CN102851384A - Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof - Google Patents

Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof Download PDF

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CN102851384A
CN102851384A CN2012103567255A CN201210356725A CN102851384A CN 102851384 A CN102851384 A CN 102851384A CN 2012103567255 A CN2012103567255 A CN 2012103567255A CN 201210356725 A CN201210356725 A CN 201210356725A CN 102851384 A CN102851384 A CN 102851384A
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escherichia coli
primer
pcr
gene
coli
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徐义刚
李丹丹
刘忠梅
吴岩
刘新亮
李苏龙
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HEILONGJIANG CENTRY-EXIT INSPECTION AND QUARANTINE TECHNICAL CENTER
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HEILONGJIANG CENTRY-EXIT INSPECTION AND QUARANTINE TECHNICAL CENTER
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Abstract

The invention relates to a multi-PCR (polymerase chain reaction)-based method for quickly detecting four diarrhoeic Escherichia coli and special primers thereof. The method comprises the following steps: respectively selecting enterohemorrhagic Escherichia coli O157:H7O-antigen gene, enterotoxigenic Echerichia coli LT gene, enteropathogenic Echerichia coli bfpA gene and enteroinvasive Echerichia coli invasion plasmid gene as target genes, carrying out comparative analysis on the gene sequences, selecting the conserved region of the target gene sequence to design and synthesize the primers which are disclosed as SEQ ID NO.1-8 in the sequence table, and establishing a multi-PCR detection method to carry out qualitative detection on the four diarrhoeic Escherichia coli. The method provided by the invention has strong detection specificity. The invention also relates to a kit for detection. The invention is quick and simple and has the advantages of high accuracy and high sensitivity.

Description

Four kinds of multi-PCR detection method and primer sets thereof that cause diarrhoeic Escherichia coli
Technical field
The invention belongs to technical field of biological, be specifically related to a kind of method and primer special thereof that causes diarrhoeic Escherichia coli based on four kinds of multiple PCR technique rapid detection.
Background technology
Food safety is global great public health problem, and the food origin disease virulence factor of having reported has kind more than 250, and wherein pathogen enterobacteria is modal biological paathogenic factor in the food origin disease.According to the World Health Organization (WHO), the annual diarrhoea case that causes because of the microbial contamination of food source property in the whole world reaches several hundred million, and 0 ~ 15 years old dead children are 1,700,000 people approximately.Cause at present diarrhoeic Escherichia coli and be still one of important pathogen that causes diarrhoea, particularly infantile diarrhea causes diarrhoeic Escherichia coli recall rate, constituent ratio, Dominant Types and serotype different in China and external diarrhea patient, makes troubles to clinical diagnosis.
Escherichia coli ( Escherichia coli, E.coli) be commonly called as intestinal bacteria, found first in 1885 by Escherich, originally be considered to the normal integral part of intestinal microflora always, belong to conditioned pathogen.20 middle of century, it is pathogenic that people recognize that some special serotype intestinal bacteria has humans and animals, especially to baby and cub, often causes severe diarrhea and septicemia.The intestinal bacteria relevant with human diseases are referred to as and cause diarrhoeic Escherichia coli (diarrheogenic E.coli), mainly be divided into four classes according to biological characteristics: produce enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive E.Coli (EIEC) and enterohemorrhagic Escherichia coli (EHEC).Cause diarrhoeic Escherichia coli as the infecting both domestic animals and human pathogenic bacterium, have important public health meaning.
ETEC main infection children and traveller, developing country is particularly serious, and contaminated water and food are main source of infection, and main pathogenic is enterotoxins of Escherichia coli LT or ST, be mildness diarrhoea when the clinical symptom that infects is slight, can develop into the choleraic diarrhea symptom when serious; EPEC is the main pathogenic fungi of infantile diarrhea, has hyperinfection, and severe patient can cause death, and pathogenic bacteria main infection intestinal epithelial cells or tissue culture cells surface form distinctive histopathology damage; EIEC mainly causes big-age-child and grownup diarrhoea, eruption and prevalence once, and clinical symptom is watery diarrhea, sometimes presents the dysentery symptom; The main serotype of EHEC is O157:H7, causes sporadic or the fulminant hemorrhagic colitis, can produce shiga toxin like cell toxin.At present China for the detection method that causes diarrhoeic Escherichia coli mainly take traditional detection method as main, the concrete operations flow process is: sample to be checked → increase bacterium → separation and Culture → gram's staining microscopy/biochemistry and the inspection of bacterium colony observation/biochemical reaction test/enterotoxin etc., complicated operation is loaded down with trivial details, detection time is long, finishing whole trace routine needs 5～10d, and it is single to detect target.
In the case, adopt that multiple PCR method is disposable to detect single pathogen infection or polyinfection, to ensuring food safety and human health has realistic meaning.The present invention has synthesized 4 pairs of specific PCR primers according to 4 kinds that the choose conserved regions design that cause the diarrhoeic Escherichia coli target-gene sequence, by system and reaction condition optimization, has set up single step reaction and has detected simultaneously 4 kinds of multiple PCR methods that cause diarrhoeic Escherichia coli.Put into practice confirming, the bacterium isolation identification susceptibility of the inventive method ratio routine is high and detection is quick, can be used for quick diagnosis and the epidemiology survey of clinical case, has certain practicality.
Summary of the invention
The object of the present invention is to provide a kind ofly have fast, accurately, sensitive, special single step reaction detects enterorrhagia Bacillus coil 0157: H7, enteroinvasive E.Coli, enteropathogenic Escherichia coli and 4 kinds of multiple PCR methods that cause diarrhoeic Escherichia coli of enterotoxigenic escherichia coli simultaneously.
