CN108796098A - A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin - Google Patents
A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin Download PDFInfo
- Publication number
- CN108796098A CN108796098A CN201810606462.6A CN201810606462A CN108796098A CN 108796098 A CN108796098 A CN 108796098A CN 201810606462 A CN201810606462 A CN 201810606462A CN 108796098 A CN108796098 A CN 108796098A
- Authority
- CN
- China
- Prior art keywords
- detection
- shiga toxin
- primer
- colon bacillus
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12Q1/6876—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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses primer, kit and its methods that a kind of PSR isothermal amplifications detect colon bacillus shiga toxin.The primer includes detection primer Ft and detection primer Bt；Wherein, the nucleotide sequence of detection primer Ft is as shown in SEQ ID NO.1；The nucleotide sequence of detection primer Bt is as shown in SEQ ID NO.2.The present invention also provides a kind of PSR isothermal amplifications to detect colon bacillus shiga toxin kit, the kit includes above-mentioned primer, Bst archaeal dna polymerases, and the mixed solution etc. of calcein and manganese chloride, the detection of polymerase spiral response can be carried out to colon bacillus shiga toxin, it will not be caused because of the change of temperature the loss of time, time-consuming short, reaction process is easy, detection cycle is short, high specificity, can detect by an unaided eye testing result.The present invention has great importance to the Site Detection of exploitation and microorganism that novel constant-temperature amplification technique expands.
Description
Technical field
The invention belongs to biotechnology, more particularly to a kind of PSR isothermal amplifications detection colon bacillus shiga poison
Primer, kit and its method of element.
Background technology
Shiga toxin is mainly to be generated by Escherichia coli, shiga toxin producing escherichia coli (shiga toxin-
Producing Escheriachia coli, STEC)-it is one of food main pathogenic bacteria.STEC Major Virulence Factors are located at
On stx1, stx2 gene on bacteriophage, both genes are separately encoded shiga toxin Stx1, Stx2.Shiga toxin Stx1,
The dosage that Stx2 causes patient to infect is very low, can lead to patient's tormina and diarrhea, and acute renal failure occurs for severe patient and dead
It dies.
Mainly there are traditional biochemical identification method and molecular biotechnology to the detection method of shiga toxin at present
PCR method, the detection method of all kinds of biochemical automatic assessing instruments of enzyme linked immunological kit and application also based on immunology, with
And most emerging genechip detection means.Conventional detection identification method workload is relatively large and testing result general 4~5
It can just obtain a result, and time-consuming, judge qualification result according to experimental phenomena, it is difficult to meet the needs of Rapid identification.In recent years
The genechip detection and PCR amplification method to grow up has stronger specific, quick, sensitive, but cost is higher.Exempt from
Epidemiology detection method, such as ELISA, immunochromatography etc., high sensitivity, but operating process is complicated, high expensive, is unsuitable for big
Scale detects sample.Current most widely used isothermal amplification technology-LAMP also has its limitation, as design of primers is complicated,
False positive rate is high, reagent valence height is higher, and due to the protection of Japanese intellectual property, China's limitation in Transformation Application is strong.It is poly-
Technology is compared with other nucleic acid amplification technologies, Ke Yi for synthase spiral response (Polymerase Spiral Reaction, PSR)
Target sequence is fast, efficiently, specifically expanded under isothermy, and easy to operate, need not accurately alternating temperature equipment, cost compared with
It is low, show vast potential for future development in food-borne microorganism detection field.Therefore, it establishes a kind of for colon bacillus shiga poison
The novel isothermal nucleic acid amplification method with independent intellectual property rights of element has great importance.
Invention content
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of PSR isothermal amplifications
Detect the primer of colon bacillus shiga toxin.
Another object of the present invention is to provide the reagents that a kind of PSR isothermal amplifications detect colon bacillus shiga toxin
Box.
