CN101824468A - Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method - Google Patents
Primer group for detecting Yersinia pestis, rapid diagnosis kit and detection method Download PDFInfo
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Abstract
The invention discloses a primer group for detecting Yersinia pestis, a rapid diagnosis kit and a detection method, wherein the primer group consists of the following four primers: an external primer F3, an external primer B3, an inner primer FIP and an inner primer BIP. The kit consists of the primer group, Bst DNA polymerase, sample pretreatment solution, stabilizing solution, reaction solution, colored solution and positive control solution, and the seven kinds of solutions are placed in a vessel. Both the primer group and the kit can detect the Yersinia pestis with high efficiency and high specificity, as well as are based on loop-mediated isothermal amplification technology, apply six segments, four primers and one constant temperature, complete an amplification reaction within 1 hour, and are low in detection cost, short in detection time, high in yield and specificity, obvious in negative and positive result color developing difference, high in verification rate, obvious and reliable in verification.
Description
Technical field
The present invention relates to the biological assay of yersinia pestis, be specifically related to a kind of yersinia pestis and detect with primer sets, quick diagnosis reagent kit and detection method.
Background technology
Yersinia's genus (Yersinia) now is included into enterobacteriaceae, and former is the pathogenic bacteria of animal infectious disease, and the people is by contact infection animal or contaminated food and ill.Yersinia's genus roughly is divided into yersinia pestis (Yersinia pestis), yersinia pseudotuberculosis (Y.pseudotuberculosis) at present, Yersinia enterocolitica (Y.enterocolitica) and Yersinia intermedia (Y.intermedia), preceding 3 kinds have more pathogenic to the mankind.Wherein, yersinia pestis causes is disease of natural focus in the rodent, and infectivity is strong, and the case fatality rate height easily leads to and is very popular.
At present yersinia pestis there is multiple detection method, from with the pathogenic micro-organism isolation identification, morphology identifies with serological identification to be master's national standard (GB/T 18936-2003), immunology detection technology to differential protein, nucleic acid probe, polymerase chain reaction (PCR) technology equimolecular biological detection method (GB/T 22287-2008), wherein the cause of disease detection of nucleic acids is in rapidity, security, aspect such as accuracy and susceptibility all improves a lot, these new technologies attempt to break through traditional morphology, microbiologies such as biochemical reaction detect 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, direction sensitive 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.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.
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, LAMP mainly is 6 particular section utilizing 4 kinds of different Auele Specific Primers (primers F 3, primer B3, primers F IP and primer BIP) identification target gene, efficient under polysaccharase and isothermal condition, fast, high amplified target sequence specifically, amplified reaction normally adopts staining agent that reaction product is analyzed after finishing.Used polysaccharase is all selected the archaeal dna polymerase with strand displacement characteristic usually for use among the LAMP, as the Bst archaeal dna polymerase.Concerning LAMP, the design of 4 species-specific primers is its key points.
LAMP is because of the advantage of himself, the detection of pathogenic micro-organism and the diagnosis of communicable disease now have been used to, as: the detection of severe acute respiratory syndrome coronavirus (SARS-CoV), mycobacterium detect, the adenovirus membranous conjunctivitis detects, fungi detects etc., and now not seeing as yet has LAMP to be used for the relevant report that hepatitis A virus detects.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of high specific primer sets that loop-mediated isothermal amplification technique detects yersinia pestis that can be used for is provided.
Another object of the present invention be to provide by above-mentioned primer sets and other LAMP reacted constituents form can be fast, the quick diagnosis reagent kit of efficient, specific detection yersinia pestis.
Another object of the present invention is to provide the detection method of above-mentioned quick diagnosis reagent kit.
Above-mentioned purpose of the present invention is achieved by following scheme:
One, yersinia pestis of the present invention detects and uses primer sets, be based on loop-mediated isothermal amplification technique, according to disclosed yersinia pestis gene order, choose the specific sequence of yersinia pestis gene, analysis is designed then, the energy specificity is differentiated the Auele Specific Primer group of yersinia pestis, identifies the yersinia pestis gene by PCR, and this primer sets is made up of following four primers:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:1;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:2;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:3;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:4.