The object of the present invention is achieved like this: select respectively enterorrhagia Bacillus coil 0157: H7 O-antigen gene, enterotoxigenic escherichia coli LT gene, enteropathogenic Escherichia coli BfpA gene and enteroinvasive E.Coli invasion plasmid gene are target gene, compare of analysis by gene order, choose the conserved regions design synthetic primer of target-gene sequence, set up multi-PCR detection method, cause diarrhoeic Escherichia coli to 4 kinds and carry out qualitative detection.
A kind of four kinds of multiple PCR detection primer groups that cause diarrhoeic Escherichia coli, nucleotides sequence is classified as:
Enterohemorrhagic Escherichia coli O 157: H 7Primer pair is:
OF：5′-ATTGCGCTGAGGCCTTTG-3′，
OR：5′-CGAGTACATTGGCATCGTG-3′；
The enterotoxigenic escherichia coli primer pair is:
LF：5′-GCACACGCAGCTCCTCAGTC-3′，
LR：5′-TCCTTCATCCTTTCAATGGCTTT-3′；
The enteropathogenic Escherichia coli primer pair is:
BF：5′-GGAAGTCAAATTCATGGGGGTAT-3′，
BR：5′-GGAATCAGACGCAGACTGGTAGT-3′；
The enteroinvasive E.Coli primer pair is:
IF：5′-CTGGATGGTATGGTGAGG-3′，
IR：5′-GGAGGCCAACAATTATTTCC-3。
The present invention also has following feature:
1, the interpolation mol ratio of described primer pair is OF/OR :LF/LR :BF/BR :IF/IR is 1 :1 :1 :1.3.
2, a kind of four kinds of multiplex PCRs that cause diarrhoeic Escherichia coli detect the preparation pcr amplification primer group of using positive reference substance, and nucleotides sequence is classified as:
Enterohemorrhagic Escherichia coli O 157: H 7The preparation pcr amplification primer of positive reference substance:
EHEC-F：5′-CTATAGAAATTGCTGCTGTAG-3′，
EHEC-R：5′-TCTAACCCTTCAGCATTATTA-3′；
The preparation pcr amplification primer of enterotoxigenic escherichia coli positive reference substance:
ETEC-F：5′-AACCCTGGATTCATCATGCAC-3′，
ETEC-R：5′-TAGTTTTCCATGCTGATTGCC-3′；
The preparation pcr amplification primer of enteropathogenic Escherichia coli positive reference substance:
EPEC-F：5′-AAATCATGAATAAGAAATACG-3′，
EPEC-R：5′-CAGTATTTTTACATGCAGTTG-3′；
The preparation pcr amplification primer of enteroinvasive E.Coli positive reference substance:
EIEC-F：5′-CTGGAAAAACTCAGTGCCTCT-3′，
EIEC-R：5′-AGCTTCCGTACGCTTCAGTAC-3′。
3, four kinds of multi-PCR detection methods that cause diarrhoeic Escherichia coli of a kind of non-diagnosis or therapeutic purpose comprise the steps:
(1) preparation of pcr template
Carry out extraction and the purifying of bacterial genomes DNA with reference to day root TIANamp Bacteria DNA Kit specification sheets;
(2) design PCR primer
Select enterohemorrhagic Escherichia coli O 157: H 7LT gene, the enteropathogenic Escherichia coli of O-antigen gene, enterotoxigenic escherichia coli BfpThe invasion plasmid gene of A gene and enteroinvasive E.Coli is as target gene, and the synthetic following primer of design carries out the amplification of target gene:
Enterohemorrhagic Escherichia coli O 157: H 7The pcr amplification primer of O-antigen gene:
EHEC-F：5′-CTATAGAAATTGCTGCTGTAG-3′，
EHEC-R：5′-TCTAACCCTTCAGCATTATTA-3′；
The pcr amplification primer of enterotoxigenic escherichia coli LT gene:
ETEC-F：5′-AACCCTGGATTCATCATGCAC-3′，
ETEC-R：5′-TAGTTTTCCATGCTGATTGCC-3′；
Enteropathogenic Escherichia coli BfpThe pcr amplification primer of A gene:
EPEC-F：5′-AAATCATGAATAAGAAATACG-3′，
EPEC-R：5′-CAGTATTTTTACATGCAGTTG-3′；
The pcr amplification primer of enteroinvasive E.Coli invasion plasmid gene:
EIEC-F：5′-CTGGAAAAACTCAGTGCCTCT-3′，
EIEC-R：5′-AGCTTCCGTACGCTTCAGTAC-3′；
Utilize the synthetic target gene PCR primer of design, respectively with enterohemorrhagic Escherichia coli O 157: H 7The genomic dna of (ATCC 35150), enterotoxigenic escherichia coli (ATCC 35401), enteropathogenic Escherichia coli (ATCC 11775) and enteroinvasive E.Coli (ATCC 43893) is the template amplification target gene, the target-gene sequence of amplification is carried out the BLAST compare of analysis, chooses respectively every kind of conserved regions design that causes the diarrhoeic Escherichia coli target-gene sequence and synthesized 4 pairs of specificity identification with multi-plex PCR primers:
Enterohemorrhagic Escherichia coli O 157: H 7:
OF：5′-ATTGCGCTGAGGCCTTTG-3′，
OR：5′-CGAGTACATTGGCATCGTG-3′；
Enterotoxigenic escherichia coli:
LF：5′-GCACACGCAGCTCCTCAGTC-3′，
LR：5′-TCCTTCATCCTTTCAATGGCTTT-3′；
Enteropathogenic Escherichia coli:
BF：5′-GGAAGTCAAATTCATGGGGGTAT-3′，
BR：5′-GGAATCAGACGCAGACTGGTAGT-3′；
Enteroinvasive E.Coli:
IF：5′-CTGGATGGTATGGTGAGG-3′，
IR：5′-GGAGGCCAACAATTATTTCC-3′；
(3) preparation of positive quality control standard substance:
Adopt the PCR method enterorrhagia Bacillus coil 0157 that increases respectively: H7 O-antigen gene, enterotoxigenic escherichia coli LT gene, enteropathogenic Escherichia coli BfpA gene and enteroinvasive E.Coli invasion plasmid gene, respectively the PCR product is connected with cloning vector pMD-19-T vector, the transformed competence colibacillus e. coli jm109 obtains positive recombinant bacterium, prepares positive plasmid with reference to magnificent Shun's a small amount of extraction of plasmid DNA test kit specification sheets;
(4) PCR reaction system:
Through the comparative analysis to the various combination experimental result, determined best substance PCR reaction system and reaction conditions, corresponding PCR product agarose gel electrophoresis detected result is seen Fig. 1.
