CN101307351A - Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof - Google Patents
Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof Download PDFInfo
- Publication number
- CN101307351A CN101307351A CNA2008100277593A CN200810027759A CN101307351A CN 101307351 A CN101307351 A CN 101307351A CN A2008100277593 A CNA2008100277593 A CN A2008100277593A CN 200810027759 A CN200810027759 A CN 200810027759A CN 101307351 A CN101307351 A CN 101307351A
- Authority
- CN
- China
- Prior art keywords
- liquid
- reaction
- listeria monocytogenes
- sample
- reagent box
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a mononuclear hyperplasia Listeria monocytogenes gene quick diagnosis reagent box based on a loop-mediated isothermal amplification technology and a method for detecting the same. The reagent box consists of two pairs of primers, DNA polymerase, reaction liquid, sample pretreatment liquid, developing liquid and a positive contrast solution, and the six kinds of liquid are respectively contained in a container. The gene quick diagnosis reagent box adopts six segments and four primers, and can judge whether a target substance exists or not according to the fact whether amplification occurs or not, thereby having high specificity. Moreover, the reagent box has quickness, high efficiency and high sensitivity, and can carry out amplification reaction just at a constant temperature without adopting special reagent and equipment. In addition, the reagent box can realize simple identification; therefore, pyrophosphate radical ion separated out from dNTP is combined with Mg<2+> in a reaction solution so as to generate byproduct magnesium pyrophosphate precipitate which can be identified through unaided viewing; moreover, after the developing liquid is added, the remarkable positive and negative result color developing difference is more obvious and reliable.
Description
Technical field
The present invention relates to biological detection reagent, be specifically related to a kind of Listeria monocytogenes rapid diagnostic kit and detection method thereof based on loop-mediated isothermal amplification technique.
Background technology
At present listeria monocytogenes there is multiple detection method, from being accredited as main national standard (GB/T4789.7-2003) with pathogenic micro-organism isolation identification, morphology evaluation and automatic biochemical, immunology detection technology, nucleic acid probe, polymerase chain reaction (PCR) technology equimolecular biological detection method [food safety detection and modern biotechnology to differential protein, Chemical Industry Press, 2004].Wherein the cause of disease detection of nucleic acids all improves a lot at aspects such as rapidity, security, accuracy and susceptibilitys, these new technologies are attempted to break through microbiologies such as traditional morphology, biochemical reaction and are detected old model, do not need microorganism is separated purification, and directly its gene and gene product are carried out rapid detection with the enrichment liquid of sample or sample, and combine with Protocols in Molecular Biology and information biology means, to accurately, fast, the direction of sensitivity and automatization develops.
With polymerase chain reaction (PCR) technology is that the cause of disease nucleic acid detection technique of representative also exists some problems in actual applications, as the special instrument of common polymerase chain reaction (PCR) Technology Need, and there are easy crossed contamination and the loaded down with trivial details shortcoming of operating process.And fluorescence real-time quantitative polymerase chain reaction (real time PCR) though technology has solved the problem of crossed contamination preferably, and has simplified operating process, needs more complicated quantitative assay instrument, therefore is not suitable for field quick detection.And the cost of fluorescent probe is higher in the real-time quantitative polymerase chain reaction PCR technology, has strengthened the difficulty of applying.The immunology detection technology is fast and convenient with low cost, but requires the monoclonal antibody of high quality high stability, otherwise because of accuracy is not enough, can only be the auxiliary detection means at present.So in time use the newest fruits of biotech development significant to satisfying improving constantly of pathogenic micro-organism detection requirement.Wherein isothermal duplication (Isothermal Amplification) nucleic acid Fast Detection Technique is the rapid progress on the cause of disease nucleic acid detection technique, the loop-mediated isothermal amplification technique of now having set up (LAMP) has a lot of superiority, and does not also see that useful loop-mediated isothermal amplification technique detects monokaryon hyperplasia Liszt's gene quick diagnosis kit at present.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of detect cost low, easy to use, detect rapidly and efficiently, highly sensitive Listeria monocytogenes rapid diagnostic kit based on loop-mediated isothermal amplification technique, this test kit is based on loop-mediated isothermal amplification technique and detects listeria monocytogenes.
Another object of the present invention provides the detection method of above-mentioned Listeria monocytogenes rapid diagnostic kit.
