CN106755332A - A kind of Shigella detection method of content - Google Patents
A kind of Shigella detection method of content Download PDFInfo
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- CN106755332A CN106755332A CN201611069304.9A CN201611069304A CN106755332A CN 106755332 A CN106755332 A CN 106755332A CN 201611069304 A CN201611069304 A CN 201611069304A CN 106755332 A CN106755332 A CN 106755332A
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- 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
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a kind of Shigella detection method of content, comprise the following steps：（1）The measuring samples containing Shigella are taken, genomic DNA is extracted：3g measuring samples to 5 10min of concussion that in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, are vortexed are weighed, is well mixed to obtain mixture；Take 0.5mLGN enrichment liquids dilution mix mixture be added to through sterilizing 4.5mLGN enrichment liquids in obtain each dilution gradient, if 57 dilution factors；Using three pipes or five pipe parallel methods, each dilution factor sets 35 repeating samples, and 18 24h are cultivated in 37 DEG C, until mixed liquor only stops culture, referred to as bacterium solution in slight haze in test tube.The shortcomings of the inventive method can overcome the detection cycle that traditional microculture detection method is present long, complex steps, waste time and energy, detection time is set to greatly shorten by 23 days, operating procedure and labour intensity also greatly reduce, and reach time saving and energy saving, rapid sensitive requirement.
Description
Technical field
The present invention relates to a kind of detection method, specifically a kind of Shigella detection method of content.
Background technology
Shigella（Shigella）Bacterium is generally called shigella dysenteriae, is triggering each opportunistic pathogen institute of China's infectious diarrhea disease
Ranked first in accounting example, be that a class has hyperinfection and the serious Gram-negative enteric pathogenic bacteria of harm.The annual whole world
Therefore bacterium infection is ill 1.6 hundred million people, there are about 1,100,000 people death, and the overwhelming majority is less than 5 years old children.Due in sanitary sewage
Containing many pathogenic microorganisms, such as bacterium from enteron aisle, virus, protozoan, parasite etc., these microorganisms portion big absolutely
Divide all will enter excess sludge with sewage disposal, during the Agricultural land use of sludge, by polluted surface water, underground
Water, form the number of ways such as aerosol and be diffused into environment.These are with sludge into the pathogenic microorganism in environment to human body
Health and Environmental security cause huge potential threat, therefore, the Shigella in sludge is quickly and accurately detected, to preventing
Shigella Spreading and diffusion tool is of great significance.The detection of Shigella generally uses microbiological test of food hygiene
National standard（GB4789.5–2003）, i.e., using the identification of differential medium combination Physiology and biochemistry.But this method is applied to sludge
It is another on the one hand because sludge composition is complicated, produce interference to the sensitivity for detecting containing a large amount of microorganisms during sample detection
There is the shortcomings of detection cycle is long, loss is high, program is complicated, required reagent is various in aspect.
The content of the invention
It is an object of the invention to provide a kind of Shigella detection method of content, to solve to be proposed in above-mentioned background technology
Problem.
