test for for detecting antigens or detecting antigens or antibodies, antibodies, becausethe because theinert inert vector vectorbacteria bacteria SE1 SE1is isananinsoluble insoluble particleantigen, particle antigen,andand hashas thethe bearing bearing
function of an indirect agglutination test vector and inert vector. function of an indirect agglutination test vector and inert vector.
The content of the present invention also includes use of the inert vector Escherichia coli The content of the present invention also includes use of the inert vector Escherichia coli
as a reagent for an indirect agglutination test for detecting antigens or antibodies, because the as a reagent for an indirect agglutination test for detecting antigens or antibodies, because the
inert vector bacteria SE1 can be used as an inert vector for an indirect agglutination test for inert vector bacteria SE1 can be used as an inert vector for an indirect agglutination test for
detecting antigens detecting antigens ororantibodies, antibodies,and andcancan also also be used be used as anas an inert inert vectorvector in an indirect in an indirect
agglutination test for detecting antigens or antibodies. agglutination test for detecting antigens or antibodies.
The content of the present invention also includes use of the inert vector Escherichia coli The content of the present invention also includes use of the inert vector Escherichia coli
as aa reagent, as reagent, technique techniqueandand platform platform for for detecting detecting a chicken-related a chicken-related pathogen pathogen infection, infection,
because the because the inert inert vector vector bacteria bacteria SE1 can be SE1 can be used usedasasananinert inertvector vectorininananindirect indirect agglutination test for detecting antigens or antibodies, and can also be used as an inert vector agglutination test for detecting antigens or antibodies, and can also be used as an inert vector
for detecting chicken-related pathogen infections (antibodies). for detecting chicken-related pathogen infections (antibodies).
Compared with the prior art, the characteristic advantages of the present invention are: Compared with the prior art, the characteristic advantages of the present invention are:
Thepresent The presentinvention inventionprovides provides an inert an inert vector vector Escherichia Escherichia coli coli strain strain SE1, SE1, and and the the
bacteria number bacteria number ofofthe thestrain strainatat aa working workingconcentration concentration hashas no no agglutination agglutination reaction reaction with with different kinds different of chicken kinds of chickensera. sera.Based Based on the on the inert inert vector vector bacteria bacteria has properties has the the properties of of expressing and presenting antigen factors on the bacterial surface, it provides an inert vector expressing and presenting antigen factors on the bacterial surface, it provides an inert vector bacteria and bacteria and potential potential use usethereof thereofforfora asimple simple andand rapid rapid indirect indirect agglutination agglutination detection detection method. It innovatively improves and perfects the specificity and sensitivity bottleneck of the method. It innovatively improves and perfects the specificity and sensitivity bottleneck of the wholecell whole cell agglutination agglutination test test in in the the existing existing agglutination agglutination antigen antigen antibody detection, and antibody detection, and isis expectedtoto bebedeveloped expected developedandand used used as aasnovel a novel detection detection reagent, reagent, andgreat and has has great application application value and value and market marketprospects. prospects.