Substance PCR reaction system, such as following table:
10×PCR Buffer (Mg 2+ free) 2.5μL
dNTP 0.2mmol/L
Mg 2+ 2.5mmol/L
Upstream primer 0.2μmol/L
Downstream primer 0.2μmol/L
Taq DNA Polymerase 1.5U
Dna profiling 1μL
Aseptic deionized water Be supplemented to 25 μ L
Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 60s carry out 35 circulations; 72 ℃ are extended 10min;
On the basis of substance PCR reaction system, optimize and determined multi-PRC reaction system and reaction conditions, wherein PCR primer concentration ratio is ETEC:EHEC:EIEC:EPEC=1 :1 :1.3 :1, corresponding multiple PCR products agarose gel electrophoresis detected result is seen Fig. 2.
The multi-PRC reaction system, such as following table:
10×PCR Buffer (Mg 2+ free) 2.5μL
dNTP 0.3mmol/L
Mg 2+ 4mmol/L
Primer OF/OR 0.2μmol/L
Primer LF/LR 0.2μmol/L
Primer BF/BR 0.2μmol/L
Primer I F/IR 0.26μmol/L
Taq DNA Polymerase 2.0U
Dna profiling 1.5μL
Aseptic deionized water Be supplemented to 25 μ L
Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 60s carry out 35 circulations; 72 ℃ are extended 10min, and wherein, the primer concentration ratio is ETEC:EHEC:EIEC:EPEC=1:1:1.3:1;
(5) utilize agarose gel electrophoresis method for detecting to judge detected result.
4, the invention provides a kind of four kinds of multiplex PCR detections that cause diarrhoeic Escherichia coli and use test kit, comprise the primer sets of above-mentioned detection usefulness.
The present invention is respectively with Aeromonas hydrophila (ATCC 7966), campylobacter jejuni (ATCC 33560), intestinal bacteria (ATCC 25922, ATCC 8739), enterorrhagia Bacillus coil 0157 :(ATCC 35150 for H7, ATCC43889, ATCC43895, strain isolated 5 strains), (ATCC 35401 for enterotoxigenic escherichia coli, strain isolated 1 strain), (ATCC 43893 for enteroinvasive E.Coli, strain isolated 1 strain), (ATCC 11775 for enteropathogenic Escherichia coli, ATCC 43887, strain isolated 2 strains), Enterobacter sakazakii (ATCC 51329), Listeria monocytogenes (ATCC 19111), sheep listeria bacteria (ATCC 33090), Ying Nuoke listeria bacteria (ATCC 19119), this listeria bacteria of Weir (ATCC 35897), Xi Er listeria bacteria (ATCC 35967), Plesiomonas shigelloides (ATCC 14030), proteus vulgaris (ATCC49027), Salmonella choleraesuls (ATCC 10708), shigella flexneri (ATCC 12022), streptococcus aureus (ATCC 29213), Hemolytic streptococcus (CMCC32121), vibrio cholerae (ATCC 14035), Vibrio parahemolyticus (ATCC 27519), Vibrio alginnolyficus (ATCC33839), the genomic dna of Vibrio vulnificus (ATCC 33149) and yersinia entero-colitica (ATCC 9610) is tested, the specificity that detects to probe into the method.The result shows, the inventive method can be carried out specific detection to reference culture and the isolated strains of enterorrhagia Bacillus coil 0157: H7, enterotoxigenic escherichia coli, enteropathogenic Escherichia coli and enteroinvasive E.Coli, and no cross reaction between the synthetic primer pair of design and between primer and other bacteriums proves that the inventive method has stronger detection specificity.Test kit of the present invention also has simple fast, and accuracy is high, the advantage that sensitivity is good.
Adopt test kit to extract the enterorrhagia Bacillus coil 0157 of incubated overnight: the genomic dna of H7, enterotoxigenic escherichia coli, enteropathogenic Escherichia coli and enteroinvasive E.Coli, measure 4 kinds of extracting and cause diarrhoeic Escherichia coli genomic dna concentration, then carry out respectively 2 times of gradient dilutions, from every kind of every grade of diluent that causes the diarrhoeic Escherichia coli genomic dna, get 1 μ L and mix, carry out multiplex PCR as template and detect.The result shows, this multiple PCR method detects simultaneously 4 kinds of minimum detectabilities that cause diarrhoeic Escherichia coli genomic dna concentration and is respectively: enterorrhagia Bacillus coil 0157: H7 is 32pg/ μ L; Enteroinvasive E.Coli 128pg/ μ L; Enteropathogenic Escherichia coli 128pg/ μ L; Enterotoxigenic Escherichia coli is 32pg/ μ L, illustrates that the inventive method has the susceptibility of height.
