CN106399562A - Detection method of Shigella in food - Google Patents
Detection method of Shigella in food Download PDFInfo
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- CN106399562A CN106399562A CN201611025690.1A CN201611025690A CN106399562A CN 106399562 A CN106399562 A CN 106399562A CN 201611025690 A CN201611025690 A CN 201611025690A CN 106399562 A CN106399562 A CN 106399562A
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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
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- 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
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Abstract
The invention discloses a detection method of Shigella in food. An isothermal amplification method is adopted, the Shigella ipaC gene specific sequence is adopted as a target sequence, SYBER Green I is adopted as a fluorescent dye, and then fluorescent quantitative detection and analysis are performed. The specific process comprises the following steps: (1) pretreatment; (2) establishment of an isothermal amplification reaction system; (3) fluorescent quantitative reaction; and (4) detection and analysis. The detection method disclosed by the invention has the advantages of high sensitivity, strong specificity and sensitivity, low requirements on template DNA, less time consumption and simple and convenient process.
Description
Technical field
The present invention relates to a kind of method of detecting bacterium, the detection method of shigella in specifically a kind of food.
Background technology
Shigella is a class gram negative bacilli, is the most commonly seen pathogen of human bacterial's property dysentery, common name
Dysentery bacterium.Bacillary dysentery is modal infectious intestinal disease, and two season of autumn in summer, patient was most.The source of infection is mainly patient and band
Bacterium person, by polluting the peroral infections such as food, the drinking-water of dysentery bacterium.The mankind are susceptible to shigella, 10～200 antibacterials
10～50% volunteers can be made to cause a disease.It is, in general, that the state of an illness of bacillary dysentery is heavier caused by shigella.Acute bacillary dysentery is common
Yu children, various dysentery bacterium all can cause.Morbidity is anxious, does not often occur in stomachache, diarrhoea, assumes serious systemic toxicity profiles disease
Shape.Acute bacillary dysentery treatment is thorough, or Abwehrkraft des Koepers are low, malnutrition or during with other chronic diseases, easily switch to chronic.Disease
Journey many more than two months, protracted course of disease or when send out when healing.
In current food, traditional detection method Main Basiss standard GB/T 4789.5-2012 of shigella, needs to detest
Oxygen culture, separation, screening and biochemical identification, though testing result is accurately, complex operation, detection cycle are long, can not meet quick
The demand of detection.Protocols in Molecular Biology because having the advantages that detection sensitivity height, high specificity, easy and simple to handle, in food-borne
Play huge effect in the detection of pathogenic bacterium, be also applied to the detection of shigella.With food safety detection standard
Raising, find more quick, accurately, easily detection technique seems most important.
The cultural method of traditional detection shigella simultaneously, needs to separate, screens and biochemical identification, also need to blood if necessary
Clear learn identification, generally 4～6d, waste time and energy, have the shortcomings that sensitivity is low, poor specificity, false positive, time and effort consuming.Respectively
Plant conventional PCR method and have the advantages that sensitivity is strong, specificity is high, easy, quick, have more application to detect in shigella
Report, but limit due to haveing such problems as PCR post processing to produce the false positive that leads to of pollution and the special instrument and equipment of needs
Make its application in grass-roots unit.Accurate, sensitive, quick, free of contamination clinical test method therefore to be developed.
Content of the invention
It is an object of the invention to provide in a kind of structure food simple, easy to use shigella detection method,
To solve the problems, such as to propose in above-mentioned background technology.
For achieving the above object, the present invention provides following technical scheme：
The detection method of shigella in a kind of food, using constant-temperature amplification method, with shigella ipaC gene specific sequence
For target sequence, SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Detailed process includes walking as follows
Suddenly：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 30-40 DEG C, incubation time
For 20-28h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.7-1.9；
Described SYBER Green I fluorescent dye dilutes for 120-150 times；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 2-6mmol/L；
The concentration of described metabisulfite solution is 10-20mmol/L；
The OD260/OD280 value of described DNA primer is 1.7-1.9；
The concentration of described glutamic acid solution is 70-90mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 92-96 DEG C of degeneration 4-6min, 92-96
DEG C degeneration 0.5-1.5min；50-70 DEG C of annealing 20-40s；68-76 DEG C of extension 0.5-1.5min；32 circulations, last 68-74 DEG C
Extend 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
As the further scheme of the present invention：Concrete steps（1）Middle cultivation temperature is 35 DEG C, and incubation time is 24h.