Brief Description Brief DescriptionofofFigures Figures
Figure 11 is Figure is the the colony colony morphology morphologyofofthetheinert inertvector vector Escherichia Escherichia coli coli SE1 SE1and and
Escherichia coli Escherichia coli standard standard strain strainEDL933 EDL933 ononLBLB agar agar medium; medium;
Figure 22 is Figure is the the colony colony morphology morphologyofofthetheinert inertvector vector Escherichia Escherichia coli coli SE1 SE1and and Escherichia coli Escherichia coli standard standard strain strainEDL933 EDL933 ononeosin-methylene eosin-methylene blue blue agar agar medium; medium;
Figure 33 isis the Figure the species-specific species-specific PCR identification diagram PCR identification of the diagram of the inert inert vector vector Escherichia coli Escherichia coliofofthethe present present application, application, laneslanes 1-5 molecular 1-5 are: are: molecular weight weight marker, marker,
Escherichia coli Escherichia coli SE1, SE1, Escherichia Escherichia coli coliEDL933, EDL933, Escherichia Escherichia coli coliDH5α DH5a andSalmonella and DH5 and Salmonella Salmonella
negative control; negative control;
Figure 4 is the agglutination reaction diagram, the detection results of the agglutination Figure 4 is the agglutination reaction diagram, the detection results of the agglutination
reaction test reaction test of of the thevector vectorbacteria bacteriaSE1 SE1 bacteria bacteria suspension with 10 suspension with 10 billion billion CFU/mL bacteria CFU/mL bacteria
concentration and chicken sera of different types and different genetic backgrounds: concentration and chicken sera of different types and different genetic backgrounds:
1: SPFchicken 1: SPF chicken serum; serum; 2: Infected 2: Infected chicken chicken positive positive serum Escherichia serum (avian (avian Escherichia coli coli positive serum); positive 3: Infected serum); 3: Infected chicken positive serum chicken positive serum(Salmonella (Salmonella positive positive serum); serum); 4: 4: Infected Infected
positive serum chicken positive chicken serum(chicken (chicken coccidiosis coccidiosis positive positive serum); serum); 5: Infected 5: Infected chicken chicken positive positive
serum(taeniasis serum (taeniasis positive positive serum); 6: Viral serum); 6: infected positive Viral infected positiveserum serum (chicken Newcastledisease (chicken Newcastle disease positive serum); positive 7: Viral serum); 7: infected positive Viral infected positive serum serum (chicken Newcastledisease (chicken Newcastle diseasepositive positiveserum); serum);
8: 8: Viral infectedpositive Viral infected positiveserum serum (avian (avian influenza influenza positive positive serum);serum); 9: Vaccinated 9: Vaccinated yellow feather yellow feather
type of type of local local breed breedbroiler broilerserum; serum; 10:10: white white feather feather broiler broiler clinical clinical immune immune serum; serum; 11: 11: VaccinatedGuangxi Vaccinated Guangxi Yuanfeng-based Yuanfeng-based Lingshan Lingshan fragrantfragrant chicken chicken breedserum; breed broiler broiler12:serum; 12: clinical immune clinical serumfrom immune serum from Changzhou Changzhou Sandeli Sandeli Golden Golden Grass.Grass.
Note: the Note: the negative negativecontrol controlfor foragglutination agglutinationreaction reactionisisthe thereaction reactionbetween betweenSE1SE1 and and
physiological saline; physiological saline; the thepositive positivecontrol controlforfor agglutination agglutination reaction reaction is the is the agglutination agglutination
- 66 - reaction of reaction of the the Salmonella pullorumreference Salmonella pullorum referencestrain strainCVCC526 CVCC526 and SPF and the the chicken SPF chicken positive positive serum (infected with Salmonella enteritidis). serum (infected with Salmonella enteritidis).
DetailedDescription Detailed Description Thepresent The presentinvention inventionwill willbe be further further described described in detail in detail below below in conjunction in conjunction with with
specific embodiments. specific embodiments.
Before further Before further describing describing the the specific specific embodiments embodiments ofofthe thepresent presentinvention, invention,it it should be should be
understoodthat understood that the the protection protection scope scopeofofthe thepresent presentinvention inventionisisnot notlimited limitedtotothe thefollowing following specific embodiments; specific embodiments; itit should shouldalso also be be understood understoodthat thatthe the terms terms used usedinin the the embodiments embodiments of of
the present the invention are present invention are to to describe specific embodiments, describe specific nottotolimit embodiments, not limitthe theprotection protectionscope scope
of the of the present present invention. invention. Unless otherwisedefined, Unless otherwise defined,all all technical technical and and scientific scientific terms used in terms used in the present the present invention invention have the same have the samemeaning meaningas as commonly commonly understood understood by those by those skilled skilled in in the the art. InIn addition art. addition to tothe thespecific specificmethods, methods, equipment, andmaterials equipment, and materialsused usedininthe theembodiments, embodiments, based on based onthe the knowledge knowledgeof of thethe priorart prior artand andthe thedescription descriptionofofthe thepresent presentinvention inventionbybythose those skilled in skilled in the the art, art,any any method, equipmentandand method, equipment material material of of thethe prior prior artart thatisissimilar that similartotooror
equivalent to equivalent to the the method, equipmentand method, equipment andmaterial materialininthe theembodiments embodimentsof of thethe present present invention invention
can also be used to achieve the present invention. can also be used to achieve the present invention.