Another object of the present invention is to provide the sides that a kind of PSR isothermal amplifications detect colon bacillus shiga toxin
Method.It is good with high sensitivity, specificity, and easy to operate quickly as a result accurately and reliably, testing cost is low, is suitble to Site Detection
Using the characteristics of.
The purpose of the invention is achieved by the following technical solution：A kind of PSR isothermal amplifications detection colon bacillus shiga
The primer of toxin, including detection primer Ft and detection primer Bt, nucleotide sequence it is as follows：
Detection primer Ft:
5'-CTCTTCAGCCAGTCGTCGTGCAACAGCGACATCATCCG-3'(SEQ ID NO.1)；
Detection primer Bt：
5’-GTGCTGCTGACCGACTTCTCATTCCTTCCCGTAACAACT-3’(SEQ ID NO.2)。
A kind of kit of PSR isothermal amplifications detection colon bacillus shiga toxin, including above-mentioned PSR isothermal duplications are anti-
The primer of colon bacillus shiga toxin should be detected.
Detection primer Ft and detection primer in the primer of the PSR isothermal amplifications detection colon bacillus shiga toxin
The concentration of Bt is 50 μM.
The kit of the PSR isothermal amplifications detection colon bacillus shiga toxin, also comprises the following components：
A, 2 × reaction buffer：The ammonium sulfate of the Tris-HCl of 40.0mM, 20.0mM, the potassium chloride of 20.0mM, 16.0mM
Magnesium sulfate, the Tween 20 of 0.2% (v/v), the glycine betaine of 1.4M, the dNTPs (each) of 10.0mM；
B, Bst archaeal dna polymerases；
C, the mixed solution of calcein and manganese chloride.
Bst archaeal dna polymerases described in component B are preferably the Bst archaeal dna polymerase aqueous solutions of a concentration of 8U/ μ L.
The mixed solution of calcein and manganese chloride described in component C is prepared via a method which to obtain：
(i) calcein is dissolved in dimethyl sulfoxide (DMSO) (DMSO), the calcein solution of 50 μM of configuration；Manganese chloride is molten
Yu Shuizhong configures the manganese chloride aqueous solution of 1mM；
(ii) it takes the calcein solution of 50 μM of 25 μ L to be uniformly mixed with the manganese chloride aqueous solution of 10 μ L1mM, obtains calcium Huang
(concentration ratio of calcein solution and manganese chloride solution is 1 to the mixed solution of green element and manganese chloride:8).
The kit of the PSR isothermal amplifications detection colon bacillus shiga toxin is in detection colon bacillus shiga poison
Application in element.
A kind of method of PSR isothermal amplifications detection colon bacillus shiga toxin, includes the following steps：
(1) DNA of bacteria of extraction measuring samples is as template DNA, and controls the OD of template DNA aqueous solution260/OD280Value
It is 1.8~2.0；
(2) 40~45 minutes are kept the temperature in 65 DEG C of water-baths carries out polymerase spiral amplified reaction；Wherein, polymerase spiral expands
Increasing reaction system is 26 μ L reaction systems：2 × reaction buffer 12.5 μ L, 50 μM of detection primer Ft and 50 μM of detection primer
Each 0.8 μ L of Bt, the 1.0 μ L of Bst DNA polymerases of 2.0 μ l, 8U/ μ L of DNA profiling, deionized water complement to 25 μ L；It is eventually adding
The mixed solution of the calcein and manganese chloride of 1 μ L；
(3) wait that keeping the temperature 2 minutes in 80 DEG C of water-baths after the completion of reacting terminates reaction, then detect by an unaided eye color change,
If color is yellow, illustrate not containing colon bacillus shiga toxin in measuring samples；If color becomes green, illustrate measuring samples
In contain colon bacillus shiga toxin.
The nucleotide sequence of detection primer Ft described in step (2) is as shown in SEQ ID NO.1, the core of detection primer Bt
Nucleotide sequence is as shown in SEQ ID NO.2.
