CN104212902B - Composition for detecting tetracycline-resistant Escherichia coli - Google Patents
Composition for detecting tetracycline-resistant Escherichia coli Download PDFInfo
- Publication number
- CN104212902B CN104212902B CN201410453400.8A CN201410453400A CN104212902B CN 104212902 B CN104212902 B CN 104212902B CN 201410453400 A CN201410453400 A CN 201410453400A CN 104212902 B CN104212902 B CN 104212902B
- Authority
- CN
- China
- Prior art keywords
- tetracycline
- escherichia coli
- resistant
- detection
- tetb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a composition for detecting tetracycline-resistant Escherichia coli. The composition comprises a primer pair as shown in SEQ ID NO.1/2 and a primer pair as shown in SEQ ID NO.3/4. On the basis, an mPCR-DHPLC/electrophoresis detection kit and method are further established to detect tetracycline-resistant Escherichia coli strains carrying tetracycline-resistant genes tetA and/or tetB in foods. By using the established method, the two tetracycline-resistant genes tetA and tetB of the tetracycline-resistant Escherichia coli in a sample can be simultaneously detected at high specificity and high sensitivity in one-time detection, and furthermore whether a sample to be detected is polluted by the tetracycline-resistant Escherichia coli carrying the tetracycline-resistant genes tetA and tetB is determined. In addition, the method has the advantages of short detection time consumption, simplicity and rapidness in operation, high speed, high throughput and the like and is suitable for meeting the requirement for rapid detection and capable of saving a great number of labors and financial resources and meeting the requirement for rapidly and accurately detecting microorganisms in the foods.
Description
Technical field
The invention belongs to technical field of biological, is related in food and biological product entrained by drug resistance Escherichia coli
The detection of drug resistant gene, detects especially for the mPCR of tetracycline-resistant gene entrained by Escherichia coli.
Technical background
Escherichia coli are the pathogenic bacteria of common caused zoonosis, and the bacterial strain in food animal source has drug resistance
May there is huge potential hazard to the mankind, the research for detecting the Drug Resistance of E. coli of food source, animal sources etc. is particularly weighed
Will.Because the research of bacterial drug resistance is mainly clinical medicine domain, and for animal derived and food borne bacteria drug resistance
Research is started late, and often due to bacterium simultaneously food chain is broadcast to the mankind, people to having in mind the intermediate link of food chain more
In the detection of Pathogenicity of Bacteria, and have ignored bacterial drug resistance.
In livestock rearing, TCs is widely used in the prevention work to all kinds of bacteriosises
In, antibiotic usage dosage is blindly increased, tetracycline extended residence time in livestock and poultry body is made, increase the off-drug period.Exist simultaneously
Livestock products production process also be used to improve the production performance of domestic animal, the random or raising addition in feed as growth promoter
Tetracycline；Additionally, ill poultry clinical symptoms are covered using TCs before butchering escapes ante-mortem inspection.Dynamic
Excessive or substantial amounts of tetracycline residue, promotes bacterium to produce drug resistance in object or in animality relevant food, causes with resistance to
When the pathogen of the property of medicine causes human foods to be poisoned or catch, medicine cannot be treated, or the drug-resistance factor of endurance strain
(such as plasmid, integron) is propagated by the way that the approach such as food chain are popular, causes rate of resistant strains to improve, and antibiotic failure etc. is serious
Consequence.
Escherichia coli are almost most serious in all antibiotic to the drug resistance of tetracycline, and there are some researches show ill dynamic
Apparently higher than healthy animal separation strains, this phenomenon is likely to result in infected animal to the drug resistance of Escherichia coli separation strains in object
Treatment difficulty increase, or even can produce pathogenic bacteria resistant rate improve hidden danger.Therefore, tetracycline resistance gene is detected, and
Its popularity in animal derived food is monitored, and in other non-animal derived food popularities for prevention tetracycline
Antibody-resistant bacterium causes health and harm economically to the mankind and animal, and understanding tetracycline resistance gene is in Escherichia coli
Carrying rate, the use of even tetracycline is all significant.Resistant rate of the ox source Escherichia coli to tetracycline medication
For 30.5%, swine Escherichia coli is up to 96.3% to tetracycline medication resistant rate, and research finds that Escherichia coli are modal
Tolerance medicine is tetracycline.Bryan etc. (2004) have studied the people from the U.S. and the detached Escherichia coli of various animal bodies, find
TetA and tetB are topmost Escherichia coli tetracycline resistance genes, and its carrying rate is respectively 35% and 63%.