Example 1: Isolation and identification of Escherichia coli SE1 Example 1: Isolation and identification of Escherichia coli SE1
360-day-oldhealthy 360-day-old healthylaying laying hens hens werewere collected collected from from healthy healthy chickenchicken flocks flocks of the of the buildings 22 and buildings and 44 in in the the first firstpoultry poultryfarm farmofofJiangsu JiangsuWuxi Wuxi Mashan PoultryGroup Mashan Poultry Groupon on October October
18, 18, 2016, andplaced 2016, and placedininultra-clean ultra-cleanbenches. benches.TheThe chicken chicken bodybody surface surface was sterilized was sterilized with with
alcohol, and alcohol, and chicken's chicken'sliver, liver, spleen, spleen, intestine intestine and andother otherorgans organsandand tissues tissues were were removed removed
aseptically, placed in a sterile culture dish, shredded and ground at the same time and weighed, aseptically, placed in a sterile culture dish, shredded and ground at the same time and weighed,
and homogenate and homogenate was was drawn drawn intointo a steriletest a sterile testtube tube for use. Buffered for use. peptonewater Buffered peptone water(BPW) (BPW)waswas
weighed,autoclaved weighed, autoclavedand andsubpackaged, subpackaged, and and the the collected collected tissue tissue samples samples werewere addedadded to BPWto BPW
and incubated and incubated at at 37°C 37°C overnight. overnight. 11 mL mLwas was drawn drawn for for enrichment enrichment culture culture at at 37°C 37°C in in Escherichia coli Escherichia coli detection detection broth broth (EC (ECbroth), broth),after after overnight overnight culture, culture, 20uL 20μLofofthe 20µL theenrichment enrichment solution was solution drawnfor was drawn forstreak streak culture culture on on macconkey macconkey agar agar medium medium at 37°C. at 37°C. The colonies The colonies with with suspicious characteristics suspicious characteristics was pickedfor was picked forstreak streakculture cultureonon eosin-methylene eosin-methylene blue blue agar,agar, the the black and black andpurple purplesingle singlecolonies coloniesonon eosin-methylene eosin-methylene blueblue agar agar was picked was picked for purification for purification
culture at culture at 37°C on LB 37°C on LBagar agarmedium. medium. The The purified purified colonies colonies onagar on LB LB medium agar medium were were picked picked and inoculated and inoculatedininLBLBliquid liquidmedium, medium, the the suspicious suspicious Escherichia Escherichia coli coli colonies colonies were were further further
-7 7- biochemicalidentified biochemical identified and and species-specific species-specific PCR identified. PCR identified.
11 mL ofthe mL of the above aboveSE1 SE1 bacteriasolution bacteria solutioncultured culturedovernight overnightwas was taken taken to to prepare prepare a DNA a DNA
template by template byaa boiling boiling method. method.The TheEscherichia Escherichia coliB-glucuronidase coli β-glucuronidase ß-glucuronidase gene gene uidA uidA was was amplified amplified
by PCR, by PCR,and andobserved observed and and identifiedbyby1.5% identified 1.5% gelgel electrophoresis,and electrophoresis, and theexpected the expected sizeofofthe size the
target DNA target fragment DNA fragment was was 162bp; 162bp; the the synthetic synthetic primer primer sequences sequences in reference in reference were were as follows: as follows:
F: F: 5′ TGGTAATTACCGACGAAAACGGC TGGTAATTACCGACGAAAACGGC3' 3′(SEQ (SEQ ID ID ID NO. NO.NO. 1); 5' 1); 1); R: R: 5′ F: 5' TGGTAATTACCGACGAAAACGGC 3' (SEQ R: 5' ACGCGTGGTTACAGTCTTGCG ACGCGTGGTTACAGTCTTGCG 3' (SEQ ID 3′ (SEQ NO. 2); ID theNO. 2); the system reaction reaction20system µL, 20 μL, including uL, including
10 10 μL of 22 ×X Taq µL of uL Master Mix Taq Master Mix (Dye (DyePlus), Plus), each each 1 1 μL µL of uL of uidA-F/R (10 μM), uidA-F/R (10 µM), 22 uL uM), μL of µL of DNA DNA template, 66 uL template, μLofofsterile µL sterileultrapure ultrapurewater watertotomake make upuL; up 20 20 the µL; μL;optimized the optimized PCR reaction PCR reaction
conditions: 94°C conditions: for 55 min; 94°C for min;94°C 94°Cforfor3030S, s,s,60°C 60°Cforfor 3030 s, s,72°C S, 72°C forfor 30 30 s, s, S, 3030 cycles;72°C cycles; 72°C forfor
10 min.The 10 min. Theresults resultsshowed showed thatthat SE1 SE1 was to was able able to amplify amplify bands bands of of the the same same size size as the as the
reference Escherichia reference Escherichia coli coli strain strain EDL933 (O157: EDL933 (0157: (O157: H7-type H7-type Escherichia Escherichia coli coli weakened weakened strain,strain,
lack of lack of eae eae and and stx stx genes) genes) and and Escherichia Escherichia coli coli DH5α DH5a (Figure (Figure DH5 (Figure 3). 3). 3).
