CN108796099A - A kind of Escherichia coli O 157:PSR detection primers, kit and its detection method of H7 - Google Patents
A kind of Escherichia coli O 157:PSR detection primers, kit and its detection method of H7 Download PDFInfo
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Abstract
The invention discloses a kind of Escherichia coli O 157s:PSR detection primers, kit and its detection method of H7.The primer includes detection primer Ft, Bt, and accelerates primer I F, IB；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；Accelerate the nucleotide sequence of primer I F as shown in SEQ ID NO.3；Accelerate the nucleotide sequence of primer I B as shown in SEQ ID NO.4.The present invention also provides a kind of Escherichia coli O 157s:The PSR detection kits of H7, testing result can be obtained at 40 minutes or so, while ensuring reliability, specificity and the sensitivity of detection, particularly suitable middle-size and small-size unit and Site Detection, and for improving great group's medical diagnosis on disease rate, the medical diagnosis on disease of early stage has very important significance.
Description
Technical field
The invention belongs to biotechnology, more particularly to a kind of Escherichia coli O 157:The PSR detection primers of H7, reagent
Box and its detection method.
Background technology
Pathogenic microorganism is accurately identified, for the food security accident caused by food-borne microorganism and guidance
Rational therapy mode has great importance.The Testing and appraisal method of microorganism is broadly divided into following three categories at present：Separation training
Support identification method, immunology detection and detection of nucleic acids.It is separately cultured that identification method is cumbersome, experimental period is long, it is special to experimenter
Industry level requirement is high.Immunology detection means then need to prepare expensive monoclonal antibody, more demanding to sample, for complexity
Sample can not detect.And detection of nucleic acids is then mostly based on PCR (PCR) method and fluorescence quantitative PCR method, both
Method needs expensive instrument and equipment, and testing cost is higher, is not suitable for the application of middle-size and small-size unit and Site Detection.
PCR and loop-mediated isothermal amplification technique are mainly used for the detection of nucleic acids of Escherichia coli O 157 at present.And it is 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
It is high higher, and due to the protection of Japanese intellectual property, China's limitation in Transformation Application is strong.Polymerase spiral response
(Polymerase Spiral Reaction, PSR) technology, can be fast under isothermal conditions compared with other nucleic acid amplification technologies
Speed, efficiently, specifically expand target sequence, and it is easy to operate, need not accurately alternating temperature equipment, cost is relatively low, food-borne micro-
Field of biological detection shows vast potential for future development.Therefore, it establishes a kind of for Escherichia coli O 157:H7 is novel to be had solely
The isothermal nucleic acid amplification method of vertical intellectual property has great importance.
Escherichia coli O157:H7 is the most important serotype of enterorrhagia Escherichia coli, mainly passes through edible dirt
The food of dye, such as giving birth to or minced steak product, raw milk and contaminated vegetables and bean sprout are propagated to the mankind, after infecting human body
Show as corresponding clinical symptoms.Low-grade infection can cause cramp and diarrhea, fever and vomiting that may also occur, incubation period
It generally 3~8 days, 10 days can self-healing.Severe infection then shows as hemorrhagic colitis, hemolytic uremic syndrome.
E.coliO157:H7 infective doses are extremely low, and disease can be caused by eating less than five bacteriums, and progression of the disease is fast.From nineteen eighty-two
The U.S. is found for the first time because of E.coli O157:Since food poisoning caused by H7, epidemic situation starts to spread and spread, in succession China,
Multiple countries such as Britain, Canada, Japan cause to fall ill, and significant threat is constituted to human health.At present by E.coli
O157:Food origin disease has become global hygienic issues caused by H7 infection.It is a kind of quickly low it is therefore desirable to establish
The detection E.coliO157 of cost:The detection method and its kit of H7.
Invention content
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of Escherichia coli O 157:H7
PSR detection primers.
Another object of the present invention is to provide a kind of Escherichia coli O 157s:The PSR detection kits of H7.
Another object of the present invention is to provide the Escherichia coli O 157:The application of the PSR detection kits of H7.
It is still another object of the present invention to provide a kind of Escherichia coli O 157s:The PSR detection methods of H7.This method has spirit
The characteristics of sensitivity is high, specificity is good, and easy to operate quickly as a result accurately and reliably, testing cost is low, suitable Site Detection application.