Two, quick diagnosis reagent kit of the present invention is made up of above-mentioned primer sets, Bst archaeal dna polymerase, sample pretreatment liquid, stable liquid, reaction solution, colour developing liquid and positive control solution, more than seven kinds of liquid place container respectively, wherein:
The prescription of described reaction solution is: contain 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, 0.01～0.04mol/L 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;
The preferred version of above-mentioned reaction solution prescription is: contain 2mmol/L dNTP, 25mmol/L Tris-HCl, 12.5mmol/L Repone K, 12.5mmol/L ammonium sulfate, 10mmol/L sal epsom, 0.04mol/LTritonX-100,1mol/L trimethyl-glycine, each 2mol/L of inner primer FIP/BIP and each 0.25mol/L of outer primer F3/B3.
Described sample pretreatment prescription is: Tris-HCl, the 1～2mmol/L EDTA and the 0.01～0.04mol/L Triton X-100 that contain 10～20mmol/L pH8.0.
The screening formulation of above-mentioned sample pretreatment liquid is: Tris-HCl, the 2mmol/L EDTA and the 0.04mol/L Triton X-100 that contain 20mmol/L pH8.0.
Described positive control is the yersinia pestis genomic dna.
Described colour developing liquid can be selected any fluorescence dye commonly used, preferred SYBR Green I or EvaGreen.
Described stable liquid can be selected any stable liquid commonly used, preferred paraffinic oils.
Used Bst archaeal dna polymerase, fluorescence dye and all kinds of SOLVENTS are commercially available in the test kit of the present invention.
Three, the production technique of quick diagnosis reagent kit of the present invention comprises the steps:
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.
Four, the detection method of quick diagnosis reagent kit of the present invention comprises the steps:
1, sample preparation
After testing sample increased bacterium by international standard (GB/T4789.8-2003), get that to increase bacteria suspension 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 the sample template DNA;
2, loop-mediated isothermal amplification technique reaction process
In reaction tubes, add reaction solution 38～40 volume %, big fragment 0.9～1.8 volume % of Bst archaeal dna polymerase, stable liquid 52～54.5 volume %, sample template DNA4.5～9 volume %, isothermal reaction.Described volume percent is meant the volume percent that accounts for the total system of reaction.
Popular response condition when the reaction conditions of above-mentioned loop-mediated isothermal amplification can adopt those skilled in the art to operate loop-mediated isothermal amplification, preferred isothermal reaction temperature is 63～65 ℃, the isothermal reaction time is 45～90min.
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.
Compared with prior art, the present invention has following beneficial effect:
1. design of primers is the key point of LAMP technology, for design of primers many requirements are arranged, as the distance between each primer, the terminal stability of Tm value and primer etc., therefore there is very big difficulty in four satisfactory primers of design from the target sequence of 200～300 bases, and the present invention has designed a kind of norovirus detection primer sets according to target-gene sequence, six isolated areas on the energy specific recognition target sequence, under the effect of Bst archaeal dna polymerase, start the endless chain replacement(metathesis)reaction, start complementary strand in target DNA district synthetic, complementary sequence goes round and begins again and is formed with the very stem of polycyclic Cauliflower structure-circular DNA mixture on same chain, because the LAMP technology only could be carried out under four primers are discerned the situation of six lands of target sequence fully smoothly, so primer sets of the present invention has reduced the background influence of amplified reaction to a great extent, improved the specificity that norovirus detects greatly;
2. adopt primer sets of the present invention that yersinia pestis is detected, because high specific, so the existence that can just can judge target gene according to whether increasing whether, thereby the typing that can carry out yersinia pestis detects;
3. quick diagnosis reagent kit of the present invention is to utilize loop-mediated isothermal amplification technique rapid detection yersinia pestis, and detection sensitivity height, amplification template only need 10 copies or still less;
4. quick diagnosis reagent kit of the present invention is to finish the LAMP reaction under constant temperature (63～65 ℃) condition in 45～90 minutes, reaction conditions gentleness not only, and required instrument is simple, do not need special reagent yet, overcome conventional P CR inherent detection time long, pollute easily and detect shortcoming such as cost height;
5. quick diagnosis reagent kit of the present invention amplification fast and efficient can be finished amplification less than 1 hour, and the productive rate height;
6. quick diagnosis reagent 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;
7. quick diagnosis reagent kit of the present invention is simple to operate, and is lower to testing staff's technical quality requirement, can set up rapid screening system with low cost, realizes on-the-spot high-throughput rapid detection;
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; And adopt quick diagnosis reagent kit of the present invention only to need 2 hours, and it also is equivalent to or is better than the traditional detection technology on methodology indexs such as sensitivity, specificity and sensing range.