Utilize the inventive method to repeat 20 detections same with 4 kinds of mixing positive criteria product that cause the preparation of diarrhoeic Escherichia coli minimum detectability genomic dna concentration, detected result is all identical; Twice (30 days timed intervals) detects same batch with 4 kinds of mixing positive criteria product that cause the preparation of diarrhoeic Escherichia coli minimum detectability genomic dna concentration, and detected result is all identical, and visible the inventive method has good repeatability and stable.
The inventive method is applied to inspection and quarantine to be put into practice in the work, its result compares with causing diarrhoeic Escherichia coli national standard detection method (GB 4789.6-1994 microbiological test of food hygiene-Diarrheogenil Escherichia coli check), verifies reliability and practicality that the method detects.The result shows, utilize the diarrhoeic Escherichia coli multi-PCR detection method that causes of setting up that 35 parts of beef samples, 30 parts of milk fish samples, 27 parts of pork samples, 40 parts of chicken samples, 75 parts of water samples and 25 parts of infantile diarrhea thing samples are detected, detect altogether 11 parts and cause the diarrhoeic Escherichia coli positive sample, wherein enterorrhagia Bacillus coil 0157: H7 detects 4 strains, and enteroinvasive E.Coli detects 1 strain; Enteropathogenic Escherichia coli detects 7 strains; Enterotoxigenic Escherichia coli detects 5 strains, and detected result and National Standard Method detected result coincidence rate are 100%.Put into practice confirming, the bacterium isolation identification susceptibility of the inventive method ratio routine is high and detection is quick, has potential practical value.
Description of drawings
Fig. 1 is the foundation (1:EPEC that causes diarrhoeic Escherichia coli substance PCR detection method; 2:ETEC; 3:EHEC O157:H7; 4:EIEC; 5:DNA Marker DL 2000);
Fig. 2 is four kinds of foundation (1:EHEC O157:H7 and ETEC of causing the diarrhoeic Escherichia coli multi-PCR detection method; 2:EHEC O157:H7, EPEC and ETEC; 3:EHEC O157:H7, EIEC, EPEC and ETEC; 4:DNA Marker DL 2000).
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment:
1.PCR the preparation of template
Carry out extraction and the purifying of bacterial genomes DNA with reference to day root TIANamp Bacteria DNA Kit specification sheets, be sequentially added into corresponding reagent:
1.1 get in the 1.5mL sample to be checked the bacterium enrichment liquid that spends the night, the centrifugal 1min of 10000r/min abandons supernatant, adds 200 μ L damping fluid GA, thalline thoroughly suspends;
1.2 add 20 μ L Proteinase Ks (20mg/mL), and add 220 μ L damping fluid GB, abundant mixing, 70 ℃ of water-bath effect 10min;
1.3 add 220 μ L dehydrated alcohols, abundant mixing 15s is transferred to gained solution (comprising flocks) among the adsorption column CB3 after brief centrifugal, the centrifugal 30s of 12000r/min discards the liquid in the collection tube;
Contain dehydrated alcohol 1.4 add 500 μ L damping fluid GD(in the adsorption column CB3), the centrifugal 30s of 12000r/min discards the liquid in the collection tube;
Contain dehydrated alcohol 1.5 add 600 μ L rinsing liquid PW(in the adsorption column CB3), the centrifugal 30s of 12000r/min discards the liquid in the collection tube;
The work 1.6 repeat to drill: add 600 μ L rinsing liquid PW(in the adsorption column CB3 and contain dehydrated alcohol), the centrifugal 30s of 12000r/min discards the liquid in the collection tube;
1.7 adsorption column CB3 is put back in the collection tube, the centrifugal 2min of 12000r/min, room temperature is placed 2-5min, dries residual rinsing liquid;
1.8 adsorption column CB3 is changed in the new collection tube, and central authorities add 100 μ L damping fluid TE at adsorption film, room temperature is placed 2-5min, and the centrifugal 2min of 12000r/min collects the DNA elutriant.
2. PCR primer
Select LT gene, the enteropathogenic Escherichia coli of O-antigen gene, the enterotoxigenic escherichia coli of enterorrhagia Bacillus coil 0157: H7 BfpThe invasion plasmid gene of A gene and enteroinvasive E.Coli is as target gene, and the synthetic following primer of design carries out the amplification of target gene:
Enterorrhagia Bacillus coil 0157: the PCR primer of H7 O-antigen gene:
EHEC-F：5′-CTATAGAAATTGCTGCTGTAG-3′，
EHEC-R：5′-TCTAACCCTTCAGCATTATTA-3′；
The PCR primer of enterotoxigenic escherichia coli LT gene:
ETEC-F：5′-AACCCTGGATTCATCATGCAC-3′，
ETEC-R：5′-TAGTTTTCCATGCTGATTGCC-3′；
Enteropathogenic Escherichia coli BfpThe PCR primer of A gene:
EPEC-F：5′-AAATCATGAATAAGAAATACG-3′，
EPEC-R：5′-CAGTATTTTTACATGCAGTTG-3′；
The PCR primer of enteroinvasive E.Coli invasion plasmid gene:
EIEC-F：5′-CTGGAAAAACTCAGTGCCTCT-3′，
EIEC-R：5′-AGCTTCCGTACGCTTCAGTAC-3′；
Utilize the synthetic target gene PCR primer of design, respectively take enterorrhagia Bacillus coil 0157: the genomic dna of H7 (ATCC 35150), enterotoxigenic escherichia coli (ATCC 35401), enteropathogenic Escherichia coli (ATCC 11775) and enteroinvasive E.Coli (ATCC 43893) is the template amplification target gene, the target-gene sequence of amplification is carried out the BLAST compare of analysis, chooses respectively every kind of conserved regions design that causes the diarrhoeic Escherichia coli target-gene sequence and synthesized 4 pairs of specificity identification with multi-plex PCR primers (seeing Table 1):