The purpose of the invention is achieved by the following technical solution：A kind of Escherichia coli O 157:The PSR detection primers of H7, packet
Detection primer Ft, Bt is included, and accelerates primer I F, IB, nucleotide sequence is as follows：
Detection primer Ft:
5'-TTGGCATCGTGTGGACAGGGTAGGACCGCAGAGGAAAGA-3'(SEQ ID NO.1)；
Detection primer Bt：
5'-TGGGACAGGTGTGCTACGGTTTCCACGCCAACCAAGATC-3'(SEQ ID NO.2)；
Accelerate primer I F:5'-GTTTCGATGAGTTTATCTGC-3'(SEQ ID NO.3)；
Accelerate primer I B:5'-TCAAAAGCACCCTATAGCTG-3'(SEQ ID NO.4).
A kind of Escherichia coli O 157:The PSR detection kits of H7, including above-mentioned Escherichia coli O 157:The PSR detections of H7 are drawn
Object.
The above-mentioned Escherichia coli O 157:Each primer of PSR detection primers of H7 (draw by detection primer Ft, Bt, and acceleration
Object IF, IB) concentration be 50 μM.
The Escherichia coli O 157:The PSR detection kits of H7, also comprise 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 Escherichia coli O 157:The PSR detection kits of H7 are in detection Escherichia coli O 157:Application in H7.
A kind of Escherichia coli O 157:The PSR detection methods of H7, include 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
Bt each 0.8 μ L, 50 μM of acceleration primer I F and 50 μM of each 0.4 μ L of acceleration primer I B, the Bst of 2.0 μ L, 8U/ μ L of DNA profiling
1.0 μ L of archaeal dna polymerase, add water to 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 Escherichia coli O 157 in measuring samples:H7；If color becomes green, illustrate measuring samples
In contain Escherichia coli O 157:H7.
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 accelerates the nucleotide sequence of primer I F as shown in SEQ ID NO.3 as shown in SEQ ID NO.2, accelerates primer I B
Nucleotide sequence as shown in SEQ ID NO.4.
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) it is directed to E.coliO157 provided in the present invention:Polymerase spiral shell designed by H7 specific target sequences rfbE
Testing and appraisal system is revolved, solves that the period needed for method in the prior art is long, and sensitivity is low, of high cost, field application difficulty etc.
Defect.Pass through the conservative of the specific sequence rfbE (Genebank Accession No.JN578671.1) of selection target bacterial strain
Region, designs a pair of of detection primer and a pair of of acceleration primer and addition fluorescent dye builds polymerase spiral shell isothermal amplification body
System, can obtain testing result to shorten the period of traditional Escherichia coli at 40 minutes or so, while ensure the reliability of detection, spy
Anisotropic and sensitivity.This has very important significance for improving great group's medical diagnosis on disease rate, the medical diagnosis on disease of early stage.
(2) detection time can be reduced to 40~45 minutes in the present 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.
(3) present invention expands under isothermal conditions, will not be caused because of the change of temperature the loss of time, time-consuming short, in addition,
The technology does not need special, expensive instrument and reagent, amplified production do not need gel electrophoresis, directly can with fluorescent dye colour developing
Simple and efficient to handle with naked eyes judging result, testing cost is relatively low.The particularly suitable middle-size and small-size list of kit and method of the present invention
Position and Site Detection.
Description of the drawings
Fig. 1 is that polymerase spiral response technology detects E.coliO157:The result and Gel electrophoresis results figure of H7；Wherein,
It is that polymerase spiral response technology detects E.coliO157 to scheme A:(NG is blank control to the result of H7, and 1 is Escherichia coli O 157:
H7ATCC43895,2 be Escherichia coli O 157:H7ATCC43894,3 be Escherichia coli E019)；Figure B is polymerase spiral response
Technology detects E.coliO157:(swimming lane NG is blank control to the Gel electrophoresis results of H7, and swimming lane 1 is Escherichia coli O 157:
H7ATCC43895, swimming lane 2 are Escherichia coli O 157:H7ATCC43894, swimming lane 3 are Escherichia coli E019).
Fig. 2 is the experimental result picture of specific detection；Wherein, 1 is staphylococcus aureus ATCC23235；2 be cheese breast
Bacillus GBHM-21；3 be salmonella ATCC14028；4 be pseudomonas aeruginosa ATCC27853；5 be Listeria monocytogenes
ATCC19118；6 be Escherichia coli O 157:H7ATCC43895.
Fig. 3 is sensitivity experiments result figure；Wherein, 1 is 112ng/ μ L；2 be 11.2ng/ μ L；3 be 1.12ng/ μ L；4 are
112pg/μL；5 be 11.2pg/ μ L；6 be 1.12pg/ μ L；NG is 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.
Each raw material used in following embodiments and reagent can be obtained from commercially available in addition to particularly pointing out.
Embodiment 1 is based on polymerase spiral response isothermal amplification technique and detects E.coliO157:The microbial process of H7
1, based on polymerase spiral isothermal amplification technique detect pathogenic microorganism method, the present embodiment with
E.coliO157:It is as follows using reagent for H7：
A. concentration is respectively 50 μM of detection primer Ft aqueous solutions and Bt aqueous solutions, accelerates primer I F and IB aqueous solution, primer
Sequence is following (5 ' -3 ')：
Detection primer Ft:TTGGCATCGTGTGGACAGGGTAGGACCGCAGAGGAAAGA(SEQ ID NO.1)；
Detection primer Bt：TGGGACAGGTGTGCTACGGTTTCCACGCCAACCAAGATC(SEQ ID NO.2)；
Accelerate primer I F:GTTTCGATGAGTTTATCTGC(SEQ ID NO.3)；
Accelerate primer I B:TCAAAAGCACCCTATAGCTG(SEQ ID NO.4)；
B.2 × reaction liquid storage：By the Tris-HCl of a concentration of 40.0mM, the ammonium sulfate of 20.0mM, the potassium chloride of 20.0mM,
The magnesium sulfate of 16.0mM, the Tween 20 of 0.2% (v/v), the glycine betaine of 1.4M, dNTPs (each) compositions of 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, polymerase spiral response amplification technique detection E.coli O157 are utilized using mentioned reagent:H7, including walk as follows
Suddenly:
(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 three plants of E.coli O157:H7 distinguishes
It is Escherichia coli O 157:H7ATCC43895, ATCC43894 and E019；Wherein：E.coli O157:H7ATCC43895 and
ATCC43894 is purchased from American Type Culture Collecti；E019 can (all Rong cryopreservations be to enterorrhagia Escherichia coli according to document
The induction of VBNC states and influence research [D] the South China Science & Engineering University of toxin expression quantity, 2015.) it obtains；It is extracted and is tried using DNA
Agent box (Guangdong Dongsheng bio tech ltd) extracts each group DNA of bacteria, is operated according to kit specification, obtained by experimental group
The OD of DNA of bacteria aqueous solution260/OD280Value (260nm and 280nm under absorption photometric ratio) be 1.8.
(2)E.coli O157:The polymerase spiral amplified reaction of H7：
The polymerase spiral amplification reaction system that total volume is 26 μ L is configured in reaction tube：2 × reaction liquid storage is added
The isometric 1.6 μ L of mix primer mixed liquor of 12.5 μ L, Ft and Bt accelerate primer I F and IB isometric mixed liquor 0.8 μ L, Bst
1 μ L of archaeal dna polymerase, 2.0 μ L of DNA profiling supplement volume to 25 μ L with deionized water, are eventually adding the calcein of above-mentioned concentration
And 1 μ L of manganese chloride mixed liquor aqueous solution, mixing.Each material concentration is at this time：Tris-HCl 20.0mM, ammonium sulfate
10.0mM, potassium chloride 10.0mM, magnesium sulfate 8.0mM, Tween 20 0.1% (v/v), glycine betaine 0.7M, dNTPs (each)
1.4mM, Bst archaeal dna polymerase 8U, each 1.6 μM of primers F t, Bt, each 0.8 μM of primer I F and IB.Reaction tube is placed in 65 DEG C of water-baths
Middle insulation reaction 45 minutes keeps the temperature 2 minutes 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 not containing E.coli O157:
H7 bacterium；The color of experimental group becomes green, illustrates containing E.coli O157:H7 bacterium then carry out amplified production in 2% fine jade
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 E.coli O157:H7 specific tests
By Escherichia coli O 157:H7ATCC43895 is with the genomic DNA of non-Escherichia coli according to reacting in embodiment 1
System and condition establish polymerase spiral response detection method, carry out specific test；Wherein, non-Escherichia coli are：It is golden yellow
(the peaceful Lactobacillus caseis GBHM-21 identifications of Bao Zhi, pilot scale prepares and hair by staphylococcus A TCC23235, Lactobacillus casei GBHM-21
Ferment technical research [D] South China Science & Engineering University, 2015.), salmonella ATCC14028, pseudomonas aeruginosa ATCC27853 are single
Increase Listeria ATCC19118.Escherichia coli O 157 is set:H7 genomes are positive control, and ultra-pure water is that negative control is (negative
Result of the result of control with blank control in Figure 1A), the results are shown in Figure 2.The reaction system of genome of E.coli is added
Become green, is positive findings.And it is orange that non-genome of E.coli, which is added, still, reaction presents negative.It is thus shown that being based on
Polymerase spiral response detects E.coliO157:The primer of H7 has higher specificity.
Embodiment 3PSR detection E.coli O157:The sensitivity tests of H7
By Escherichia coli O 157:The genome of H7 carries out 10 times of concentration gradient dilutions, respectively 112ng/ μ L, 11.2ng/ μ
L, 1.12ng/ μ L, 112pg/ μ L, 11.2pg/ μ L and 1.12pg/ μ L, while negative control (deionized water) is set, according to implementation
Reaction system in example 1 builds polymerase spiral response amplification method, to determine the sensibility of detection method, as a result such as Fig. 3 institutes
Show.It can be seen from the figure that occur reaction system of the concentration higher than 11.2pg/ μ L of e. coli dna becomes in sample
Positive findings are presented in green.The result shows that：The E.coli O157 polymerase spiral response methods of foundation can detect in sample
The e. coli dna of 11.2pg/ μ L reactions.
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 40 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.
High sensitivity:The sensitivity of Standard PCR detection method is 100pg/ μ L, detection method using the present invention, detection
Lower limit is 11.2pg/ μ L, is 10 times or so of routine PCR reaction.
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.
Sequence table
<110>South China Science & Engineering University
<120>A kind of Escherichia coli O 157:PSR detection primers, kit and its detection method of H7
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tgggacaggt gtgctacggt ttccacgcca accaagatc 39
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gtttcgatga gtttatctgc 20
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Claims (8)
1. a kind of Escherichia coli O 157:The PSR detection primers of H7, it is characterised in that：Including detection primer Ft, Bt, and accelerate
Primer I F, IB；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；
Accelerate the nucleotide sequence of primer I F as shown in SEQ ID NO.3；
Accelerate the nucleotide sequence of primer I B as shown in SEQ ID NO.4.
2. a kind of Escherichia coli O 157:The PSR detection kits of H7, it is characterised in that：Including large intestine bar described in claim 1
Bacterium O157:The PSR detection primers of H7.
3. Escherichia coli O 157 according to claim 2:The PSR detection kits of H7, it is characterised in that：
The above-mentioned Escherichia coli O 157:The concentration of each primer of PSR detection primers of H7 is 50 μM.
4. Escherichia coli O 157 according to claim 3:The PSR detection kits of H7, which is characterized in that further include as follows
Component：
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. Escherichia coli O 157 according to claim 4:The PSR detection kits of H7, which is characterized in that institute in component C
The mixed solution of the calcein and manganese chloride stated is to be 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 L1mM, obtains calcein
With the mixed solution of manganese chloride.
6. Escherichia coli O 157 according to claim 4:The PSR detection kits of H7, it is characterised in that：
The Bst archaeal dna polymerase aqueous solutions that Bst archaeal dna polymerases described in component B are a concentration of 8U/ μ L.
7. claim 2~6 any one of them Escherichia coli O 157:The PSR detection kits of H7 are in detection Escherichia coli
O157:Application in H7.
8. a kind of Escherichia coli O 157:The PSR detection methods of H7, 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
The Bst DNA of 0.8 μ L, 50 μM of acceleration primer I F and 50 μM of each 0.4 μ L of acceleration primer I B, 2.0 μ L, 8U/ μ L of DNA profiling are poly-
1.0 μ L of synthase, add water to 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, such as face
Color is yellow, illustrates not containing Escherichia coli O 157 in measuring samples:H7；If color becomes green, illustrate to contain in measuring samples
There is Escherichia coli O 157:H7；
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 accelerates the nucleotide sequence of primer I F as shown in SEQ ID NO.3 as shown in SEQ ID NO.2, accelerates the core of primer I B
Nucleotide sequence is as shown in SEQ ID NO.4.
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| CN104862394A (en) * | 2015-05-11 | 2015-08-26 | 昆明理工大学 | Primer for detecting Escherichia coli, and method and application of primer |
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| WO2020168950A1 (en) * | 2019-02-22 | 2020-08-27 | 华南理工大学 | Cpa primer for escherichia coli o157:h7, kit, and detection method |
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