At present, domestic method Main Basiss clinic and the Laboratory Standard committee to animal derived bacterial drug resistance
(Clinical and Laboratory Standards Institute, CLSI) is worked out《Bacterium is resistance in animal and its product
The measure disk diffusion method of the property of medicine》SN/T1944-2007 carries out external Bacteria Culture drug susceptibility test, and lacks quick detection food
The detection method of Escherichia coli antibody-resistant bacterium in product.External Bacteria Culture drug susceptibility test is cumbersome, is unsuitable for a large amount of samples
Detection, and the drug-resistant phenotype of bacterial strain can only be detected, and for the still unexpressed antibody-resistant bacterium of drug resistant gene easily causes missing inspection.And
And complete susceptibility identification full-automatic biochemical assessing instrument it is expensive, qualification result is affected larger by operating personnel, is also easily made
Into false resistance or false sensitivity phenomenon；In addition the result of the drug sensitivity testing in vitro cannot assess danger of the bacterial drug resistance to human body
Evil.Therefore it is badly in need of setting up the detection method of more precise and high efficiency, can quickly realizes the discriminating of antibody-resistant bacterium, and can be prevented effectively from
False resistance or false sensitivity phenomenon that tested bacterial strain is produced because of drug sensitivity testing in vitro.
The content of the invention
An object of the present invention, is to provide the composition for the E. coli detection of resistance to tetracycline, including primer pair
SEQ ID NO.1/2 and primer pair SEQ ID NO.3/4.
Table is described as follows with regard to these primer pairs：
The composition is applied to PCR reactions, and specific aim expands the genes of interest fragment that may contain in testing sample,
These genes of interest fragments include：Escherichia coli tetracycline resistance gene tetA and tetB.
The second object of the present invention is to provide a kind of kit for the E. coli detection of resistance to tetracycline, including above-mentioned
The composition for the E. coli detection of resistance to tetracycline of the present invention.
Obviously, the kit can be applicable to PCR amplifications, in the kit, except including above-mentioned specific primer group
Compound, can also include other components for PCR reactions, and the component includes but is not limited to Taq archaeal dna polymerases, dNTP,
10 × PCR buffer solutions and water.
The kit reagent box preservation condition：- 20 DEG C of preservations.
The purpose of further aspect of the present invention is to provide a kind of resistance to tetracycline of detection colibacillary method, including PCR anti-
The step of answering, the PCR reactions are mPCR for the E. coli detection of resistance to tetracycline composition using the present invention mentioned above
Amplimer.
PCR reactants in specific embodiment, described in the colibacillary method of the resistance to tetracycline of detection of the present invention
It is the μ L of cumulative volume 25, including：The μ L of Taq archaeal dna polymerases 0.25 of the μ l of testing sample DNA solution 1,5U/ μ L, the μ L of PCR reactant liquors 12
With the water of surplus；
TrisHCl containing 10mM, 50mM KCl, 25mM MgCl in the PCR reactant liquors2, dNTP each 2.5mM and 0.1 μ
The primer pair SEQ ID NO.1/2 (SEQ ID NO.1 and SEQ ID NO.2 are each 0.1 μM) of M and 0.1 μM of primer pair SEQ ID
NO.3/4 (SEQ ID NO.3 and SEQ ID NO.4 are each 0.1 μM).