Above-mentioned purpose of the present invention is achieved by following technical solution:
One, Listeria monocytogenes rapid diagnostic kit of the present invention, form by two pairs of primers, Bst archaeal dna polymerase, reaction solution, stable liquid, sample pretreatment liquid, colour developing liquid and positive control solution, more than six kinds of liquid place container respectively, wherein:
Described two pairs of primers are:
Outer primer F3:ACAAGACTTCACCAATCCA is shown in SEQ ID NO:1;
Outer primer B3:GTCTTTTAAGTGGAGTAAACCTT is shown in SEQ ID NO:2;
Inner primer FIP:TAAGTCTCTTTGCAATTGACCGACTTTTACGTGTACACAGAAAAGCG is shown in SEQ ID NO:3;
Inner primer BIP:CCTGTGCCAAAGCATTTTTACATTTTTTAGGCAAGTCATCTTGTTCG is shown in SEQ ID NO:4;
Above-mentioned reaction solution contains 1.6～2mmol/L dNTP, 20～25mmol/L Tris-HCl, 10～12.5mmol/L Repone K, 10～12.5mmol/L ammonium sulfate, 8～10mmol/L sal epsom, 0.1～0.125%TritonX-100,0.8～1mol/L trimethyl-glycine, each 1.6～2mol/L of inner primer FIP/BIP and each 0.2～0.25mol/L of outer primer F3/B3.Preferred ratio is: reaction solution contains 2mmol/L dNTP, 25mmol/L Tris-Cl, 12.5mmol/L Repone K, 12.5mmol/L ammonium sulfate, 10mmol/L sal epsom, 0.125%TritonX-100,1mol/L trimethyl-glycine, each 2mol/L of inner primer FIP/BIP and each 0.25mol/L of outer primer F3/B3.(0.1～0.125%TritonX-100 is: the volume percent that Triton X-100 accounts for reaction solution is 0.1～0.125%)
Above-mentioned sample pretreatment liquid contains Tris-HCl, 1～2mmol/L EDTA and 1～1.2% Triton X-100 of 10～20mmol/L pH 8.0.Preferred ratio is: sample pretreatment liquid contains Tris-HCl, 2mmol/L EDTA and 1.2% TritonX-100 of 20mmol/L pH 8.0.(1～1.2%Triton X-100 is: the volume percent that Triton X-100 accounts for sample pretreatment liquid is 1～1.2%)
Above-mentioned positive control is Listeria monocytogenes group DNA.
Above-mentioned colour developing liquid is preferably SYBR Green I.
Aforementioned stable liquid is preferably paraffin oil.
Two, the production technique of gene quick diagnosis kit of the present invention
1, with behind inner primer FIP/BIP and the outer primer F3/B3 synthesizing and purifying, quantitatively preparation, concentration detects, the sampling quality inspection;
2, reaction solution is aseptic subpackaged, and determine the quality inspection of sampling with carrying out concentration according to experiment;
3, stable liquid is aseptic subpackaged, the sampling quality inspection;
4, with the positive control sample preparations, packing, sampling quality inspection;
5, assembling test kit.
Three, the detection method of gene quick diagnosis kit of the present invention
1, sample preparation:
Testing sample is centrifugal in centrifuge tube, remove supernatant, add sample pretreatment liquid in the precipitation and mix, cooled on ice after the boiling water bath deactivation, behind the high speed centrifugation, supernatant is stand-by sample template DNA;
2, loop-mediated isothermal amplification technique reaction process
In reaction tubes, add reaction solution 38～40 volume %, Bst archaeal dna polymerase 0.9～1.8 volume %, stable liquid 52～54.5 volume %, sample template DNA4.5～9 volume %, 63～65 ℃ of isothermal reaction 45～90min.Described volume percent is meant the volume percent that accounts for four component cumulative volumes.
3, post-reaction treatment
In above-mentioned reaction tubes and positive controls, add colour developing liquid respectively, mixing, the sample sets colour developing is identical with control group then positive, otherwise negative.