The mixed solution of calcein and manganese chloride described in step (2) is prepared via a method which to obtain：
(i) calcein is dissolved in dimethyl sulfoxide (DMSO) (DMSO), the calcein solution of 50 μM of configuration；Manganese chloride is molten
Yu Shuizhong configures the manganese chloride aqueous solution of 1mM；
(ii) it takes the calcein solution of 50 μM of 25 μ L to be uniformly mixed with the manganese chloride aqueous solution of 10 μ L1mM, obtains calcium Huang
(concentration ratio of calcein solution and manganese chloride solution is 1 to the mixed solution of green element and manganese chloride:8).
The present invention has the following advantages and effects with respect to the prior art：
(1) reflecting for the polymerase spiral detection designed by shiga toxin opposite sex target sequence stx2 provided in the present invention
Determine system, solves the defects of period needed for method in the prior art is long, and sensitivity is low, of high cost, field application is difficult.
(2) detection time can be reduced to 60 minutes by method of the invention, with tradition loop-mediated isothermal amplification technique phase
Than quick-break detection cycle, the Site Detection of exploitation and microorganism to the amplification of novel constant-temperature amplification technique has great importance.
Meanwhile the invention also discloses specific target sequence stx2 (the Genebank Accession for shiga toxin
No.AF162758.1) specific regions of conserved region devise a pair of of detection primer, to ensure that the reliability of testing result.
Secondly, the present invention expands under isothermal conditions, will not be caused because of the change of temperature the loss of time, time-consuming short, just at 60 minutes
It can complete result interpretation.In addition, the technology does not need special, expensive instrument and reagent, amplified production does not need gel electrophoresis,
Directly can be with naked eyes judging result with fluorescent dye colour developing, simple and efficient to handle, testing cost is relatively low.The present invention kit and
The particularly suitable middle-size and small-size unit of method and Site Detection.
Description of the drawings
Fig. 1 is the result and Gel electrophoresis results figure that polymerase spiral response technology detects shiga toxin；Wherein, figure A is
(NG is blank control to the result of polymerase spiral response technology detection shiga toxin；1 is Escherichia coli O 157:
H7ATCC43895,2 be Escherichia coli E019, and 3 be Escherichia coli E020, and 4 be Escherichia coli E043, and 5 be Escherichia coli
E044)；Scheming the Gel electrophoresis results that B is polymerase spiral response technology detection shiga toxin, (swimming lane NG is blank control；Swimming
Road 1 is Escherichia coli O 157:H7ATCC43895, swimming lane 2 are Escherichia coli E019, and swimming lane 3 is Escherichia coli E020, swimming lane 4
For Escherichia coli E043, swimming lane 5 is Escherichia coli E044).
Fig. 2 is specific detection experimental result picture；Wherein, 1：Escherichia coli E019；2：Escherichia coli E020； 3:Large intestine
Bacillus E043；4:Escherichia coli E044；5：Escherichia coli ATCC43895；6：Salmonella ATCC29629；7：Salmonella
ATCC19585；8：Salmonella ATCC14028；9：Salmonella ATCC13076；10:Listeria monocytogenes ATCC19116；
11：Listeria monocytogenes ATCC19114,12：Listeria monocytogenes ATCC19115；13：Pseudomonas aeruginosa ATCC27853；
14：Pseudomonas aeruginosa ATCC9027；15：Staphylococcus aureus ATCC23235；16：Staphylococcus aureus
ATCC6358； 17：Vibrio parahemolyticus ATCC17802.
