CN104878081A - Aeromonas hydrophila tetracycline drug-resistance gene detection method - Google Patents
Aeromonas hydrophila tetracycline drug-resistance gene detection method Download PDFInfo
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- CN104878081A CN104878081A CN201510087983.1A CN201510087983A CN104878081A CN 104878081 A CN104878081 A CN 104878081A CN 201510087983 A CN201510087983 A CN 201510087983A CN 104878081 A CN104878081 A CN 104878081A
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Abstract
The invention discloses an aeromonas hydrophila tetracycline drug-resistance gene detection method. The method comprises the following steps: (1) collecting 0.5-0.7 g of aeromonas hydrophila living bacteria, resuspending with 5-7 mL of deionized water, repeatedly freeze-thawing to break cells, centrifuging and collecting a supernatant to obtain a template; (2) mixing the template with tetracycline drug-resistance gene detection primers TetA-F, TetA-R, TetC-F, TetC-R, TetG-F and TetG-R, and carrying out PCR amplification; and (3) carrying out agarose gel electrophoresis after PCR amplification in the step (2) and sending for sequencing. The detection method provided by the invention is sensitive, specific and rapid and can be adopted to detect a trace amount of target gene without the time-consuming aeromonas hydrophila culture stage.
Description
Technical field
The invention belongs to detection of pathogens technical field, be specifically related to a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method.
Background technology
Tetracycline antibiotics is the class Broad spectrum antibiotics produced by actinomycetes, comprises duomycin, terramycin and semi-synthetic derivative methacycline, doxycycline, dimethylin tsiklomitsin etc.This product is broad-spectrum antibacterial agent, tool germicidal action during high density.Except common gram positive organism, gram-negative bacteria and anerobe, most rickettsiae, Mycoplasma, chlamydiaceae, anonymous mycobacteria belong to, spirochete is also responsive to this product.For many years due to the widespread use of tetracyclines, common clinical pathogenic bacteria comprises the gram negative bacillis such as gram positive organism and enterobacter such as staphylococcus to the most resistance of tsiklomitsin, and, there is crossing drug resistant with between veriety.
Large quantity research both domestic and external shows, because tsiklomitsin medicine is long-term and a large amount of use, Aeromonas hydrophila is very serious to the resistance of tetracycline medication.What common tetracycline resistance gene had been found that has: TetA, TetB, TetC, TetD, TetE, TetG etc.The cross transmission that tetracycline resistance bacterial strain is caused by approach such as aquatic products product and food chains, directly constitutes a threat to HUMAN HEALTH.
Current China aquatic products product pathogenic bacterium Resistance detection in the research of tsiklomitsin medicine Resistance detection, is carried out few to the research of pathogenic bacteria resistance to drugs gene in controlling.Traditional method is by measuring the minimum inhibitory concentration of Aeromonas hydrophila or the resistance of inhibition zone size measurement Aeromonas hydrophila.Traditional drug sensitive test has to pass through the step such as loaded down with trivial details Aeromonas hydrophila separation and purification, breeding amplification, and sense cycle is long, wants about 48h at the soonest.Be unfavorable for selecting medicine to treat timely.Meanwhile, drug sensitive test is in vitro by the phenotypic resistance characteristic of medicine tentative inspection Aeromonas hydrophila, can not detect the implicit type resistance of Aeromonas hydrophila.
Summary of the invention
The object of the invention is to overcome prior art defect, a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method is provided.