Indole (indole, I) test, methyl red (MR) test, VP test and citrate (C) utilization test were Indole (indole, I) test, methyl red (MR) test, VP test and citrate (C) utilization test were
used totoconduct used conduct biochemical biochemical identification identification for inert for the the inert vector vector bacteria bacteria SE1the SE1 using using the commercialmicro-biochemical commercial micro-biochemical tube, tube, andand the the results results were were analyzed analyzed and and determined determined according according
to the to the instructions instructions ofof the themicro micro biochemical biochemical tube,tube, andresults and the the results met met the the biochemical biochemical
characteristics ofof Escherichia characteristics Escherichia coli coli described describedin in the the national national standard standard method method
(GB4789.38-2012) (Table (GB4789.38-2012) (Table 1). 1). The The above above results results indicated indicated that that we isolated we isolated and identified and identified a a
strain of Escherichia coli and named it SE1. strain strain of of Escherichia Escherichia coli coli and and named named it it SE1. SE1.
Table 1: Table 1: SE1 biochemicalcharacteristics SE1 biochemical characteristics Indole Indole Methyl Methyl VP test VP test Citrate Citrate Glucose Glucose Lacto Lacto Maltos Maltos Manno Manno Sucro Sucro
(I) (I) red red (VP) (VP) (C) (C) gas gas se se e e se se se se
(MR) (MR) production production
(37℃) (37°C)
SE1 SE1 + + + + - - - - + + + + + + + + + + EDL933 EDL933 + + + + - - - - + + + + + + + + + + DH5α DH5a + + + + - - + + - + + + + + + DH5 -- - --
Note: "-" represents negative; "+" represents positive. Note: "_" represents negative; "+" represents positive.
- 88 -
Example2 2Verification Example Verificationthat thatinert inert Escherichia Escherichia coli coli SE1 hasnononon-specific SE1 has non-specificagglutination agglutinationwith with different kinds different kinds of ofgenetic geneticbackground chickensera background chicken sera The inert The inert vector vector bacteria bacteria SE1 cultured overnight SE1 cultured overnight was centrifuged at was centrifuged at 4°C with aa 4°C with
centrifuge at centrifuge at 4000 rpmfor 4000 rpm for 10 10 min, min,and andthe thesupernatant supernatantwas wasdiscarded. discarded.The Thebacteria bacteriapellet pelletwas was
resuspendedininsterile resuspended sterile PBS PBSandand washed washed threethree timestimes beforebefore being being resuspended resuspended to different to different
concentrations of concentrations of bacteria bacteria (500 million to (500 million to 10 10 billion billion CFU/mL) quantity CFU/mL) quantity gradient gradient to to bebe tested. tested.
