CN104195250B - For detecting escherichia coli and the compositions of I class integron thereof - Google Patents

For detecting escherichia coli and the compositions of I class integron thereof Download PDF

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CN104195250B
CN104195250B CN201410453110.3A CN201410453110A CN104195250B CN 104195250 B CN104195250 B CN 104195250B CN 201410453110 A CN201410453110 A CN 201410453110A CN 104195250 B CN104195250 B CN 104195250B
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escherichia coli
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gene
integron
detection
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郑秋月
曹际娟
徐杨
徐君怡
战晓微
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Abstract

Does the present invention provide a kind of for detecting escherichia coli and the compositions of I class integron thereof, including primer pair SEQ? ID? NO.1/2 and primer pair SEQ? ID? NO.3/4. Set up mPCR-DHPLC/ electrophoresis detection test kit and method on this basis further, identify specific gene 16S～23SrRNA gene and integron genes Integrase1 in order to detect E. coli species in food. The method set up in single detects can simultaneously high specific, identify escherichia coli in sample and carry integron genes Integrase1 escherichia coli in high sensitivity. The detection of described method is consuming time short, simple to operation, and has the advantages such as fast high-flux, it is possible to save substantial amounts of labour and financial resources, is suitable for the requirement of quickly detection, meets microorganism in food and detects rapidly demand accurately.

Description

For detecting escherichia coli and the compositions of I class integron thereof
Technical field
The invention belongs to technical field of biological, relate in food and biological product the detection of the device relevant to drug resistant gene entrained by drug resistance escherichia coli, the mPCR especially for escherichia coli and entrained I class integron thereof detects.
Technical field
Escherichia coli are the pathogenic bacterium of common caused zoonosis, and the bacterial strain in food animal source has drug resistance and is likely to the mankind are existed huge potential hazard, and the research of the Drug Resistance of E. coli of detection food source, animal sources etc. is particularly important. Owing to the research of bacterial drug resistance is mainly clinical medicine domain, and animal derived and food borne bacteria drug resistance research is started late, and often due to antibacterial food chain simultaneously is broadcast to the mankind, the intermediate link of food chain is conceived to the detection of Pathogenicity of Bacteria by people more, and have ignored bacterial drug resistance.
At present, the domestic method Main Basis clinic to animal derived bacterial drug resistance and Laboratory Standard committee (ClinicalandLaboratoryStandardsInstitute, CLSI) " animal and the mensuration disk diffusion method of bacterial drug resistance in the goods thereof " SN/T1944-2007 worked out carries out external antibacterial culturing drug susceptibility test, and lacks and quickly detect the detection method of escherichia coli Resistant strain in food. External antibacterial culturing drug susceptibility test complex operation, is unsuitable for the detection of a large amount of sample, and can only detect the drug-resistant phenotype of bacterial strain, and easily causes missing inspection for the still unexpressed Resistant strain of drug resistant gene.And it is expensive to complete the full-automatic biochemical assessing instrument that susceptibility identifies, qualification result is relatively big by the impact of operator, also easily causes false drug resistance or false sensitivity phenomenon; In addition the result of this drug sensitivity testing in vitro cannot assess the bacterial drug resistance harm to human body.
The drug resistance of most of antibacterials produces owing to obtaining exogenous drug resistant gene. Integron, by catching one or more drug resistant gene box, makes antibacterial show corresponding antibiotic drug resistance or multidrug resistant. Integron system is as a moveable Genetic elements, and location on chromosome, makes antibacterial produce drug resistance by vertical transmission. Integron can carry one or more drug resistant gene box, owing to antibacterial constantly can catch drug resistant gene box by integrase gene from environment, and makes it have drug resistance or multi-drug resistant. Meanwhile, integron can pass through to be present in the removable Genetic elements such as plasmid, transposon, carries out Horizontal transfer, thus causing the propagation of bacterial drug resistance between same kind or different genera. Having been found that have at least 8 class integrons so far, wherein I type integron is the most general, and they can integrate 70 several genes boxes, and the overwhelming majority is drug resistant gene box. The I type integron carrying drug resistant gene box in escherichia coli is popular relatively broad, and escherichia coli I type integron can be integrated and carry aadA, dhfr, sat, bla and ereA gene. It is therefore contemplated that, to escherichia coli and to carry the precisely detection of I class integron situation to research drug tolerance of strain further be very important foundation.