Fig. 3 is sensitivity experiments result figure；Wherein, 20 be 68ng/ μ L, and 21 be 6.8ng/ μ L, and 22 be 680pg/ μ L, 23
It is 6.8pg/ μ L for 68pg/ μ L, 24,25 be 680fg/ μ L, and 26 be negative control.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Embodiment 1 is based on polymerase spiral response isothermal amplification technique detection E.coli O157:The microbial process of H7
1, the method for detecting pathogenic microorganism based on polymerase spiral isothermal amplification technique, the present embodiment is with E.coli
O157:It is as follows using reagent for H7：
A. concentration is respectively following (5 ' -3 ') for 50 μM of detection primer Ft aqueous solutions and Bt aqueous solutions primer sequence：
Detection primer Ft: CTCTTCAGCCAGTCGTCGTG-CAACAGCGACATCATCCG(SEQ ID NO.1)；
Detection primer Bt： GTGCTGCTGACCGACTTCTC-ATTCCTTCCCGTAACAACT(SEQ ID NO.2)；
B.2 × reaction liquid storage：By the Tris-HCl of a concentration of 40.0mM, ammonium sulfate 20.0m M, 20.0 m M of potassium chloride,
Magnesium sulfate 16.0m M, 0.2% (v/v) Tween 20, the mixed liquor composition of glycine betaine 1.4M, dNTPs (each) 10.0mM；
C. Bst archaeal dna polymerases (large fragment, NEB companies) aqueous solution of a concentration of 8U/ μ l；
D. the mixed solution of calcein and manganese chloride：(dimethyl is sub- for the calcein solution that first configuration concentration is 50 μM
Sulfone dissolves)；Then the calcein solution for taking 50 μM of 25 μ L, is uniformly mixed that (calcium is yellowish green with the manganese chloride aqueous solution of 10 μ L1mM
The concentration ratio of plain solution and manganese chloride solution is 1:8).
2, shiga toxin is detected using polymerase spiral response amplification technique using mentioned reagent, included the following steps:
(1) DNA of bacteria of extraction measuring samples is as template DNA：
Experimental group and blank control group is arranged in the present embodiment simultaneously, and wherein experimental group is five plants of Escherichia coli O 157s:
H7ATCC43895 (American Type Culture Collecti), E019[1], E020[1], E043[1], E044[1]；Using DNA extraction kit
(Guangdong Dongsheng bio tech ltd) extracts each group DNA of bacteria, is operated according to kit specification, bacterium obtained by experimental group
The OD of aqueous dna260/OD280Value (260nm and 280nm under absorption photometric ratio) be 1.8.
(2) the polymerase spiral amplified reaction of colon bacillus shiga toxin：It is 26 μ l's that total volume is configured in reaction tube
Polymerase spiral amplification reaction system；2 × reaction liquid storage isometric 1.6 μ l of mix primer mixed liquor of 12.5 μ l, Ft and Bt are added,
1 μ l of Bst archaeal dna polymerases, 2.0 μ l of DNA profiling supplement volume to 25 μ l with deionized water, are eventually adding the calcium of above-mentioned concentration
Yellowish green element and 1 μ l of manganese chloride mixed liquor aqueous solution, mixing.Each material concentration is at this time：Tris-HCl 20.0m M, sulfuric acid
Ammonium 10.0m M, potassium chloride 10.0m M, magnesium sulfate 8.0m M, Tween 20 0.1% (v/v), glycine betaine 0.7M, dNTPs
(each) 1.4m M, Bst archaeal dna polymerase 8U, each 1.6 μM of primers F t, Bt.Reaction tube is placed in insulation reaction in 65 DEG C of water-baths
60 minutes, 2 minutes were kept the temperature in 80 DEG C of water-baths and terminates reaction.
(3) color developing detection：Wait for that after reaction, detect by an unaided eye color change
The results are shown in Figure 1, as a result shows：The color of blank control group is yellow, illustrates that detecting bacterial strain is free of strong-willed he
Toxin gene；The color of experimental group becomes green, illustrates containing shiga toxin gene, and 2% fine jade is then carried out to amplified production
Sepharose electrophoresis, the positive group present trapezoid-shaped strips, negative group without amplified band, it is consistent with expected results.