The present invention is said based on loop-mediated isothermal amplification technique (loop-mediated isotherma1 amplication of DNA, abbreviation LAMP) method of rapid detection listeria monocytogenes, be utilize the Bst archaeal dna polymerase and according to two couple of target-gene sequence design special in, outer primer (being inner primer FIP/BIP and outer primer F3/B3), six isolated areas on the specific recognition target sequence, start the endless chain replacement(metathesis)reaction, start complementary strand in target DNA district synthetic, and result's complementary sequence on same chain goes round and begins again and is formed with the very stem of polycyclic Cauliflower structure-circular DNA mixture.In the LAMP reaction process, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product---magnesium pyrophosphate milky white precipitate, can be by the visual inspection result of determination.The LAMP reaction is to finish in 45～90 minutes under constant temperature (63～65 ℃) condition.This comparatively gentle temperature condition and do not have temperature cycle that required instrument is oversimplified, overcome conventional P CR inherent detection time long, pollute easily and detect shortcoming such as cost height.In addition, this detection method is lower to testing staff's technical quality requirement, and actually operating is very easy, does not need special reagent and plant and instrument, helps setting up rapid screening system with low cost.The LAMP method is a kind of gene amplification method of easy, quick, high degree of specificity.Constant temperature gene amplification technology and round pcr (comprising the fluorescence real-time quantitative PCR technology) are compared, can find that this technology is equivalent to or is better than round pcr on methodology indexs such as sensitivity, specificity and sensing range, and do not rely on any special plant and instrument and can realize on-the-spot high-throughput rapid detection, and detect cost far below fluorescent quantitative PCR technique.Domestic do not have the test kit of this respect to sell at present as yet.At present in the national standard with microorganism separation and Culture and morphology be accredited as main, in conjunction with the passing method that biochemical analysis and serological typing are identified, preliminary evaluation needs 2～3 days, finishes probation report and needs 10～15 days; Adopt gene quick diagnosis kit of the present invention only to need 2 hours.And, having added colour developing liquid in the reaction solution of the present invention, qualification result is more visual and clear.
Compared with prior art, the present invention has following beneficial effect: 1. gene quick diagnosis kit of the present invention only needs just energy amplified reaction of a steady temperature, does not need special reagent and equipment, and it is low to detect cost; 2. gene quick diagnosis kit of the present invention is used six sections, four primers, and whether the existence that just can judge target substance according to whether increasing to be, so has high specific; 3. gene quick diagnosis kit of the present invention amplification fast and efficient can be finished amplification less than 1 hour, and the productive rate height; 4. gene quick diagnosis kit of the present invention is highly sensitive, and amplification template only needs 10 copies or still less; 5. gene quick diagnosis kit of the present invention is identified easy, pyrophosphate ion of separating out from dNTP and the Mg the reaction soln
2+In conjunction with, produce by product-magnesium pyrophosphate precipitation, can identify by visual inspection, and after adding colour developing liquid, yin and yang attribute colour development difference as a result is remarkable, checking rate height, more obviously reliable.
Embodiment
The preparation of embodiment 1 test kit
(1) in the following sequence through the synthetic oligomerization picodna primer of dna synthesizer:
Outer primer F3:ACAAGACTTCACCAATCCA is shown in SEQ ID NO:1;
Outer primer B3:GTCTTTTAAGTGGAGTAAACCTT is shown in SEQ ID NO:2;
Inner primer FIP:TAAGTCTCTTTGCAATTGACCGACTTTTACGTG TACACAGAAAAGCG is shown in SEQ ID NO:3;
Inner primer BIP:CCTGTGCCAAAGCATTTTTACATTTTTTAGGCAAGTCATCTTGTTCG is shown in SEQ ID NO:4;
(2) purchase archaeal dna polymerase: the BstDNA polysaccharase places container.
(3) preparation reaction solution: reaction solution contains 2mmol/LdNTP, 25mmol/L Tris-Cl, 12.5mmol/L Repone K, 12.5mmol/L ammonium sulfate, 10mmol/L sal epsom, 0.125 volume %TritonX-100,1mol/L trimethyl-glycine, each 2mol/L of inner primer FIP/BIP and each 0.25mol/L of outer primer F3/B3, places container.
(4) preparation sample pretreatment liquid: sample pretreatment liquid contains 20mmol/L Tris-HCl (pH 8.0), 2mmol/L EDTA and 1.2 volume % Triton X-100, places container.
(5) purchase stable liquid: paraffin oil places container.
(6) purchase colour developing liquid: SYBR Green I places container.
(7) extract positive control: Listeria monocytogenes group DNA places container.