PCR reaction ginsengs in specific embodiment, described in the colibacillary method of the resistance to tetracycline of detection of the present invention
Number is：
Denaturation：94 DEG C, 1min；
Into circulation：94 DEG C of denaturation 30s, 57 DEG C of annealing 90s, 72 DEG C of extension 90s, 30 circulations；
Terminate extending：72 DEG C, 10min.
Product to mPCR, can adopt DHPLC or the method for electrophoresis to carry out further interpretation of result：
One of specific embodiment, the step of carrying out DHPLC to PCR product and analyze, analysis condition is as follows：
Chromatographic column：PS-DVB ＆ C18 DNASep chromatographic columns, 4.6mm × 50mm, 3 μm of granularity；
Column temperature：50℃；
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 50ml TEAA and 250 μ l acetonitriles mix, plus sterilizing ultra-pure water is settled to 1000ml resulting solutions；B is 50ml
TEAA and 250ml acetonitriles mix, plus sterilizing ultra-pure water is settled to 1000ml resulting solutions；
Flow velocity：0.9mL/min；
Detector：Fluorescence detector, light source：150W Xenon lamps；PLE bandwidth：15nm；Emission spectra bandwidth：
15.3nm；Detection sensitivity：2s is integrated in wavelength 350nm；
Applied sample amount：The μ L of PCR primer 10.
To the DHPLC testing results, judged according to following standard：
Detection sample occurs without amplification absworption peak, can determine that sample result is feminine gender, and directly report does not detect corresponding
Pathogenic bacteria；
Occur the typical PCR primer absworption peak of any one and the above in two kinds of genes of detection sample tetA, tetB, and absorb
When peak value is more than 3mV, can determine that the sample carries Escherichia coli tetracycline resistance gene, the bacterium is the positive strain of resistance to tetracycline；
When in two kinds of genes of detection sample tetA, tetB without typical PCR primer absworption peak, can determine that the sample is not carried greatly
Enterobacteria tetracycline resistance gene, the bacterium is the negative strain of resistance to tetracycline；
When there is typical PCR primer absworption peak, but absorption peak less than 3mV in detection sample, it is proposed that sample is reformed, and reforms
Results peaks absorption value is then feminine gender still less than 3mV, is otherwise probable positive.
Analysis to the mPCR amplifications, the two of specific embodiment, the step of be electrophoretic analysis：10 μ L PCR are produced
Thing, in 3% agarose gel electrophoresis, 100V, after 20min, on ultraviolet transmission analyzer result is observed.
To the electrophoresis detection result, judged according to following standard：
Detection sample tetA, tetB genes can determine that the sample does not carry tetA without correspondingly sized amplified band,
Two kinds of tetracycline resistance genes of tetB；
Detection sample tetA gene there is correspondingly sized amplified band and tetB genes without correspondingly sized amplified band,
Or tetB genes there is correspondingly sized amplified band and tetA genes without correspondingly sized amplified band, or tetA, tetB base
Because there is correspondingly sized amplified band, can determine that the sample is tetracycline resistant bacterial strain.
The present invention is for Escherichia coli tetracycline resistance gene tetA and tetB common in food, and design specificity is multiple
PCR probes and kit, and further set up mPCR-DHPLC/ electrophoretic detections, to detect food in the large intestine of resistance to tetracycline
Bacillus.By the method set up is in the one-time detection while resistance to Fourth Ring in high specific, in high sensitivity complete paired samples
The detection of two kinds of drug resistant genes of plain Escherichia coli tetA and tetB, and then determine whether measuring samples are carried tetA and tetB is resistance to
The tetracycline resistance Escherichia coli of medicine gene are polluted.Additionally, methods described detection is time-consuming short, it is simple to operation, and with fast
The advantages of fast high flux, substantial amounts of labour and financial resources can be saved, be adapted to the requirement of quick detection, meet microorganism in food
The rapid accurately demand of detection.
Description of the drawings
The width of accompanying drawing of the present invention 8, be respectively：
Fig. 1：Escherichia coli tetracycline resistance gene mPCR- electrophoresis detection results.
Fig. 2：Escherichia coli tetracycline resistance gene mPCR-DHPLC testing results.