Summary of the invention
An object of the present invention, is in that to provide for detecting escherichia coli and the compositions of I class integron thereof, including primer pair SEQIDNO.1/2 and primer pair SEQIDNO.3/4.
Description such as following table about these primer pairs:
Described compositions be applied to PCR reaction, specific aim amplification testing sample in be likely to containing genes of interest fragment, these genes of interest fragments include: escherichia coli integron Integrase1 gene and E. coli 16S～23SrRNA gene.
The two of the purpose of the present invention be in that provide a kind of for detecting escherichia coli and the test kit of I class integron thereof, including the invention described above for detecting escherichia coli and the compositions of I class integron thereof.
Obviously, described test kit can be applicable to pcr amplification, in described test kit, except including above-mentioned specific primer sets thing, it is also possible to including other component for PCR reaction, described component includes but not limited to Taq DNA polymerase, dNTP, 10 × PCR buffer and water.
Described kit reagent box preservation condition :-20 DEG C of preservations.
The purpose of further aspect of the present invention is in that to provide a kind of method detecting escherichia coli and I class integron thereof, including the step of PCR reaction, described PCR reaction uses the present invention's mentioned above to be mPCR amplimer for detecting the compositions of escherichia coli and I class integron thereof.
In specific embodiment, the detection escherichia coli of the present invention and the PCR reaction system cumulative volume 25 μ L described in method of I class integron thereof, including: Taq DNA polymerase 0.25 μ L, the PCR reactant liquor 12 μ L of testing sample DNA solution 1 μ l, 5U/ μ L and the water of surplus;
Containing 10mMTris HCl, 50mMKCl, 25mMMgCl in described PCR reactant liquor2, the primer pair SEQIDNO.1/2 (each 0.1 μM of SEQIDNO.1 and SEQIDNO.2) of each 2.5mM of dNTP and 0.1 μM and 0.1 μM primer pair SEQIDNO.3/4 (each 0.1 μM of SEQIDNO.3 and SEQIDNO.4).
In specific embodiment, the PCR response parameter described in method detecting escherichia coli and I class integron thereof of the present invention is:
Denaturation: 94 DEG C, 1min;
Enter circulation: 94 DEG C of degeneration 30s, 57 DEG C of annealing 90s, 72 DEG C extend 90s, 30 circulations;
Terminate extending: 72 DEG C, 10min.
Product to mPCR, it is possible to adopt the method for DHPLC or electrophoresis to carry out further interpretation of result:
One of specific embodiments, carries out the step of DHPLC analysis to PCR product, and analysis condition is as follows:
Chromatographic column: PS-DVB&C18DNASep chromatographic column, 4.6mm × 50mm, granularity 3 μm;
Column temperature: 50 DEG C;
According to volume ratio, mobile phase is:
0min:55.0%A, 45.0%B;
0.5min:50.2%A, 49.8%B;
3.6min:41.8%A, 58.2%B;
6.8min:38.2%A, 61.8%B;
9.9min:36.3%A, 63.7%B;
13.0min:35.0%A, 65.0%B;
A is 50mlTEAA and 250 μ l acetonitrile mixing, adds sterilizing ultra-pure water and is settled to 1000ml gained solution; B is the mixing of 50mlTEAA and 250ml acetonitrile, adds sterilizing ultra-pure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorescence detector, light source 150WXenon lamp; Excitation spectrum bandwidth 15nm; Emission spectra bandwidth 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 10 μ L.
7. the method described in claim 6, it is characterised in that judge described DHPLC testing result according to following standard:
Detection sample occurs without amplification absworption peak, can determine that sample result is negative, and direct report does not detect corresponding pathogenic bacterium;
There is typical PCR primer absworption peak in detection sample E.coli16S～23SrRNA gene, and absorption peak more than 3mV time, can determine that this sample is escherichia coli, and Integrase1 gene be without amplification absworption peak, can determine that this bacterium does not carry I class integron;
There is typical PCR primer absworption peak in detection sample E.coli16S～23SrRNA gene, and absorption peak more than 3mV time, can determine that this sample is escherichia coli, there is typical PCR primer absworption peak in Integrase1 gene simultaneously, can determine that this bacterium carries I class integron;
Detection sample occur typical PCR primer absworption peak, but absorption peak less than 3mV time, it is proposed that after sample Zengjing Granule again detect; Results peaks of reforming absorption value then for negative, is otherwise probable positive still less than 3mV.