As the further scheme of the present invention：Concrete steps（2）Described in the OD260/OD280 value of DNA profiling be 1.8.
As the further scheme of the present invention：Concrete steps（2）Described in SYBER Green I fluorescent dye be 135 times
Dilution.
As the further scheme of the present invention：Concrete steps（2）Described in alkali solution of beet concentration be 4mmol/L.
As the further scheme of the present invention：Concrete steps（2）Described in metabisulfite solution concentration be 15mmol/L.
As the further scheme of the present invention：Concrete steps（2）Described in the OD260/OD280 value of DNA primer be 1.8.
As the further scheme of the present invention：Concrete steps（2）Described in glutamic acid solution concentration be 80mmol/L.
As the further scheme of the present invention：Concrete steps（3）In 94 DEG C of degeneration 5min, 94 DEG C of degeneration 1.0min；60℃
Annealing 3s；72 DEG C of extension 1.0min；32 circulations, last 71 DEG C of extension 10min.
Compared with prior art, the invention has the beneficial effects as follows：
Using constant-temperature amplification method, with shigella ipaC gene specific sequence as target sequence, SYBER Green I contaminates for fluorescence
Material, subsequently carries out fluorogenic quantitative detection analysis；Detailed process comprises the steps：（1）Pretreatment；（2）Build constant-temperature amplification anti-
Answer system；（3）Fluorescent quantitation reacts；（4）Detection and analysis.The detection method sensitivity of the present invention is high, and specificity and sensitivity are strong,
Template DNA is required relatively low, take short, method is easy.
Specific embodiment
With reference to specific embodiment, the technical scheme of this patent is described in more detail.
Embodiment 1
The detection method of shigella in a kind of food, using constant-temperature amplification method, with shigella ipaC gene specific sequence
For target sequence, SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Detailed process includes walking as follows
Suddenly：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 30 DEG C, incubation time is
20h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.7；
Described SYBER Green I fluorescent dye is 120 times of dilutions；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 2mmol/L；
The concentration of described metabisulfite solution is 10mmol/L；
The OD260/OD280 value of described DNA primer is 1.7；
The concentration of described glutamic acid solution is 70mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 92 DEG C of degeneration 4min, 92 DEG C of degeneration
0.5min；50 DEG C of annealing 20s；68 DEG C of extension 0.5min；32 circulations, last 68 DEG C of extension 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
Embodiment 2
The detection method of shigella in a kind of food, using constant-temperature amplification method, with shigella ipaC gene specific sequence
For target sequence, SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Detailed process includes walking as follows
Suddenly：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 33 DEG C, incubation time is
22h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.7；
Described SYBER Green I fluorescent dye is 130 times of dilutions；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 3mmol/L；
The concentration of described metabisulfite solution is 12mmol/L；
The OD260/OD280 value of described DNA primer is 1.7；
The concentration of described glutamic acid solution is 70mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 93 DEG C of degeneration 4min, 93 DEG C of degeneration
0.8min；55 DEG C of annealing 25s；70 DEG C of extension 1.0min；32 circulations, last 69 DEG C of extension 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
Embodiment 3
The detection method of shigella in a kind of food, using constant-temperature amplification method, with shigella ipaC gene specific sequence
For target sequence, SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Detailed process includes walking as follows
Suddenly：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 35 DEG C, incubation time is
24h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.8；
Described SYBER Green I fluorescent dye is 135 times of dilutions；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 4mmol/L；
The concentration of described metabisulfite solution is 15mmol/L；
The OD260/OD280 value of described DNA primer is 1.8；
The concentration of described glutamic acid solution is 80mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 94 DEG C of degeneration 5min, 94 DEG C of degeneration
1.0min；60 DEG C of annealing 30s；72 DEG C of extension 1.0min；32 circulations, last 71 DEG C of extension 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
Embodiment 4
The detection method of shigella in a kind of food, using constant-temperature amplification method, with shigella ipaC gene specific sequence
For target sequence, SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Detailed process includes walking as follows
Suddenly：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 38 DEG C, incubation time is
27h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.9；
Described SYBER Green I fluorescent dye is 140 times of dilutions；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 5mmol/L；
The concentration of described metabisulfite solution is 18mmol/L；
The OD260/OD280 value of described DNA primer is 1.9；
The concentration of described glutamic acid solution is 85mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 95 DEG C of degeneration 6min, 95 DEG C of degeneration
1.3min；65 DEG C of annealing 35s；74 DEG C of extension 1.3min；32 circulations, last 72 DEG C of extension 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
Embodiment 5
The detection method of shigella in a kind of food, using constant-temperature amplification method, with shigella ipaC gene specific sequence
For target sequence, SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Detailed process includes walking as follows
Suddenly：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 40 DEG C, incubation time is
28h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.9；
Described SYBER Green I fluorescent dye is 150 times of dilutions；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 6mmol/L；
The concentration of described metabisulfite solution is 20mmol/L；
The OD260/OD280 value of described DNA primer is 1.9；
The concentration of described glutamic acid solution is 90mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 96 DEG C of degeneration 6min, 96 DEG C of degeneration
1.5min；70 DEG C of annealing 40s；76 DEG C of extension 1.5min；32 circulations, last 74 DEG C of extension 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
Above the better embodiment of this patent is explained in detail, but this patent is not limited to above-mentioned embodiment party
Formula, in the ken that one skilled in the relevant art possesses, can also be on the premise of without departing from this patent objective
Various changes can be made.