To achieve the above object, the present invention provides following technical scheme：
A kind of Shigella detection method of content, comprises the following steps：（1）The measuring samples containing Shigella are taken, gene is extracted
Group DNA：3g measuring samples to the concussion 5-10min that in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, is vortexed are weighed, mixing is equal
It is even to obtain mixture；Take 0.5mLGN enrichment liquids dilution mix mixture be added to through sterilizing 4.5mLGN enrichment liquids in obtain
Each dilution gradient, if 5-7 dilution factor；Using three pipes or five pipe parallel methods, each dilution factor sets 3-5 repeating sample, in
37 DEG C of culture 18-24h, until mixed liquor only stops culture, referred to as bacterium solution in slight haze in test tube；（2）Selective enrichment
Culture：Removing step（1）Bacterium solution 0.5mL is added in Shigella increasing bacterial context soup in each pipe of gained, 37 DEG C of culture 18-24h；
（3）DNA is extracted：Removing step（2）Bacterium solution 2mL is added in 10mL centrifuge tubes in each pipe of gained, 10000g centrifugation 3min, is discarded
Supernatant；1mL sterile PBS buffers are added, uniform, 10000g centrifugation 3min, abandoning supernatant is gently blown and beaten；Add 10 μ L
Ultra-pure water, liquid nitrogen frozen and boiling water bath multigelation 3 times, then 10000g centrifugations 3min take supernatant, complete DNA and extract；Carry
4 DEG C of preservations in the DNA short time after taking, for a long time -20 DEG C of preservations；（4）Set up the real-time fluorescence list primer isothermal of Shigella
Expand 25 μ L reaction systems：RNA/DNA combination primers 5.6 μm of ol/L, Blocker0.36 μm of ol/L, 10 × BstBuffer2.5 μ
L、BstDNApolymerase20U、10×RNaseHBuffer2.5μL、RNaseH5U、dNTPs0.2mmol/L、
MgCl23.5mmol/L, RNaseInhibitor16U, the μ L of DNA profiling 1, the μ L of SYBERGreen II 0.3, remaining uses sterilizing DEPC
Water supplies system；The SYBERGreen II is 300 times of dilutions；By DNA profiling, combination primer, Blocker and reaction buffer
Mixed liquor through 99 DEG C, be cooled to 60 DEG C after 90s treatment, be rapidly added RNaseH and BstDNA polymerases, it is fixed in real-time fluorescence
58 DEG C of reaction 40min, real-time monitoring fluorescence signal in course of reaction in amount PCR instrument；（5）Detection and analysis：Analysis detecting data.
As further scheme of the invention：The primer selects GCUUAGUGA-TTTGATGGTGTC, 5 ' end 9nt synthesis
It is RNA sequence, 3 ' end 12nt synthesize DNA sequence dna；Blocker selects TTAGATAATGTGGTA, 3 ' end biotin modifications,
It is middle random plus five XNA modifications.
As further scheme of the invention：The IXNA includes the one kind in A, C, G, T, U or mC Liu Zhong yu bases.
As further scheme of the invention：Step（1）The extraction DNA methods use common pyrolysis method, albumen
Enzyme K methods, saturated phenol method or WATER-WASHING METHOD combination commercial kit method.
Compared with prior art, the beneficial effects of the invention are as follows：The inventive method can overcome traditional microculture to examine
Survey method exist detection cycle is long, complex steps, the shortcomings of waste time and energy, detection time is greatly shortened 2-3 days, operation
Step and labour intensity also greatly reduce, and reach time saving and energy saving, rapid sensitive requirement.
Specific embodiment
The technical scheme in the embodiment of the present invention is clearly and completely described below.
In the embodiment of the present invention, a kind of Shigella detection method of content comprises the following steps：（1）Take containing Shigella
Measuring samples, extract genomic DNA：3g measuring samples are weighed in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, whirlpool
Rotation concussion 5-10min, is well mixed to obtain mixture；The mixture for taking the dilution mixing of 0.5mLGN enrichment liquids is added to by sterilizing
4.5mLGN enrichment liquids in obtain each dilution gradient, if 5-7 dilution factor；Using three pipes or five pipe parallel methods, each is dilute
Degree of releasing sets 3-5 repeating sample, and 18-24h is cultivated in 37 DEG C, until mixed liquor only stops culture in slight haze in test tube, referred to as
It is bacterium solution；（2）Selective enrichment culture：Removing step（1）Bacterium solution 0.5mL is added to Shigella increasing bacterial context soup in each pipe of gained
In, 37 DEG C of culture 18-24h；（3）DNA is extracted：Removing step（2）Bacterium solution 2mL is added in 10mL centrifuge tubes in each pipe of gained,