Analysis to described mPCR amplification, the two of specific embodiments, are the steps of electrophoretic analysis, and described agarose gel electrophoresis condition is: agarose 3%, Marker100bp, voltage 200V, electric current 190mA, time 20min, applied sample amount 10 μ L; Observed result on ultraviolet transmission analyser.
To described electrophoresis detection result, judge according to following standard:
As correspondingly sized amplified band does not occur in testing sample, then can report that this sample survey result is for feminine gender;
There is correspondingly sized amplified band in detection sample E.coli16S～23SrRNA gene, can determine that this sample is escherichia coli, and Integrase I gene is without correspondingly sized amplified band, can determine that this bacterium does not carry I class integron;
All there is correspondingly sized amplified band in detection sample E.coli16S～23SrRNA, Integrase I gene, can determine that this bacterium is the escherichia coli carrying I class integron.
The present invention is directed to E. coli species in food and identify that specific gene 16S～23SrRNA gene and integron genes Integrase1 design specificity multiple PCR primer and test kit, and set up mPCR-DHPLC/ electrophoretic detection further, in order to detect escherichia coli and integron genes Integrase1 escherichia coli Resistant strain in food.The method set up in single detects can simultaneously high specific, identify escherichia coli in sample and carry integron genes Integrase1 escherichia coli in high sensitivity. The detection of described method is consuming time short, simple to operation, and has the advantages such as fast high-flux, it is possible to save substantial amounts of labour and financial resources, is suitable for the requirement of quickly detection, meets microorganism in food and detects rapidly demand accurately.
Accompanying drawing explanation
Accompanying drawing 8 width of the present invention, respectively:
Fig. 1: escherichia coli 16S～23SrRNA gene and integron genes Integrase1mPCR-electrophoresis detection result,
Fig. 2: escherichia coli 16S～23SrRNA gene and integron genes Integrase1mPCR-DHPLC testing result,
In Fig. 1 and Fig. 2: 1:E.coli16S～23SrRNA; 2:Integrase1.
The method specificity test result of Fig. 3: mPCR-electrophoresis detection escherichia coli and I class integron thereof,
The method specificity test result of Fig. 4: mPCR-DHPLC detection escherichia coli and I class integron thereof,
In Fig. 3 and Fig. 4,1. staphylococcus aureus; 2. Listeria Monocytogenes; 3. Salmonella typhimurium; 4. enterococcus faecalis; 5. escherichia coli ATCC25922; 6. escherichia coli ATCC51446
The method sensitivity test result of Fig. 5: mPCR-electrophoresis detection escherichia coli and I class integron thereof,
The method sensitivity test result of Fig. 6: mPCR-electrophoresis detection escherichia coli and I class integron thereof,
In Fig. 5 and Fig. 6: 1.5.4 × 105Cfu/mL; 2.5.4 × 104Cfu/mL; 3.5.4 × 103Cfu/mL; 4.5.4 × 102cfu/mL。
Fig. 7: the mPCR-electrophoresis detection result of escherichia coli actual sample separation strain,
Fig. 8: the mPCR-DHPLC testing result of escherichia coli actual sample separation strain,
In Fig. 7 and Fig. 8: 1. escherichia coli I class integron negative strain; 2. escherichia coli I class integron positive strain; 3. escherichia coli I class integron positive strain.
In above-mentioned accompanying drawing, abscissa is retention time (unit: minute min), and vertical coordinate represents absworption peak signal intensity (unit: millivolt mV).
Detailed description of the invention
Following non-limiting example, foundation and the application thereof of this method are further described by it, it is possible to make those of ordinary skill in the art more fully understand the present invention, but do not limit the present invention in any way.
If without specified otherwise, main agents, instrument and merchandise resources thereof that this part uses be: the reagent such as bacterial genomes DNA small scale purification test kit (TakaRaMiniBESTBacterialGenomicDNAExtractionkit), Taq enzyme and PCR buffer are all purchased from precious biological engineering (Dalian) company limited; Triethylamine acetyl salt (TEAA, chromatographically pure) is purchased from Transgenomic company; Acetonitrile (chromatographically pure) is purchased from Fisher company; Regular-PCR instrument PE24000 (PerkinElmer company, the U.S.); Denaturing high-performance chromatography instrument NAV-99-4500 (Transgenomic company, the U.S.); High speed centrifuge centrifuge5804 (Eppendorf company, Germany).
