CN105112519A - CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method - Google Patents
CRISPR-based Escherichia coli O157:H7 strain detection reagent box and detection method Download PDFInfo
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Abstract
The invention discloses a CRISPR-based Escherichia coli O157:H7 strain detection reagent box and a detection method and belongs to the technical field of molecular biology. The detection reagent box comprises primers designed for a CRISPR1 locus gene sequence or a homologous sequence with homology reaching higher than 95% of an Escherichia coli O157:H7 strain and shown as SEQ ID NO.1 and SEQ ID NO.2 in a sequence table. The primers are utilized for PCR amplification, and three unique spacer sequences (shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7) of CRISPR1 are detected to be bacterially positive in an amplification product. The detection method is simple to operate, high in sensitivity and specificity, uniform with serotyping result of Escherichia coli O157:H7 and low in detection expense and has good popularization and application value.
Description
Technical field
The present invention relates to a kind of Escherichia coli O 157 based on CRISPR: H7 bacterial strain detection kit, the invention still further relates to a kind of Escherichia coli O 157 based on CRISPR simultaneously: H7 bacterial strain detection method, belongs to technical field of molecular biology.
Background technology
Escherichia coli O 157: H7 is a colibacillary principal causative bacterial strain, O157:H7 infects not only causing bleeding property colitis, ecphyaditis, the serious gastrointestinal such as esophagostenosis and perforation of colon complication (PaiCH, GordonR, SimsHV, etal.SporadicCasesofHemorrhagicColitisAssociatedwithEsch erichiaColiO157:H7.Clinical, Epidemiologic, andBacteriologicFeatures [J] .AnnalsofInternalMedicine, 1984, 101 (6): 738-742), and in children and elderly population, also can cause hemolytic urinary tract syndromes (hemolyticuremicsyndrome, and thrombotic thrombocytic purpura (thromboticthrombocytopenicpurpura HUS), the systemic complications such as TTP), severe patient renal failure causes death (MoriY, WadaH, TamakiS, etal.OutcomeofThromboticThrombocytopenicPurpuraandHemoly ticUremicSyndromeinJapan [J] .ClinicalandAppliedThrombosis/hemostasis:OfficialJournal oftheInternationalAcademyofClinicalandAppliedThrombosis/ hemostasis, 2000, 5 (2): 110-112).At present, Escherichia coli O 157: H7 detection method mainly contains conventional detection method, immunological detection and molecular biological assay.The cost of routine monitoring method is higher, and molecular biological assay is easy, fast, normal with specific encoding gene as rfbE gene, flagellar antigen H7 specific gene flic, stx (shiga-like toxin), hly (hemolysin), eaeA (encoding adhesin) etc. are as target gene (PinaM.FRATAMICO, ChitritaDEBROY.DetectionofEscherichiaColiO157:H7inFoodUs ingReal-timeMultiplexPcrAssaysTargetingtheStx1, Stx2, WzyO157, andtheFlicH7OrEaeGenes [J] .FoodAnalyticalMethods, 2010, 3 (4): 330-337), there is high specificity, easy and simple to handle, the advantages such as result is accurate, it is the common method detecting Escherichia coli O 157: H7.