Concrete technical scheme of the present invention is as follows:
A kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method, comprises the steps:
(1) Aeromonas hydrophila viable bacteria body 0.5 ~ 0.7g is collected, resuspended with 5 ~ 7mL deionized water, multigelation smudge cells, collected after centrifugation supernatant liquor, obtains template;
(2) this template and tetracycline medication drug resistant gene are detected primer TetA-F, TetA-R, TetC-F, TetC-R, after TetG-F and TetG-R mixes jointly, carry out pcr amplification, the reaction system of this pcr amplification comprises ultrapure water, PCR damping fluid, final concentration is the dNTPs (dGTP of 0.25 ~ 0.35mmol/L, dCTP, dATP and dTTP), final concentration is the TetA-F of 0.5 ~ 0.6mmol/L, final concentration is the TetA-R of 0.5 ~ 0.6mmol/L, final concentration is the TetC-F of 0.3 ~ 0.4mmol/L, final concentration is the TetC-R of 0.3 ~ 0.4mmol/L, final concentration is the TetG-F of 0.4 ~ 0.5mmol/L, final concentration is the TetG-R of 0.4 ~ 0.5mmol/L and final concentration is the Taq DNA polymerase of 0.01 ~ 0.02U/uL, wherein TetA-F, TetA-R, TetC-F, TetC-R, TetG-F and TetG-R is respectively as SEQ ID 1, SEQID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 and SEQ ID 6,10 times of mother liquors of above-mentioned PCR damping fluid comprise 50 ~ 55mM sodium-chlor, the 10mMTris-HCl of pH8.3, 0.01 ~ 0.02% glycerine and 0.01 ~ 0.02% ammonium sulfate, 25 ~ 30mM MgCl
2,
(3), after the pcr amplification of step (2) terminates, carry out agarose gel electrophoresis and deliver order-checking.
In a preferred embodiment of the invention, described step (1) is: collect Aeromonas hydrophila viable bacteria body 0.5 ~ 0.6g, resuspended with 5 ~ 6mL deionized water, multigelation smudge cells, collect supernatant liquor after the centrifugal 10 ~ 15min of 10000 ~ 12000rpm, obtain template.
In a preferred embodiment of the invention, the TetG-R that the TetC-R that the TetC-F that the TetA-R that the TetA-F that the dNTPs that the reaction system of described pcr amplification comprises ultrapure water, PCR damping fluid, final concentration are 0.3mmol/L, final concentration are 0.5mmol/L, final concentration are 0.5mmol/L, final concentration are 0.3mmol/L, final concentration are 0.3mmol/L, final concentration are the TetG-F of 0.4mmol/L, final concentration is 0.4mmol/L and final concentration are the Taq DNA polymerase of 0.01U/uL.
In a preferred embodiment of the invention, 10 times of mother liquors of described PCR damping fluid comprise 50mM sodium-chlor, the 10mMTris-HCl of pH8.3,0.01% glycerine and 0.01% ammonium sulfate, 25mM MgCl
2.
In a preferred embodiment of the invention, the reaction conditions of described pcr amplification is as follows: 94 ~ 95 DEG C of denaturation 10 ~ 12min, then following circulation is entered: 94 ~ 95 DEG C of sex change 45 ~ 50s, 54 ~ 55 DEG C of annealing 1 ~ 1.5min, 72 ~ 74 DEG C of extension 45 ~ 50s, totally 30 ~ 35 circulations, 7 ~ 8min is extended in 72 DEG C, 2 ~ 4 DEG C of preservations after loop ends.
Preferred further, the reaction conditions of described pcr amplification is as follows: 94 DEG C of denaturation 10min, then enter following circulation: 94 DEG C of sex change 45s, 54 DEG C of annealing 1min, 72 DEG C of extensions 45s, totally 30 circulations, extend 7min in 72 DEG C after loop ends, 4 DEG C of preservations.
In a preferred embodiment of the invention, the deposition condition of described step (3) is: 2% sepharose, 80V voltage, time 40min.
The invention has the beneficial effects as follows:
1, detection method of the present invention is responsive, special, quick, and the goal gene that can detect denier detects, and does not need through time-consuming Aeromonas hydrophila cultivation stage;
2, detection method of the present invention adds the multiple tetracycline resistance gene of multipair primer pair and increases simultaneously in same reaction system, can set up internal contrast in detection gene process; The quantity of template can be indicated; Can be increased several goal gene simultaneously: time and the reagent of consumption are few, reduces setup time and raise the efficiency; The detection of sample and goal gene can be carried out in large quantities;
It is some that 3 detection methods of the present invention can indicate in many resistant organisms, or distinguish the kind in agreeing to belong to or strain.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection result figure of the embodiment of the present invention 1.
Embodiment
By reference to the accompanying drawings below by way of embodiment technical scheme of the present invention is further detailed and is described.
