CN103173536B - Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli - Google Patents
Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli Download PDFInfo
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- CN103173536B CN103173536B CN201210594019.4A CN201210594019A CN103173536B CN 103173536 B CN103173536 B CN 103173536B CN 201210594019 A CN201210594019 A CN 201210594019A CN 103173536 B CN103173536 B CN 103173536B
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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, 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 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 twenty-nine, Nyfeldt isolates Listeria Monocytogenes (Listeriamonocytogenes) for the first time in patient body, and it is the pathogenic bacteria of a kind of important zoonosis and food origin disease.The sickness rate of listeriosis is very low, but lethality rate up to 20%～30%, in the crowd of newborn infant and immunocompromised more up to 70%.Listeria Monocytogenes is widely distributed at occurring in nature, research shows, the food poisoning being caused by Listeria Monocytogenes mainly occurs in the middle of the food that water-activity is higher, for example, and meat, bird, milk and milk products, fishery products etc.Wherein, the pollution rate of milk-product is 5%～10%, and the pollution rate of meat and meat product is 30%, and the pollution rate of poultry is 15%, and the pollution rate of fishery products is 4%～8%.Gai Jun China is listed in 21 century affects one of 12 kinds of pathogenic micro-organisms of Chinese's health.In 2000, WHO food safety work program proposed Listeria Monocytogenes to classify as one of food-borne pathogens of emphasis prosecution.Therefore, need to set up a kind of effective Listeria Monocytogenes 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 Listeria Monocytogenes, 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 Listeria Monocytogenes, and wherein forward primer sequence is SEQ ID NO:1:
5-GCAAAACCTGGTGAT
gCTCTTCtCTTTGACTATGGTAGCGGAAT-3; Reverse primer sequence is SEQ ID NO:2:
5-GCTGTCTAGATATA
GCTCTTCGCCAGAGCCGTGGATGTTA-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-TGACTATGGTAGCGGAATTTCTCACG-3 5 end mark vitamin Hs;
The sequence of P2 probe is SEQ ID NO:4:
P2:5-GTTAAATACGATAACATCCACGGCTCTG-3 3 end flag F ITC.
Another aspect of the present invention relates to a kind of detection kit of Listeria Monocytogenes, 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 Listeria Monocytogenes 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 Listeria Monocytogenes reference culture
By following formula, make the nicking restriction endonuclease nucleic acid constant-temperature amplification kit of Listeria Monocytogenes:
1) template pretreatment reaction liquid:
Every 23 μ L contain 2.5 μ L 10 * Taq Platinum bufferII, 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-GCAAAACCTGGTGAT
GCTCTTCTCTTTGACTATGGTAGCGGAAT-3；
Reverse primer: SEQ ID NO:2:
5-GCTGTCTAGATATA
GCTCTTCGCCAGAGCCGTGGATGTTA-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-GCAAAACCTGGTGAT
GCTCTTCTCTTTGACTATGGTAGCGGAAT-3；
Reverse primer: SEQ ID NO:2:
5-GCTGTCTAGATATA
GCTCTTCGCCAGAGCCGTGGATGTTA-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
5-TGACTATGGTAGCGGAATTTCTCACG-3 5 end mark vitamin Hs;
The sequence of probe P2 is SEQ ID NO:4:
5-GTTAAATACGATAACATCCACGGCTCTG-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 Listeria Monocytogenes reference culture ATCC 7644DNA:
Listeria Monocytogenes reference culture ATCC 7644 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 Listeria Monocytogenes, 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 Listeria Monocytogenes:
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-GCAAAACCTGGTGAT
GCTCTTCTCTTTGACTATGGTAGCGGAAT-3；
Reverse primer: SEQ ID NO:2:
5-GCTGTCTAGATATA
GCTCTTCGCCAGAGCCGTGGATGTTA-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-GCAAAACCTGGTGAT
GCTCTTCTCTTTGACTATGGTAGCGGAAT-3；
Reverse primer: SEQ ID NO:2:
5-GCTGTCTAGATATA
GCTCTTCGCCAGAGCCGTGGATGTTA-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
5-TGACTATGGTAGCGGAATTTCTCACG-3 5 end mark vitamin Hs;
The sequence of probe P2 is SEQ ID NO:4:
5-GTTAAATACGATAACATCCACGGCTCTG-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 Listeria Monocytogenes.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 Listeria Monocytogenes 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 Listeria Monocytogenes.