The short palindrome tumor-necrosis factor glycoproteins (Clusteredregularlyinterspacedshortpalindromicrepeats of the regular intervals of cluster, CRISPR) be by one section of discontinuous direct repetitive sequence (directrepeat, and insert intervening sequence (spacer wherein DR), S) (TouchonMARIE is formed, RochaEduardoPC.TheSmall, SlowandSpecializedCrisprandAnti-crisprofEscherichiaandSa lmonella [J] .PlOSOne, 2010,5 (6): e11126).First CRISPR finds (IshinoY in intestinal bacteria, ShinagawaH, MakinoK, etal.NucleotideSequenceoftheIapGene, ResponsibleforAlkalinePhosphataseIsozymeConversioninEsch erichiaColi, andIdentificationoftheGeneProduct [J] .JournalofBacteriology, 1987,169 (12): 5429-5433).(GuoXJ, WangYF, DuanGC, etal.DetectionandAnalysisofCrisprsofShigella [J] .CurrentMicrobiology, 2015,70 (1): 85-90 are all found at present in many bacteriums and archeobacteria; HorvathPHILIPPE, RomeroDennisA,
aNNE-CLAIRE, etal.Diversity, Activity, andEvolutionofCrisprLociinStreptococcusThermophilus [J] .JournalofBacteriology, 2007,190 (4): 1401-1412).Tumor-necrosis factor glycoproteins is almost completely the same in a CRISPR site, sequence is relatively conservative, its significant feature can be transcribed and form RNA secondary structure, it embodies the conservative property (MakarovaKiraS of bacterium in evolution, HaftDanielH, BarrangouRODOLPHE, etal.EvolutionandClassificationoftheCrispr-casSystems [J] .NatureReviews.Microbiology, 2011,9 (6): 467-477).Intervening sequence is alterable height in CRISPR structure, almost can not find two identical intervening sequences in same CRISPR site.Generally believe that intervening sequence derives from external source Mobile Genetic Elements at present, as plasmid, phage etc. (MojicaFranciscoJM,
c é SAR, Garc í a-Mart í nezJES ú S, etal.InterveningSequencesofRegularlySpacedProkaryoticRep eatsDeriveFromForeignGeneticElements [J] .JournalofMolecularEvolution, 2005,60 (2): 174-182).4 CRISPR sites have been described in intestinal bacteria, CRISPR1 and CRISPR2 is extensively present in intestinal bacteria, there is consistent tumor-necrosis factor glycoproteins, it lays respectively at core gene iaP and cysH, between ygcE and ygcF, CRISPR3 with CRISPR4 has consistent tumor-necrosis factor glycoproteins equally, it is (Diez-VillasenorC between core gene clpA and infA, AlmendrosC, Garcia-MartinezJ, etal.DiversityofCRISPRlociinEscherichiacoli [J] .Microbiology, 2010, 156 (Pt5): 1351-1361).The research of current CRISPR in intestinal bacteria mainly concentrates on the functionally active research (PulUMIT of intestinal bacteria CRISPR, WurmREINHILD, ArslanZIHNI, etal.IdentificationandCharacterizationofE.ColiCrispr-cas PromotersandTheirSilencingByH-NS [J] .MolecularMicrobiology, 2010,75 (6): 1495-1512, PougachKSENIA, SemenovaEKATERINA, BogdanovaEKATERINA, etal.Transcription, ProcessingandFunctionofCrisprCassettesinEscherichiaColi [J] .MolecularMicrobiology, 2010, 77 (6): 1367-1379), relation (the TouchonM that intestinal bacteria CRISPR and R-plasmid shift, CharpentierS, PognardD, PicardB, ArletG, RochaEP, etal.Antibioticresistanceplasmidsspreadamongnaturalisola tesofEscherichiacoliinspiteofCRISPRelements.Microbiology, 2012, 158:2997-3004) etc.And the quantitative PCR specific detection Escherichia coli O 157 based on CRISPR reported before this: the associated serum bacterial strains such as H7 (Useofclusteredregularlyinterspacedshortpalindromicrepeat sequencepolymorphismsforspecificdetectionofenterohemorrh agicEscherichiacolistrainsofserotypesO26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, andO157:H7byreal-timePCR) method, three pairs of primers and probe need be designed, detecting step is complicated, and cost is higher.
Summary of the invention
The object of this invention is to provide a kind of Escherichia coli O 157 based on CRISPR: H7 bacterial strain detection kit.
Meanwhile, the present invention also provides a kind of Escherichia coli O 157 based on CRISPR: H7 bacterial strain detection method, and the method is simple to operate, and Sensitivity and Specificity is high, and Escherichia coli O 157: H7 serotype result is consistent, and testing cost is low.
In order to realize above object, the technical solution adopted in the present invention is:
Escherichia coli O 157 based on CRISPR: H7 bacterial strain detection kit, it comprises for Escherichia coli O 157: the CRISPR1 locus gene sequence of H7 bacterial strain or its homology reach the primer of the homologous sequence design of more than 95%;
Described primer is as follows:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ' (as shown in SEQIDNO.1),
Downstream primer: 5 '-CGTAYYCCGGTRGATTTGGA-3 ' (as shown in SEQIDNO.2), Y are C or T, R is A or G.