The equal purchased from American Culture Collection (ATCC) of this patent institute reference culture is in Table 1. Each bacterial strain uses strain preservative tube-80 DEG C preservation, and activation culture etc. all carries out according to relevant national standard (GB), inspection and quarantine industry standard (SN) or internal authority standard method.
Table 1 test strain
Embodiment 1
(1) the assembling of the design of primer, synthesis and test kit:
Identify that specific gene 16S～23SrRNA gene and integron genes Integrase1 design specific primer for E. coli species, determined that the primer sequence for detecting and expanding fragment length are as shown in table 2:
Table 2
The design test kit for detecting on this basis.This test kit includes Taq DNA polymerase and the PCR reactant liquor that concentration is 5U/ μ L; Containing 10mMTris HCl, 50mMKCl, 25mMMgCl in PCR reactant liquor therein2, dNTP (dATP, dGTP, dCTP and dTTP) each 2.5mM and above-mentioned 2 pairs of primer pairs (concentration is as shown in table 2).
(2) mPCR:
Use the (1) designed multi-primers of above-mentioned steps and correspondingly test kit, carry out pcr amplification, comprise the steps:
1. the preparation of measuring samples: adopt isolation kit method to prepare testing sample DNA genome:
A, food samples:
Product 25g (position of clip sample is with reference to national standard method) is taken food with sterile working, it is equipped with in aseptic triangular flask or the slap type homogenizing bag of 225mLBPW after pulverizing, rock 3～5min or bounce 1min, after triangular flask or homogenizing bag being sealed, cultivating 18～24h at 36 DEG C.
Use bacterial genomes to extract test kit and extract its genomic DNA, produce pcr template. Labelling, directly as pcr template or-20 DEG C of preservations.
B, reference culture: the single bacterium colony of picking cultivates 18～24h in nutrient broth, use bacterial genomes to extract test kit and extract its genomic DNA, produce pcr template. Labelling, directly as pcr template or-20 DEG C of preservations.
2. pcr amplification:
Take 1 μ l testing sample DNA solution, add the PCR reactant liquor in 12 μ l test kits, 0.25 μ LTaqDNA polymerase and sterilizing ultra-pure water to cumulative volume 25 μ L; 5000r/min is centrifuged 10s, then carries out pcr amplification by following parameters:
Denaturation: 94 DEG C, 1min;
Enter circulation: 94 DEG C of degeneration 30s, 57 DEG C of annealing 90s, 72 DEG C extend 90s, 30 circulations;
Terminate extending: 72 DEG C, 10min;
PCR primer 4 DEG C preservation is to be measured;
(3) mPCR product analysis:
1. 10 μ L step (2) mPCR products therefroms are taken after 3% agarose gel electrophoresis (200V) detects 20min, observed result on ultraviolet transmission analyser, as shown in Figure 1. Visible, the pcr amplification product of E. coli 16S～23SrRNA, int I gene is detected by agarose gel electrophoresis method and all can obtain single band, and size is consistent with intended. Wherein E.coli16S～23SrRNA gene (swimming lane 1) amplifies specific band consistent with the clip size of expection amplification 232bp clearly; (swimming lane 2 amplifies specific band consistent with the clip size of expection amplification 494bp clearly to int I gene. (such as Fig. 1)
2. PCR primer being carried out DHPLC analysis, DHPLC analysis condition is as follows:
Chromatographic column: PS-DVB&C18DNASep chromatographic column, 4.6mm × 50mm, granularity 3 μm;
Column temperature: 50 DEG C;
According to volume ratio, mobile phase is:
0min:55.0%A, 45.0%B;
0.5min:50.2%A, 49.8%B;
3.6min:41.8%A, 58.2%B;
6.8min:38.2%A, 61.8%B;
9.9min:36.3%A, 63.7%B;
13.0min:35.0%A, 65.0%B;
Wherein, buffer solution A is 50mlTEAA and 250 μ l acetonitrile mixing, adds sterilizing ultra-pure water and is settled to 1000ml gained solution; Buffer solution B is the mixing of 50mlTEAA and 250ml acetonitrile, adds sterilizing ultra-pure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorescence detector, light source: 150WXenon lamp; Excitation spectrum bandwidth: 15nm; Emission spectra bandwidth: 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 10 μ L.