Fig. 3：The colibacillary method specific test result of the resistance to tetracycline of mPCR- electrophoresis detections (1. Staphylococcus aureus
Bacterium；2. Listeria Monocytogenes；3. salmonella typhimurium；4. enterococcus faecalis；5. Escherichia coli sensitive tetracycline
Bacterial strain；6. the Escherichia coli tetracycline resistant bacterial strain of tetA and tetB is contained).
Fig. 4：MPCR-DHPLC detects the colibacillary method specific test result of resistance to tetracycline (1. Staphylococcus aureus
Bacterium；2. Listeria Monocytogenes；3. salmonella typhimurium；4. enterococcus faecalis；5. Escherichia coli sensitive tetracycline
Bacterial strain；6. the Escherichia coli tetracycline resistant bacterial strain of tetA and tetB is contained).
Fig. 5：The colibacillary method sensitivity test result (1 of the resistance to tetracycline of mPCR- electrophoresis detections:5.4×106cfu/
mL；2:5.4×105cfu/mL；3:5.4×104cfu/mL；4:5.4×103cfu/mL；5:5.4×102cfu/mL)。
Fig. 6：MPCR-DHPLC detects the colibacillary method sensitivity test result (1 of resistance to tetracycline:5.4×106cfu/
mL；2:5.4×105cfu/mL；3:5.4×104cfu/mL；4:5.4×103cfu/mL；5:5.4×102cfu/mL)。
Fig. 7：The mPCR- electrophoresis detection results of Escherichia coli actual sample separation strains tetracycline resistance gene (are 1. carried
The Escherichia coli tetracycline resistant bacterial strain of tetA and tetB；2. the Escherichia coli tetracycline resistant bacterial strain of tetB is carried；3. carry
The Escherichia coli tetracycline resistant bacterial strain of tetA).
Fig. 8：The mPCR-DHPLC testing results of Escherichia coli actual sample separation strains tetracycline resistance gene (are 1. carried
The Escherichia coli tetracycline resistant bacterial strain of tetA and tetB；2. the Escherichia coli tetracycline resistant bacterial strain of tetB is carried；3. carry
The Escherichia coli tetracycline resistant bacterial strain of tetA)
In above-mentioned accompanying drawing, abscissa is retention time (unit：Minute min), ordinate represents absworption peak signal strength signal intensity
(unit：Millivolt mV).
Specific embodiment
Following non-limiting examples, its foundation and its application to this method is further described, and can make ability
The those of ordinary skill in domain is more fully understood the present invention, but limits the present invention never in any form.
If without specified otherwise, main agents, instrument and its merchandise resources that this part is used is：Bacterial genomes DNA
Small scale purification kit (TakaRa MiniBEST Bacterial Genomic DNA Extraction kit), Taq enzyme and
The reagents such as PCR buffer solutions are purchased from precious bioengineering (Dalian) Co., Ltd；Triethylamine acetyl salt (TEAA, chromatographically pure) is purchased from
Transgenomic companies；Acetonitrile (chromatographically pure) is purchased from Fisher companies；Regular-PCR instrument PE24000 (PerkinElmer companies,
The U.S.)；Denaturing high-performance chromatography instrument NAV-99-4500 (Transgenomic companies, the U.S.)；Supercentrifuge
Centrifuge 5804 (Eppendorf companies, Germany).
Reference culture used herein is purchased from American Type Culture collection (ATCC) and Chinese medicine microbial bacteria
Preservation administrative center (CMCC) is planted, 1 is shown in Table.Each bacterial strain is using -80 DEG C of preservations of strain preservative tube, activation culture etc. according to correlation
National standard (GB), inspection and quarantine professional standard (SN) or internal authority standard method carry out.