Concrete, in downstream primer, Y is T, R is G.
Escherichia coli O 157 based on CRISPR: H7 bacterial strain detection kit, can also comprise 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4), sterilizing ultrapure water, positive control dna (Escherichia coli O 157: H7), 10 × PCR bacterium colony toughener and DNAMarker2000 etc.
Escherichia coli O 157 based on CRISPR: H7 bacterial strain detection method, comprise the following steps: with genome of E.coli DNA or bacterium liquid for template, upstream and downstream primer is utilized to carry out pcr amplification, check order and analyze pcr amplification product, detect that 3 of CRISPR1 unique intervening sequences are positive for bacteria, article 3, unique intervening sequence is as shown in SEQIDNO.5, SEQIDNO.6, SEQIDNO.7;
Described upstream and downstream primer is:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ' (as shown in SEQIDNO.1),
Downstream primer: 5 '-CGTAYYCCGGTRGATTTGGA-3 ' (as shown in SEQIDNO.2), Y are C or T, R is A or G.Concrete, in downstream primer, Y is T, R is G.
Described genome of E.coli DNA can adopt boiling method or DNA extraction kit to extract.
The reaction system of described pcr amplification is: 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4) 12.5 μ L, genome of E.coli DNA profiling 2 μ L, sterilizing ultrapure water 8.5 μ L, amounts to 25 μ L.
The reaction system of described pcr amplification is: 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4) 12.5 μ L, single bacterium colony, adds to 25 μ L after 10 × PCR bacterium colony toughener (commercial goods, main component is trimethyl-glycine) is diluted to 10 times with sterilizing ultrapure water.
The response procedures of described pcr amplification is: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 90s, totally 10 circulations; Sex change and annealing conditions constant, each circulation its extend step extend 10s, totally 25 circulations; 72 DEG C are continued to extend 10min.
Beneficial effect of the present invention:
The present invention is by Escherichia coli O 157 in CRISPRdatabase database: the Escherichia coli O 157 that H7 genome sequencing result is analyzed and preserved laboratory: H7 detects discovery, it has CRISPR1 site, and CRISPR1 detects 4 tumor-necrosis factor glycoproteinss and 3 unique intervening sequences.For above-mentioned CRISPR1 locus gene sequences Design upstream and downstream primer, sequencing analysis is carried out through pcr amplification after product, detect that 3 of CRISPR1 unique intervening sequences (as Suo Shi SEQIDNO.5, SEQIDNO.6, SEQIDNO.7) are positive for bacteria.This detection method is simple to operate, and Sensitivity and Specificity is high, and Escherichia coli O 157: H7 serotype result is consistent, and testing cost is low, has good application value.
Accompanying drawing explanation
Fig. 1 is the gel electrophoresis figure of pcr amplification product in test example.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Test example
One, the bacterial isolates used in test
Escherichia coli O 157: H7 was located away from Suixian County, Henan in 2000, and numbering is respectively 2000001.EscherichiacoliO157:H7 bacterial strain: EscherichiacoliO157:H7str.Sakai in CRISPRsdatabase, EscherichiacoliO157:H7str.EC4115, EscherichiacoliO157:H7str.TW14359, EscherichiacoliXuzhou21.
Two, the reagent used in test and instrument
(1) LB substratum
LB liquid nutrient medium: take 1.0g Tryptones, the leaching of 0.5g yeast powder, 1.0g sodium-chlor, be dissolved in 100mL distilled water, with 10mol/LNaOH adjust pH to 7.2,121 DEG C of autoclaving 20min, put 4 DEG C and save backup.
LB solid medium: take 1.0g Tryptones, the leaching of 0.5g yeast powder, 1.0g sodium-chlor, 1.5g agar powder, be dissolved in 100mL distilled water, with 10mol/LNaOH adjust pH to 7.2,121 DEG C of autoclaving 20min, when being cooled to about 50 DEG C, pour plate is for subsequent use.
(2) PCR reaction kit is purchased from Sangon Biotech (Shanghai) Co., Ltd., agarose is the import packing of BIOWEST company, yeast leaching powder, Tryptones are purchased from Oxoid company of Britain, agar powder available from Sigma, other common agents are domestic analytical pure level reagent.