Description of drawings
Fig. 1 is specific degree test-results figure among the embodiment 3;
Wherein, 1～26 is non-plague bacillus, and 27 is plague bacillus EV001, and 28 is the mixing of 27 kinds of DNA of bacteria;
Fig. 2 is embodiment 3 medium sensitivity test-results figure;
Wherein, 0 is plague bacillus bacterium stoste, and 1～12 is respectively 10
-1～10
-12These 12 concentration levels;
Fig. 3 is embodiment 3 medium sensitivities test electrophoresis result figure;
Wherein, M is marker, and 0 is plague bacillus bacterium stoste, and 1～12 is respectively 10
-1～10
-12These 12 concentration levels.
Embodiment
Below in conjunction with specific embodiment the present invention is done description further, but specific embodiment is not done any qualification to the present invention.
The preparation of embodiment 1 yersinia pestis quick diagnosis reagent kit
(1) adopts the synthetic following aligning primer of dna synthesizer
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:1;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:2;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:3;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:4.
(2) purchase archaeal dna polymerase: Bst DNA polymerase (big fragment) places container.
(3) preparation reaction solution: the prescription of reaction solution contains each 0.25mo1 preparation of 2mmol dNTP, 25mmol Tris-Cl, 12.5mmol Repone K, 12.5mmol ammonium sulfate, 10mmol sal epsom, 0.04mol TritonX-100,1mol trimethyl-glycine, each 2mol of inner primer FIP/BIP and outer primer F3/B3 by every 1L solution, places container.
(4) preparation sample pretreatment liquid: the prescription of sample pretreatment liquid is prepared by Tris-HCl, 2mmol EDTA and the 0.04mol Triton X-100 that every 1L solution contains 20mmol pH8.0, places container.
(5) purchase stable liquid: paraffin oil places container;
(6) purchase colour developing liquid: SYBR Green I places container.
(7) extract positive control: the yersinia pestis genomic dna places container.
(8) above-mentioned 7 containers are dressed up test kit, encapsulation.
The preparation technology of present embodiment test kit 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.
9, assembling test kit.
The preparation of embodiment 2 yersinia pestis quick diagnosis reagent kits
The prescription of reaction solution is: contain 1.6mmol dNTP, 20mmol Tris-Cl, 10mmol Repone K, 10mmol ammonium sulfate, 8mmol/L sal epsom, 0.01mol TritonX-100,0.8mol trimethyl-glycine, each 1.6mol of inner primer FIP/BIP and each 0.2mol of outer primer F3/B3 in every 1L reaction solution.
Colour developing liquid is EvaGreen.
Other are with embodiment 2.
The application of embodiment 3 yersinia pestis gene quick diagnosis kits
Present embodiment adopts embodiment 1 preparation gained test kit following 27 strain bacterial strains to be carried out the quick diagnosis of yersinia pestis gene.
One, test strain
1. Pseudomonas aeruginosa (ATCC 27853); 2. streptococcus aureus (ATCC 25923); 3. streptococcus aureus (ATCC 29533); 4. intestinal bacteria (ATCC 29522); 5. Vibrio vulnificus (ATCC 27562); 6. intestinal bacteria (8099); 7. enteroinvasive E.Coli (EIEC); 8. pathogenic colon bacillus (EPEC); 9. Shigella flexneri (51142); 10. Song Nei Shi shigella (51592); 11. enterohemorrhagic Escherichia coli (EHEC); 12. EAEC (EAggEC); 13. Corynebacterium diphtheriae (" O " type); 14. Corynebacterium diphtheriae (" H " type); 15. bacillus typhi murium; 16. Ye Ersenshi enterocolitis bacterium; 17. Pparatyphoid A; 18. Bruce Salmonella; 19. Klebsiella Pneumoniae; 20. Serratia; 21. vibrio cholerae; 22. the false unit cell of verdigris; 23. acinetobacter calcoaceticus; 24. Vibrio parahaemolyticus; 25. Europe, sea, Hong Kong bacterium; 26. Plesiomonas (302 hospitals of PLA are so kind as to give); 27. plague bacillus (the EV001 vaccine strain is economized CDC and is so kind as to give).
Above-mentioned bacterial strains is this area research bacterial strain commonly used, can obtain from biotech firm or the purchase of reagent company.