2 polymerase spiral response of embodiment detects shiga toxin specific test
By Escherichia coli (the Escherichia coli E019 containing shiga toxin[1], Escherichia coli E020[1], Escherichia coli E043[1], Escherichia coli E044[1], Escherichia coli ATCC43895) and without containing other bacterial strain (salmonellas of shiga toxin
ATCC29629, salmonella ATCC19585, salmonella ATCC14028, salmonella ATCC 13076, Listeria monocytogenes
ATCC19116, Listeria monocytogenes ATCC19114, Listeria monocytogenes ATCC19115, pseudomonas aeruginosa ATCC27853,
Pseudomonas aeruginosa ATCC9027, staphylococcus aureus ATCC23235, staphylococcus aureus ATCC6358, parahemolyticas
The genomic DNA of vibrios ATCC17802 establishes polymerase spiral response detection method according to above-mentioned reaction system and condition, carries out
Specific test.It is positive control that the genome containing shiga toxin Escherichia coli, which is arranged, and ultra-pure water is that negative control is (negative right
According to identical as the result of the blank control in Fig. 1), the results are shown in Figure 2.The result shows that only containing shiga toxin large intestine bar
There is positive reaction in bacterium genome, and negative reaction is presented in remaining.
The sensitivity tests of embodiment 3PSR detection colon bacillus shiga toxins
The genome of Escherichia coli O 157 is carried out 10 times of concentration gradients to dilute, respectively 68ng/ μ l, 6.8ng/ μ l,
680pg/ μ l, 68pg/ μ l, 6.8pg/ μ l, 680fg/ μ l, while negative control (deionized water) is set, in accordance with the above-mentioned embodiment 1
In reaction system build polymerase spiral response amplification method, to determine the sensibility of detection method, the results are shown in Figure 3.
The result shows that：The colon bacillus shiga toxin polymerase spiral response method of foundation can detect the large intestine bar of 680pg/ μ L in sample
Bacterium DNA.
Conclusion：Polymerase spiral response amplification method and Standard PCR and fluorescent PCR are can be seen that from above-mentioned experimental result
It has the following advantages that：
It operates and identifies and is simple and efficient：Standard PCR whole process can just go out in 2~4 hours as a result, quantitative fluorescent PCR
1~1.5 hour is needed, detection method provided by the present invention just may occur in which positive findings at 60 minutes.Secondly to instrument requirements
It is low, it is only necessary to a common water-bath, and testing result can be directly observed by fluorescent dye, eliminate traditional electrophoresis inspection
Survey step.Have wide practical use in quick detection and the practice of Site Detection.
High specificity：Only by whether amplification just can determine whether the presence or absence of target gene, so as to complete determining for bacterium
Property detection.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.Bibliography
[1] all Rong cryopreservations study the induction of enterorrhagia Escherichia coli VBNC states and the influence of toxin expression quantity
[D] South China Science & Engineering University, 2015.
Sequence table
<110>South China Science & Engineering University
<120>A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Detection primer Ft
<400> 1
ctcttcagcc agtcgtcgtg caacagcgac atcatccg 38
<210> 2
<211> 39
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223>Detection primer Bt
<400> 2
gtgctgctga ccgacttctc attccttccc gtaacaact 39
Claims (8)
1. a kind of primer of PSR isothermal amplifications detection colon bacillus shiga toxin, it is characterised in that：Including detection primer Ft
With detection primer Bt；Wherein, the nucleotide sequence of detection primer Ft is as shown in SEQ ID NO.1；The nucleotide of detection primer Bt
Sequence is as shown in SEQ ID NO.2.
2. a kind of kit of PSR isothermal amplifications detection colon bacillus shiga toxin, it is characterised in that：Including claim
The primer of PSR isothermal amplifications detection colon bacillus shiga toxin described in 1.
3. the kit of PSR isothermal amplifications detection colon bacillus shiga toxin according to claim 2, feature exist
In：
Detection primer Ft and detection primer Bt in the primer of the PSR isothermal amplifications detection colon bacillus shiga toxin
Concentration is 50 μM.