Before test, Before test, the the bacteria bacteria solution solution was mixedwith was mixed witha avortexer, vortexer,and and thethe agglutination agglutination testwas test was first performed first withsterile performed with sterile PBS PBSandand SPF SPF chicken chicken sera sera to to ensure ensure thatabove that the the bacteria above bacteria solution had solution had nonoself-coagulation self-coagulation and andnon-specific non-specificagglutination. agglutination. A Afewfew pieces pieces of of
surface-cleaned ordinary surface-cleaned ordinary glass glass plates plates were weretaken taken in ultra-clean in an an ultra-clean table table (room (room temperature,20-25°C), temperature,20-25°C) temperature,20-25°C),thethe the vector vector vector bacteria bacteria bacteria waswas was centrifuged, centrifuged, centrifuged, resuspended resuspended resuspendedand and with andwashed washed washed with with sterile PBS sterile pre-cooled to PBS pre-cooled to 4°C 4°Cfor for33times, times,and andthen thenresuspended resuspendedandand diluted diluted to to thethe specified specified
bacteria concentration. bacteria concentration. AAdrop (approximately drop(approximately 10 of 10 uL) µL) μL) of vector vector bacteria bacteria wasand was drawn drawn and droppedvertically dropped vertically on onthe thesurface surfaceofofa aglass glassplate plateplaced placedhorizontally.using horizontally.usinga micropipette. a micropipette.
Thenananequal Then equalamount amount of serum of serum to betotested be tested was quickly was quickly added.added. The bacteria The bacteria solution solution and and serumwere serum weremixed mixed thoroughly thoroughly by using by using a sterilized a sterilized pipette pipette tip,and tip, andcoated coatedinto intoa asheet sheetwith witha a diameter of diameter of 1-2 1-2cm, cm,and andthen thenthetheglass glassplate platewas was shaken shaken smoothly. smoothly. The The test test results results mustmust be be observed within observed within 22 min. min. The Thestandard standardjudgment judgmentstatus statuswas wasthat thatwithin within2 2min min at at room room
temperature, ifif the temperature, the bacteria bacteria solution solution and andthe theserum serum to tested to be be tested produced produced a flocculent a flocculent or or
granular precipitate visible to naked eyes, the reaction result was judged as positive, otherwise granular precipitate visible to naked eyes, the reaction result was judged as positive, otherwise
it was judged as negative. it was judged as negative.
Theresults The results showed showed that that under under different different concentration concentration conditions conditions (500 (500 million million to 10 to 10 billion CFU/mL), billion theinert CFU/mL), the inert Escherichia Escherichiacoli coli vector vector strain strain SE1 hadno SE1 had noself-coagulation, self-coagulation, and and the the results of results of agglutination agglutinationtests testswith with different different types types of chickens of chickens from genetic from various various genetic
backgrounds, including: backgrounds, including: SPF chicken serum, SPF chicken serum,healthy healthychicken chickenserum, serum,chicken chickenbacterial bacterial infection positive infection positive serum, chickenparasite serum, chicken parasiteinfection infectionpositive positiveserum, serum,chicken chicken virus virus infection infection
positive serum positive andmultiple serum and multipleimmunized immunized chicken chicken sera,sera, werewere all negative all negative (Table (Table 2, Figure 2, Figure 4), 4), indicating that indicating that the the inert inertEscherichia Escherichiacoli coliSE1 SE1 had had no non-specific agglutination no non-specific agglutination reaction reaction with with different kinds of chicken sera, and it was considered as an inert vector bacteria. different kinds of chicken sera, and it was considered as an inert vector bacteria.