Embodiment 1
(1) LB culture media shaking vase cultivates propagation Aeromonas hydrophila, shake flask culture conditions is 30 DEG C, 18h, 120rpm, shake-flask culture terminates rear collected by centrifugation Aeromonas hydrophila viable bacteria body 0.5g, resuspended with 5mL deionized water, multigelation 5 smudge cellses, collect supernatant liquor after the centrifugal 10min of 10000pm, obtain template;
(2) this 5uL template and tetracycline medication drug resistant gene are detected primer TetA-F, TetA-R, TetC-F, TetC-R, after TetG-F and TetG-R (primer concentration is respectively 25mmol/L, with the dilution of 1mM Tris-HCl--0.1mM edta buffer liquid) common mixing, carry out pcr amplification, the reaction system of this pcr amplification comprises deionized water, PCR damping fluid, final concentration is the dNTPs (dGTP of 0.3mmol/L, dCTP, dATP and dTTP), final concentration is the TetA-F of 0.5mmol/L, final concentration is the TetA-R of 0.5mmol/L, final concentration is the TetC-F of 0.3mmol/L, final concentration is the TetC-R of 0.3mmol/L, final concentration is the TetG-F of 0.4mmol/L, final concentration is the TetG-R of 0.4mmol/L and final concentration is the Taq DNA polymerase of 0.01U/uL, wherein TetA-F, TetA-R, TetC-F, TetC-R, TetG-F and TetG-R is respectively as SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 and SEQ ID6,10 times of mother liquors of above-mentioned PCR damping fluid comprise 50mM sodium-chlor, the 10mMTris-HCl of pH8.3, 0.01% glycerine and 0.01% ammonium sulfate, 25mM MgCl
2,
The reaction system of above-mentioned pcr amplification is 50uL, reaction conditions is as follows: 94 DEG C of denaturation 10min, then enter following circulation: 94 DEG C of sex change 45s, 54 DEG C of annealing 1min, 72 DEG C of extensions 45s, totally 30 circulations, 7min is extended in 72 DEG C, 4 DEG C of preservations after loop ends;
(3) after the pcr amplification of step (2) terminates, get 5uL PCR primer, after mixing with 1uL Loading buffer, point sample in 2% agarose gel electrophoresis plate hole, 80V voltage, electrophoresis 40min, to take pictures under Ultraluminescence Cheng Xiangyi judgement, as shown in Figure 1, the band of visible about the 850bp of result, about 400bp, about 550bp, indicates TetA gene, TetC gene, TetG gene to electrophoresis result respectively.
Together with trigenic to purified PCR primer 50uL and 20uL upstream and downstream primer, send Shenzhen Hua Da gene company limited to carry out DNA sequencing simultaneously.
The above, be only preferred embodiment of the present invention, therefore can not limit scope of the invention process according to this, the equivalence change namely done according to the scope of the claims of the present invention and description with modify, all should still belong in scope that the present invention contains.
Claims (7)
1. an Aeromonas hydrophila tetracycline medication drug resistant gene detection method, is characterized in that: comprise the steps:
(1) Aeromonas hydrophila viable bacteria body 0.5 ~ 0.7g is collected, resuspended with 5 ~ 7mL deionized water, multigelation smudge cells, collected after centrifugation supernatant liquor, obtains template;
(2) this template and tetracycline medication drug resistant gene are detected primer TetA-F, TetA-R, TetC-F, TetC-R, after TetG-F and TetG-R mixes jointly, carry out pcr amplification, the reaction system of this pcr amplification comprises ultrapure water, PCR damping fluid, final concentration is the dNTPs of 0.25 ~ 0.35mmol/L, final concentration is the TetA-F of 0.5 ~ 0.6mmol/L, final concentration is the TetA-R of 0.5 ~ 0.6mmol/L, final concentration is the TetC-F of 0.3 ~ 0.4mmol/L, final concentration is the TetC-R of 0.3 ~ 0.4mmol/L, final concentration is the TetG-F of 0.4 ~ 0.5mmol/L, final concentration is the TetG-R of 0.4 ~ 0.5mmol/L and final concentration is the Taq DNA polymerase of 0.01 ~ 0.02U/uL, wherein TetA-F, TetA-R, TetC-F, TetC-R, TetG-F and TetG-R is respectively as SEQ ID 1, SEQ ID 2, SEQ ID 3, SEQ ID 4, SEQ ID 5 and SEQ ID 6,10 times of mother liquors of above-mentioned PCR damping fluid comprise 50 ~ 55mM sodium-chlor, the 10mMTris-HCl of pH8.3, 0.01 ~ 0.02% glycerine and 0.01 ~ 0.02% ammonium sulfate, 25 ~ 30mM MgCl
2,
(3), after the pcr amplification of step (2) terminates, carry out agarose gel electrophoresis and deliver order-checking.