4. the kit of PSR isothermal amplifications detection colon bacillus shiga toxin according to claim 3, feature exist
In also comprising the following components：
A, 2 × reaction buffer：The ammonium sulfate of the Tris-HCl of 40.0mM, 20.0mM, the potassium chloride of 20.0mM, the sulphur of 16.0mM
Sour magnesium, the Tween 20 of 0.2% (v/v), the glycine betaine of 1.4M, the dNTPs of 10.0mM；
B, Bst archaeal dna polymerases；
C, the mixed solution of calcein and manganese chloride.
5. the kit of PSR isothermal amplifications detection colon bacillus shiga toxin according to claim 4, feature exist
In the mixed solution of calcein and manganese chloride described in component C is prepared via a method which to obtain：
(i) calcein is dissolved in dimethyl sulfoxide (DMSO), the calcein solution of 50 μM of configuration；Manganese chloride is soluble in water, match
Set the manganese chloride aqueous solution of 1mM；
(ii) it takes the calcein solution of 50 μM of 25 μ L to be uniformly mixed with the manganese chloride aqueous solution of 10 μ L1 mM, it is yellowish green to obtain calcium
The mixed solution of element and manganese chloride.
6. the kit of PSR isothermal amplifications detection colon bacillus shiga toxin according to claim 4, feature exist
In：
The Bst archaeal dna polymerase aqueous solutions that Bst archaeal dna polymerases described in component B are a concentration of 8U/ μ L.
7. the kit of claim 2~6 any one of them PSR isothermal amplifications detection colon bacillus shiga toxin is being examined
Survey the application in colon bacillus shiga toxin.
8. a kind of method of PSR isothermal amplifications detection colon bacillus shiga toxin, which is characterized in that include the following steps：
(1) DNA of bacteria of extraction measuring samples is as template DNA；
(2) 40~45 minutes are kept the temperature in 65 DEG C of water-baths carries out polymerase spiral amplified reaction；Wherein, the amplification of polymerase spiral is anti-
It is 26 μ L reaction systems to answer system：2 × reaction buffer 12.5 μ L, 50 μM of detection primer Ft and 50 μM of detection primer Bt are each
0.8 μ L, the 1.0 μ L of Bst archaeal dna polymerases of 2.0 μ l, 8U/ μ L of DNA profiling, deionized water complement to 25 μ L；It is eventually adding 1 μ L's
The mixed solution of calcein and manganese chloride；
(3) wait that keeping the temperature 2 minutes in 80 DEG C of water-baths after the completion of reacting terminates reaction, then detect by an unaided eye color change, such as face
Color is yellow, illustrates not containing colon bacillus shiga toxin in measuring samples；If color becomes green, illustrate to contain in measuring samples
There is colon bacillus shiga toxin；
The nucleotide sequence of detection primer Ft described in step (2) is as shown in SEQ ID NO.1, the nucleotide of detection primer Bt
Sequence is as shown in SEQ ID NO.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810606462.6A CN108796098A (en) | 2018-06-13 | 2018-06-13 | A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810606462.6A CN108796098A (en) | 2018-06-13 | 2018-06-13 | A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108796098A true CN108796098A (en) | 2018-11-13 |
Family
ID=64086999
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810606462.6A Pending CN108796098A (en) | 2018-06-13 | 2018-06-13 | A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108796098A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951896A (en) * | 2019-12-26 | 2020-04-03 | 华南理工大学 | CPA detection primer, kit and method for Escherichia coli Shiga toxin II |
| CN110982916A (en) * | 2020-01-02 | 2020-04-10 | 刘思洁 | Primer combination and detection kit for detecting shiga toxin-producing escherichia coli |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487057A (en) * | 2009-01-23 | 2009-07-22 | 浙江省疾病预防控制中心 | Loop-mediated isothermal amplification fast detecting reagent kit and method for O157:H7 coliform |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
-
2018
- 2018-06-13 CN CN201810606462.6A patent/CN108796098A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487057A (en) * | 2009-01-23 | 2009-07-22 | 浙江省疾病预防控制中心 | Loop-mediated isothermal amplification fast detecting reagent kit and method for O157:H7 coliform |