(8) above-mentioned 7 containers are dressed up test kit, encapsulation.
Preparation technology is summarized as follows:
1, with behind inner primer FIP/BIP and the outer primer F3/B3 synthesizing and purifying, quantitatively preparation, concentration detects, the sampling quality inspection;
2, reaction solution is aseptic subpackaged, and determine the quality inspection of sampling with carrying out concentration according to experiment;
3, with the stable liquid packing, the sampling quality inspection;
4, with the positive control sample preparations, packing, sampling quality inspection;
5, assembling test kit.
The preparation of embodiment 2 test kits
The prescription of reaction solution is: reaction solution contains 1.8mmol/LdNTP, 20mmol/LTris-Cl, 10mmol/L Repone K, 10mmol/L ammonium sulfate, 8mmol/L sal epsom, 0.1 volume %TritonX-100,0.8mol/L trimethyl-glycine, each 1.6mol/L of inner primer FIP/BIP and each 0.2mol/L of outer primer F3/B3.
The prescription of sample pretreatment liquid is: sample pretreatment liquid contains 10mmol/L Tris-HCl (pH 8.0), 1mmol/L EDTA and 1 volume %Triton X-100.
Other are with embodiment 1.
The application of embodiment 3 Listeria monocytogenes rapid diagnostic kits
1, sample preparation (template DNA extraction)
1) enrichment liquid of getting the 1ml incubated overnight is in the eppendorf pipe, and centrifugal 2 minutes of 1000rpm removes supernatant;
2) add 100 μ l sample pretreatment liquid in centrifuge tube, mix with precipitation gained thalline;
3) boil in the boiling water after 10 minutes and placed cooled on ice immediately 10 minutes;
4) 10000rpm is centrifugal 2 minutes, and supernatant is as stand-by template DNA.
2, the reaction process of loop-mediated isothermal amplification technique
1) in 200 μ l reaction tubess preparation reaction system: reaction solution 22 μ l, Bst archaeal dna polymerase 0.5 μ l (4U), stable liquid 30 μ l, template DNA 2.5 μ l.
2) with the reaction tubes for preparing in 64 ℃ of isothermal reaction 1h.
3, post-reaction treatment
In above-mentioned reaction tubes, add 2 μ l SYBR Green I, mixing, also add SYBR Green I mixing in the heliotropism control tube (Listeria monocytogenes group DNA) simultaneously, if the same shows green with control tube of reaction tubes is then positive, if reaction tubes manifests orange then negative.
Listeria monocytogenes rapid diagnostic kit and detection method sequence table .txtSEQUENCE LISTING thereof based on loop-mediated isothermal amplification technique
＜110〉Guangzhou Huafeng Biotech Co., Ltd.
＜120〉based on the Listeria monocytogenes rapid diagnostic kit and the detection thereof of loop-mediated isothermal amplification technique
Method
<130>
<160>4
<170>PatentIn?version?3.2
<210>1
<211>19
<212>DNA
＜213〉artificial sequence
<400>1
Outer primer F3:ACAAGACTTCACCAATCCA
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<400>2
Outer primer B3:GTCTTTTAAGTGGAGTAAACCTT
<210>3
<211>47
<212>DNA
＜213〉artificial sequence
<400>3
Inner primer F1P:TAAGTCTCTTTGCAATTGACCGACTTTTACGTGTACACAGAAAAGCG
<210>4
<211>47
<212>DNA
＜213〉artificial sequence
<400>4
Inner primer BIP:CCTGTGCCAAAGCATTTTTACATTTTTTAGGCAAGTCATCTTGTTCG
Claims (7)
1, a kind of Listeria monocytogenes rapid diagnostic kit is characterized in that being made up of two pairs of primers, Bst archaeal dna polymerase, reaction solution, stable liquid, sample pretreatment liquid, colour developing liquid and positive control solution, more than seven kinds of liquid place container respectively,
Above-mentioned two pairs of primers are:
Outer primer F3:ACAAGACTTCACCAATCCA;
Outer primer B3:GTCTTTTAAGTGGAGTAAACCTT;
Inner primer FIP:TAAGTCTCTTTGCAATTGACCGACTTTTACGTGTACACAGAAAAGCG;
Inner primer BIP:CCTGTGCCAAAGCATTTTTACATTTTTTAGGCAAGTCATCTTGTTCG;
Above-mentioned reaction solution contains 1.6～2mmol/L dNTP, 20～25mmol/L Tris-Cl, 10～12.5mmol/L Repone K, 10～12.5mmol/L ammonium sulfate, 8～10mmol/L sal epsom, 0.1～0.125%TritonX-100,0.8～1mol/L trimethyl-glycine, each 1.6～2mol/L of inner primer FIP/BIP and each 0.2～0.25mol/L of outer primer F3/B3;
Above-mentioned sample pretreatment liquid contains Tris-HCl, 1～2mmol/L EDTA and the 1～1.2%Triton X-100 of 10～20mmol/L pH 8.0;
Above-mentioned positive control is Listeria monocytogenes group DNA.