Table 2: agglutination reaction results of different concentrations of SE1 bacteria suspension Table 2: agglutination reaction results of different concentrations of SE1 bacteria suspension
with chicken with chickensera sera Vector bacteria Vector bacteria SE1 SE1 bacteria bacteria suspension suspension with with different concentrations different concentrations (CFU/mL) (CFU/mL)
- 99 -
500 11 billion billion 22 billion billion 5 5billion billion 1010 billion billion
million million
SPF Chicken SPF ChickenSerum Serum (provided (provided by by --- - -- - - - -- - -
ShandongAgricultural Shandong AgriculturalUniversity) University) SPF Chicken SPF ChickenSerum Serum (provided (provided by by --- - -- - - - -- - -
HarbinMedical Harbin MedicalUniversity) University) Avian Escherichia Avian Escherichiacoli colipositive positive -- - - - - - - - - - - I
serum serum
Avian Escherichia Avian Escherichiacoli coliclinical clinical- - - - - - - - - - -
negative serum negative serum SPFchickenserum SPFchicken serum infected infected with with 01 O1 --- - - - - - - - -
Escherichiacoli Escherichia Escherichia coli coli
SPFchicken SPF chickenserum serum infected infected with with O2 O2 - - - - -- - - - - - - - I
Escherichia coli Escherichia coli
SPF chicken SPF chickenserum serum infectedwith infected with - - - - - -- - - - -
O78Escherichia 078 Escherichiacoli coli Salmonellaclinical Salmonella clinical positive positive serum serum - - - - - -- - - - -
SPF chicken SPF chickenserum seruminfected infectedwith with - - - - - - - - -- - -
Salmonellagallinarum Salmonella gallinarum SPF Chicken SPF Chickenserum serum infectedwith infected with- - - - -- - - - - - I - -
Salmonella enteritidis Salmonella enteritidis
Salmonella pullorum/typhimurium Salmonella pullorum/typhimurium- - - - - - - - - - -I
clinical positive serum clinical positive serum
Salmonella pullorum/typhimurium Salmonella pullorum/typhimurium- - - - -I - - - -- - -
clinical negative clinical negative serum serum
AvianPasteurellosis Avian Pasteurellosis positive positive serum serum - - - -- - - I - - - -
Chicken Staphylococcus Chicken Staphylococcuspositive positive - - - - -- - - - - - - - - I
serum serum
Chickencoccidiosis Chicken coccidiosispositive positive serum serum - - I - - I - - -I - -- - -
Chickentaeniasis Chicken taeniasis positive positive serum serum - - - -- - -- - -- - -
- 10 - - -10-
Avianleukosis Avian leukosis virus virus positive positive serum serum -- -- - -- - -- - --
Chickennewcastle Chicken newcastle disease disease positive positive -- - -- - -I - -I - -I
serum serum
Avianinfluenza Avian influenzapositive positive serum serum - - - - - - - -I - -
Chicken Marek's Chicken Marek'sdisease diseasepositive positive - -- - -- - - - -- - -
serum serum
Chicken infectious Chicken infectious bursal bursal disease disease - - - -- - - - -- - -
positive serum positive serum
Mycoplasmagallisepticum Mycoplasma gallisepticum positive positive - - - -- - - - -- - -I
serum serum
Chickenegg Chicken eggdrop dropsyndrome syndrome positive positive - - - - - - - -- - - I
serum serum
Avian encephalomyelitis Avian encephalomyelitis virus virus - - - - -- - - - - -- - -I
infection positive infection positive serum serum
ChickenInfectious Chicken Infectiousbronchitis bronchitisvirus virus - - - - - -- - - --
positive serum positive serum
Yellow feather Yellow feather broiler broiler(rooster) (rooster) - - - - - -- - -- - -I
serum serum
Yellowfeather Yellow feather broiler broiler (hen) (hen) serum serum - -- - -- - - - - - - - -
White feather broiler (rooster) serum White feather broiler (rooster) serum -- - -- - - - - - -I
Whitefeather White feather broiler broiler (hen) (hen) serum serum -- - -- - -- - -- - -
Hailan Brown Hailan Browncommercial commercial layer layer serum serum - -- - - - - I - -I - -I
Guangxi Yuanfeng Guangxi Yuanfengspiced spicedchicken chicken - -- - -- - -- - -- - -I
(Lingshanfragrant (Lingshan fragrant chicken) chicken)serum serum ChangzhouSandeli Changzhou SandeliGolden Golden Grass Grass - - - - - - - -- - -I
chicken serum chicken serum Jiangsu Lihua Jiangsu mountain -- snow mountain Lihua snow - -- - - - -I - -
chicken serum chicken serum Beijing fatty Beijing fatty chicken chicken serum serum - - - -- - -- - -I - - I
YunlingChahuachicken Xishuangbanna YunlingChahua Xishuangbanna chicken - - - -- - -I - -- - - I
- 11 - - -11- serum serum
Luhuachickenserum Luhua chickenserum - - - -- - - - - - -
Jiangsu Luyuan Jiangsu Jiangsu Luyuanchicken Luyuan chicken chicken Serum Serum Serum - - - - - I - - - - -
Note: "-" represents negative; "+" represents positive "-" represents negative; "+" represents positive Note: "_"
In summary, the basic principle of the invention of the present application, the main In summary, the basic principle of the invention of the present application, the main
identification characteristics of the inert vector bacteria SE1, and an inert vector provided by identification characteristics of the inert vector bacteria SE1, and an inert vector provided by
the inert vector bacteria in the development of an indirect agglutination test detection method the inert vector bacteria in the development of an indirect agglutination test detection method
and potential application prospects are described. Those skilled in the art should understand and potential application prospects are described. Those skilled in the art should understand
that the that the present presentinvention invention isisnot notlimited bybythethe limited above aboveembodiments, embodiments, and the above and the above
embodiments embodiments and and specificationonly specification only describethetheinvention describe inventionprinciples principlesofofthe the present present application. Without departing from the functions, principles and scope of the present application. Without departing from the functions, principles and scope of the present
invention, invention, thepresent invention, the present present invention willwill invention invention will continue to be to continue continue tobe beimproved improved improved and perfected, and these and and perfected, and perfected, and these
improvements improvements arerequired are requiredtotobebewithin withinthe theprotection protectionscope scopeofofthe the present present invention. invention. The The
claimedprotection claimed protection scope scopeof of the the present present invention is defined invention is defined by by the the appended claimsand appended claims and equivalents thereof. equivalents thereof.