Claims (9)
1. in a kind of food shigella detection method it is characterised in that adopt constant-temperature amplification method, with shigella ipaC
Gene specific sequence is target sequence, and SYBER Green I is fluorescent dye, subsequently carries out fluorogenic quantitative detection analysis；Concrete mistake
Journey comprises the steps：
（1）Pretreatment：
In an aseptic environment, food is added in TSB pancreas peptone soybean broth, cultivation temperature is 30-40 DEG C, incubation time
For 20-28h, after culture terminates, Reusability normal saline is carried out, and subsequently extracts STb gene；
（2）Build isothermal amplification reactions system：
DNA profiling 1 μ L, SYBER Green I fluorescent dye 0.1 μ L, 10 × Buffer 3.0 μ L, archaeal dna polymerase 10U are sweet
Dish aqueous slkali 0.5 μ L, metabisulfite solution 0.1 μ L, dNTP 3 μ L, DNA primer 3 μ L, IPTG 2 μ L, glutamic acid solution 2 μ L,
Remaining uses dd H2O complements to 30 μ L；
The OD260/OD280 value of described DNA profiling is 1.7-1.9；
Described SYBER Green I fluorescent dye dilutes for 120-150 times；
KCl, Tris-HCl is contained in described Buffer（pH 8.3）, sucrose；
Described archaeal dna polymerase adopts Pfu polymerase；
The concentration of described alkali solution of beet is 2-6mmol/L；
The concentration of described metabisulfite solution is 10-20mmol/L；
The OD260/OD280 value of described DNA primer is 1.7-1.9；
The concentration of described glutamic acid solution is 70-90mmol/L；
（3）Fluorescent quantitation reacts：
Isothermal amplification reactions system is added in fluorescent quantitation machine, is subsequently expanded, 92-96 DEG C of degeneration 4-6min, 92-96
DEG C degeneration 0.5-1.5min；50-70 DEG C of annealing 20-40s；68-76 DEG C of extension 0.5-1.5min；32 circulations, last 68-74 DEG C
Extend 10min；
（4）Detection and analysis：
Quantitative analyses are carried out according to quantitative fluorescence analysis software, result counts.
2. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（1）Middle training
Foster temperature is 35 DEG C, and incubation time is 24h.
3. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（2）Middle institute
The OD260/OD280 value stating DNA profiling is 1.8.
4. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（2）Middle institute
Stating SYBER Green I fluorescent dye is 135 times of dilutions.
5. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（2）Middle institute
The concentration stating alkali solution of beet is 4mmol/L.
6. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（2）Middle institute
The concentration stating metabisulfite solution is 15mmol/L.
7. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（2）Middle institute
The OD260/OD280 value stating DNA primer is 1.8.
8. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（2）Middle institute
The concentration stating glutamic acid solution is 80mmol/L.
9. in food according to claim 1 the detection method of shigella it is characterised in that concrete steps（3）In 94
DEG C degeneration 5min, 94 DEG C of degeneration 1.0min；60 DEG C of annealing 3s；72 DEG C of extension 1.0min；32 circulations, last 71 DEG C of extensions
10min.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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2016
- 2016-11-22 CN CN201611025690.1A patent/CN106399562A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 孙素霞等: "福氏志贺氏菌毒力基因ipaC在大肠杆菌中表达的初步研究，29-31,41", 《中国人兽共患病杂质》 * |
| 李伟，黄彬主编: "《分子诊断学》", 30 September 2015, 中国医药科技出版社 * |
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