2. a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method as claimed in claim 1, it is characterized in that: described step (1) is: collect Aeromonas hydrophila viable bacteria body 0.5 ~ 0.6g, resuspended with 5 ~ 6mL deionized water, multigelation smudge cells, collect supernatant liquor after the centrifugal 10 ~ 15min of 10000 ~ 12000rpm, obtain template.
3. a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method as claimed in claim 1, is characterized in that: the TetG-R that the TetC-R that the TetC-F that the TetA-R that the TetA-F that the dNTPs that the reaction system of described pcr amplification comprises ultrapure water, PCR damping fluid, final concentration are 0.3mmol/L, final concentration are 0.5mmol/L, final concentration are 0.5mmol/L, final concentration are 0.3mmol/L, final concentration are 0.3mmol/L, final concentration are the TetG-F of 0.4mmol/L, final concentration is 0.4mmol/L and final concentration are the Taq DNA polymerase of 0.01U/uL.
4. a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method as claimed in claim 1, it is characterized in that: 10 times of mother liquors of described PCR damping fluid comprise 50mM sodium-chlor, the 10mMTris-HCl of pH8.3,0.01% glycerine and 0.01% ammonium sulfate, 25mM MgCl
2.
5. a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method as described in claim arbitrary in Claims 1-4, it is characterized in that: the reaction conditions of described pcr amplification is as follows: 94 ~ 95 DEG C of denaturation 10 ~ 12min, then following circulation is entered: 94 ~ 95 DEG C of sex change 45 ~ 50s, 54 ~ 55 DEG C of annealing 1 ~ 1.5min, 72 ~ 74 DEG C of extension 45 ~ 50s, totally 30 ~ 35 circulations, 7 ~ 8min is extended in 72 DEG C, 2 ~ 4 DEG C of preservations after loop ends.
6. a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method as claimed in claim 5, it is characterized in that: the reaction conditions of described pcr amplification is as follows: 94 DEG C of denaturation 10min, then following circulation is entered: 94 DEG C of sex change 45s, 54 DEG C of annealing 1min, 72 DEG C of extension 45s, totally 30 circulations, 7min is extended in 72 DEG C, 4 DEG C of preservations after loop ends.
7. a kind of Aeromonas hydrophila tetracycline medication drug resistant gene detection method as claimed in claim 1, is characterized in that: the deposition condition of described step (3) is: 2% sepharose, 80V voltage, time 40min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944669A (en) * | 2006-10-20 | 2007-04-11 | 四川大学 | Multiple PCR detecting technology for drug resistant gene on tetracyline medicines of zoogenous bacteria |
| CN102876772A (en) * | 2012-06-20 | 2013-01-16 | 山东省海洋水产研究所 | Method for detecting tetracycline resistance gene in seawater |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944669A (en) * | 2006-10-20 | 2007-04-11 | 四川大学 | Multiple PCR detecting technology for drug resistant gene on tetracyline medicines of zoogenous bacteria |
| CN102876772A (en) * | 2012-06-20 | 2013-01-16 | 山东省海洋水产研究所 | Method for detecting tetracycline resistance gene in seawater |
Non-Patent Citations (3)
| Title |
|---|
| L.-K. NG ET AL.: "Multiplex PCR for the detection of tetracycline resistant genes", 《MOLECULAR AND CELLULAR PROBES》 * |
| 党宏月等: "海水养殖环境细菌耐药性的危害", 《海洋科学集刊》 * |
| 李国烈等: "水产动物源细菌耐药性与预防控制", 《渔业信息与战略》 * |
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