All there is single absworption peak when 50 DEG C of non denatured by DHPLC detection in the pcr amplification product of E. coli 16S～23SrRNA, int I gene. Wherein E.coli16S～23SrRNA gene amplification goes out the typical absorption peak consistent with the clip size of expection amplification 232bp, and baseline is smooth; Int I gene amplification goes out the typical absorption peak consistent with the clip size of expection amplification 494bp, and baseline is smooth. (such as Fig. 2)
Embodiment 2. specific test
The DNA of all reference strain in extraction, the method set up by embodiment 1 carries out mPCR amplification, go forward side by side row agarose gel electrophoresis and DHPLC detection. Testing result is as shown in figures 3 and 4.
Specific test is it is shown that the method set up of the present invention, and under agarose gel electrophoresis and DHPLC analysis condition, it is possible to detection Drug Resistance of E. coli, and non-false positive and false negative result produce. DHPLC analyzes result and shows (Fig. 4), only two kinds of Resistant genetype E. coli detections are to positive absworption peak, and two absworption peaks meet with amplified fragments size, it was shown that the DHPLC detection method of foundation is to escherichia coli in food in food and carries integron genes Integrase1 escherichia coli and has good specificity.
Embodiment 3 detection sensitivity is tested
Escherichia coli ATCC51446 is inoculated in TSB culture medium, cultivates 24h for 36 ± 1 DEG C, increase bacterial context soup and obtain 10 through gradient dilution-1,10-2,10-3,10-4,10-5,10-6Bacterium solution, corresponding bacterial number respectively 5.4 × 107Cfu/mL, 5.4 × 106Cfu/mL, 5.4 × 105Cfu/mL, 5.4 × 104Cfu/mL, 5.4 × 103Cfu/mL, 5.4 × 102Cfu/mL. To gradient dilution 5.4 × 10 respectively5Cfu/mL, 5.4 × 104Cfu/mL, 5.4 × 103Cfu/mL, 5.4 × 102Cfu/mL bacterium solution extracts DNA, will carry out mPCR amplification according to the method set up according to embodiment 1, go forward side by side row agarose gel electrophoresis and DHPLC detection. Testing result is as depicted in figures 5 and 6.
Agarose gel electrophoresis detection shows, detection sensitivity is 5.4 × 103Cfu/mL; DHPLC testing result shows, detection sensitivity also can reach 5.4 × 103cfu/mL。
Embodiment 4. actual sample detects
The escherichia coli that 3 strains separate from actual sample utilize the mPCR method that embodiment 1 is set up carry out pcr amplification, go forward side by side row agarose gel electrophoresis and DHPLC detection, and the result of result with the susceptibility test methods gained of detection drug-resistant phenotype is compared. Shown in result of the test such as accompanying drawing 7 and accompanying drawing 8.
Result shows, 3 strain escherichia coli all carry E.coli16S～23SrRNA gene, 2 strain escherichia coli carry Integrase1 gene, qualification result is consistent with GB4789.38-2012 method testing result, and 2 strain carry the bacterial strain of I class integron Integrase1 gene and be multiple antibiotic resistant strain, illustrate that the method can be Rapid identification escherichia coli, and provide foundation for bacterium drug resistance.
In the present embodiment, the Resistant strain that traditional method as a comparison is identified, after bacteria genus is identified, need to just can confirm that its drug resistance then through antibacterial 24h incubated overnight, and the method and Bacteria Identification susceptibility instrument are both needed to measure antibacterial reduced turbidity, it is subject to the impact of testing staff's operation; And disk diffusion method identifies and consuming time need 3-5 days, workload is big, and qualification result influence factor is many. And use the method that this patent sets up can while identifying bacteria genus, detect its entrained I class integron, method on average total (including sample preparation) 6h consuming time, detects (not including sample preparation) consuming time 2.5h, easy and simple to handle quickly.

Claims (9)

1. for detecting escherichia coli and the compositions of I class integron thereof, including primer pair SEQIDNO.1/2 and primer pair SEQIDNO.3/4.
