CN108715889A - A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7 - Google Patents
A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7 Download PDFInfo
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Abstract
The invention discloses a kind of state enterorrhagia Bacillus coil 0157s living:The step of rapid detection method of H7, the detection method is：By Escherichia coli O 157:H7 cultivates to obtain bacteria suspension；Nitrine ethidium bromide solution is added to bacteria suspension under the conditions of being protected from light, is stood in dark, then uncaps and is placed on ice, expose, centrifugation is abandoned supernatant, is resuspended with ultra-pure water, metal bath, centrifugation, and supernatant is the strain gene group DNA extracted；The design and synthesis of primer and probe；The sterile ultra-pure waters of DNA are diluted, double fluorescent PCR amplification is carried out；" maximum second derivative method " is selected to be analyzed, with CP value judging results.It has the beneficial effect that：Detection method is specific, reproducible, and sensitivity, accuracy rate are high, can quickly differentiate enterohemorrhagic escherichia coli 0157:The dead states of H7 and state bacterium living, testing cost are low.
Description
Technical field
The present invention relates to technical field of microbial detection more particularly to a kind of state enterorrhagia Bacillus coil 0157s living:H7
Rapid detection method.
Background technology
In recent years, with the improvement of people's living standards, China's food hygiene situation is greatly improved, but food source
Property pathogenic microorganisms prevalence break out accident and still appear in the newspapers repeatly, in food pathogenic bacteria pollution be food security it is most important endanger because
Element, and food poisoning main reason.In recent years, salmonella, listeria spp, Escherichia coli O 157, campylobacter
Etc. food pathogenics have become the number one killer for endangering food security.Wherein, Escherichia coli O 157:H7 is enterohemorrhagic large intestine bar
Bacterium（EHEC）A most important serotype, as a kind of pathogenic entero becteria of infecting both domestic animals and human, Escherichia coli O 157:H7 mainly leads to
The animal derived food infection mankind for crossing pollution, can cause diarrhea, hemorrhagic enteritis or even hemolytic uremic syndrome etc. a variety of
Disease, the bacterium pathogenicity is strong, and infective dose is extremely low, and less than 10 bacteriums can cause disease, harm extremely serious.
Currently, enterorrhagia Bacillus coil 0157:The detection of H7 mainly has bacterium isolated culture and PCR methods, Qian Zhezuo
For the goldstandard in laboratory, need to increase repeatedly bacterium, bacterium colony separation and a variety of biochemistry, serology identification experiment etc., it is complicated for operation, consume
Duration, the DNA nucleic acid that the latter is based primarily upon microorganism carries out augmentation detection, and bacterium is either under viable bacteria or dead bacterium state
All there is DNA nucleic acid, so as to cause pathogenic bacteria living are actually had no in the positive sample of some PCR detections, this and existing biography
Uniting, pathogenic bacteria examination criteria is inconsistent, i.e., existing PCR detection method and standard based on DNA cannot be distinguished present in sample
State living and dead state microorganism become the bottleneck that pathogenic bacteria are detected using round pcr.It is based on this reason, has been issued at present
In the relevant criterion of cloth, round pcr is not used as routine diagnostic method, and is used only to the pathogenic bacteria to being separately cultured
Rapid identification is carried out, the pathogenic bacteria in sample cannot directly be detected, this is all there's no one who doesn't or isn't such in developed countries such as the U.S..Cause
This, speeds passage through customs the needs tested and put to adapt to import and export cargo in inspection and quarantine, shortens detection time, increase and have molecular biosciences
The accuracy for learning detection method, avoids the unnecessary wasting of resources, effectively reduces workload, work out a kind of state enterohemorrhagic living
Escherichia coli O 157:The rapid detection method of H7 is imperative.
The prior art such as Authorization Notice No. is the Chinese invention patent of 105527428 B of CN, discloses a kind of quickly detection
Escherichia coli O 157：The detection method of H7.This method is by Fe3O4/Ru(bqy)3 2+Nanoparticle enrichment of bacterial, prepares test paper
Item, sample detection.It eliminates Escherichia coli O 157：The step of H7 is eluted from immunomagnetic beads improves capture rate；
It eliminates Ru (bqy)3 2+Nanoparticle is sprayed on the step on bonding pad, and immunological response is more uniform, makes a variation when quantitatively detecting
Coefficient is small；Reduce workload and living contaminants probability.Detection scheme sensitivity is very high, stability is fine.But this method
State living and dead state microorganism present in sample cannot be distinguished.
