CN102605091A - EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment - Google Patents
EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in farm environment Download PDFInfo
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Abstract
The invention discloses an EMA-PCR (Ethidium Monoazide-Polymerase Chain Reaction) detection method of viable pathogen in a farm environment. The EMA-PCR detection method mainly comprises the following steps of: (1) pre-processing a sample to obtain bacterial liquid; (2) adding 50-100 micrograms of azided ethidium bromide in 1 ml of the bacterial liquid, standing the bacterial liquid for 5-10 min in a dark condition, and carrying out illumination treatment for 1-3 min by using a 650 W halogen light source; (3) extracting a template DNA (Deoxyribonucleic Acid) from the treated bacterial liquid; and (4) carrying out PCR detection by using the template DNA. The EMA-PCR detection method provided by the invention has the advantages of effectively removing interference of dead cell DNA, greatly improving the detection accuracy, rapidly and effectively detecting the normal viable pathogen in the sample, obtaining more accurate detection result and the like.
Description
Technical field
The present invention relates to the detection method of pathogenic bacteria viable bacteria in a kind of plant environment, what be specifically related to is the EMA-PCR detection method of viable bacteria.
Background technology
Along with Developing of Animal Industry, China cultures structure and cultures to modern times breed, breeding scale transformation from tradition just gradually, also is the important symbol of modernized livestock industry.But breeding scale also exists a lot of drawbacks, and wherein, scale raising used indiscriminate in breeding process to microbiotic, causes antibiotic medicine residual in a large number for a long time in animal body, is having a strong impact on the quality of animal products.Pathogenic bacteria is not accomplished monitoring at any time, thereby cause a large amount of antibiotic abuses or excessively use, cause chemical sproof generation on the one hand, make medicine residual in a large number in animal body on the one hand, influence the livestock product quality, thereby the infringement people's is healthy.So extremely important to monitoring of plant environment pathogenic bacteria, on the one hand for reducing the pollution of pathogenic micro-organism to environment, on the one hand can rational use of drug in therapeutic process.
The detection method of plant environment pathogenic bacterium mainly adopts technology such as increasing bacterium, selectivity cultivation, Physiology and biochemistry evaluation, conventional P CR equimolecular biological method and serological reaction at present.Round pcr has been widely used with its high specificity, highly sensitive and accurate advantage such as quick in the detection of plant pathogenic bacteria; But conventional P CR technology can't be distinguished the dead bacterium and the bacterium that lives in the sample, and in viable bacteria DNA and dead bacterium DNA, all can amplify goal gene.Though dead bacterium is being safe from harm property, its DNA long-term existence is detected too, too high on the one hand estimation the quantity that exists of viable bacteria, do not having to make detected result false positive occur under the situation of viable bacteria on the one hand, thereby disturbing the verity that detects.
Summary of the invention
The present invention provides the EMA-PCR detection method of pathogenic bacteria viable bacteria in a kind of plant's environment that can detect pathogenic bacteria fast and effectively.
To achieve these goals, technical scheme of the present invention is following:
The EMA-PCR detection method of pathogenic bacteria viable bacteria in plant's environment mainly is made up of following steps:
(1) sample is through obtaining bacterium liquid after the pre-treatment;
(2) in the bacterium liquid of 1ml, add the nitrine ethidium bromide of 50～100ug, at dark condition held 5～10min, with 650W halogen light source photo-irradiation treatment 1～3min;
(3) extract template DNA the bacterium liquid after handling;
(4) carrying out PCR with template DNA detects.
Further, the pre-treatment step of said bacterium liquid is:
(a1) get 1.5g weight in wet base faecal samples;
(a2) add the aseptic PBS damping fluid of 30ml, vortex oscillation suspends, and the centrifugal 4min of 500 * g gets supernatant;
(a3) the deposition repeating step (a2) of centrifugal back formation; With the supernatant of obtaining high speed centrifugation 5min under the condition of 9000 * g;
(a4) collecting precipitation, and use 30ml concentration to suspend as the PBS damping fluid of 0.1M, centrifugal under the condition of 9000 * g;
(a5) repeating step (a4) obtains thalline, and thalline is suspended in the TE damping fluid of 3ml, and vibration mixes and promptly makes bacterium liquid.