Two, 27 strain bacterial strains to present embodiment carry out the template DNA extraction, and its step is as follows:
(1) adopts the about 200ml of aseptic technique water sampling (or samples such as settling, ooze), can staticly settle or the centrifugal 1min removal of 1000r/min large particulate matter if any impurity;
(2) will pass through aperture 0.22 μ m～0.45 μ m membrane filtration through precipitation or centrifuged sample (supernatant), and take off filter membrane and place the 15ml aqua sterilisa, fully wash-out;
(3) get the 5ml elution samples, the centrifugal 2min of 10000rpm obtains bacterial sediment;
(4) add 80 μ L DNA extraction liquid, 100 ℃ of water-bath 10min put 10min on ice behind the mixing;
(5) the centrifugal 2min of 10000r/min, supernatant is nucleic acid-templated.
Three, loop-mediated isothermal amplification
In 200 μ l reaction tubess preparations 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, with the reaction tubes for preparing in 64 ℃ of isothermal reaction 1h.
In above-mentioned reaction product, add 2 μ l SYBR Green I, mixing, also add SYBR Green I mixing in the heliotropism control tube (plague bacillus genomic dna) simultaneously, if the same shows green with control tube of reaction tubes is then positive, if reaction tubes manifests orange then negative.
Four, specific degree test
Above-mentioned LAMP method increases respectively to 27 strain bacterial strains, by last colour developing result as can be seen 27 strain bacterial strains have only plague bacillus the colour developing result for green, positive bacterial strain, it is orange that the colour developing result of all the other 26 strains is, negative bacterial strain, as shown in Figure 1.
The DNA extraction liquid of plague bacillus is mixed with the DNA extraction liquid of other 26 strain bacterial strain respectively, respectively get 2ul then and carry out the detection of LAMP method respectively, and do two groups parallel group, last colour developing result shows, all hybrid dnas are all positive, and the high specificity of test kit of the present invention to plague bacillus is described thus.
Five, sensitivity test
Plague bacillus in pure culture on the ordinary nutrient agar flat board after 18 hours, is chosen two and completely encircles bacterium colony and be suspended in the 5ml stroke-physiological saline solution, obtain original bacterium liquid, its bacterial concentration is 1.06 * 10
9Cfu/ml carries out 10 to original bacterium liquid respectively with physiological saline
-1～10
-12This original bacterium liquid to 10 of the dilution of these 12 concentration levels
-1～10
-12These 12 concentration levels promptly obtain diluting 12 parts of bacterium liquid, and its concentration is respectively 1.06 * 10
8, 1.06 * 10
7, 1.06 * 10
6, 1.06 * 10
5, 1.06 * 10
4, 1.06 * 10
3, 1.06 * 10
2, 1.06 * 10
1, 1.06,1.06 * 10
-1, 1.06 * 10
-2, 1.06 * 10
-3, the bacterium colony after will diluting is then got 100ul respectively and is tiled on the ordinary nutrient agar flat board, cultivates 24h for 25 ℃, gets the cultivation bacterium liquid 1ml of each concentration level then respectively, extracts DNA respectively, makes LAMP and detects.
By the colour developing result as can be seen, 1.06 * 10
8, 1.06 * 10
7, 1.06 * 10
6, 1.06 * 10
5, 1.06 * 10
4The result of these five bacterium liquid weaker concns is positive, and the result of remaining 7 bacterium liquid weaker concn is negative, as shown in Figure 2, illustrates that thus test kit of the present invention can detect the 5th extent of dilution, and promptly bacterial concentration is 1.06 * 10
4
To 10
-1～10
-12These 12 concentration levels carry out electrophoresis detection respectively, the result as shown in Figure 3,1.06 * 10
8, 1.06 * 10
7, 1.06 * 10
6, 1.06 * 10
5, 1.06 * 10
4The electrophoresis banding pattern of these five bacterium liquid weaker concns is identical with the electrophoresis banding pattern of plague bacillus stoste, and the electrophoresis banding pattern of the electrophoresis banding pattern of all the other 7 bacterium liquid weaker concns and plague bacillus stoste is inequality, illustrates that its detection sensitivity of test kit of the present invention can reach 1.06 * 10
4
Yersinia pestis detects with primer sets, quick diagnosis reagent kit and detection method sequence table
SEQUENCE?LISTING
＜110〉Guangzhou Huafeng Biotech Co., Ltd.