Table 1 multiple PCR primer is to sequence
Figure 2012103567255100002DEST_PATH_IMAGE002
3. the preparation of positive quality control standard substance
Adopt PCR method amplification enterorrhagia Bacillus coil 0157: H7 O-antigen gene, enterotoxigenic escherichia coli LT gene, enteropathogenic Escherichia coli BfpA gene and enteroinvasive E.Coli invasion plasmid gene, respectively the PCR product is connected with cloning vector pMD-19-T vector, the transformed competence colibacillus e. coli jm109 obtains positive recombinant bacterium, prepares positive plasmid with reference to magnificent Shun's a small amount of extraction of plasmid DNA test kit specification sheets:
3.1 picking positive colony list bacterium colony is inoculated in 5mL and contains 100 μ g/mL Amp rThe LB substratum in, 37 ℃ of overnight incubation;
3.2 get 1～3mL incubated overnight bacterium liquid, the centrifugal 5min of 12000r/min abandons supernatant, the Buffer P1(that adds 250 μ L contains RNase) bacterial sediment that fully vibrates is to its thorough suspension;
3.3 add the Buffer P2 of 250 μ L, centrifuge tube 6-10 time of leniently turning upside down immediately, mixing, room temperature leaves standstill 2-4min;
3.4 add the Buffer P3 of 350 μ L, gentleness is put upside down centrifuge tube 6-10 time repeatedly, mixing, the centrifugal 10min of 12000r/min;
3.5 supernatant liquor is moved in the adsorption column, and the centrifugal 30s of 12000r/min abandons filtrate, and adsorption column is put into collection tube;
3.6 add 500 μ L B1 liquid, the centrifugal 30s of 12000r/min abandons filtrate, and adsorption column is put into collection tube;
3.7 add 500 μ L W1 liquid (containing dehydrated alcohol), the centrifugal 30s of 12000r/min abandons filtrate, and adsorption column is put into collection tube;
3.8 add 500 μ L W1 liquid (containing dehydrated alcohol), room temperature leaves standstill 1min, the centrifugal 30s of 12000r/min abandons filtrate, and adsorption column is put into collection tube, the unloaded centrifugal 1min of 12000r/min;
3.9 adsorption column is transferred in the 1.5mL centrifuge tube of a cleaning, central authorities add 150 μ L deionized waters at adsorption film, room temperature leaves standstill 2min, the centrifugal 1min of 12000r/min, and wash-out is collected plasmid DNA.
The plasmid DNA of extracting is carried out quantitatively, ℃ save backup as test kit positive quality control standard substance-20.The quantitative Analysis formula is: positive plasmid copy number copies/ μ L=(OD 260* 50 * 10 -9* extension rate * 6.02 * 10 23)/(660 * base number), in the formula: 50 representatives use diameter 1cm cuvette at OD 260Equaling 1 o'clock corresponding double-stranded DNA concentration is 50 μ g/mL; 660 represent double-stranded DNA base pair molecular-weight average.
4. the Establishment and optimization of reaction system
Adopt 4 kinds of test kit extraction incubated overnight to cause the diarrhoeic Escherichia coli genomic dna, with the reaction template of its isoconcentration mixing as system optimization.Optimization principles is to determine first substance PCR reaction system and reaction conditions, optimizes multi-PRC reaction system and reaction conditions on its basis again.
4.1 the optimization of substance PCR reaction system and condition
The PCR reaction system is 25 μ L:10 * PCR Buffer (Mg 2+Free) 2.5 μ L are with Mg 2+, dNTP, TaqDNA Polymerase and primer are prepared into the combination of different concns, are supplemented to 25 μ L with deionized water.Mg 2+, dNTP, TaqThe concentration range of DNA Polymerase and primer is followed successively by: Mg 2+Concentration range is set in 1.0mmol/L-8mmol/L, increases progressively with 0.5mmol/L; The dNTP concentration range is set in 0.1 mmol/L-0.8mmol/L, increases progressively with 0.05mmol/L; TaqDNA Polymerase concentration range is set in 0.5U-3.5U, with the 0.5U incremented; The primer concentration scope is set in 0.1 μ mol/L-0.6 μ mol/L, increases progressively with 0.1 μ mol/L, adopts matrix method to compare test, to determine best reaction composition.Reaction conditions: 95 ℃ of 5min; 95 ℃ of 20s, in order to improve sensitivity and the specificity of PCR reaction, according to the primer annealing temperature of design, take 55 ℃ for basic, progressively increase until 62 ℃ with 0.5 ℃, time 30s carries out 35 circulations.
4.2 the optimization of multi-PRC reaction system and condition
On the basis of the substance PCR reaction system after the optimization, reaction system and the condition that causes the diarrhoeic Escherichia coli method based on four kinds of multiple PCR technique rapid detection is optimized.In the optimizing process, according in the multiple PCR products every kind cause what of diarrhoeic Escherichia coli amplification gene content of fragment, suitably adjust the primer pair concentration ratio.The reaction system that causes the diarrhoeic Escherichia coli method based on four kinds of multiple PCR technique rapid detection of determining is:
10×PCR Buffer (Mg 2+ free) 2.5μL
dNTP 0.3mmol/L
Mg 2+ 4mmol/L
Primer OF/OR 0.2μmol/L
Primer LF/LR 0.2μmol/L
Primer BF/BR 0.2μmol/L
Primer I F/IR 0.26μmol/L
Taq DNA Polymerase 2.0U
Dna profiling 1.5μL
Aseptic deionized water Be supplemented to 25 μ L
Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 60s carry out 35 circulations; 72 ℃ are extended 10min.The primer concentration ratio is ETEC:EHEC:EIEC:EPEC=1:1:1.3:1.