Further, the extraction step of said template DNA is: get the bacterium liquid after 1ml handles, centrifugal 5min under the speed of 12000rpm; Get deposition, should precipitate 100 ℃ of water-bath 5min with 100ul sterilization deionized water is resuspended; The centrifugal 5min of 10000rpm gets supernatant and promptly makes template DNA.
Temperature is too high when preventing illumination exerts an influence to sample, and said halogen light source is positioned on ice.
As the distance of optimum, the distance of halogen light source and bacterium liquid is 16cm～20cm in the said step (2).
As a kind of preferred, said pathogenic bacteria is enterotoxigenic E.Coli, EHEC, Salmonellas or streptococcus aureus.
The present invention compared with prior art has the following advantages and beneficial effect:
(1) in the testing process of the present invention through the nitrine ethidium bromide optionally with the mixt bacteria bacterium colony in dead cell DNA covalent attachment, the DNA that removes dead cell disturbs, and has improved the accuracy that detects greatly;
(2) result of the present invention shows, uses method of the present invention can detect the common pathogen viable bacteria in the sample fast and effectively;
(3) the present invention can effectively detect the pathogenic bacterium viable bacterias such as enterotoxigenic Escherichia coli, streptococcus aureus, Salmonellas and EHEC in common pathogen viable bacteria, the especially sample of handling in the sample of back;
(4) halogen light source of the present invention is positioned on ice, can prevent that the heat that light source sends from impacting the viable bacteria in the sample, makes detected result more accurate;
(5) distance of halogen light source of the present invention and bacterium liquid adopts 16cm～20cm, when maximization reduction thermal source is to the influence of bacterium liquid, guarantees nitrine ethidium bromide and dead cell DNA covalent attachment, and the DNA that removes dead cell disturbs;
(6) the used time weak point of the present invention's process simple to operate, whole is suitable for promoting.
Description of drawings
Fig. 1 is the gel electrophoresis figure of the EMA-PCR of enterotoxigenic E.Coli in the sample.
Fig. 2 is the different concns EMA-PCR gel electrophoresis figure of cytomixis bacteria suspension anyway.
Fig. 3 is the EMA-PCR gel electrophoresis figure of the mixed bacterium suspension of different viable cell ratios among the embodiment 1.
Fig. 4 is the interference experiment gel electrophoresis figure of EMA-PCR detection sensitivity.
Fig. 5 is the gel electrophoresis figure of the EMA-PCR of enterorrhagia Bacillus coil 0157 in the sample: H7.
Fig. 6 is the EMA-PCR gel electrophoresis figure of the mixed bacterium suspension of different viable cell ratios among the embodiment 2.
Fig. 7 is the gel electrophoresis figure of the EMA-PCR of streptococcus aureus in the sample.
Fig. 8 is the EMA-PCR gel electrophoresis figure of the mixed bacterium suspension of different viable cell ratios among the embodiment 3.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described further, but embodiment of the present invention is not limited to the following example.
Present embodiment mainly is made up of following steps:
(1) sample is through obtaining bacterium liquid after the pre-treatment;
(a1) get 1.5g weight in wet base faecal samples;
(a2) add the aseptic PBS damping fluid of 30ml, vortex oscillation suspends, and the centrifugal 4min of 500 * g gets supernatant;
(a3) the deposition repeating step (a2) of centrifugal back formation; With the supernatant of obtaining high speed centrifugation 5min under the condition of 9000 * g;
(a4) collecting precipitation, and use 30ml concentration to suspend centrifugal 5min under the condition of 9000 * g as the PBS damping fluid of 0.1M;
(a5) behind the repeating step (a4), obtain thalline, thalline is suspended in the TE damping fluid of 3ml, vibration mixes, and promptly makes bacterium liquid.