Invention content
The purpose of the present invention is to provide a species specificity, reproducible, sensitivity, accuracy rate are high, can quickly differentiate intestines
Enterohemorrhagic E.coli 0157:The dead states of H7 and state bacterium living, the low work state enterorrhagia Bacillus coil 0157 of testing cost:H7's
Rapid detection method.
The present invention is directed to the problem of being mentioned in background technology, and the technical solution taken is：
A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7, including Zengjing Granule, strain gene group DNA
Extraction, primer and probe design reacted with synthesis, PCR amplification, result judge, be as follows：
Zengjing Granule：By Escherichia coli O 157:H7 is inoculated into improvement E.C ovobiocins and increases in bacterial context soup m (EC) n, at 38-45 DEG C
Lower Zengjing Granule 15-20h is to get bacteria suspension, and spare, the step enriching effect is notable so that Escherichia coli O 157:H7 reaches pair
Growth period is counted, bacterial structure is stablized at this time, and metabolism, physiological property are than more consistent and good, and defence capability is most strong, nitrine ethidium bromide
It is maximum to act on difficulty；
The extraction of strain gene group DNA：Culture bacteria suspension 1ml is taken, the nitrine bromine of a concentration of 0.05mg/mL is added under the conditions of being protected from light
Second ingot solution（EMA）, so that EMA final concentrations is reached 2.0-4.0mg/L, slight oscillatory mixing stands 14-16min, then in dark
Bacterium solution is taken out, apart from fluorescent tube 14-16cm, uncaps and is placed on ice, persistently exposing 5-10min with 600-700W halogen tungsten lamps makes nitrine
Sample is taken out and is placed on rotating speed to centrifuge 2-5min in the centrifuge of 10000-15000r/min, abandons supernatant by ethidium bromide photodissociation,
It is resuspended 1 time, then 98-103 DEG C of metal bath 8-12min with ultra-pure water, then is placed in the centrifugation that rotating speed is 10000-15000r/min
2-5min is centrifuged in machine, supernatant is the strain gene group DNA extracted, and spare, EMA is as a kind of novel DNA in the step
Insert type fluorescent dye can be inserted into cell wall or the incomplete dead cell DNA of cell membrane, be exposed when with the visible light of high intensity
When photoactivation EMA, active nitrene intermediate is will produce, intermediate forms compound with the close covalent bonds of DNA and makes it
Continuation amplification ability is lost, the EMA nitrenes reactive intermediate to dissociate in system is combined to form azanol with the water in system, to make
EMA is consumed completely, does not interfere with follow-up DNA cloning, and the complete cell wall of living cells can prevent EMA from contaminating with cell membrane
The entrance of material effectively prevents the influence to viable bacteria DNA cloning due to the formation of azanol, so as to not have to viable bacteria and dead bacterium
It separates and directly detects viable bacteria, while the step does not expend reagent with metal bath method, it is easy to operate, direct, quick, it obtains
Amount of DNA is more, and purity also can reach test requirements document, while at a temperature of same purpose, metal bath compared to water-bath, inactivation of bacterial
Effect is more preferable；
The design and synthesis of primer and probe：It is comprehensive according to testing goal using primer-design software Primer Premier 5.0
Close many indexs such as PCR assay sensitivities, specificity, screen target gene and corresponding primer, then by the primer filtered out and
Probe carries out back inspection analysis in Genbank, while doing tetraploid rice, the primer for selecting homology low, and structure double PCR draws
Object, probe combinations rfbE genes or fliC genes, spare, the high sensitivity of rfbE genes or fliC genes, amplification efficiency are high, and two
The minimum detectability of gene reaches 116CFU/mL, and Escherichia coli O 157 is detected less than national standard:The rfbE single-genes of H7 are glimmering
Light PCR minimum detectability degree 291CFU/mL, meet the requirements；
PCR reaction amplifications：By the strain gene group DNA of extraction, 10 times of gradient dilutions are done with sterile ultra-pure water, therefrom choose 5
Gradient dilution DNA solution carries out double fluorescent PCR amplification as template, and double fluorescent PCR reacts 25 μ L of total volume, includes
Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of upstream and downstream primers, each 1 μ L of 5 μM of probes, ddH2O 7.5
μ L, 1 μ L, PCR response parameters of DNA profiling：37 DEG C of 5min, 94 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extension 45s
（Detect fluorescence signal）, 40 cycles；
As a result judge：According to LightCycler480 fluorescence quantitative PCR instrument operation manuals, " maximum second derivative method " is selected to carry out
Analysis, with CP values（Crossing point refer to cycle-index when amplification curve generates fluorescence jumping）Judging result, CP values≤
35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP values should suitably increase template quantity and reform experiment between 35-40, the detection
Method specificity, reproducible, high sensitivity, can quickly differentiate enterohemorrhagic escherichia coli 0157:The dead states of H7 and state bacterium living,
Testing result meets the existing conventional bacteria isolated culture examination criteria in China, before having higher application value and promoting
Scape.