10000g is centrifuged 3min, abandoning supernatant；1mL sterile PBS buffers are added, uniform, 10000g centrifugation 3min are gently blown and beaten,
Abandoning supernatant；Add 10 μ L ultra-pure waters, liquid nitrogen frozen and boiling water bath multigelation 3 times, then 10000g centrifugations 3min take
Clear liquid, completes DNA and extracts；4 DEG C of preservations in the DNA short time after extraction, for a long time -20 DEG C of preservations；（4）Set up Shigella
The μ L reaction systems of real-time fluorescence list primer isothermal duplication 25：5.6 μm of ol/L, Blocker0.36 μm of ol/ of RNA/DNA combination primers
L、10×BstBuffer2.5μL、BstDNApolymerase20U、10×RNaseHBuffer2.5μL、RNaseH5U、
DNTPs0.2mmol/L, MgCl23.5mmol/L, RNaseInhibitor16U, the μ L of DNA profiling 1, the μ L of SYBERGreen II 0.3,
Remaining supplies system with sterilizing DEPC water；The SYBERGreen II is 300 times of dilutions；By DNA profiling, combination primer,
The mixed liquor of Blocker and reaction buffer is cooled to 60 DEG C through 99 DEG C after 90s treatment, is rapidly added RNaseH and BstDNA
Polymerase, 58 DEG C of reaction 40min, real-time monitoring fluorescence signal in course of reaction on real-time fluorescence quantitative PCR instrument；（5）Detection
Analysis：Analysis detecting data.The primer selects GCUUAGUGA-TTTGATGGTGTC, 5 ' end 9nt to synthesize RNA sequence, 3 '
End 12nt synthesizes DNA sequence dna；Blocker selects TTAGATAATGTGGTA, and 3 ' end biotin modifications, centre adds at random
Five XNA modifications.The IXNA includes the one kind in A, C, G, T, U or mC Liu Zhong yu bases.Step（1）The extraction DNA methods
Using common pyrolysis method, proteinase-K pathway, saturated phenol method or WATER-WASHING METHOD combination commercial kit method.
Embodiment 1：
Shigella detection method of content of the present invention, comprises the following steps：（1）The measuring samples containing Shigella are taken, base is extracted
Because of a group DNA：3g measuring samples to the concussion 5min that in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, is vortexed are weighed, mixing is equal
It is even to obtain mixture；Take 0.5mLGN enrichment liquids dilution mix mixture be added to through sterilizing 4.5mLGN enrichment liquids in obtain
Each dilution gradient, if 5-7 dilution factor；Using three pipes or five pipe parallel methods, each dilution factor sets 3 repeating samples, in 37
DEG C culture 18-24h, until mixed liquor only stops culture, referred to as bacterium solution in slight haze in test tube；（2）Selective enrichment is trained
Support：Removing step（1）Bacterium solution 0.5mL is added in Shigella increasing bacterial context soup in each pipe of gained, 37 DEG C of culture 18h；（3）DNA
Extract：Removing step（2）Bacterium solution 2mL is added in 10mL centrifuge tubes in each pipe of gained, 10000g centrifugation 3min, supernatant discarded
Liquid；1mL sterile PBS buffers are added, uniform, 10000g centrifugation 3min, abandoning supernatant is gently blown and beaten；Add 10 μ L ultrapure
Water, liquid nitrogen frozen and boiling water bath multigelation 3 times, then 10000g centrifugations 3min take supernatant, complete DNA and extract；After extraction
The DNA short time in 4 DEG C preservation, for a long time -20 DEG C preservation；（4）Set up the real-time fluorescence list primer isothermal duplication of Shigella
25 μ L reaction systems：RNA/DNA combinations primer 5.6 μm of ol/L, Blocker0.36 μm of ol/L, 10 × BstBuffer2.5 μ L,
BstDNApolymerase20U、10×RNaseHBuffer2.5μL、RNaseH5U、dNTPs0.2mmol/L、
MgCl23.5mmol/L, RNaseInhibitor16U, the μ L of DNA profiling 1, the μ L of SYBERGreen II 0.3, remaining uses sterilizing DEPC
Water supplies system；The SYBERGreen II is 300 times of dilutions；By DNA profiling, combination primer, Blocker and reaction buffer
Mixed liquor through 99 DEG C, be cooled to 60 DEG C after 90s treatment, be rapidly added RNaseH and BstDNA polymerases, it is fixed in real-time fluorescence
58 DEG C of reaction 40min, real-time monitoring fluorescence signal in course of reaction in amount PCR instrument；（5）Detection and analysis：Analysis detecting data.Institute
Stating primer selects GCUUAGUGA-TTTGATGGTGTC, 5 ' end 9nt to synthesize RNA sequence, and 3 ' end 12nt synthesize DNA sequence dna；
Blocker selects TTAGATAATGTGGTA, and 3 ' end biotin modifications are middle random plus five XNA modifications.The IXNA
Including the one kind in A, C, G, T, U or mC Liu Zhong yu bases.Step（1）The extraction DNA methods use common pyrolysis method, albumen
Enzyme K methods, saturated phenol method or WATER-WASHING METHOD combination commercial kit method.