5. specific test
Cause the method for diarrhoeic Escherichia coli to Aeromonas hydrophila (ATCC 7966), campylobacter jejuni (ATCC 33560), intestinal bacteria (ATCC 25922, ATCC 8739), enterorrhagia Bacillus coil 0157 with what set up based on four kinds of multiple PCR technique rapid detection :(ATCC 35150 for H7, ATCC43889, ATCC43895, strain isolated 5 strains), (ATCC 35401 for enterotoxigenic escherichia coli, strain isolated 1 strain), (ATCC 43893 for enteroinvasive E.Coli, strain isolated 1 strain), (ATCC 11775 for enteropathogenic Escherichia coli, ATCC 43887, strain isolated 2 strains), Enterobacter sakazakii (ATCC 51329), Listeria monocytogenes (ATCC 19111), sheep listeria bacteria (ATCC 33090), Ying Nuoke listeria bacteria (ATCC 19119), this listeria bacteria of Weir (ATCC 35897), Xi Er listeria bacteria (ATCC 35967), Plesiomonas shigelloides (ATCC 14030), proteus vulgaris (ATCC49027), Salmonella choleraesuls (ATCC 10708), shigella flexneri (ATCC 12022), streptococcus aureus (ATCC 29213), Hemolytic streptococcus (CMCC32121), vibrio cholerae (ATCC 14035), Vibrio parahemolyticus (ATCC 27519), Vibrio alginnolyficus (ATCC33839), Vibrio vulnificus (ATCC 33149) and yersinia entero-colitica (ATCC 9610) detect, the specificity that detects to probe into the method.The result shows, the inventive method can be carried out specific detection to reference culture and the isolated strains of enterorrhagia Bacillus coil 0157: H7, enterotoxigenic escherichia coli, enteropathogenic Escherichia coli and enteroinvasive E.Coli, and no cross reaction between the synthetic primer pair of design and between primer and other bacteriums proves that the inventive method has stronger detection specificity (seeing Table 2).
Table 2 specificity experimental result
Figure 2012103567255100002DEST_PATH_IMAGE001
6. sensitivity test
Adopt test kit to extract the enterorrhagia Bacillus coil 0157 of incubated overnight: the genomic dna of H7, enterotoxigenic escherichia coli, enteropathogenic Escherichia coli and enteroinvasive E.Coli, measure 4 kinds of extracting and cause diarrhoeic Escherichia coli genomic dna concentration, then carry out respectively 2 times of gradient dilutions, getting 1 μ L from every kind of every grade of diluent that causes the diarrhoeic Escherichia coli genomic dna mixes, carry out multiplex PCR as template and detect, to determine the detection sensitivity of the method.Result's demonstration, the inventive method has the detection sensitivity (the sensitivity detected result sees Table 3) of height.
Table 3 sensitivity test experience result
Figure 2012103567255100002DEST_PATH_IMAGE006
7. replica test
7.1 it is same with 4 kinds of mixing positive criteria product that cause the preparation of diarrhoeic Escherichia coli minimum detectability genomic dna concentration that the method that causes diarrhoeic Escherichia coli based on four kinds of multiple PCR technique rapid detection of utilize setting up repeats to detect for 20 times, investigates repeatability that the method detects and stable.Result's demonstration, each detected result is all identical.
7.2 the method interval that causes diarrhoeic Escherichia coli based on four kinds of multiple PCR technique rapid detection of utilize setting up was detected same batch in 30 days with 4 kinds of mixing positive criteria product that cause the preparation of diarrhoeic Escherichia coli minimum detectability genomic dna concentration, investigated repeatability that the method detects and stable.Result's demonstration, twice detected result is all identical.
8. practical proof test
The method that causes diarrhoeic Escherichia coli based on four kinds of multiple PCR technique rapid detection of setting up is applied to inspection and quarantine to be put into practice in the work, 35 parts of beef samples, 30 parts of milk fish samples, 27 parts of pork samples, 40 parts of chicken samples, 75 parts of water samples and the 25 parts of infantile diarrhea thing samples that gather are detected, its result compares with causing diarrhoeic Escherichia coli national standard detection method (GB 4789.6-1994 microbiological test of food hygiene-Diarrheogenil Escherichia coli check), investigates reliability and practicality that the method detects.Result's demonstration detects altogether 11 parts and causes the diarrhoeic Escherichia coli positive sample, and detected result and National Standard Method detected result coincidence rate are that 100%(sees Table 4).Put into practice confirming, the bacterium isolation identification susceptibility of the inventive method ratio routine is high and detection is quick, has potential practical value.