(2) the nitrine ethidium bromide of adding 100ug in the bacterium liquid of 1ml is at dark condition held 5min, with 650W halogen light source photo-irradiation treatment 1min.Halogen light source is positioned on ice in the present embodiment, and the distance of halogen light source and bacterium liquid is 20cm.
(3) extract template DNA the bacterium liquid after handling; Get the bacterium liquid after 1ml handles, centrifugal 5min gets deposition under the speed of 12000rpm, should precipitate with 100ul sterilization deionized water is resuspended, and 100 ℃ of water-bath 5min, the centrifugal 5min of 10000rpm gets supernatant, and this supernatant is template DNA.
(4) carrying out PCR with template DNA detects.The template DNA that adopts step (3) to make carries out PCR and detects, and present embodiment is the detection to enterotoxigenic E.Coli.With the LTa specificity amplification primer is example, and primer sequence is: F:5 '-GCGACAGATTATACCGTGC-3 '; R:5 '-GGTCTCTATATTCCCTGTT-3 ', amplification purpose stripe size is 438bp, detected result such as Fig. 1.
The detection of control strain is carried out PCR through above-mentioned steps again after deadly processing of bacterium liquid process with step (1) acquisition and is detected detected result such as Fig. 1.
The process of cause death handling is following: bacterium liquid is positioned under 72 ℃ of water bath condition, and using final concentration behind the 10min is that 70% Virahol is handled 20min and got final product.
Detected result: 1～3 is the detected result of enterotoxigenic E.Coli among Fig. 1, and 4～12 is the detected result of control strain.Can draw through Fig. 1, can effectively detect viable bacteria in the sample, avoid the interference of dead bacterium, improve the accuracy that detects through method of the present invention.
Control experiment:
1, the different concns EMA-PCR amplification of cytomixis bacteria suspension anyway: get enterotoxigenic E.Coli viable cell bacteria suspension and each 250ul of dead cell bacteria suspension of same concentrations, pass with the PBS damping fluid and doubly dilute 10
-1～10
-9Each weaker concn is got 50ul and is carried out coated plate cultivation back counting, and remaining part is carried out EMA-PCR with the method for embodiment 1 and detected.The result is through agarose gel electrophoresis, detected result such as Fig. 2.
Detected result: the gradient dilution liquid 10 of 1～10 respectively corresponding enterotoxigenic E.Coli life or death cytomixis bacteria suspension among Fig. 2
0～10
-9, detect its sensitivity and be respectively 19CFU/ml, 12 CFU/ml, 15 CFU/ml, 11 CFU/ml.
2, the EMA-PCR of the mixed bacterium suspension of different viable cell ratios amplification: in the mixing suspension of enterotoxigenic E.Coli viable cell bacteria suspension and dead cell bacteria suspension; The mixing suspension that contains 100%, 75%, 50%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0% viable cell bacteria suspension is respectively carried out EMA-PCR with the method for embodiment 1 to be detected; The result is through agarose gel electrophoresis, detected result such as Fig. 3.1～12 corresponding respectively mixing suspension that contains 100%, 75%, 50%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0% enterotoxigenic E.Coli viable cell bacteria suspension among Fig. 3.
3, the interference experiment of EMA-PCR detection sensitivity: get enterotoxigenic E.Coli viable cell bacteria suspension and each 250ul of dead cell bacteria suspension of same concentrations, pass with the PBS damping fluid and doubly dilute 10
-1～10
-9, get 50ul respectively and sneak in other assorted bacterium of 450ul, carry out EMA-PCR with the method for embodiment 1 and detect, the result is through agarose gel electrophoresis, detected result such as Fig. 4.
The result shows: the interference of assorted bacterium does not influence the sensitivity of its detection.
Present embodiment only is that with the difference of embodiment 1 bacterial classification that detects is different; The bacterial classification that present embodiment detects is enterorrhagia Bacillus coil 0157: H7; With the rfbE specificity amplification primer is example, and primer sequence is: F:5 '-AGATTGCGCTGAAGCCTTTG-3 '; R:5 '-ATTGGCATCGTGTGGACAG-3 ', amplification purpose stripe size is 495bp, detected result such as Fig. 5.