PTC-100 type gene amplification in vitro instrument purchased from MJRESEARCH company, DYY-8C type voltage stabilization and current stabilization timing electrophoresis apparatus purchased from Liuyi Instruments Plant, Beijing, Genesnap image reading apparatus purchased from American Syngene company.
The test materials be not specifically noted and method are known technology, can draw together at common tool school bag " Molecular Cloning: A Laboratory guide " (J. Pehanorm Brooker, D.W Russell work, the third edition, Science Press, 2002) etc. in search.
Three, Escherichia coli O 157: the CRISPR site of H7 bacterial strain is detected
(1) preparation of DNA profiling
Boiling method is adopted to extract complete genome DNA from Escherichia coli bacteria liquid.Escherichia coli O 157 is taken out: the frozen conservation pipe of H7 in-80 DEG C of refrigerators, be put in 4 DEG C of refrigerator rewarmings 5 hours (4 ~ 6 hours), with aseptic inoculation ring fast fetching bacterium liquid in super clean bench, with segmentation method of scoring transferred species on LB solid medium flat board, 37 DEG C of thermostat containers are hatched 21 hours (18 ~ 24 hours), single bacterium colony on picking LB agar plate, be inoculated in LB liquid nutrient medium, 37 DEG C of shaking culture 7 hours (6 ~ 8 hours), get 1mL bacterium liquid in the Eppdorf pipe of 1.5mL, under rotating speed 14000r/min centrifugal 1 minute, abandon supernatant, add ultrapure water 100 μ L, concussion mixing, boil 10min, recentrifuge 10 minutes under rotating speed 14000r/min, get supernatant, obtain genome of E.coli DNA, be placed in-20 DEG C of storages for subsequent use.
(2) PCR primer Design and synthesis
According to intestinal bacteria CRISPR1 locus gene primers such as Touchon, as follows:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ',
Downstream primer: 5 '-CGTATTCCGGTGGATTTGGA-3 '.
Expection amplified production length is about 500bp, and primer is synthesized by match Parkson, Beijing gene engineering company limited.
(3) pcr amplification
The reaction system of pcr amplification: with reference to PCR reaction kit (Beijing Tian Gen biochemical technology company limited) description of product, prepare 25 μ L reaction systems, wherein 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4) 12.5 μ L, genome of E.coli DNA profiling 2 μ L, add sterilizing ultrapure water to 25 μ L.
The response procedures of pcr amplification: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 90s, totally 10 circulations; Sex change and annealing conditions constant, each circulation its extend step extend 10s, totally 25 circulations; 72 DEG C are continued to extend 10min.
(4) pcr amplification product analysis
Get 5 μ LPCR amplified productions, electrophoresis in 20g/L sepharose, does molecular weight marker with DNAMarker2000, voltage 5V/cm, after electrophoresis 20min, analyzes with gel images scanner.The electrophoresis result of pcr amplification CRISPR1 gene product in 20g/L sepharose is shown in Fig. 1 (in figure, M is DNAMarker, and 1 is pcr amplification product).As can be seen from Figure 1, the length of pcr amplification product and the length of expection meet, and confirm the success of pcr amplification CRISPR1 gene.
(5) pcr amplification result order-checking qualification
Transfer to Sangon Biotech (Shanghai) Co., Ltd. to check order pcr amplification product, total length sequencing result is as shown in SEQIDNO.3.The CRIAPR1 locus gene sequence (GeneBank:NC_002695.1) that in the gene order and Genebank that obtain checking order, EscherichiacoliO157:H7str.Sakai announces carries out BLAST comparison, and both result displays 99% are identical.
(6) pcr amplification result sequencing analysis
CRISPR1 locus gene sequence in sequencing result is analyzed, result is presented at Escherichia coli O 157: the CRISPR1 site of H7 can detect 4 tumor-necrosis factor glycoproteinss (as shown in SEQIDNO.4) and 3 unique intervening sequences (seeing the following form 1), these 3 unique intervening sequences are carried out BLAST comparison at Genebank database, finds that it mates completely with 3598252-3598283,3598313-3598344,3598374-3598405 of EscherichiacoliO157:H7str.Sakaichromosome respectively.