- 12 - - 12
Claims (4)
1. 1. An An inert inert vector vectorEscherichia Escherichia coli, coli,which whichhas hasbeen been deposited deposited in in the theChina China General General
MicrobiologicalCulture Microbiological CultureCollection CollectionCenter Center(CGMCC) (CGMCC) on March on March 18,with 18, 2019 2019the with the accession accession
numberofofCGMCC number CGMCC No.17339, No. 17339, and and is is classified classified as Escherichia as Escherichia colicoli withwith a straincode a strain code of of SE1. SE1.
2. Use of the inert vector Escherichia coli of claim 1 in the preparation of an inert vector 2. Use of the inert vector Escherichia coli of claim 1 in the preparation of an inert vector
of an indirect agglutination test for detecting target antigens or in the preparation of an inert of an indirect agglutination test for detecting target antigens or in the preparation of an inert
vector of an indirect agglutination test for detecting antigen-targeted antibodies. vector of an indirect agglutination test for detecting antigen-targeted antibodies.
3. Use of the inert vector Escherichia coli of claim 1 as a reagent, technique and platform 3. Use of the inert vector Escherichia coli of claim 1 as a reagent, technique and platform
for an indirect agglutination test for detecting antigens or antibodies. for an indirect agglutination test for detecting antigens or antibodies.
4. Use of the inert vector Escherichia coli of claim 1 as a reagent, technique and platform 4. Use of the inert vector Escherichia coli of claim 1 as a reagent, technique and platform
for detecting a chicken-related pathogen infection. for detecting a chicken-related pathogen infection.
- 13 - - 13
SE1 EDL933 EDL933 Figure 11 Figure
SE1 LAS EDL933 Figure 22 Figure
uidA SE1 SE1 EDL933 EDL933DH5a Negaive DH5Negaive Negaive M
GEBER 2000bp
1000bp 750bp 500bp
250bp 162bp 100bp
Figure 33 Figure
1/2 1/2
1 3 4 2
5 6 7 8
9 10 11 12 12
Positive Positive Positive Negtivecontorl Negtive contorl contorl contorl
Figure 44 Figure
2/2 2/2 2/2
Sequencelist Sequence list
<110> Yangzhou <110> YangzhouUniversity University
<120>Inert <120> InertVector VectorEscherichia EscherichiaColi Coliand andUse UseThereof Thereof
<130> xhx2019050901 <130> xhx2019050901
<141> <141> 2019-05-09 2019-05-09
<160> 22 <160>
<170> SIPOSequenceListing <170> SIPOSequenceListing 1.01.0
<210> 11 <210> <211> 23 <211> 23 <212> DNA <212> DNA <213>Artificial <213> Artificial sequence sequence
<400> 11 <400> tggtaattac cgacgaaaac tggtaattac ggc cgacgaaaac ggc 23 23
<210> 22 <210> <211> 21 <211> 21 <212> DNA <212> DNA <213>Artificial <213> Artificial sequence sequence
<400> 22 <400> acgcgtggtt acagtcttgc acgcgtggtt acagtcttgc gg 21 21 Page 11 Page
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