2. for detecting escherichia coli and the test kit of I class integron thereof, including the compositions described in claim 1.
3. the method for escherichia coli and I class integron thereof in detection food, including the step of PCR reaction, it is characterised in that described PCR reaction uses the compositions described in claim 1 to be amplimer.
4. the method described in claim 3, it is characterised in that described PCR reaction system cumulative volume 25 μ L, including: Taq DNA polymerase 0.25 μ L, the PCR reactant liquor 12 μ L of testing sample DNA solution 1 μ l, 5U/ μ L and the water of surplus;
Containing 10mMTris HCl, 50mMKCl, 25mMMgCl in described PCR reactant liquor2, the primer pair SEQIDNO.1/2 of each 2.5mM of dNTP and 0.1 μM and 0.1 μM primer pair SEQIDNO.3/4.
5. the method described in claim 3, it is characterised in that described PCR response parameter is:
Denaturation: 94 DEG C, 1min;
Enter circulation: 94 DEG C of degeneration 30s, 57 DEG C of annealing 90s, 72 DEG C extend 90s, 30 circulations;
Terminate extending: 72 DEG C, 10min.
6. the method described in claim 3, it is characterised in that also including the step that PCR product carries out DHPLC analysis, analysis condition is as follows:
Chromatographic column: PS-DVB&C18DNASep chromatographic column, 4.6mm × 50mm, granularity 3 μm;
Column temperature: 50 DEG C;
According to volume ratio, mobile phase is:
0min:55.0%A, 45.0%B;
0.5min:50.2%A, 49.8%B;
3.6min:41.8%A, 58.2%B;
6.8min:38.2%A, 61.8%B;
9.9min:36.3%A, 63.7%B;
13.0min:35.0%A, 65.0%B;
A is 50mlTEAA and 250 μ l acetonitrile mixing, adds sterilizing ultra-pure water and is settled to 1000ml gained solution; B is the mixing of 50mlTEAA and 250ml acetonitrile, adds sterilizing ultra-pure water and is settled to 1000ml gained solution;
Flow velocity: 0.9mL/min;
Detector: fluorescence detector, light source 150WXenon lamp; Excitation spectrum bandwidth 15nm; Emission spectra bandwidth 15.3nm; Detection sensitivity: at wavelength 350nm integration 2s;
Applied sample amount: PCR primer 10 μ L.
7. the method described in claim 6, it is characterised in that judge described DHPLC testing result according to following standard:
Detection sample occurs without amplification absworption peak, can determine that sample result is negative, and direct report does not detect corresponding pathogenic bacterium;
There is typical PCR primer absworption peak in detection sample E.coli16S～23SrRNA gene, and absorption peak more than 3mV time, can determine that this sample is escherichia coli, and Integrase1 gene be without amplification absworption peak, can determine that this bacterium does not carry I class integron;
There is typical PCR primer absworption peak in detection sample E.coli16S～23SrRNA gene, and absorption peak more than 3mV time, can determine that this sample is escherichia coli, there is typical PCR primer absworption peak in Integrase1 gene simultaneously, can determine that this bacterium carries I class integron;
Detection sample occur typical PCR primer absworption peak, but absorption peak less than 3mV time, it is proposed that sample is reformed; Results peaks of reforming absorption value then for negative, is otherwise probable positive still less than 3mV.
8. the method described in claim 3, it is characterised in that also include the step that PCR product is carried out electrophoretic analysis, described agarose gel electrophoresis condition is: agarose 3%, Marker100bp, voltage 200V, electric current 190mA, time 20min, applied sample amount, 10 μ L;Observed result on ultraviolet transmission analyser.
9. the method described in claim 8, it is characterised in that judge described electrophoresis result according to following standard:
As correspondingly sized amplified band does not occur in testing sample, then can report that this sample survey result is for feminine gender;
There is correspondingly sized amplified band in detection sample E.coli16S～23SrRNA gene, can determine that this sample is escherichia coli, and Integrase I gene is without correspondingly sized amplified band, can determine that this bacterium does not carry I class integron;
All there is correspondingly sized amplified band in detection sample E.coli16S～23SrRNA, Integrase I gene, can determine that this bacterium is the escherichia coli carrying I class integron.
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