Table 4 application in practice result
Figure 2012103567255100002DEST_PATH_IMAGE008
＜110〉Heilungkiang Entry-Exit Inspection and Quarantine Bureau inspection and quarantine technique center
＜120〉four kinds of multi-PCR detection method and primer sets thereof that cause diarrhoeic Escherichia coli
<140>
<141>
<160>16
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<400>1
ATTGCGCTGA GGCCTTTG 18
<210>2
<211>19
<212>DNA
＜213〉artificial sequence
<400>2
CGAGTACATT GGCATCGTG 19
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<400>3
GCACACGCAG CTCCTCAGTC 20
<210>4
<211>23
212>DNA
＜213〉artificial sequence
<400>4
TCCTTCATCC TTTCAATGGC TTT 23
<210>5
<211>23
212>DNA
＜213〉artificial sequence
<400>5
GGAAGTCAAA TTCATGGGGG TAT 23
<210>6
<211>23
212>DNA
＜213〉artificial sequence
<400>6
GGAATCAGAC GCAGACTGGT AGT 23
<210>7
<211>18
212>DNA
＜213〉artificial sequence
<400>7
CTGGATGGTA TGGTGAGG 18
<210>8
<211>20
212>DNA
＜213〉artificial sequence
<400>8
GGAGGCCAAC AATTATTTCC 20
<210>9
<211>21
212>DNA
＜213〉artificial sequence
<400>9
CTATAGAAAT TGCTGCTGTA G 21
<210>10
<211>21
212>DNA
＜213〉artificial sequence
<400>10
CTATAGAAAT TGCTGCTGTA G 21
<210>11
<211>21
212>DNA
＜213〉artificial sequence
<400>11
AACCCTGGAT TCATCATGCA C 21
<210>12
<211>21
212>DNA
＜213〉artificial sequence
<400>12
TAGTTTTCCA TGCTGATTGC C 21
<210>13
<211>21
212>DNA
＜213〉artificial sequence
<400>13
AAATCATGAA TAAGAAATAC G 21
<210>14
<211>21
212>DNA
＜213〉artificial sequence
<400>14
CAGTATTTTT ACATGCAGTT G 21
<210>15
<211>21
212>DNA
＜213〉artificial sequence
<400>15
CTGGAAAAAC TCAGTGCCTC T 21
<210>16
<211>21
212>DNA
＜213〉artificial sequence
<400>16
AGCTTCCGTA CGCTTCAGTA C 21

Claims (5)

1. primer sets is used in the one kind four kinds multiplex PCR detections that cause diarrhoeic Escherichia coli, it is characterized in that, nucleotides sequence is classified as:
Enterohemorrhagic Escherichia coli O 157: H 7Primer pair is:
OF：5′-ATTGCGCTGAGGCCTTTG-3′，
OR：5′-CGAGTACATTGGCATCGTG-3′；
The enterotoxigenic escherichia coli primer pair is:
LF：5′-GCACACGCAGCTCCTCAGTC-3′，
LR：5′-TCCTTCATCCTTTCAATGGCTTT-3′；
The enteropathogenic Escherichia coli primer pair is:
BF：5′-GGAAGTCAAATTCATGGGGGTAT-3′，
BR：5′-GGAATCAGACGCAGACTGGTAGT-3′；
The enteroinvasive E.Coli primer pair is:
IF：5′-CTGGATGGTATGGTGAGG-3′，
IR：5′-GGAGGCCAACAATTATTTCC-3。
2. primer sets is used in a kind of four kinds of multiplex PCR detections that cause diarrhoeic Escherichia coli according to claim 1, and it is characterized in that: the interpolation mol ratio of described primer pair is that OF/OR:LF/LR:BF/BR:IF/IR is 1:1:1:1.3.
3. the one kind four kinds multiplex PCRs that cause diarrhoeic Escherichia coli detect the preparation pcr amplification primer group of using positive reference substance, it is characterized in that, nucleotides sequence is classified as:
Enterohemorrhagic Escherichia coli O 157: H 7The preparation pcr amplification primer of positive reference substance:
EHEC-F：5′-CTATAGAAATTGCTGCTGTAG-3′，
EHEC-R：5′-TCTAACCCTTCAGCATTATTA-3′；
The preparation pcr amplification primer of enterotoxigenic escherichia coli positive reference substance:
ETEC-F：5′-AACCCTGGATTCATCATGCAC-3′，
ETEC-R：5′-TAGTTTTCCATGCTGATTGCC-3′；
The preparation pcr amplification primer of enteropathogenic Escherichia coli positive reference substance:
EPEC-F：5′-AAATCATGAATAAGAAATACG-3′，
EPEC-R：5′-CAGTATTTTTACATGCAGTTG-3′；
The preparation pcr amplification primer of enteroinvasive E.Coli positive reference substance:
EIEC-F：5′-CTGGAAAAACTCAGTGCCTCT-3′，
EIEC-R：5′-AGCTTCCGTACGCTTCAGTAC-3′。
4. four kinds of multi-PCR detection methods that cause diarrhoeic Escherichia coli of a non-diagnosis or therapeutic purpose is characterized in that, comprise the steps:
(1) preparation of pcr template
Carry out extraction and the purifying of bacterial genomes DNA with reference to day root TIANamp Bacteria DNA Kit specification sheets;
(2) design PCR primer
Select enterohemorrhagic Escherichia coli O 157: H 7LT gene, the enteropathogenic Escherichia coli of O-antigen gene, enterotoxigenic escherichia coli BfpThe invasion plasmid gene of A gene and enteroinvasive E.Coli is as target gene, and the synthetic following primer of design carries out the amplification of target gene:
Enterohemorrhagic Escherichia coli O 157: H 7The pcr amplification primer of O-antigen gene:
EHEC-F：5′-CTATAGAAATTGCTGCTGTAG-3′，
EHEC-R：5′-TCTAACCCTTCAGCATTATTA-3′；
The pcr amplification primer of enterotoxigenic escherichia coli LT gene:
ETEC-F：5′-AACCCTGGATTCATCATGCAC-3′，
ETEC-R：5′-TAGTTTTCCATGCTGATTGCC-3′；
Enteropathogenic Escherichia coli BfpThe pcr amplification primer of A gene:
EPEC-F：5′-AAATCATGAATAAGAAATACG-3′，
EPEC-R：5′-CAGTATTTTTACATGCAGTTG-3′；
The pcr amplification primer of enteroinvasive E.Coli invasion plasmid gene:
EIEC-F：5′-CTGGAAAAACTCAGTGCCTCT-3′，
EIEC-R：5′-AGCTTCCGTACGCTTCAGTAC-3′；