Primer of the present invention, probe and test kit can also carry out to other sample the detection of Listeria Monocytogenes.
Claims (4)
1. primer and the probe of the nicking restriction endonuclease nucleic acid constant-temperature amplification rapid detection of Listeria Monocytogenes, wherein:
Forward primer sequence be SEQ ID NO:1,
Reverse primer sequence be SEQ ID NO:2,
The sequence of P1 probe be 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 Listeria Monocytogenes 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.
3. a nicking endonuclease nucleic acid isothermal amplification rapid detection kit for Listeria Monocytogenes, 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 * NE Buffer3 reaction buffer, 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;
Wherein said 10 * NEBuffer3 reaction buffer composition is: 100mmol/L NaCl, 50mmol/L Tris-HCl, 10mmol/L MgCl
2, 1mmol/L dithiothreitol pH7.9;
(3) Nt.BspQI nicking restriction endonuclease: 4U/ μ L;
(4) Bst archaeal dna polymerase: 2U/ μ L;
Each 10 μ mol/L of (5) 5 ' end biotinylated probe P1 and 3 end FITC label probe P2
(6) universal nucleic acid amplified material fast testing plate;
Wherein the sequence of forward primer, reverse primer and probe P1, P2 as claimed in claim 1.
4. test kit claimed in claim 3 is for detection of the Listeria Monocytogenes in food.
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|---|---|---|---|
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| CN201210594019.4A CN103173536B (en) | 2012-12-21 | 2012-12-21 | Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli |
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| CN108410952A (en) * | 2018-05-11 | 2018-08-17 | 重庆出入境检验检疫局检验检疫技术中心 | The sandwich DNA hybridization of Listeria Monocytogenes, which quickly detects, uses probe, kit and detection method |
Citations (2)
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|---|---|---|---|---|
| WO2007140506A1 (en) * | 2006-06-02 | 2007-12-13 | Human Genetic Signatures Pty Ltd | Modified microbial nucleic acid for use in detection and analysis of microorganisms |
| CN102373284A (en) * | 2011-11-17 | 2012-03-14 | 山东出入境检验检疫局检验检疫技术中心 | Kit capable of quickly detecting amplification of incising incision enzyme nucleic acid of salmonella at constant temperature |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007140506A1 (en) * | 2006-06-02 | 2007-12-13 | Human Genetic Signatures Pty Ltd | Modified microbial nucleic acid for use in detection and analysis of microorganisms |
| CN102373284A (en) * | 2011-11-17 | 2012-03-14 | 山东出入境检验检疫局检验检疫技术中心 | Kit capable of quickly detecting amplification of incising incision enzyme nucleic acid of salmonella at constant temperature |
Non-Patent Citations (2)
| Title |
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| 王建广等.单核细胞增生李斯特氏菌依赖解旋酶DNA恒温扩增检测方法的建立.《中国预防兽医学报》.2011,第33卷(第2期),130-131. * |
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Inventor after: Jiang Yinghui Inventor after: Zhang Xiaomei Inventor after: Shao Xiuling Inventor after: Lei Zhiwen Inventor after: Li Zhengyi Inventor after: Zhao Liqing Inventor after: Zhu Suzhen Inventor after: Wang Jianguang Inventor before: Jiang Yinghui Inventor before: Shao Xiuling Inventor before: Zhang Xiaomei Inventor before: Lei Zhiwen Inventor before: Li Zhengyi Inventor before: Zhao Liqing Inventor before: Zhu Suzhen Inventor before: Wang Jianguang |
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Free format text: CORRECT: INVENTOR; FROM: JIANG YINGHUI SHAO XIULING ZHANG XIAOMEI LEI ZHIWEN LI ZHENGYI ZHAO LIQING ZHU SUZHEN WANG JIANGUANG TO: JIANG YINGHUI ZHANG XIAOMEI SHAO XIULING LEI ZHIWEN LI ZHENGYI ZHAO LIQING ZHU SUZHEN WANG JIANGUANG |
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