Preferably, containing 0.012-0.016% deoxysodium cholate in the extraction step of strain gene group DNA in EMA solution
With ‰ carnitines of 0.022-0.025, in EMA solution the presence of deoxysodium cholate and carnitine can significantly change dead bacterium cell membrane table
Surface properties, the extent of damage for increasing dead bacterium membrane structure so that EMA can be quickly through the membrane structure of dead bacterium cell, in turn
With DNA tertiary structures covalent bond therein, the DNA of dead bacterium is made to lose continuation amplification ability, effectively eliminates part in result
Film cracks insufficient dead bacterium false positive phenomenon caused by result, final to improve state enterorrhagia Bacillus coil 0157 living:H7 is examined
The accuracy rate and rate of survey method, and the dosage of EMA can be reduced, and then reduce testing cost.
Compared with prior art, beneficial effects of the present invention are：
1）Detection method is specific, reproducible, and sensitivity, accuracy rate are high, can quickly differentiate enterohemorrhagic large intestine bar
Bacterium 0157:The dead states of H7 and state bacterium living, testing cost is low, and testing result meets the existing conventional bacteria isolated culture inspection in China
Mark is accurate, has higher application value and promotion prospect；
2）Detection method loses continuation amplification ability with EMA solution energy dead cells DNA, and can effectively prevent to viable bacteria
The influence of DNA cloning directly detects viable bacteria so as to not have to separate viable bacteria and dead bacterium；
3）Detection method effectively eliminates part film in result with EMA solution and cracks insufficient dead bacterium caused by result
False positive phenomenon, it is final to improve state enterorrhagia Bacillus coil 0157 living:The accuracy rate and rate of H7 detection methods, and can subtract
The dosage of few EMA, and then reduce testing cost；
4）The high sensitivity of detection method gene, amplification efficiency are high, and the minimum detectability of two genes reaches
116CFU/mL detects Escherichia coli O 157 less than national standard:The rfbE single-gene fluorescent PCR minimum detectability degree of H7
291CFU/mL meets the requirements.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7, including Zengjing Granule, strain gene group DNA
Extraction, primer and probe design reacted with synthesis, PCR amplification, result judge, be as follows：
1）Zengjing Granule：By Escherichia coli O 157:H7 is inoculated into improvement E.C ovobiocins and increases in bacterial context soup m (EC) n, at 38 DEG C
Lower Zengjing Granule 20h is to get bacteria suspension, and spare, the step enriching effect is notable so that Escherichia coli O 157:H7 reaches logarithm
Growth period, at this time bacterial structure stablize, metabolism, physiological property than more consistent and good, defence capability is most strong, nitrine ethidium bromide make
With difficulty maximum；
2）The extraction of strain gene group DNA：Culture bacteria suspension 1ml is taken, the nitrine of a concentration of 0.05mg/mL is added under the conditions of being protected from light
Ethidium bromide solution（EMA）, so that EMA final concentrations is reached 4.0mg/L, slight oscillatory mixing stands 14min, then by bacterium in dark
Liquid takes out, and apart from fluorescent tube 16cm, uncaps and is placed on ice, and persistently exposing 10min with 600W halogen tungsten lamps makes nitrine ethidium bromide photodissociation, will
Sample take out is placed on rotating speed be 10000r/min centrifuge in centrifuges 5min, abandon supernatant, with ultra-pure water be resuspended 1 time, then
98 DEG C of metal bath 12min, then be placed in the centrifuge that rotating speed is 10000r/min and centrifuge 5min, supernatant is the bacterial strain base extracted
Spare because of a group DNA, EMA can be inserted into cell wall or cell as a kind of novel DNA insert type fluorescent dyes in the step
In the incomplete dead cell DNA of film, when activating EMA with the visible light exposure of high intensity, it will produce among active nitrene