Utilize the synthetic target gene PCR primer of design, respectively with enterohemorrhagic Escherichia coli O 157: H 7The genomic dna of (ATCC 35150), enterotoxigenic escherichia coli (ATCC 35401), enteropathogenic Escherichia coli (ATCC 11775) and enteroinvasive E.Coli (ATCC 43893) is the template amplification target gene, the target-gene sequence of amplification is carried out the BLAST compare of analysis, chooses respectively every kind of conserved regions design that causes the diarrhoeic Escherichia coli target-gene sequence and synthesized 4 pairs of specificity identification with multi-plex PCR primers:
Enterohemorrhagic Escherichia coli O 157: H 7:
OF：5′-ATTGCGCTGAGGCCTTTG-3′，
OR：5′-CGAGTACATTGGCATCGTG-3′；
Enterotoxigenic escherichia coli:
LF：5′-GCACACGCAGCTCCTCAGTC-3′，
LR：5′-TCCTTCATCCTTTCAATGGCTTT-3′；
Enteropathogenic Escherichia coli:
BF：5′-GGAAGTCAAATTCATGGGGGTAT-3′，
BR：5′-GGAATCAGACGCAGACTGGTAGT-3′；
Enteroinvasive E.Coli:
IF：5′-CTGGATGGTATGGTGAGG-3′，
IR：5′-GGAGGCCAACAATTATTTCC-3′；
(3) preparation of positive quality control standard substance:
Adopt the PCR method enterorrhagia Bacillus coil 0157 that increases respectively: H7 O-antigen gene, enterotoxigenic escherichia coli LT gene, enteropathogenic Escherichia coli BfpA gene and enteroinvasive E.Coli invasion plasmid gene, respectively the PCR product is connected with cloning vector pMD-19-T vector, the transformed competence colibacillus e. coli jm109 obtains positive recombinant bacterium, prepares positive plasmid with reference to magnificent Shun's a small amount of extraction of plasmid DNA test kit specification sheets;
(4) PCR reaction system:
Substance PCR reaction system, such as following table:
10×PCR Buffer (Mg 2+ free) 2.5μL dNTP 0.2mmol/L Mg 2+ 2.5mmol/L Upstream primer 0.2μmol/L Downstream primer 0.2μmol/L Taq DNA Polymerase 1.5U Dna profiling 1μL Aseptic deionized water Be supplemented to 25 μ L
Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 60s carry out 35 circulations; 72 ℃ are extended 10min;
The multi-PRC reaction system, such as following table:
10×PCR Buffer (Mg 2+ free) 2.5μL dNTP 0.3mmol/L Mg 2+ 4mmol/L Primer OF/OR 0.2μmol/L Primer LF/LR 0.2μmol/L Primer BF/BR 0.2μmol/L Primer I F/IR 0.26μmol/L Taq DNA Polymerase 2.0U Dna profiling 1.5μL Aseptic deionized water Be supplemented to 25 μ L
Reaction conditions is: 95 ℃ of 5min; 95 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 60s carry out 35 circulations; 72 ℃ are extended 10min, and wherein, the primer concentration ratio is ETEC:EHEC:EIEC:EPEC=1:1:1.3:1;
(5) utilize agarose gel electrophoresis method for detecting to judge detected result.
5. test kit is used in the one kind four kinds multiplex PCR detections that cause diarrhoeic Escherichia coli, it is characterized in that: comprise primer sets claimed in claim 1.
CN2012103567255A 2012-09-24 2012-09-24 Multi-PCR (polymerase chain reaction) detection method of four diarrhoeic Escherichia coli and primer group thereof Pending CN102851384A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103361414A (en) * 2013-03-20 2013-10-23 冯家望 LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for pathogenic Escherichia coli
CN105154582A (en) * 2015-10-29 2015-12-16 大连民族大学 Detection kit and method for four kinds of Escherichia coli
CN106399486A (en) * 2016-08-31 2017-02-15 北京卓诚惠生生物科技股份有限公司 Primer group and kit for detecting diarrhea-causing parasites through multi-PCR technology
CN112646906A (en) * 2020-12-30 2021-04-13 广东省微生物研究所（广东省微生物分析检测中心） Diarrhea-causing escherichia coli standard reference strain containing specific molecular target and detection and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
徐义刚 等: "多重PCR-DHPLC检测4种主要致腹泻性大肠埃希氏菌方法的建立与应用", 《食品科学》 *

Cited By (7)

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Publication number Priority date Publication date Assignee Title
CN103361414A (en) * 2013-03-20 2013-10-23 冯家望 LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for pathogenic Escherichia coli
CN103361414B (en) * 2013-03-20 2015-01-07 冯家望 LAMP (loop-mediated isothermal amplification) detection primer group, kit and detection method for pathogenic Escherichia coli
CN105154582A (en) * 2015-10-29 2015-12-16 大连民族大学 Detection kit and method for four kinds of Escherichia coli
CN106399486A (en) * 2016-08-31 2017-02-15 北京卓诚惠生生物科技股份有限公司 Primer group and kit for detecting diarrhea-causing parasites through multi-PCR technology
CN106399486B (en) * 2016-08-31 2019-05-17 北京卓诚惠生生物科技股份有限公司 The primer sets and kit for causing diarrhea helminth are detected for multiplex PCR
CN112646906A (en) * 2020-12-30 2021-04-13 广东省微生物研究所（广东省微生物分析检测中心） Diarrhea-causing escherichia coli standard reference strain containing specific molecular target and detection and application thereof
CN112646906B (en) * 2020-12-30 2022-06-14 广东省微生物研究所（广东省微生物分析检测中心） Diarrhea-causing escherichia coli standard reference strain containing specific molecular target and detection and application thereof

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