2,, it is characterized in that described colour developing liquid is SYBRGreen I according to the described test kit of claim 1.
3,, it is characterized in that described sample pretreatment liquid contains the Tris-HCl of 20mmol/L pH 8.0,2mmol/L EDTA and 1.2% Triton X-100 according to the described test kit of claim 1.
4,, it is characterized in that described reaction solution contains 2mmol/L dNTP, 25mmol/L Tris-Cl, 12.5mmol/L Repone K, 12.5mmol/L ammonium sulfate, 10mmol/L sal epsom, 0.125%TritonX-100,1mol/L trimethyl-glycine, each 2mol/L of inner primer FIP/BIP and each 0.25mol/L of outer primer F3/B3 according to the described test kit of claim 1.
5, a kind of method that detects listeria monocytogenes is characterized in that using the described test kit of claim 1, follows these steps to carry out:
(1) testing sample is centrifugal in centrifuge tube, remove supernatant, add sample pretreatment liquid in the precipitation and mix, cooled on ice after the boiling water bath deactivation, behind the high speed centrifugation, supernatant is stand-by sample template DNA;
(2) in reaction tubes, add reaction solution 38～40 volume %, Bst archaeal dna polymerase 0.9～1.8 volume %, stable liquid 52～54.5 volume %, sample template DNA 4.5～9 volume %, isothermal reaction;
(3) in above-mentioned reaction tubes and positive controls, add colour developing liquid respectively, mixing, the sample sets colour developing is identical with control group then positive, otherwise negative.
6,, it is characterized in that in the step (2) that the reaction conditions of isothermal reaction is 63～65 ℃ of temperature, reaction times 45～90min according to the described detection method of claim 5.
7,, it is characterized in that described stable liquid is paraffin oil according to the described detection method of claim 1.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100277593A CN101307351A (en) | 2008-04-29 | 2008-04-29 | Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof |
| CN200910138319XA CN101555529B (en) | 2008-04-29 | 2009-04-28 | Loop-mediated isothermal amplification technology-based Listeria monocytogenes rapid diagnostic kit and testing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100277593A CN101307351A (en) | 2008-04-29 | 2008-04-29 | Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101307351A true CN101307351A (en) | 2008-11-19 |
Family
ID=40124029
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100277593A Pending CN101307351A (en) | 2008-04-29 | 2008-04-29 | Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof |
| CN200910138319XA Active CN101555529B (en) | 2008-04-29 | 2009-04-28 | Loop-mediated isothermal amplification technology-based Listeria monocytogenes rapid diagnostic kit and testing method thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910138319XA Active CN101555529B (en) | 2008-04-29 | 2009-04-28 | Loop-mediated isothermal amplification technology-based Listeria monocytogenes rapid diagnostic kit and testing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN101307351A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555529B (en) * | 2008-04-29 | 2012-01-11 | 广州华峰生物科技有限公司 | Loop-mediated isothermal amplification technology-based Listeria monocytogenes rapid diagnostic kit and testing method thereof |
| CN101748201B (en) * | 2008-11-28 | 2012-06-27 | 中华人民共和国黑龙江出入境检验检疫局检验检疫技术中心 | Method of loop-mediated isothermal amplification (LAMP) for detecting Listeria monocytogenes |
| CN103725763A (en) * | 2012-10-12 | 2014-04-16 | 苏州四同医药科技有限公司 | High-specificity high-sensitivity listeria monocytogene detection method |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110964787B (en) * | 2015-09-02 | 2022-08-26 | 上海旺旺食品集团有限公司 | Rapid constant-temperature detection method and kit for cronobacter sakazakii |
| CN108865922A (en) * | 2018-05-02 | 2018-11-23 | 山东出入境检验检疫局检验检疫技术中心 | listeria monocytogenes standard sample and preparation method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101082579A (en) * | 2006-05-29 | 2007-12-05 | 广州华峰生物科技有限公司 | Gene rapid diagnosis method based on annular mediated isothermal amplification technology |