The test strain of table 1
Embodiment 1
(1) the design of primer, synthesis and the assembling of kit：
Primer is designed for Escherichia coli tetracycline resistance gene tetA and tetB, the primer sequence for detection is determined
And expanding fragment length is as shown in table 2：
Table 2
The kit for detecting is designed on this basis.The kit includes that the Taq DNA that concentration is 5U/ μ L are polymerized
Enzyme and PCR reactant liquors；TrisHCl containing 10mM, 50mM KCl, 25mM MgCl in PCR reactant liquors therein2、dNTP
(concentration is such as the primer pair of (dATP, dGTP, dCTP and dTTP) each 2.5mM and above-mentioned 2 pairs of colibacillary tetracycline resistance genes
Shown in table 2).
⑵mPCR：
Using the (1) designed multi-primerses of above-mentioned steps and correspondingly kit, enter performing PCR amplification, including following step
Suddenly：
1. the preparation of measuring samples：Testing sample DNA genomes are prepared using isolation kit method：
A, food samples：
Product 25g (position of clip sample is with reference to national standard method) is taken food with sterile working, is added after crushing and is equipped with
In the aseptic triangular flask or slap type homogenizing bag of 225mLBPW, rock 3～5min or bounce 1min, triangular flask or homogenizing bag are sealed
18～24h is cultivated after mouthful at 36 DEG C.
Its genomic DNA is extracted using bacterial genomes extracts kit, pcr template is produced.Mark, directly as PCR
Template or -20 DEG C of preservations.
B, reference culture：Picking single bacterium colony cultivates 18～24h in nutrient broth, using bacterial genomes extracts reagent
Box extracts its genomic DNA, produces pcr template.Mark, directly as pcr template or -20 DEG C of preservations.
2. PCR amplifications：
1 μ l testing sample DNA solutions are taken, PCR reactant liquors, 0.25 μ L Taq archaeal dna polymerases in 12 μ l kits is added
And sterilizing ultra-pure water is to the μ L of cumulative volume 25；5000r/min is centrifuged 10s, then enters performing PCR amplification by following parameters：
Denaturation：94 DEG C, 1min；
Into circulation：94 DEG C of denaturation 30s, 57 DEG C of annealing 90s, 72 DEG C of extension 90s, 30 circulations；
Terminate extending：72 DEG C, 10min；
4 DEG C of preservations of PCR primer are to be measured；
(3) mPCR product analysis：
1. take 10 μ L steps (2) mPCR products therefroms 3% agarose gel electrophoresis (200V) detection 20min after, ultraviolet
Result is observed on tem analysis instrument, as shown in Figure 1.It can be seen that, amplified fragments size is Escherichia coli Fourth Ring for the band of 363bp
Plain drug resistant gene tetA；Amplified fragments size is Escherichia coli tetracycline resistance gene tetB for the band of 216bp.
2. PCR primer is carried out into DHPLC analyses, DHPLC analysis conditions are as follows：
Chromatographic column：PS-DVB ＆ C18 DNASep chromatographic columns, 4.6mm × 50mm, 3 μm of granularity；
Column temperature：50℃；
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, cushioning liquid A is 50ml TEAA and 250 μ l acetonitriles mix, plus sterilizing ultra-pure water is settled to 1000ml gained
Solution；Cushioning liquid B is the mixing of 50ml TEAA and 250ml acetonitrile, plus sterilizing ultra-pure water is settled to 1000ml resulting solutions；
Flow velocity：0.9mL/min；
Detector：Fluorescence detector, light source：150W Xenon lamps；PLE bandwidth：15nm；Emission spectra bandwidth：
15.3nm；Detection sensitivity：2s is integrated in wavelength 350nm；
Applied sample amount：The μ L of PCR primer 10.
As shown in Figure 2, the pcr amplification product of two kinds of primers can produce positive absorption to mPCR-DHPLC testing results
Peak, and absworption peak meets with amplified fragments size.
The specific test of embodiment 2.