Body, intermediate form compound with the close covalent bonds of DNA and it are made to lose continuation amplification ability, the EMA nitrogen to dissociate in system
Guest's reactive intermediate is combined to form azanol with the water in system, to make EMA be consumed completely, is not interfered with follow-up DNA and is expanded
Increase, and the complete cell wall of living cells can prevent the entrance of EMA dyestuffs from effectively being avoided due to the formation of azanol with cell membrane
Influence to viable bacteria DNA cloning directly detects viable bacteria so as to not have to separate viable bacteria and dead bacterium, while the step is golden
Belong to bath method and do not expend reagent, easy to operate, direct, quick, acquisition amount of DNA is more, and purity also can reach test requirements document, simultaneously
At a temperature of same purpose, for metal bath compared to water-bath, the effect of inactivation of bacterial is more preferable；
3）The design and synthesis of primer and probe：Using primer-design software Primer Premier 5.0, according to testing goal,
The many indexs such as comprehensive PCR assay sensitivities, specificity, screen target gene and corresponding primer, then the primer that will be filtered out
Inspection analysis is carried out back in Genbank with probe, while doing tetraploid rice, the primer for selecting homology low builds double PCR
Primer, probe combinations fliC genes, spare, the high sensitivity of rfbE genes or fliC genes, amplification efficiency are high, and two genes are most
Low detection limits reach 116CFU/mL, and Escherichia coli O 157 is detected less than national standard:The rfbE single-genes fluorescent PCR of H7 is most
Low detection limits degree 291CFU/mL, meets the requirements；
4）PCR reaction amplifications：By the strain gene group DNA of extraction, 10 times of gradient dilutions are done with sterile ultra-pure water, therefrom choose 5
A gradient dilution DNA solution carries out double fluorescent PCR amplification as template, and double fluorescent PCR reacts 25 μ L of total volume, includes
Premix Ex Taq (Probe qPCR) 12.5 μ L, each 0.5 μ L of 10 μM of upstream and downstream primers, each 1 μ L of 5 μM of probes, ddH2O 7.5
μ L, 1 μ L, PCR response parameters of DNA profiling：37 DEG C of 5min, 94 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extension 45s
（Detect fluorescence signal）, 40 cycles；
5）As a result judge：According to LightCycler480 fluorescence quantitative PCR instrument operation manuals, select " maximum second derivative method " into
Row analysis, with CP values（Crossing point refer to cycle-index when amplification curve generates fluorescence jumping）Judging result, CP values
≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP values should suitably increase template quantity and reform experiment between 35-40, the inspection
Survey method specificity, reproducible, high sensitivity, can quickly differentiate enterohemorrhagic escherichia coli 0157:The dead states of H7 and state living are thin
Bacterium, testing result meet the existing conventional bacteria isolated culture examination criteria in China, have higher application value and popularization
Foreground.
Contain 0.012% deoxysodium cholate and 0.025 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution
Alkali, in EMA solution the presence of deoxysodium cholate and carnitine can significantly change that dead bacterium cell membrane surface property, to increase dead bacterium thin
The extent of damage of after birth structure so that EMA can quickly through the membrane structure of dead bacterium cell, and then with DNA three-levels knot therein
Structure covalent bond, makes the DNA of dead bacterium lose continuation amplification ability, effectively eliminates part film in result and cracks insufficient dead bacterium pair
As a result false positive phenomenon caused by, it is final to improve state enterorrhagia Bacillus coil 0157 living:The accuracy rate and speed of H7 detection methods
Rate, and the dosage of EMA can be reduced, and then reduce testing cost.
Embodiment 2：
A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7, including Zengjing Granule, strain gene group DNA
Extraction, primer and probe design reacted with synthesis, PCR amplification, result judge, be as follows：
1）Zengjing Granule：By Escherichia coli O 157:H7 is inoculated into improvement E.C ovobiocins and increases in bacterial context soup m (EC) n, at 45 DEG C
Lower Zengjing Granule 15h is spare to get bacteria suspension；
2）The extraction of strain gene group DNA：Culture bacteria suspension 1ml is taken, the nitrine of a concentration of 0.05mg/mL is added under the conditions of being protected from light
Ethidium bromide solution（EMA）, so that EMA final concentrations is reached 2.0mg/L, slight oscillatory mixing stands 16min, then by bacterium in dark
Liquid takes out, and apart from fluorescent tube 14cm, uncaps and is placed on ice, and persistently exposing 5min with 700W halogen tungsten lamps makes nitrine ethidium bromide photodissociation, will
Sample take out is placed on rotating speed be 15000r/min centrifuge in centrifuges 2min, abandon supernatant, with ultra-pure water be resuspended 1 time, then
103 DEG C of metal bath 8min, then be placed in the centrifuge that rotating speed is 15000r/min and centrifuge 2min, supernatant is the bacterial strain base extracted
It is spare because of a group DNA；
3）The design and synthesis of primer and probe：Using primer-design software Primer Premier 5.0, according to testing goal,
The many indexs such as comprehensive PCR assay sensitivities, specificity, screen target gene and corresponding primer, then the primer that will be filtered out
Inspection analysis is carried out back in Genbank with probe, while doing tetraploid rice, the primer for selecting homology low builds double PCR
Primer, probe combinations fliC genes, it is spare；
4）PCR reaction amplifications：By the strain gene group DNA of extraction, 10 times of gradient dilutions are done with sterile ultra-pure water, are carried out dual glimmering
Light PCR amplification, double fluorescent PCR react 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, 10 μM
Each 0.5 μ L of upstream and downstream primer, each 1 μ L of 5 μM of probes, ddH27.5 μ L of O, 1 μ L, PCR response parameters of DNA profiling：37 DEG C of 5min, 94
DEG C pre-degeneration 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extend 45s（Detect fluorescence signal）, 40 cycles；
5）As a result judge：According to LightCycler480 fluorescence quantitative PCR instrument operation manuals, select " maximum second derivative method " into
Row analysis, with CP values（Crossing point refer to cycle-index when amplification curve generates fluorescence jumping）Judging result, CP values
≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP values should suitably increase template quantity and reform experiment between 35-40.
Contain 0.016% deoxysodium cholate and 0.022 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution
Alkali.
Embodiment 3：
A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7, including Zengjing Granule, strain gene group DNA
Extraction, primer and probe design reacted with synthesis, PCR amplification, result judge, be as follows：
1）Zengjing Granule：By Escherichia coli O 157:H7 is inoculated into improvement E.C ovobiocins and increases in bacterial context soup m (EC) n, at 41 DEG C
Lower Zengjing Granule 18h is spare to get bacteria suspension；
2）The extraction of strain gene group DNA：Culture bacteria suspension 1ml is taken, the nitrine of a concentration of 0.05mg/mL is added under the conditions of being protected from light
Ethidium bromide solution（EMA）, so that EMA final concentrations is reached 3.0mg/L, slight oscillatory mixing stands 15min, then by bacterium in dark
Liquid takes out, and apart from fluorescent tube 15cm, uncaps and is placed on ice, and persistently exposing 8min with 650W halogen tungsten lamps makes nitrine ethidium bromide photodissociation, will
Sample take out is placed on rotating speed be 12000r/min centrifuge in centrifuges 4min, abandon supernatant, with ultra-pure water be resuspended 1 time, then
100 DEG C of metal bath 10min, then be placed in the centrifuge that rotating speed is 12000r/min and centrifuge 4min, supernatant is the bacterial strain extracted
Genomic DNA, it is spare；
3）The design and synthesis of primer and probe：Using primer-design software Primer Premier 5.0, according to testing goal,
The many indexs such as comprehensive PCR assay sensitivities, specificity, screen target gene and corresponding primer, then the primer that will be filtered out
Inspection analysis is carried out back in Genbank with probe, while doing tetraploid rice, the primer for selecting homology low builds double PCR
Primer, probe combinations rfbE genes, it is spare；
4）PCR reaction amplifications：By the strain gene group DNA of extraction, 10 times of gradient dilutions are done with sterile ultra-pure water, are carried out dual glimmering
Light PCR amplification, double fluorescent PCR react 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, 10 μM
Each 0.5 μ L of upstream and downstream primer, each 1 μ L of 5 μM of probes, ddH27.5 μ L of O, 1 μ L, PCR response parameters of DNA profiling：37 DEG C of 5min, 94
DEG C pre-degeneration 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extend 45s（Detect fluorescence signal）, 40 cycles；
5）As a result judge：According to LightCycler480 fluorescence quantitative PCR instrument operation manuals, select " maximum second derivative method " into
Row analysis, with CP values（Crossing point refer to cycle-index when amplification curve generates fluorescence jumping）Judging result, CP values
≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP values should suitably increase template quantity and reform experiment between 35-40.
Contain 0.015% deoxysodium cholate and 0.024 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution
Alkali.
Embodiment 4：
A kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7, including Zengjing Granule, strain gene group DNA
Extraction, primer and probe design reacted with synthesis, PCR amplification, result judge, be as follows：
1）Zengjing Granule：By Escherichia coli O 157:H7 is inoculated into improvement E.C ovobiocins and increases in bacterial context soup m (EC) n, at 41 DEG C
Lower Zengjing Granule 18h is spare to get bacteria suspension；
2）The extraction of strain gene group DNA：Culture bacteria suspension 1ml is taken, the nitrine of a concentration of 0.05mg/mL is added under the conditions of being protected from light
Ethidium bromide solution（EMA）, so that EMA final concentrations is reached 3.0mg/L, slight oscillatory mixing stands 15min, then by bacterium in dark
Liquid takes out, and apart from fluorescent tube 15cm, uncaps and is placed on ice, and persistently exposing 8min with 650W halogen tungsten lamps makes nitrine ethidium bromide photodissociation, will
Sample take out is placed on rotating speed be 12000r/min centrifuge in centrifuges 4min, abandon supernatant, with ultra-pure water be resuspended 1 time, then
100 DEG C of metal bath 10min, then be placed in the centrifuge that rotating speed is 12000r/min and centrifuge 4min, supernatant is the bacterial strain extracted
Genomic DNA, it is spare；
3）The design and synthesis of primer and probe：Using primer-design software Primer Premier 5.0, according to testing goal,
The many indexs such as comprehensive PCR assay sensitivities, specificity, screen target gene and corresponding primer, then the primer that will be filtered out
Inspection analysis is carried out back in Genbank with probe, while doing tetraploid rice, the primer for selecting homology low builds double PCR
Primer, probe combinations rfbE genes, it is spare；
4）PCR reaction amplifications：By the strain gene group DNA of extraction, 10 times of gradient dilutions are done with sterile ultra-pure water, are carried out dual glimmering
Light PCR amplification, double fluorescent PCR react 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, 10 μM
Each 0.5 μ L of upstream and downstream primer, each 1 μ L of 5 μM of probes, ddH27.5 μ L of O, 1 μ L, PCR response parameters of DNA profiling：37 DEG C of 5min, 94
DEG C pre-degeneration 3min, 94 DEG C of denaturation 20s, 60 DEG C of annealing extend 45s（Detect fluorescence signal）, 40 cycles；
5）As a result judge：According to LightCycler480 fluorescence quantitative PCR instrument operation manuals, select " maximum second derivative method " into
Row analysis, with CP values（Crossing point refer to cycle-index when amplification curve generates fluorescence jumping）Judging result, CP values
≤ 35 are judged to the positive, and value >=40 CP are judged to feminine gender, and CP values should suitably increase template quantity and reform experiment between 35-40.
Contain 0.015% deoxysodium cholate and 0.024 ‰ meat in the extraction step of above-mentioned bacterial strains genomic DNA in EMA solution
Alkali, containing 2.36% dextrorotation carnitine in carnitine, the dextrorotation carnitine that above-mentioned carnitine contains special proportioning can accelerate the photodissociation of EMA
De- rate improves the dispersibility of reactive intermediate containing nitrene in water, and then fully activates and inactivated with water in conjunction with azanol is formed,
Make EMA thoroughly inactivate no longer to be combined with DNA molecular, avoids EMA to state enterorrhagia Bacillus coil 0157 living:The genome of H7
The influence of DNA extracted amounts ensures the extracted amount of DNA, so that state enterorrhagia Bacillus coil 0157 living:The DNA cloning of H7
It is smoothed out, the final sensitivity for improving detection method and accuracy rate.