1～3 is the detected result of enterorrhagia Bacillus coil 0157: H7 among Fig. 5, and 4～12 is the detected result of control strain.
The control experiment of present embodiment is: the EMA-PCR amplification of the mixed bacterium suspension of different viable cell ratios, detected result such as Fig. 6.
Present embodiment only is that with the difference of embodiment 1 bacterial classification that detects is different, and the bacterial classification that present embodiment detects is a streptococcus aureus, is example with the nuc specificity amplification primer, and primer sequence is: F:5 '-TTAGCCAAGCCTTGACGAAC-3 '; R:5 '-AGGGCAATACGCAAAGAGGT-3 ', amplification purpose stripe size is 480bp, detected result such as Fig. 7.
1～4 is the detected result of streptococcus aureus among Fig. 7, and 5～12 is the detected result of control strain.
The control experiment of present embodiment is: the EMA-PCR amplification of the mixed bacterium suspension of different viable cell ratios, detected result such as Fig. 8.
According to the method described above, just can realize the present invention well.
Claims (6)
1. the EMA-PCR detection method of pathogenic bacteria viable bacteria in plant's environment is characterized in that, mainly is made up of following steps:
(1) sample is through obtaining bacterium liquid after the pre-treatment;
(2) in the bacterium liquid of 1ml, add the nitrine ethidium bromide of 50～100ug, at dark condition held 5～10min, with 650W halogen light source photo-irradiation treatment 1～3min;
(3) extract template DNA the bacterium liquid after handling;
(4) carrying out PCR with template DNA detects.
2. the EMA-PCR detection method of pathogenic bacteria viable bacteria is characterized in that in the plant according to claim 1 environment, and the pre-treatment step of said bacterium liquid is:
(a1) get 1.5g weight in wet base plant environmental sample;
(a2) add the aseptic PBS damping fluid of 30ml, vortex oscillation suspends, and the centrifugal 4min of 500 * g gets supernatant;
(a3) the deposition repeating step (a2) of centrifugal back formation; With the supernatant of obtaining high speed centrifugation 5min under the condition of 9000 * g;
(a4) collecting precipitation, and use 30ml concentration to suspend as the PBS damping fluid of 0.1M, centrifugal under the condition of 9000 * g;
(a5) repeating step (a4) obtains thalline, and thalline is suspended in the TE damping fluid of 3ml, and vibration mixes and promptly makes bacterium liquid.
3. the EMA-PCR detection method of pathogenic bacteria viable bacteria is characterized in that in the plant according to claim 2 environment, and the extraction step of said template DNA is: get the bacterium liquid after 1ml handles; Centrifugal 5min under the speed of 12000rpm; Get deposition, should precipitate 100 ℃ of water-bath 5min with 100ul sterilization deionized water is resuspended; The centrifugal 5min of 10000rpm gets supernatant and promptly makes template DNA.
4. according to the EMA-PCR detection method of pathogenic bacteria viable bacteria in each described plant environment of claim 1～3, it is characterized in that: said halogen light source is positioned on ice.
5. the EMA-PCR detection method of pathogenic bacteria viable bacteria in the plant according to claim 4 environment is characterized in that: the distance of halogen light source and bacterium liquid is 16cm～20cm in the said step (2).
6. the EMA-PCR detection method of pathogenic bacteria viable bacteria in the plant according to claim 5 environment is characterized in that: said pathogenic bacteria is enterotoxigenic E.Coli, EHEC, Salmonellas or streptococcus aureus.
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| CN107164229A (en) * | 2016-12-14 | 2017-09-15 | 四川省畜牧科学研究院 | A kind of method of the total bacterium of the extraction purification from environmental sample |
| CN108715889A (en) * | 2018-04-27 | 2018-10-30 | 曾小敏 | A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7 |
| CN109652509A (en) * | 2018-12-27 | 2019-04-19 | 新乡学院 | A kind of method of lactic acid bacterium number in detection fermented feed |
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Application publication date: 20120725 |