Embodiment 2：
Shigella detection method of content of the present invention, comprises the following steps：（1）The measuring samples containing Shigella are taken, base is extracted
Because of a group DNA：3g measuring samples to the concussion 10min that in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, is vortexed are weighed, mixing is equal
It is even to obtain mixture；Take 0.5mLGN enrichment liquids dilution mix mixture be added to through sterilizing 4.5mLGN enrichment liquids in obtain
Each dilution gradient, if 7 dilution factors；Using three pipes or five pipe parallel methods, each dilution factor sets 5 repeating samples, in 37 DEG C
Culture 24h, until mixed liquor only stops culture, referred to as bacterium solution in slight haze in test tube；（2）Selective enrichment culture：Move
Take step（1）Bacterium solution 0.5mL is added in Shigella increasing bacterial context soup in each pipe of gained, 37 DEG C of culture 24h；（3）DNA is extracted：
Removing step（2）Bacterium solution 2mL is added in 10mL centrifuge tubes in each pipe of gained, 10000g centrifugation 3min, abandoning supernatant；Again plus
Enter 1mL sterile PBS buffers, gently blow and beat uniform, 10000g centrifugation 3min, abandoning supernatant；Add 10 μ L ultra-pure waters, liquid nitrogen
Freezing and boiling water bath multigelation 3 times, then 10000g centrifugations 3min take supernatant, complete DNA and extract；DNA after extraction is short
4 DEG C of preservations in time, for a long time -20 DEG C of preservations；（4）Set up the μ L of real-time fluorescence list primer isothermal duplication 25 reactions of Shigella
System：RNA/DNA combinations primer 5.6 μm of ol/L, Blocker0.36 μm of ol/L, 10 × BstBuffer2.5 μ L,
BstDNApolymerase20U、10×RNaseHBuffer2.5μL、RNaseH5U、dNTPs0.2mmol/L、
MgCl23.5mmol/L, RNaseInhibitor16U, the μ L of DNA profiling 1, the μ L of SYBERGreen II 0.3, remaining uses sterilizing DEPC
Water supplies system；The SYBERGreen II is 300 times of dilutions；By DNA profiling, combination primer, Blocker and reaction buffer
Mixed liquor through 99 DEG C, be cooled to 60 DEG C after 90s treatment, be rapidly added RNaseH and BstDNA polymerases, it is fixed in real-time fluorescence
58 DEG C of reaction 40min, real-time monitoring fluorescence signal in course of reaction in amount PCR instrument；（5）Detection and analysis：Analysis detecting data.Institute
Stating primer selects GCUUAGUGA-TTTGATGGTGTC, 5 ' end 9nt to synthesize RNA sequence, and 3 ' end 12nt synthesize DNA sequence dna；
Blocker selects TTAGATAATGTGGTA, and 3 ' end biotin modifications are middle random plus five XNA modifications.The IXNA
Including the one kind in A, C, G, T, U or mC Liu Zhong yu bases.Step（1）The extraction DNA methods use common pyrolysis method, albumen
Enzyme K methods, saturated phenol method or WATER-WASHING METHOD combination commercial kit method.