Extract Escherichia coli tetracycline resistant bacterial strain (carrying tetA, the Escherichia coli positive strain of tetB), golden yellow Portugal
Grape coccus, salmonella, Listeria monocytogenes, vibrio parahemolyticus, enterococcus faecalis, pseudomonas aeruginosa (being shown in Table 1) DNA, root
The detection method set up according to claim 1 carries out double PCR amplification, and with electrophoresis and DHPLC analysis results, respectively such as Fig. 3
With shown in Fig. 4.Only Escherichia coli positive strain detects positive absworption peak, and two absworption peaks are accorded with amplified fragments size
Close, show the detection method of primer designed by the present invention and foundation to the large intestine that carries tetA and tetB drug resistant genes in food
Bacillus has preferably specificity.
The detection sensitivity of embodiment 3 is tested
Escherichia coli tetracycline resistant bacterial strain (positive strain) are inoculated in TSB culture mediums, 36 ± 1 DEG C of culture 24h increase
Bacterial context soup Jing gradient dilutions obtain 10-1,10-2,10-3,10-4,10-5,10-6,10-7Bacterium solution, correspondence bacterial number be 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.Respectively to gradient dilution 5.4 × 106Cfu/mL, 5.4 × 105Cfu/mL, 5.4 × 104Cfu/mL, 5.4 ×
103Cfu/mL, 5.4 × 102The bacterium solution of cfu/mL extracts DNA, and according to the method for embodiment 1 multiplexed PCR amplification is carried out, and with electricity
Swimming and DHPLC analysis results, respectively as shown in accompanying drawing 5 and accompanying drawing 6.
Electrophoresis detection result (Fig. 5) shows that the sensitivity of the method is 10-5(i.e. bacterial number is 5.4 × 103cfu/
mL)；DHPLC testing results (Fig. 6) show that DHPLC detection sensitivities can reach 5.4 × 102Cfu/mL, therefore, this test is built
Vertical method sensitivity can reach 5.4 × 103cfu/mL。
The actual sample of embodiment 4. is detected
By 3 plants of mPCR- electrophoresis/DHPLC methods that detached Escherichia coli are set up using embodiment 1 from actual sample
Detected.Result of the test is respectively as shown in accompanying drawing 7 and accompanying drawing 8.No. 1 bacterial strain is the Escherichia coli for carrying tetA and tetB genes
Tetracycline resistant bacterial strain；No. 2 bacterial strains are the Escherichia coli tetracycline resistant bacterial strain for carrying tetB genes；No. 2 bacterial strains are carrying
The Escherichia coli tetracycline resistant bacterial strain of tetA genes.This 3 plants of Escherichia coli are carried out into disk diffusion method and full automatic microorganism
Identification and susceptibility system drug sensitive test checking drug tolerance of strain, drug sensitivity tests are tetracycline resistant bacterial strain, explanation for this 3 plants of bacterium
MPCR primer pairs and corresponding method of detection designed by the present invention is good for detection Escherichia coli tetracycline resistant bacterial strain has
Effect.
In the present embodiment, the antibody-resistant bacterium identified using traditional disk diffusion method need to be after bacteria genus identification, then Jing is thin
Bacterium 24h incubated overnights just can confirm that its drug resistance, and the method is both needed to determine bacterium reduced turbidity with Bacteria Identification susceptibility instrument, easily receives
The impact of testing staff's operation；And disk diffusion method identification takes and needs 3-5 days, workload is big, and qualification result influence factor is more.
And the mPCR- electrophoresis/DHPLC methods set up using the present invention can detect its drug resistant gene while bacteria genus are identified,
Method average total time-consuming (including sample preparation) 6h, the time-consuming 2.5h (not including sample preparation) of detection, it is easy to operate quick.