| CN104232622A (en) * | 2014-09-24 | 2014-12-24 | 中国人民解放军疾病预防控制所 | Nucleic acid isothermal amplification method and application thereof by polymerase spiral reaction |
Non-Patent Citations (1)
| Title |
|---|
| XIHONG ZHAO ET AL.: "Development and application of a loop-mediated isothermal amplification method on rapid detection Escherichia coli O157 strains from food samples", 《MOL BIOL REP》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110951896A (en) * | 2019-12-26 | 2020-04-03 | 华南理工大学 | CPA detection primer, kit and method for Escherichia coli Shiga toxin II |
| CN110982916A (en) * | 2020-01-02 | 2020-04-10 | 刘思洁 | Primer combination and detection kit for detecting shiga toxin-producing escherichia coli |
| CN110982916B (en) * | 2020-01-02 | 2023-06-09 | 刘思洁 | Primer combination and detection kit for detecting shiga toxin-producing escherichia coli |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110938677B (en) | Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid and application | |
| CN113322338B (en) | CDA primer group and kit for detecting Shigella and application of CDA primer group and kit | |
| WO2021003878A1 (en) | Cpa primer and kit for detecting methicillin-resistant staphylococcus aureus, and detection method | |
| WO2021129118A1 (en) | Primer, kit, and method for cpa detection of pseudomonas aeruginosa | |
| Ranjbar et al. | Visual detection of Enterohemorrhagic Escherichia coli O157: H7 using loop-mediated isothermal amplification | |
| CN108796098A (en) | A kind of primer, kit and its method of PSR isothermal amplifications detection colon bacillus shiga toxin | |
| CN109355407B (en) | Primer, kit and method for detecting pseudomonas aeruginosa through PSR isothermal amplification reaction | |
| CN103571943A (en) | Nucleic acid detection method for salmonella typhimurium | |
| CN101343661A (en) | Constant temperature nucleic acid amplification fast detecting reagent kit for bacillus coli O157 and use method thereof | |
| CN101368203A (en) | Primer, reagent kit and detection method for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique fast detection | |
| CN111073986B (en) | Rapid constant-temperature detection method for salmonella, primer group and application | |
| CN109355408A (en) | A kind of primer, kit and its method of PSR detection Escherichia coli type I shiga toxin | |
| CN109355403A (en) | A kind of primer, kit and the method for PSR detection Methicillin-resistant Staphylococcus aureus | |
| CN109355405B (en) | Primer, kit and method for detecting vibrio parahaemolyticus by PSR isothermal amplification reaction | |
| CN109735636A (en) | A kind of primer, kit and the method for PSR detection staphylococcus aureus leucocytic toxin | |
| CN109355404B (en) | Primer, kit and detection method for isothermal detection of listeria monocytogenes based on polymerase helix reaction | |
| CN106434916A (en) | LAMP primer group and kit for enterobacter sakazakii, and use method of kit | |
| CN109517913B (en) | Primer, kit and method for PSR (phosphoenolpyruvate carboxylase) detection of heat-resistant direct hemolysin and heat-resistant related hemolysin | |
| CN111004854B (en) | Rapid constant temperature detection method, primer set and kit for vibrio vulnificus and vibrio cholerae simultaneously | |
| CN111073985B (en) | Rapid constant-temperature detection method, primer group and kit for salmonella | |
| CN108754001A (en) | Primer, kit and detection method based on polymerase spiral response Constant Temperature Detection salmonella | |
| CN108796099A (en) | A kind of Escherichia coli O 157:PSR detection primers, kit and its detection method of H7 | |
| CN112111584A (en) | Method for rapidly detecting escherichia coli in water | |
| CN108728560A (en) | The quick detection primer of rolling circle amplification, kit and its detection method of staphylococcus aureus | |
| CN114540516B (en) | LAMP double-strand detection probe, kit and detection method for staphylococcus aureus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |