CN103173537B - Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli O157:H7 - Google Patents
Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli O157:H7 Download PDFInfo
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- CN103173537B CN103173537B CN201210594142.6A CN201210594142A CN103173537B CN 103173537 B CN103173537 B CN 103173537B CN 201210594142 A CN201210594142 A CN 201210594142A CN 103173537 B CN103173537 B CN 103173537B
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Abstract
The invention relates to primers and probes for the nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli O157:H7, wherein a sequence of a forward primer is SEQ ID NO: 1, a sequence of a reverse primer is SEQ ID NO: 2, a sequence of a probe P1 is SEQ ID NO: 3, and a sequence of a probe P2 is SEQ ID NO: 4. Meanwhile, the invention further provides a kit, which contains the sequences of the primers and probes, for the isothermal-amplification rapid detection of Escherichia coli O157:H7 and an application method thereof. According to the kit provided by the invention, the primers and the probes are designed according to conserved gene sequences of strains to be detected, so that the specificity of a detection method is guaranteed. The detection method has sensitivity similar with that of PCR (Polymerase Chain Reaction) detection methods, an expensive PCR instrument is not required, and only an ordinary water bath is required. Results are not required for being observed by a gel electrophoresis method and are detected by using a universal nucleic acid amplimer rapid detection plate, so that the detection is simple, rapid, safe and pollution-free, and the kit is particularly applicable to food testing institutions.
Description
Technical field
The invention belongs to pathogenic microorganism detection technique field, be specifically related to a kind of nicking restriction endonuclease constant-temperature amplification (NEMA) technology of utilizing and carry out square test kit and the application thereof of the rapid detection of bacterium sample.
Background technology
Nineteen eighty-two, due to hamburger that colon bacillus O157:H7 (Escherichia coli O157:H7) pollutes, serious hemorrhagic diarrhea has been broken out once in the U.S., and since then, colon bacillus O157:H7 is confirmed to be pathogenic bacterium.Colon bacillus O157:H7 is the important foodborne bacterial pathogens in the whole world, and the main host such as infected patient and ox, sheep, pig are main contagium.Colon bacillus O157:H7 is a colibacillary hypotype, according to serum group system method, is decided to be the 157th member in O antigen family and gains the name by American scientist.Colon bacillus O157:H7 gently causes fever, stomachache, gastric disorder causing nausea, heavy occur the symptoms such as hemorrhagic diarrhea, oedema, even because of renal failure and many internal organs impaired and dead.1996, the serious epidemic situation being caused by colon bacillus O157:H7 was broken out in Japan, causes global concern.In recent years, colon bacillus O157:H7 infects in Asia, Europe, Africa, South America, Oceania happens occasionally.Some areas of China also once had epidemic situation to occur.At present, colon bacillus O157:H7 has become one of the main pathogenic fungi of countries in the world import and export meat poultry product detection.Therefore, need to set up a kind of effective detection method.
Summary of the invention
The nicking endonuclease nucleic acid isothermal amplification rapid detection kit and the detection method thereof that the object of this invention is to provide a kind of colon bacillus O157:H7, to overcome the deficiencies in the prior art, thereby provide foundation and the directive function of science for food safety.
Cardinal principle of the present invention is as follows:
(1) design a pair of 5 ' end with the primer of nicking restriction endonuclease recognition sequence and a pair of respectively by the probe of vitamin H and FITC mark;
(2) template DNA of primer and object bacteria is joined to template pretreatment reaction liquid, under the effect of Taq PlatinumDNA polysaccharase, by the working cycle of several denaturations and amplification, nicking restriction endonuclease recognition sequence is incorporated in sequence to be amplified, as the template of NEMA constant-temperature amplification;
(3) recognition sequence in nicking restriction endonuclease identification NEMA template, in a nucleic acid chains, cutting forms otch therein, exposes its 3 ' end;
(4) under the effect of Bst archaeal dna polymerase with strand displacement activity, take expose 3 ' hold as starting point, not cut another nucleic acid chains of take is template, synthetic new double-strandednucleic acid, old chain is stripped from and gets off to become the synthetic template of next round nucleic acid, recovers nicking restriction endonuclease recognition site on new synthetic double-strandednucleic acid simultaneously;
(5) step of cutting, extending and peeling off repeats, and amplifies a large amount of target nucleic acid sequences;
(6) amplified production utilizes universal nucleic acid amplified material fast testing plate to detect.
After nucleic acid amplification completes, in reaction tubes, add probe and amplified production hybridization.Check-out console is lain in a horizontal plane on operator's console, get hybridization product 10 μ L and add in the sample well of check-out console, then add 100 μ L damping fluids to carry out chromatography, 10min left and right sentence read result.
The upper C of universal nucleic acid amplified material fast testing plate is Quality Control district, and T is detection zone.All there is red stripes at Quality Control district C and detection zone T in check-out console, positive result shows the target compound that contains detection in sample; There is red stripes in Jin Quality Control district C, negative result shows in sample for detecting target compound; In Quality Control district C and detection zone T, all redfree band occurs, shows that fast testing plate lost efficacy.
One aspect of the present invention relates to primer and the probe of the nicking restriction endonuclease nucleic acid constant-temperature amplification rapid detection of colon bacillus O157:H7, and wherein forward primer sequence is SEQ ID NO:1:
5-AATAGAATAGTTA
GCTCTTCGTCTGGACTCAACGTGGATTT-3
Reverse primer sequence is SEQ ID NO:2:
5-CTAACATAGCTAA
GCTCTTCGCAACCGTTCCATTACTTACA-3
Wherein in forward primer and reverse primer, underscore is partly nicking restriction endonuclease recognition site;
The sequence of P1 probe is SEQ ID NO:3:
P1:5-TGGACTCAACGTGGATTTCATCAAAA-3 5 end mark vitamin Hs;
The sequence of P2 probe is SEQ ID NO:4:
P2:5-TATGCAACTACTGTAAGTAATGGAACGG-3 3 end flag F ITC.
Another aspect of the present invention relates to the detection kit of a kind of colon bacillus O157:H7, comprises following component:
(1) template pretreatment reaction liquid:
Comprising 10 * Taq Platinum buffer II reaction buffer, 0.1～1.0mmol/L dNTP, 0.1～1.0 μ mol/L forward primer, 0.1～1.0 μ mol/L reverse primer, Taq Platinum DNA Polymerase2.5U;
Described 10 * Taq Platinum buffer II reaction buffer composition is: 200mM Tris-HCl pH8.8,100mM KCl, 100mM (NH
4)
2sO
4, 20mM MgCl
2;
(2) NEMA isothermal amplification reactions liquid:
Comprise 10 * NEBuffer, 3 reaction buffers, 0.1～1.0mmol/L dNTP, 5%～30%DMSO, 0.1～1.0 μ mol/L forward primer, 0.1～1.0 μ mol/L reverse primer and 100 μ g/mL bovine serum albumins (BSA);
Wherein said 10 * NEBuffer, 3 reaction buffer compositions are: 100mmol/L NaCl, 50mmol/LTris-HCl, 10mmol/L MgCl
2, 1mmol/L dithiothreitol pH 7.9;
(3) Nt.BspQI nicking restriction endonuclease: 4U/ μ L;
(4) Bst archaeal dna polymerase: 2U/ μ L;
Each 10 μ mol/L of probe P2 of (5) 5 ' end mark biotinylated probes P1 and 3 ' end flag F ITC
(6) universal nucleic acid amplified material fast testing plate.Model: No. 3.
Mentioned reagent box is applied to detect the colon bacillus O157:H7 in food, and its using method comprises the following steps (1)-(4) successively:
(1) extraction of sample to be checked or DNA of bacteria:
Extract sample nucleic acid to be checked, the sample DNA OD wherein extracting
260/ OD
280in 1.6～2.0 scopes, concentration is within the scope of 10～100ng/ μ L;
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added to 2 μ L template DNA to be checked, after 94 ℃ of water-bath 7min, put into rapidly the water-bath 50s lower than 2 ℃ of examined bacterium primer Tm value temperature; Then 94 ℃ of water-bath 20s and lower than 2 ℃ of water-bath 50s of examined bacterium primer Tm value temperature, this process is carried out 5 repeatedly with cocycle; Product is for NEMA template;
(3) NEMA isothermal amplification reactions:
In the reaction tubes that 46 μ L NEMA amplification reaction solutions are housed, add the pretreated template DNA of 2 μ L, after 1.0 μ L Nt.BspQI nicking restriction endonucleases and 1.0 μ L Bst archaeal dna polymerases, put into rapidly the water-bath 60min lower than 2 ℃ of examined bacterium primer Tm value temperature.Reaction finishes rear taking-up;
(4) product detects:
After reaction finishes, add each 0.5 μ L of probe P1 and P2 in reaction tubes, 90 ℃ of water-bath 3min naturally cool to constant temperature, with universal nucleic acid amplified production fast testing plate, detect product.Detection line T and nature controlling line C all show redness, illustrate that sample to be checked is positive.
Test kit of the present invention is the primer with nicking restriction endonuclease recognition sequence according to the conservative gene sequences Design 5 of bacterial strain to be checked ' end, guarantees the specificity of detection method.The present invention adopts improved nicking restriction endonuclease nucleic acid constant-temperature amplification (NEMA) technology, this technology high specificity, have and the similar sensitivity of PCR detection method, and do not need expensive PCR instrument, the only common water-bath of need, and result needn't be observed with gel electrophoresis method, uses universal nucleic acid amplified material fast testing plate to detect, simply, fast, safe, pollution-free, be specially adapted to food inspection mechanism.
Embodiment
Below in conjunction with example, method of the present invention is described further, but example only limits to explanation, be not limited to this, the experimental technique of unreceipted actual conditions wherein, conventionally condition routinely, condition described in the < < molecular cloning experiment guide > > writing as J. Pehanorm Brooker (Sambrook) etc., or the condition of advising according to manufacturer operation.
Embodiment 1: the detection to colon bacillus O157:H7 reference culture
By following formula, make the nicking restriction endonuclease nucleic acid constant-temperature amplification kit of colon bacillus O157:H7:
1) template pretreatment reaction liquid:
Every 23 μ L contain 2.5 μ L 10 * Taq Platinum buffer II, 1.0 μ L 2.5mmol/L dNTP, 1.0 μ L10 μ mol/L forward primers, 1.0 μ L 10 μ mol/L reverse primers, 1.0 μ L 2.5U/ μ L Taq Platinum DNAPloymerase and 16.5 μ L ddH
2o (sterilizing distilled water).
Wherein said forward primer SEQ ID NO:1:
5-AATAGAATAGTTA
GCTCTTCGTCTGGACTCAACGTGGATTT-3
Reverse primer sequence is SEQ ID NO:2:
5-CTAACATAGCTAA
GCTCTTCGCAACCGTTCCATTACTTACA-3
Wherein in forward primer and reverse primer, underscore is partly nicking restriction endonuclease recognition site;
2) NEMA isothermal amplification reactions liquid:
Every 46 μ L contain 5 μ L 10 * NEBuffer 3,1.0 μ L 2.5mmol/L dNTP, 1.0 μ L 10 μ mol/L forward primers, 1.0 μ L 10 μ mol/L reverse primers, 0.5 μ L 10mg/ml BSA, 4 μ L DMSO and 33.5 μ L ddH
2o (sterilizing distilled water).
Wherein said forward primer SEQ ID NO:1:
5-AATAGAATAGTTA
GCTCTTCGTCTGGACTCAACGTGGATTT-3
Reverse primer sequence is SEQ ID NO:2:
5-CTAACATAGCTAA
GCTCTTCGCAACCGTTCCATTACTTACA-3
Wherein in forward primer and reverse primer, underscore is partly nicking restriction endonuclease recognition site;
3) Nt.BspQI nicking restriction endonuclease: 4U/ μ L;
4) Bst archaeal dna polymerase: 2U/ μ L;
5) probe P1 and P2:10 μ mol/L;
The sequence of wherein said probe P1 is SEQ ID NO:3
P1:5-TGGACTCAACGTGGATTTCATCAAAA-3 5 end mark vitamin Hs;
The sequence of P2 probe is SEQ ID NO:4:
P2:5-TATGCAACTACTGTAAGTAATGGAACGG-3 3 end flag F ITC.
6) nucleic acid thin-layer chromatography check-out console.Model: No. 3;
According to following (1)-(4) program, detect:
(1) extraction of sample colon bacillus O157:H7 reference culture ATCC 35150DNA:
Colon bacillus O157:H7ATCC 35150 is increased after bacterium cultivation, extract sample nucleic acid to be checked, the sample DNA OD wherein extracting
260/ OD
280in 1.6～2.0 scopes, concentration is within the scope of 10～100ng/ μ L.
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added to 2 μ L template DNA to be checked, after 94 ℃ of water-bath 7min, put into rapidly the water-bath 50s of 60 ℃; Then 94 ℃ of water-bath 20s and 60 ℃ of water-bath 50s processes are carried out 5 repeatedly with cocycle; Product is for NEMA template.
(3) carry out NEMA isothermal amplification reactions:
By being equipped with in the reaction tubes of 46 μ L NEMA isothermal amplification reactions liquid, add the pretreated template DNA of 2 μ L, after 1.0 μ L Nt.BspQI nicking restriction endonucleases and 1.0 μ L Bst archaeal dna polymerases, put into rapidly the water-bath 60min of 57 ℃.Reaction finishes rear taking-up;
(4) product detects:
After reaction finishes, add each 0.5 μ L of probe P1 and P2 in reaction tubes, 90 ℃ of water-bath 3min naturally cool to constant temperature, with universal nucleic acid amplified material fast testing plate, detect product.At Quality Control district C and detection zone T, all there is red stripes, illustrate that sample to be checked is colon bacillus O157:H7, and prove that primer of the present invention and probe can not produce false negative when detecting.
Embodiment 2: the detection to colon bacillus ATCC 25922
By following formula, make the nicking restriction endonuclease nucleic acid constant-temperature amplification kit of colon bacillus O157:H7:
1) template pretreatment reaction liquid:
Every 23 μ L contain 2.5 μ L 10 * Taq Platinum buffer II, 1.0 μ L 2.5mmol/L Dntp, 1.0 μ L 10 μ mol/L forward primers, 1.0 μ L 10 μ mol/L reverse primers, 1.0 μ L 2.5U/ μ L Taq Platinum DNAPloymerase and 16.5 μ L ddH
2o (sterilizing distilled water).
Wherein said forward primer SEQ ID NO:1:
5-AATAGAATAGTTA
GCTCTTCGTCTGGACTCAACGTGGATTT-3
Reverse primer sequence is SEQ ID NO:2:
5-CTAACATAGCTAA
GCTCTTCGCAACCGTTCCATTACTTACA-3
Wherein in forward primer and reverse primer, underscore is partly nicking restriction endonuclease recognition site;
2) NEMA isothermal amplification reactions liquid:
Every 46 μ L contain 5 μ L 10 * NEBuffer 3,1.0 μ L 2.5mmol/L dNTP, 1.0 μ L 10 μ mol/L forward primers, 1.0 μ L 10 μ mol/L reverse primers, 0.5 μ L 10mg/ml BSA, 4 μ L DMSO and 33.5 μ L ddH
2o (sterilizing distilled water).
Wherein said forward primer SEQ ID NO:1:
5-AATAGAATAGTTA
GCTCTTCGTCTGGACTCAACGTGGATTT-3
Reverse primer sequence is SEQ ID NO:2:
5-CTAACATAGCTAA
GCTCTTCGCAACCGTTCCATTACTTACA-3
Wherein in forward primer and reverse primer, underscore is partly nicking restriction endonuclease recognition site;
3) Nt.BspQI nicking restriction endonuclease: 4U/ μ L;
4) Bst archaeal dna polymerase: 2U/ μ L;
5) probe P1 and P2:10 μ mol/L;
The sequence of wherein said probe P1 is SEQ ID NO:3
P1:5-TGGACTCAACGTGGATTTCATCAAAA-3 5 end mark vitamin Hs;
The sequence of P2 probe is SEQ ID NO:4:
P2:5-TATGCAACTACTGTAAGTAATGGAACGG-3 3 end flag F ITC.
6) nucleic acid thin-layer chromatography check-out console.Model: No. 3;
According to following (1)-(4) program, detect:
(1) extraction of sample to be checked (colon bacillus ATCC 25922) DNA:
Extract sample colon bacillus ATCC to be checked 25922 nucleic acid, the sample DNA OD260/OD280 wherein extracting is in 1.6～2.0 scopes, and concentration is within the scope of 10～100ng/ μ L.
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added to 2 μ L template DNA to be checked, after 94 ℃ of water-bath 7min, put into rapidly the water-bath 50s of 60 ℃; Then 94 ℃ of water-bath 20s and 60 ℃ of water-bath 50s processes are carried out 5 repeatedly with cocycle; Product is for NEMA template.
(3) carry out NEMA isothermal amplification reactions:
By being equipped with in the reaction tubes of 46 μ L NEMA isothermal amplification reactions liquid, add the pretreated template DNA of 2 μ L, after 1.0 μ L Nt.BspQI nicking restriction endonucleases and 1.0 μ L Bst archaeal dna polymerases, put into rapidly the water-bath 60min of 57 ℃.Reaction finishes rear taking-up;
(4) product detects:
After reaction finishes, add each 0.5 μ L of probe P1 and P2 in reaction tubes, 90 ℃ of water-bath 3min naturally cool to constant temperature, with universal nucleic acid amplified material fast testing plate, detect product.Quality Control district C shows red stripes, and detection zone T redfree band occurs, illustrates that sample to be checked is not colon bacillus O157:H7.This result has also confirmed that primer of the present invention and detection kit can not produce false positive in application.
Embodiment 3: the detection to doubtful infection colon bacillus O157:H7 food samples
The method that food samples is described according to embodiment 1 is carried out extraction and the augmentation detection of DNA, with universal nucleic acid amplified material fast testing plate, detects product.Result Quality Control district C shows red stripes, and detection zone T also has red stripes to occur, illustrates that sample to be checked has been subject to the infection of colon bacillus O157:H7.
Primer of the present invention, probe and test kit can also carry out to other sample the detection of colon bacillus O157:H7.
Claims (2)
1. primer and the probe of the nicking restriction endonuclease nucleic acid constant-temperature amplification rapid detection of colon bacillus O157:H7, wherein:
Forward primer sequence is SEQ ID NO:1;
Reverse primer sequence is SEQ ID NO:2;
The sequence of P1 probe is SEQ ID NO:3:
The sequence of P2 probe is SEQ ID NO:4.
2. primer and the probe of the nicking restriction endonuclease nucleic acid constant-temperature amplification rapid detection of colon bacillus O157:H7 as claimed in claim 1, is characterized in that 5 of described P1 probe ' end mark vitamin H, 3 of P2 probe ' end flag F ITC.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210594142.6A CN103173537B (en) | 2012-12-21 | 2012-12-21 | Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli O157:H7 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210594142.6A CN103173537B (en) | 2012-12-21 | 2012-12-21 | Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli O157:H7 |
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| Publication Number | Publication Date |
|---|---|
| CN103173537A CN103173537A (en) | 2013-06-26 |
| CN103173537B true CN103173537B (en) | 2014-09-10 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101952459A (en) * | 2007-07-14 | 2011-01-19 | 爱奥尼安技术公司 | Nicking and extension amplification reaction for the exponential amplification of nucleic acids |
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2012
- 2012-12-21 CN CN201210594142.6A patent/CN103173537B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101952459A (en) * | 2007-07-14 | 2011-01-19 | 爱奥尼安技术公司 | Nicking and extension amplification reaction for the exponential amplification of nucleic acids |
Non-Patent Citations (2)
| Title |
|---|
| Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA;Penghua Zhang ET AL;《Protein Expression and Purification》;20090909;第226－234页 * |
| Penghua Zhang ET AL.Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA.《Protein Expression and Purification》.2009,第226－234页. |
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Inventor after: Zhang Xiaomei Inventor after: Jiang Yinghui Inventor after: Wei Xiaotang Inventor after: Jia Juntao Inventor after: Shao Xiuling Inventor after: Lei Zhiwen Inventor after: Wang Jianguang Inventor before: Zhang Xiaomei Inventor before: Shao Xiuling Inventor before: Jiang Yinghui Inventor before: Lei Zhiwen Inventor before: Wang Jianguang |
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Free format text: CORRECT: INVENTOR; FROM: ZHANG XIAOMEI SHAO XIULING JIANG YINGHUI LEI ZHIWEN WANG JIANGUANG TO: ZHANG XIAOMEI JIANG YINGHUI WEI XIAOTANG JIA JUNTAO SHAO XIULING LEI ZHIWEN WANG JIANGUANG |
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Granted publication date: 20140910 Termination date: 20171221 |