Embodiment 3：
Shigella detection method of content of the present invention, comprises the following steps：（1）The measuring samples containing Shigella are taken, base is extracted
Because of a group DNA：3g measuring samples to the concussion 8min that in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, is vortexed are weighed, mixing is equal
It is even to obtain mixture；Take 0.5mLGN enrichment liquids dilution mix mixture be added to through sterilizing 4.5mLGN enrichment liquids in obtain
Each dilution gradient, if 6 dilution factors；Using three pipes or five pipe parallel methods, each dilution factor sets 3-5 repeating sample, in 37
DEG C culture 20h, until mixed liquor only stops culture, referred to as bacterium solution in slight haze in test tube；（2）Selective enrichment culture：
Removing step（1）Bacterium solution 0.5mL is added in Shigella increasing bacterial context soup in each pipe of gained, 37 DEG C of culture 20h；（3）DNA is carried
Take：Removing step（2）Bacterium solution 2mL is added in 10mL centrifuge tubes in each pipe of gained, 10000g centrifugation 3min, abandoning supernatant；
1mL sterile PBS buffers are added, uniform, 10000g centrifugation 3min, abandoning supernatant is gently blown and beaten；10 μ L ultra-pure waters are added,
Liquid nitrogen frozen and boiling water bath multigelation 3 times, then 10000g centrifugations 3min take supernatant, complete DNA and extract；After extraction
4 DEG C of preservations in the DNA short time, for a long time -20 DEG C of preservations；（4）Set up the μ of real-time fluorescence list primer isothermal duplication 25 of Shigella
L reaction systems：RNA/DNA combinations primer 5.6 μm of ol/L, Blocker0.36 μm of ol/L, 10 × BstBuffer2.5 μ L,
BstDNApolymerase20U、10×RNaseHBuffer2.5μL、RNaseH5U、dNTPs0.2mmol/L、
MgCl23.5mmol/L, RNaseInhibitor16U, the μ L of DNA profiling 1, the μ L of SYBERGreen II 0.3, remaining uses sterilizing DEPC
Water supplies system；The SYBERGreen II is 300 times of dilutions；By DNA profiling, combination primer, Blocker and reaction buffer
Mixed liquor through 99 DEG C, be cooled to 60 DEG C after 90s treatment, be rapidly added RNaseH and BstDNA polymerases, it is fixed in real-time fluorescence
58 DEG C of reaction 40min, real-time monitoring fluorescence signal in course of reaction in amount PCR instrument；（5）Detection and analysis：Analysis detecting data.Institute
Stating primer selects GCUUAGUGA-TTTGATGGTGTC, 5 ' end 9nt to synthesize RNA sequence, and 3 ' end 12nt synthesize DNA sequence dna；
Blocker selects TTAGATAATGTGGTA, and 3 ' end biotin modifications are middle random plus five XNA modifications.The IXNA
Including the one kind in A, C, G, T, U or mC Liu Zhong yu bases.Step（1）The extraction DNA methods use common pyrolysis method, albumen
Enzyme K methods, saturated phenol method or WATER-WASHING METHOD combination commercial kit method.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be in other specific forms realized.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires to be limited rather than described above, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each
Implementation method only includes an independent technical scheme, and this narrating mode of specification is only this area for clarity
Specification an as entirety, the technical scheme in each embodiment should can also be formed this by technical staff through appropriately combined
Art personnel may be appreciated other embodiment.