Embodiment 5：
Viable bacteria and the detection of dead bacterium
Escherichia coli O 157 is prepared respectively:H7 viable bacterias bacteria suspension and dead bacterium bacteria suspension, test group use embodiment 3 and embodiment 4
Detection method be detected, for 1 detection method of control group without EMA processing, other steps and embodiment 3 are identical, control group 2 examine
Do not contain deoxysodium cholate and carnitine in the EMA of survey method, other steps and embodiment 3 are identical, and control group 3 uses the existing biography in China
The bacterium of system is separately cultured standard method SN/T 0973-2010《Enterohemorrhagic is big in inlet and outlet meat, meat products and other food
Enterobacteria O157:H7 detection methods》And inspection and quarantine professional standard SN/T 1870-2007《Pathogenic bacteria detection method in food
Real-time PCR methodology》It is detected.
The result shows that for O157:H7 viable bacterias, test group and control group testing result are consistent, and are that detection is positive；
For O157:There is opposite detection knot in the dead bacterium of H7, test group, 3 testing result of control group and SN/T 0973-2010 testing results
Fruit, and control group 1 and SN/T 0973-2010 testing results（The pathogenic bacteria are not detected）It is consistent.Illustrate the addition energy of EMA
Enough differentiate enterohemorrhagic escherichia coli 0157:The dead states of H7 and state bacterium living.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of state enterorrhagia Bacillus coil 0157 living:The rapid detection method of H7, including Zengjing Granule, strain gene group
Amplification is reacted in the extraction of DNA, the design of primer and probe with synthesis, PCR, result judges, it is characterised in that：The strain gene
Group DNA extraction step be：Bacteria suspension is taken, nitrine ethidium bromide solution is added under the conditions of being protected from light（EMA）, slight oscillatory mixing is black
It stands, then takes out bacterium solution in the dark, uncap and be placed on ice, persistently exposed with halogen tungsten lamp, centrifuge, abandon after sample is taken out
Clearly, it is resuspended, then metal bath, is centrifuged with ultra-pure water, supernatant is the strain gene group DNA extracted, spare, the EMA solution
In contain deoxysodium cholate and carnitine.
2. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, feature
It is：The bacteria suspension is 1ml, and EMA solution concentrations are 0.05mg/mL, the final concentration of 2.0-4.0mg/L of EMA in bacteria suspension.
3. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, feature
It is：Contain ‰ carnitine of 0.012-0.016% deoxysodium cholate and 0.022-0.025 in the EMA solution.
4. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, feature
It is：14-16min is stood in the dark, exposes 5-10min.
5. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, feature
It is：The metal bath temperature is 98-103 DEG C, time 8-12min.
6. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, feature
It is：The Zengjing Granule step is：By Escherichia coli O 157:H7 is inoculated into improvement E.C ovobiocins and increases bacterial context soup m (EC) n
In, Zengjing Granule 15-20h is spare to get bacteria suspension at 38-45 DEG C.
7. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, feature
It is：The PCR reacts amplification step：By the strain gene group DNA of extraction, 10 times of gradient dilutions are done with sterile ultra-pure water,
5 gradient dilution DNA solutions are therefrom chosen as template, carry out double fluorescent PCR amplification.
8. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 7:The rapid detection method of H7, feature
It is：The double fluorescent PCR reacts 25 μ L of total volume, include Premix Ex Taq (Probe qPCR) 12.5 μ L, 10 μM
Each 0.5 μ L of upstream and downstream primer, each 1 μ L of 5 μM of probes, ddH27.5 μ L of O, 1 μ L of DNA profiling.
9. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 7:The rapid detection method of H7, feature
It is：The PCR response parameters are：37 DEG C of 5min, 94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 20s, 60 DEG C of annealing extend 45s（Inspection
Survey fluorescence signal）, 40 cycles.
10. a kind of state enterorrhagia Bacillus coil 0157 living according to claim 1:The rapid detection method of H7, it is special
Sign is：The result judgment step is：According to LightCycler480 fluorescence quantitative PCR instrument operation manuals, " maximum two is selected
Order derivative method " is analyzed, with CP values（Crossing point refer to cycle-index when amplification curve generates fluorescence jumping）Sentence
Break as a result, value≤35 CP are judged to the positive, value >=40 CP are judged to feminine gender, and CP values should suitably increase template quantity weight between 35-40
It does experiment.
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| CN114058683A (en) * | 2021-11-17 | 2022-02-18 | 杭州优思达生物技术有限公司 | Method for judging nucleic acid amplification result |
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