Claims (2)
1. the composition of the E. coli detection of resistance to tetracycline, including primer pair SEQ ID NO.1/2 and primer pair SEQ ID are used for
NO.3/4。
2. the kit of the E. coli detection of resistance to tetracycline, including the composition described in claim 1 are used for.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410453400.8A CN104212902B (en) | 2014-09-05 | 2014-09-05 | Composition for detecting tetracycline-resistant Escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410453400.8A CN104212902B (en) | 2014-09-05 | 2014-09-05 | Composition for detecting tetracycline-resistant Escherichia coli |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104212902A CN104212902A (en) | 2014-12-17 |
| CN104212902B true CN104212902B (en) | 2017-05-10 |
Family
ID=52094804
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410453400.8A Expired - Fee Related CN104212902B (en) | 2014-09-05 | 2014-09-05 | Composition for detecting tetracycline-resistant Escherichia coli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104212902B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561342B (en) * | 2015-01-23 | 2017-11-21 | 杭州迪安医学检验中心有限公司 | A kind of primer and kit of detection bacterium Tetracyclines drug resistant gene |
| CN107164505A (en) * | 2017-06-16 | 2017-09-15 | 苏州乔纳森新材料科技有限公司 | A kind of molecular labeling and its application for being used to detect B races streptococcus tetracycline resistance gene |
-
2014
- 2014-09-05 CN CN201410453400.8A patent/CN104212902B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104212902A (en) | 2014-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101570783B (en) | Detection kit and detection method for 9 species of pathogenic organisms in marine products | |
| Hein et al. | Real-time PCR for the detection of Salmonella spp. in food: an alternative approach to a conventional PCR system suggested by the FOOD-PCR project | |
| Xiao et al. | Rapid and sensitive detection of Vibrio vulnificus using CRISPR/Cas12a combined with a recombinase-aided amplification assay | |
| CN107022644A (en) | Six kinds of multiple LAMP detection primers of food-borne pathogens, detection kit and detection method in fruits and vegetables | |
| O'mahony et al. | Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk | |
| Wang et al. | Saltatory rolling circle amplification (SRCA): a novel nucleic acid isothermal amplification technique applied for rapid detection of Shigella spp. in vegetable salad | |
| Singh et al. | Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella | |
| CN104212901B (en) | Compositions for drug resistance of Staphylococcus aureus detection | |
| Eyigor et al. | Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes | |
| Sanchez et al. | Analysis of viable vs. dead A ggregatibacter actinomycetemcomitans and P orphyromonas gingivalis using selective quantitative real‐time PCR with propidium monoazide | |
| Zang et al. | Can a visual loop-mediated isothermal amplification assay stand out in different detection methods when monitoring Campylobacter jejuni from diverse sources of samples? | |
| CN106591416A (en) | Coloring culture medium for detecting food-borne pathogenic Yersinia | |
| CN104212899A (en) | Composition for detecting four common drug-resistant genes of escherichia coli | |
| Milton et al. | Novel saltatory rolling circle amplification assay for rapid and visual detection of Campylobacter jejuni in chicken meat | |
| Gumaa et al. | Specific detection and differentiation between Brucella melitensis and Brucella abortus by a duplex recombinase polymerase amplification assay | |
| CN104212902B (en) | Composition for detecting tetracycline-resistant Escherichia coli | |
| Roumani et al. | Real-time PCR, and Recombinase Polymerase Amplification combined with SYBR Green I for naked-eye detection, along with Propidium Monoazide (PMA) for the detection of viable patulin-producing fungi in apples and by-products | |
| Du et al. | Probe-based loop-mediated isothermal amplification assay for multi-target quantitative detection of three foodborne pathogens in seafood | |
| Tamerat et al. | Application of molecular diagnostic techniques for the detection of E. coli O157: H7: a review | |
| Beaubrun et al. | Evaluation of a multiplex PCR method to serotype Salmonella in animal feeds pre-enrichment broth cultures | |
| Lin et al. | Rectal swab sampling followed by an enrichment culture-based real-time PCR assay to detect Salmonella enterocolitis in children | |
| CN104195250B (en) | For detecting escherichia coli and the compositions of I class integron thereof | |
| CN101591704B (en) | Detection kit for detecting three spore production bacteria in food and detection method thereof | |
| CN101570781B (en) | Detection kit and species-based detection method for lactobacilli | |
| Wang et al. | A CRISPR-Cas12a-based platform facilitates the detection and serotyping of Streptococcus suis serotype 2 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170510 Termination date: 20180905 |