Claims (4)
1. a kind of Shigella detection method of content, it is characterised in that comprise the following steps：（1）Take containing the to be checked of Shigella
Sample, extracts genomic DNA：Weigh 3g measuring samples to the concussion that in the centrifuge tube of the 50mL for filling 27mLGN enrichment liquids, is vortexed
5-10min, is well mixed to obtain mixture；The mixture for taking the dilution mixing of 0.5mLGN enrichment liquids is added to by sterilizing
Each dilution gradient is obtained in 4.5mLGN enrichment liquids, if 5-7 dilution factor；Using three pipes or five pipe parallel methods, each dilution
Degree sets 3-5 repeating sample, and 18-24h is cultivated in 37 DEG C, until mixed liquor only stops culture in slight haze in test tube, referred to as
Bacterium solution；（2）Selective enrichment culture：Removing step（1）Bacterium solution 0.5mL is added to Shigella increasing bacterial context soup in each pipe of gained
In, 37 DEG C of culture 18-24h；（3）DNA is extracted：Removing step（2）Bacterium solution 2mL is added in 10mL centrifuge tubes in each pipe of gained,
10000g is centrifuged 3min, abandoning supernatant；1mL sterile PBS buffers are added, uniform, 10000g centrifugation 3min are gently blown and beaten,
Abandoning supernatant；Add 10 μ L ultra-pure waters, liquid nitrogen frozen and boiling water bath multigelation 3 times, then 10000g centrifugations 3min take
Clear liquid, completes DNA and extracts；4 DEG C of preservations in the DNA short time after extraction, for a long time -20 DEG C of preservations；（4）Set up Shigella
The μ L reaction systems of real-time fluorescence list primer isothermal duplication 25：5.6 μm of ol/L, Blocker0.36 μm of ol/ of RNA/DNA combination primers
L、10×BstBuffer2.5μL、BstDNApolymerase20U、10×RNaseHBuffer2.5μL、RNaseH5U、
DNTPs0.2mmol/L, MgCl23.5mmol/L, RNaseInhibitor16U, the μ L of DNA profiling 1, the μ L of SYBERGreen II 0.3,
Remaining supplies system with sterilizing DEPC water；The SYBERGreen II is 300 times of dilutions；By DNA profiling, combination primer,
The mixed liquor of Blocker and reaction buffer is cooled to 60 DEG C through 99 DEG C after 90s treatment, is rapidly added RNaseH and BstDNA
Polymerase, 58 DEG C of reaction 40min, real-time monitoring fluorescence signal in course of reaction on real-time fluorescence quantitative PCR instrument；（5）Detection
Analysis：Analysis detecting data.
2. Shigella detection method of content according to claim 1, it is characterised in that the primer is selected
GCUUAGUGA-TTTGATGGTGTC, 5 ' end 9nt synthesize RNA sequence, and 3 ' end 12nt synthesize DNA sequence dna；Blocker is selected
TTAGATAATGTGGTA, 3 ' end biotin modifications are middle random plus five XNA modifications.
3. Shigella detection method of content according to claim 2, it is characterised in that the IXNA include A, C, G, T,
One kind in U or mC Liu Zhong yu bases.
4. Shigella detection method of content according to claim 1, it is characterised in that step（1）The extraction DNA side
Method uses common pyrolysis method, proteinase-K pathway, saturated phenol method or WATER-WASHING METHOD combination commercial kit method.
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| CN101153326A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting shigella |
| CN101993954A (en) * | 2010-12-01 | 2011-03-30 | 江南大学 | Fast quantitative detection method of shigella contained in sludge |
| EP2418286A1 (en) * | 2010-08-10 | 2012-02-15 | QIAGEN GmbH | Improved method for isothermal amplification of nucleic acids |
| CN104531860A (en) * | 2014-12-23 | 2015-04-22 | 河北出入境检验检疫局检验检疫技术中心 | Molecular detection method for shigella and application thereof |
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|---|---|---|---|---|
| CN101153326A (en) * | 2007-09-21 | 2008-04-02 | 珠海市疾病预防控制中心 | Primer, detection method and detection reagent kit for detecting shigella |
| EP2418286A1 (en) * | 2010-08-10 | 2012-02-15 | QIAGEN GmbH | Improved method for isothermal amplification of nucleic acids |
| CN101993954A (en) * | 2010-12-01 | 2011-03-30 | 江南大学 | Fast quantitative detection method of shigella contained in sludge |
| CN104531860A (en) * | 2014-12-23 | 2015-04-22 | 河北出入境检验检疫局检验检疫技术中心 | Molecular detection method for shigella and application thereof |
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Application publication date: 20170531 |
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