Table 1 Escherichia coli O 157: H7 bacterial strain CRISPR1 locus gene sequence information
There is according to antigen antibody reaction the feature of high degree of specificity, unknown antigen (bacterium) is detected by known antibodies, salt bacterial strain 2000001 uses Escherichia coli O 157 diagnostic serum and H7 diagnostic serum to carry out slide agglutination test, agglutination reaction is positive, and stroke-physiological saline solution makes negative control.
Embodiment 1
Based on the Escherichia coli O 157 of CRISPR in the present embodiment: H7 bacterial strain detection kit, comprises following primer:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ',
Downstream primer: 5 '-CGTATTCCGGTGGATTTGGA-3 '.
Embodiment 2
Ultimate principle: containing all ingredients component needed for polymerase chain reaction in detection kit, as primer, dNTPs, damping fluid, Taq enzyme etc., add detection sample in PCR reaction solution mixed solution, can pcr amplification reaction be carried out.Pcr amplification product judges through agarose electrophoresis dyeing, and the fragment place in corresponding size is occurred specific band by positive sample.
Product Overview: detection kit adopts polymerase chain reaction (PCR) technology, due to contained primer energy specific amplification Escherichia coli O 157: the CRISPR1 locus gene sequence of H7 bacterial strain, can be used for the infection/pollutional condition detecting various sample to be checked (as ight soil, food etc.).
Based on the Escherichia coli O 157 of CRISPR in the present embodiment: H7 bacterial strain detection kit, comprises: 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4) 20mL, sterilizing ultrapure water 30mL, (bacterium liquid preserved by glycerine to positive control dna (Escherichia coli O 157: H7) 1.5mL, it consists of 50% glycerine 500 μ L and 800500 μ L6 ~ 8h and cultivates bacterium liquid), 8 μm of each 5mL of ol/L up/down trip primer, and DNAMarker2000.
Upstream and downstream primer is:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ',
Downstream primer: 5 '-CGTATTCCGGTGGATTTGGA-3 '.
Operation instructions: extract sample DNA to be checked; Utilize upstream and downstream primer to carry out pcr amplification, be formulated as follows reaction system: 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4) 12.5 μ L, DNA profiling 2 μ L, add sterilizing ultrapure water to 25 μ L; Response procedures: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 90s, totally 10 circulations; Sex change and annealing conditions constant, each circulation its extend step extend 10s, totally 25 circulations; 72 DEG C are continued to extend 10min; PCR primer gel electrophoresis analysis, condition: 2.0% sepharose, voltage 5V/cm, time 20min; Observations under ultraviolet lamp, if occur, the band identical with positive control is the doubtful positive, and negative control does not have specific amplification band; Check order and analyze pcr amplification product, detecting that 3 of CRISPR1 unique intervening sequences (as Suo Shi SEQIDNO.5, SEQIDNO.6, SEQIDNO.7) are positive for bacteria.
Comparative example
Escherichia coli O 157: the serological identification of H7 bacterial strain:
1, principle: the feature according to antigen antibody reaction with high degree of specificity, detects unknown antigen (bacterium) by known antibodies.
2, serological identification method, step is as follows:
(1) get clean slide one piece, be divided into the little lattice of three row with glass pencil, and indicate the number of serum to be checked;
(2) use transfering loop picking 1 ring O157 diagnostic serum and H7 diagnostic serum in slide glass one end, stroke-physiological saline solution makes negative control;
(3) use O157 diagnostic serum and the mixing of H7 diagnostic serum with it of transfering loop picking a little bacterium to be measured, get bacterium to be measured at the slide the other end simultaneously and to mix with physiological saline and contrast;
(4) observations: after bacterium to be checked mixes with O157 diagnostic serum and H7 diagnostic serum, occurs in 5 minutes that macroscopic particulate state agglutinator is the positive;
(5) by following standard recording response intensity:
++++: occurs that large aggegation block, liquid are completely transparent;
+++: there are obvious aggegation block, liquid almost completely transparent, i.e. 75% aggegation;
++: have visible agglutination sheet, liquid is very not transparent, i.e. 50% aggegation;
+: opaque, has little granular substance, i.e. 25% aggegation;
-: uniform liquid is muddy, without agglutinator.
3, test-results
Result shows, and in 35 parts of testing samples, 33 parts are detected as positive for bacteria.
Embodiment 3
Based on the Escherichia coli O 157 of CRISPR in the present embodiment: H7 bacterial strain detection method, comprises the following steps:
(1) collection of sample and pre-treatment
Join in 10mL enrichment liquid by 500 μ L sample to be detected (ight soil to be checked or food etc.), 37 DEG C increase bacterium 5h (4 ~ 6h), and centrifugal 5 minutes of table model high speed centrifuge 12000rpm/min, abandons supernatant, and precipitation touches plate do detection;
(2) pcr amplification
Utilize upstream and downstream primer to carry out pcr amplification, primer is as follows:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ',
Downstream primer: 5 '-CGTATTCCGGTGGATTTGGA-3 ';
Pcr amplification reaction system: amount to 25 μ L, 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix (0.1U/ μ lTaqDNAPolymerase, 2 × PCR reaction buffer, 0.4mMdNTP, 4mMMgSO
4) 12.5 μ L, single bacterium colony, adds to 25 μ L after 10 × PCR bacterium colony toughener sterilizing ultrapure water is diluted to 10 times;
Pcr amplification reaction program: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 90s, totally 10 circulations; Sex change and annealing conditions constant, each circulation its extend step extend 10s, totally 25 circulations; 72 DEG C are continued to extend 10min;
(3) detected result
Get 5 μ LPCR amplified productions, electrophoresis in 20g/L sepharose, does molecular weight marker with DNAMarker2000, voltage 5V/cm, and after electrophoresis 20min, observations under ultraviolet lamp, occurs that the band identical with positive control is the doubtful positive; Check order and analyze pcr amplification product, detect that 3 of CRISPR1 unique intervening sequences are (as SEQIDNO.5, SEQIDNO.6, shown in SEQIDNO.7) be positive for bacteria and show that sample is subject to Escherichia coli O 157: the infection of H7 bacterial strain, in 35 parts of testing samples, 33 parts are detected as positive for bacteria, consistent with serotype result in comparative example.
Claims (6)
1. based on the Escherichia coli O 157 of CRISPR: H7 bacterial strain detection kit, is characterized in that: comprise for Escherichia coli O 157: the CRISPR1 locus gene sequence of H7 bacterial strain or its homology reach the primer of the homologous sequence design of more than 95%; Described primer is as follows:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ',
Downstream primer: 5 '-CGTAYYCCGGTRGATTTGGA-3 ', Y are C or T, R is A or G.
2. detection kit according to claim 1, is characterized in that: also comprise 2 × TaqPCRMasterMix, sterilizing ultrapure water and DNAMarker2000.
3. based on the Escherichia coli O 157 of CRISPR: H7 bacterial strain detection method, it is characterized in that: comprise the following steps: with genome of E.coli DNA or bacterium liquid for template, upstream and downstream primer is utilized to carry out pcr amplification, check order and analyze pcr amplification product, detect that 3 of CRISPR1 unique intervening sequences are positive for bacteria, article 3, unique intervening sequence is as shown in SEQIDNO.5, SEQIDNO.6, SEQIDNO.7;
Described upstream and downstream primer is:
Upstream primer: 5 '-GTTATGCGGATAATGCTACC-3 ',
Downstream primer: 5 '-CGTAYYCCGGTRGATTTGGA-3 ', Y are C or T, R is A or G.
4. detection method according to claim 3, it is characterized in that: the reaction system of described pcr amplification is: 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix12.5 μ L, genome of E.coli DNA profiling 2 μ L, sterilizing ultrapure water 8.5 μ L, amounts to 25 μ L.
5. detection method according to claim 3, it is characterized in that: the reaction system of described pcr amplification is: 8 μm of ol/L upstream primer 1 μ L, 8 μm of ol/L downstream primer 1 μ L, 2 × TaqPCRMasterMix12.5 μ L, single bacterium colony, adds to 25 μ L after 10 × PCR bacterium colony toughener sterilizing ultrapure water is diluted to 10 times.
6. the detection method according to claim 4 or 5, is characterized in that: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 90s, totally 10 circulations; Sex change and annealing conditions constant, each circulation its extend step extend 10s, totally 25 circulations; 72 DEG C are continued to extend 10min.
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