＜120〉yersinia pestis detects with primer sets, quick diagnosis reagent kit and detection method
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Claims (8)
1. a yersinia pestis detects and uses primer sets, it is characterized in that this primer sets is made up of following four primers:
Outer primer F3, its nucleotide sequence is shown in SEQ ID NO:1;
Outer primer B3, its nucleotide sequence is shown in SEQ ID NO:2;
Inner primer FIP, its nucleotide sequence is shown in SEQ ID NO:3;
Inner primer BIP, its nucleotide sequence is shown in SEQ ID NO:4.
2. quick diagnosis reagent kit, it is characterized in that this test kit is made up of the described primer sets of claim 1, Bst archaeal dna polymerase, sample pretreatment liquid, stable liquid, reaction solution, colour developing liquid and positive control solution, more than seven kinds of liquid place container respectively, wherein:
The prescription of described reaction solution is: contain 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, 0.01～0.04mol/L 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;
Described sample pretreatment prescription is: Tris-HCl, the 1～2mmol/L EDTA and the 0.01～0.04mol/LTritonX-100 that contain 10～20mmol/L pH8.0.
Described positive control is the yersinia pestis genomic dna.
3. according to the described quick diagnosis reagent kit of claim 2, it is characterized in that the prescription of described reaction solution is: contain 2mmol/L dNTP, 25mmol/L Tris-HCl, 12.5mmol/L Repone K, 12.5mmol/L ammonium sulfate, 10mmol/L sal epsom, 0.04mol/L TritonX-100,1mol/L trimethyl-glycine, each 2mol/L of inner primer FIP/BIP and each 0.25mol/L of outer primer F3/B3.
4. according to the described quick diagnosis reagent kit of claim 2, it is characterized in that the prescription of described sample pretreatment liquid is: Tris-HCl, the 2mmol/L EDTA and the 0.04mol/L TritonX-100 that contain 20mmol/L pH 8.0.
5. according to the described quick diagnosis reagent kit of claim 2, it is characterized in that described colour developing liquid is SYBR Green I or EvaGreen.
6. according to the described quick diagnosis reagent kit of claim 2, it is characterized in that described stable liquid is paraffin oil.
7. a method of utilizing the described quick diagnosis reagent kit of claim 2 to detect yersinia pestis is characterized in that this method comprises the steps:
(1) extracts testing sample DNA;
(2) in reaction tubes, add reaction solution 38～40 volume %, big fragment 0.9～1.8 volume % of Bst archaeal dna polymerase, 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 positive controls then positive, otherwise negative.
8. according to the described detection method of claim 7, it is characterized in that the isothermal reaction temperature in the described step (2) is 63～65 ℃, the isothermal reaction time is 45～90min.
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| CN103698516A (en) * | 2013-12-27 | 2014-04-02 | 天津国际旅行卫生保健中心 | Novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent |
| CN104195224A (en) * | 2014-07-02 | 2014-12-10 | 东北林业大学 | Chytrid LAMP rapid detection method and kit |
| CN110592245A (en) * | 2019-10-10 | 2019-12-20 | 中国检验检疫科学研究院 | Kit for rapidly detecting yersinia pestis |
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| CN103698516A (en) * | 2013-12-27 | 2014-04-02 | 天津国际旅行卫生保健中心 | Novel isothermal loop-mediated yersinia pestis nucleic acid mark detection reagent |
| CN104195224A (en) * | 2014-07-02 | 2014-12-10 | 东北林业大学 | Chytrid LAMP rapid detection method and kit |
| CN110592245A (en) * | 2019-10-10 | 2019-12-20 | 中国检验检疫科学研究院 | Kit for rapidly detecting yersinia pestis |
| CN112980974A (en) * | 2021-03-04 | 2021-06-18 | 中国人民解放军军事科学院军事医学研究院 | Yersinia pestis identification method based on chromosome specific probe |
| CN112980974B (en) * | 2021-03-04 | 2021-11-23 | 中国人民解放军军事科学院军事医学研究院 | Yersinia pestis identification method based on chromosome specific probe |
| WO2023077489A1 (en) * | 2021-11-06 | 2023-05-11 | 江汉大学 | Mnp marker combination of yersinia pestis, primer pair combination, kit, and application thereof |
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| CN101824468B (en) | 2012-10-10 |
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