| CN101307351A (en) * | 2008-04-29 | 2008-11-19 | 广州华峰生物科技有限公司 | Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof |
-
2008
- 2008-04-29 CN CNA2008100277593A patent/CN101307351A/en active Pending
-
2009
- 2009-04-28 CN CN200910138319XA patent/CN101555529B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101555529B (en) * | 2008-04-29 | 2012-01-11 | 广州华峰生物科技有限公司 | Loop-mediated isothermal amplification technology-based Listeria monocytogenes rapid diagnostic kit and testing method thereof |
| CN101748201B (en) * | 2008-11-28 | 2012-06-27 | 中华人民共和国黑龙江出入境检验检疫局检验检疫技术中心 | Method of loop-mediated isothermal amplification (LAMP) for detecting Listeria monocytogenes |
| CN103725763A (en) * | 2012-10-12 | 2014-04-16 | 苏州四同医药科技有限公司 | High-specificity high-sensitivity listeria monocytogene detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101555529A (en) | 2009-10-14 |
| CN101555529B (en) | 2012-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101570795B (en) | Mycobacterium tuberculosis gene rapid diagnostic primer based on loop-mediated isothermal amplification technology and use in preparing rapid diagnostic kit | |
| CN101654706B (en) | Rapid diagnostic reagent kit of legionella pneumophilia genes based on loop-mediated isothermal amplification technique and detecting method thereof | |
| CN101660005B (en) | Rapid diagnostic kit based on loop-mediated isothermal amplification technique for hepatitis A virus genes and detection method thereof | |
| CN101082581A (en) | Colon bacillus 0157 gene rapid diagnosis reagent kit of annular mediated isothermal amplification technology | |
| CN101008036A (en) | Shigella gene quick diagnosis kit based on loop-mediated equal-temperature amplification technology | |
| CN101403001B (en) | Rapid diagnosis reagent kit and detection method for pseudomonas aeruginosa gene | |
| CN101307356A (en) | Rapid diagnosis kit for staphylococcus aureus gene based on loop-mediated isothermal amplification technology and detecting method thereof | |
| CN101008039A (en) | EB virus gene quick diagnosis kit based on loop-mediated equal-temperature amplification technology | |
| CN101082579A (en) | Gene rapid diagnosis method based on annular mediated isothermal amplification technology | |
| CN101082580A (en) | vibrio parahaemolyticus gene rapid diagnosis reagent kit of annular mediated isothermal amplification technology | |
| CN101307351A (en) | Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof | |
| CN102094090A (en) | Cholera toxin virulence gene detection kit and detection method thereof | |
| CN101368204B (en) | Fast detection primer and reagent kit for enterobacter sakazakii hymenial veil mediated isothermality amplification technique | |
| CN101824468B (en) | Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method | |
| CN101942508A (en) | Escherichia coli detection kit and use method thereof | |
| CN101368203A (en) | Primer, reagent kit and detection method for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique fast detection | |
| CN101178363A (en) | Assistant hemolysis vibrion constant temperature gene amplification fast detecting reagent box and methods | |
| CN101403005B (en) | Rapid diagnosis reagent kit and detection method for cholera vibrio gene | |
| CN100478674C (en) | Salmonella gene rapid diagnosis reagent kit based on annular mediated isothermal amplification technology | |
| CN101338345A (en) | H5 avian influenza viral gene rapid diagnosis kit based on ring mediating isothermal amplification technology and detecting process thereof | |
| CN101403004B (en) | Rapid diagnosis reagent kit and detection method for vibrio vulnficus gene | |
| CN101824482B (en) | Detection kit for vibrio cholerae O1 group and detection method thereof | |
| CN101403003B (en) | Rapid diagnosis reagent kit and detection method for staphylococcus epidermidis gene | |
| CN102719548B (en) | Kit for detecting brucella and use method thereof | |
| CN101979660A (en) | Brucella detection kit and using method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |