CN103173536A - 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 PDF

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CN103173536A
CN103173536A CN2012105940194A CN201210594019A CN103173536A CN 103173536 A CN103173536 A CN 103173536A CN 2012105940194 A CN2012105940194 A CN 2012105940194A CN 201210594019 A CN201210594019 A CN 201210594019A CN 103173536 A CN103173536 A CN 103173536A
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probe
primer
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CN103173536B (en
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姜英辉
邵秀玲
张晓梅
雷质文
李正义
赵丽青
祝素珍
王建广
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Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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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

The nicking endonuclease nucleic acid isothermal amplification rapid detection kit of Listeria Monocytogenes
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 (Listeria monocytogenes) for the first time in the patient body, and it is a kind of important zoonosis and the pathogenic bacteria of 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, studies show that, the food poisoning that is caused by Listeria Monocytogenes mainly occurs in the middle of the higher food of water-activity, 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%.This bacterium is listed in 21 century in China affects one of 12 kinds of pathogenic micro-organisms of Chinese's health.In 2000, WHO food safety work program proposed Listeria Monocytogenes is classified 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 purpose of this invention is to provide a kind of Listeria Monocytogenes overcoming 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 is 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 template pretreatment reaction liquid, under the effect of Taq Platinum archaeal dna polymerase, 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 on nicking restriction endonuclease identification NEMA template, cut the formation otch on a nucleic acid chains therein, exposes its 3 ' end;
(4) under the effect of the Bst archaeal dna polymerase with strand displacement activity, take expose 3 ' hold as starting point, take not cut another nucleic acid chains as 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 simultaneously nicking restriction endonuclease recognition site on new synthetic double-strandednucleic acid;
The step of (5) 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 is completed, add the hybridization of probe and amplified production in reaction tubes.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 the Quality Control district, and T is detection zone.Check-out console red stripes all occurs at Quality Control district C and detection zone T, and positive result shows the target compound that contains detection in sample; Only red stripes occurs at Quality Control district C, negative result shows in sample for detecting target compound; All redfree band appearance in Quality Control district C and detection zone T show 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 the forward primer sequence is SEQ ID NO:1:
5-GCAAAACCTGGTGAT GCTCTTCTCTTTGACTATGGTAGCGGAAT-3; The reverse primer sequence is SEQ ID NO:2:
5-GCTGTCTAGATATA GCTCTTCGCCAGAGCCGTGGATGTTA-3。
Wherein in forward primer and reverse primer, underscore partly is 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.
The 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 that wherein extracts 260/ OD 280In 1.6～2.0 scopes, concentration is in 10～100ng/ μ L scope;
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added 2 μ L template DNAs to be checked, put into rapidly the water-bath 50s lower than 2 ℃ of inspection bacterium primer Tm value temperature after 94 ℃ of water-bath 7min; Then 94 ℃ of water-bath 20s and lower than 2 ℃ of water-bath 50s of inspection bacterium primer Tm value temperature, this process is carried out 5 repeatedly with cocycle; Product is used for the NEMA template;
(3) NEMA isothermal amplification reactions:
Add the 2 pretreated template DNAs of μ L in the reaction tubes that 46 μ L NEMA amplification reaction solutions are housed, 1.0 after μ L Nt.BspQI nicking restriction endonuclease and 1.0 μ L Bst archaeal dna polymerases, put into rapidly the water-bath 60min lower than 2 ℃ of inspection bacterium primer Tm value temperature.Reaction is taken out after finishing;
(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, detect product with universal nucleic acid amplified production fast testing plate.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 guarantees the specificity of detection method according to the primer of the conservative gene sequences Design 5 of bacterial strain to be checked ' end with nicking restriction endonuclease recognition sequence.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, only need common water-bath get final product, and result needn't observe with gel electrophoresis method, use the detection of universal nucleic acid amplified material fast testing plate to get final product, 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, usually condition routinely, condition described in " the molecular cloning experiment guide " write as J. Pehanorm Brooker (Sambrook) etc., or the condition operation of advising according to manufacturer.
Embodiment 1: to the detection of Listeria Monocytogenes reference culture
Make the nicking restriction endonuclease nucleic acid constant-temperature amplification kit of Listeria Monocytogenes by following formula:
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 primer, 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 (sterilization 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 partly is 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 (sterilization 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 partly is 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;
Detect according to following (1)-(4) program:
(1) extraction of sample Listeria Monocytogenes reference culture ATCC 7644DNA:
After Listeria Monocytogenes reference culture ATCC 7644 is increased the bacterium cultivation, extract sample nucleic acid to be checked, the sample DNA OD that wherein extracts 260/ OD 280In 1.6～2.0 scopes, concentration is in 10～100ng/ μ L scope.
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added 2 μ L template DNAs to be checked, put into rapidly the water-bath 50s of 60 ℃ after 94 ℃ of water-bath 7min; Then 94 ℃ of water-bath 20s and 60 ℃ of water-bath 50s processes are carried out 5 repeatedly with cocycle; Product is used for the NEMA template.
(3) carry out the NEMA isothermal amplification reactions:
Add the 2 pretreated template DNAs of μ L with being equipped with in the reaction tubes of 46 μ L NEMA isothermal amplification reactions liquid, 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 is taken out after finishing;
(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, detect product with universal nucleic acid amplified material fast testing plate.Red stripes all occurs at Quality Control district C and detection zone T, 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: to the detection of colon bacillus ATCC 25922
Make the nicking restriction endonuclease nucleic acid constant-temperature amplification kit of Listeria Monocytogenes by following formula:
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 primer, 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 (sterilization 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 partly is 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 (sterilization 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 partly is 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;
Detect according to following (1)-(4) program:
(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 that wherein extracts is in 1.6～2.0 scopes, and concentration is in 10～100ng/ μ L scope.
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added 2 μ L template DNAs to be checked, put into rapidly the water-bath 50s of 60 ℃ after 94 ℃ of water-bath 7min; Then 94 ℃ of water-bath 20s and 60 ℃ of water-bath 50s processes are carried out 5 repeatedly with cocycle; Product is used for the NEMA template.
(3) carry out the NEMA isothermal amplification reactions:
Add the 2 pretreated template DNAs of μ L with being equipped with in the reaction tubes of 46 μ L NEMA isothermal amplification reactions liquid, 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 is taken out after finishing;
(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, detect product with universal nucleic acid amplified material fast testing plate.Quality Control district C shows red stripes, and detection zone T redfree band occurs, and illustrates that sample to be checked is not Listeria Monocytogenes.This result has confirmed that also primer of the present invention and detection kit can not produce false positive when using.
Embodiment 3: to the detection of doubtful infection Listeria Monocytogenes food samples
Food samples is carried out extraction and the augmentation detection of DNA according to the method for embodiment 1 description, detect product with universal nucleic acid amplified material fast testing plate.The district C of Quality Control as a result shows red stripes, and detection zone T also has red stripes to occur, and 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 the detection of Listeria Monocytogenes to other sample.
Figure ISA00000834163500011
Figure ISA00000834163500021

Claims (6)

1. primer and the probe of the nicking restriction endonuclease nucleic acid constant-temperature amplification rapid detection of a Listeria Monocytogenes, wherein:
The forward primer sequence is SEQ ID NO:1;
The 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 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. primer claimed in claim 1 and probe are for detection of the Listeria Monocytogenes in food.
4. the nicking endonuclease nucleic acid isothermal amplification rapid detection kit of a Listeria Monocytogenes comprises following component:
(1) template pretreatment reaction liquid:
Comprising 10 * Taq Platinum buffer II reaction buffer, 0.1～1.0mmol/LdNTP, 0.1～1.0 μ mol/L forward primer, 0.1～1.0 μ mol/L reverse primer, Taq PlatinumDNA Polymerase 2.5U;
Described 10 * Taq Platinum buffer II reaction buffer composition is: 200mM Tris-HClpH8.8,100mM KCl, 100mM (NH 4) 2SO 4, 20mM MgCl 2
(2) NEMA isothermal amplification reactions liquid:
Comprise 10 * NE Buffer, 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;
Wherein said 10 * NEBuffer, 3 reaction buffer compositions are: 100mmol/L NaCl, 50mmol/L Tris-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 (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.
5. test kit claimed in claim 4 is for detection of the Listeria Monocytogenes in food.
6. test kit as claimed in claim 5, its using method comprises following step:
(1) extraction of sample to be checked or DNA of bacteria:
Extract sample nucleic acid to be checked, the sample DNA OD that wherein extracts 260/ OD 280In 1.6～2.0 scopes, concentration is in 10～100ng/ μ L scope;
(2) template pre-treatment:
The reaction tubes that 23 μ L template pretreatment reaction liquid are housed is added 2 μ L template DNAs to be checked, put into rapidly the water-bath 50s lower than 2 ℃ of inspection bacterium primer Tm value temperature after 94 ℃ of water-bath 7min; Then 94 ℃ of water-bath 20s and lower than 2 ℃ of water-bath 50s of inspection bacterium primer Tm value temperature, this process is carried out 5 repeatedly with cocycle.Product is used for the NEMA template;
(3) NEMA isothermal amplification reactions:
Add the 2 pretreated template DNAs of μ L in the reaction tubes that 46 μ L NEMA amplification reaction solutions are housed, 1.0 after μ L Nt.BspQI nicking restriction endonuclease and 1.0 μ L Bst archaeal dna polymerases, put into rapidly the water-bath 60min lower than 2 ℃ of inspection bacterium primer Tm value temperature, take out after reaction finishes;
(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, detect product with universal nucleic acid amplified production fast testing plate.
CN201210594019.4A 2012-12-21 2012-12-21 Kit for nicked-endonuclease-nucleic-acid-isothermal-amplification rapid detection of Escherichia coli Expired - Fee Related CN103173536B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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)

* Cited by examiner, † Cited by third party
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
Title
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》 *
王纪东: "切刻内切酶介导恒温扩增技术条件优化", 《军事医学》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

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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

COR Change of bibliographic data

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

C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20141008

Termination date: 20171221