CN102827928B - Rapid diagnosis method for plesimonas shigelloides - Google Patents
Rapid diagnosis method for plesimonas shigelloides Download PDFInfo
- Publication number
- CN102827928B CN102827928B CN201210262462.1A CN201210262462A CN102827928B CN 102827928 B CN102827928 B CN 102827928B CN 201210262462 A CN201210262462 A CN 201210262462A CN 102827928 B CN102827928 B CN 102827928B
- Authority
- CN
- China
- Prior art keywords
- seq
- primer
- lamp
- dna
- sequence
- 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.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting plesimonas shigelloides. The method comprises the following steps of: adopting a loop-mediated isothermal amplification technology; using a hug A gene as a target gene; putting designed three pairs of DNA (Deoxyribonucleic Acid) primers and a DNA template to be detected to an amplification buffer solution including MgSO4 and glycine betaine; amplifying in a constant-temperature environment of 65 DEG C, and terminating the reaction in a constant-temperature environment of 80 DEG C; and detecting the amplified product. The method disclosed by the invention is simple and easy to operate, and has good specificity, sensitivity and repeatability.
Description
Technical field
The present invention relates to the fast diagnosis method of a kind of Plesiomonas shigelloides, belong to microorganism quick diagnosis field.
Background technology
Plesiomonas shigelloides (Plesiomonas shigelloides) is the bacterium of the non-sporogenesis of a kind of Gram-negative, energy motion, amphimicrobian, oxidase positive, once with to human disease's Vibrio belonged to vibrionaceae together with Aeromonas, rear research finds that it is more approaching with Plesiomonas shigelloides and proteus at molecular level, in the second edition " systematic bacteriology uncle Jie Shi handbook ", Plesiomonas shigelloides is incorporated enterobacteriaceae into, Plesiomonas.
Water source of fresh water and river ecotope are the major storage ground of Plesiomonas shigelloides, and fish are the common hosts of Plesiomonas shigelloides.Increased gradually recent years by this microbial infant, the elderly and immunologic inadequacy patient's gastrointestinal tract infection case, is one of Etiological of mankind's gastroenteritis, has repeatly report all over the world.Plesiomonas shigelloides also can cause the Extra intestinal infections such as mankind's microbemia, cellulitis, meningitis, and this bacterium is also closely related with traveler's diarrhea.Find between 1994 to 1996 in all enterobacterias that cause traveler's diarrhea Plesiomonas shigelloides rank first place (66.7%) from one of Japan research.The mortality ratio being caused by Plesiomonas shigelloides is also increasing gradually.The infected of some Plesiomonas shigelloides is owing to failing to diagnose fast and accurately and reasonably applying microbiotic, and do not treated timely and produced serious consequence, even dead.
Do not illustrate completely although Plesiomonas shigelloides is brought out the pathogenesis of gastroenteritis, investigator conducts in-depth research some potential virulence factors.The virulence of picked-up iron ion and some pathogenic bacterias is closely related, heme in human body is an important sources of pathogenic bacteria iron ion, and itself just has heme haulage system many pathogenic bacterias, hugA gene is that coding Plesiomonas shigelloides heme iron ion utilizes one of important gene of system, and coding outer membrane protein acceptor, being an important gene that affects Plesiomonas shigelloides virulence, is also therefore a desirable testing goal gene.
The detection method of Plesiomonas shigelloides mainly contains separation and Culture, biochemical identification, regular-PCR and real-time fluorescence PCR equimolecular biological method at present.Isolation cultivation method complex operation step, sense cycle is long, generally needs within 3-5 days, complete, and can not meet the demand of rapid detection.And this bacterium lacks special screening culture medium, the bacterium colony proterties forming on some enteron aisle substratum, to have a liking for aqueous vapor unit cell similar, is difficult to difference, and this has also affected the separation rate of Plesiomonas shigelloides.Recent years, PCR and the application of real-time fluorescence PCR molecular biology for detection in detection of pathogens are more and more wider, but the testing cost of these methods is high, need accurate expensive thermal cycling instrument, and experimental situation and operator's technology is also had to strict requirement, therefore limited the general applicability of this technology, be unfavorable in Rural areas and the applying of laboratories.The greatest difficulty that Plesiomonas shigelloides is caused as the sick scholar of a kind of new communicable disease pop and clinician be exactly lack can carry out in early days it, fast, the accurate method of detection, to meet the real work demand of Rural areas and laboratories.
Loop-mediated isothermal amplification technique (Loop-mediated isothermal amplification, LAMP) is a kind of new nucleic acid amplification method of the foundation such as Notomi.LAMP is 4 primers of 6 special position designs for target sequence, utilizes Bst archaeal dna polymerase new chain of catalysis under constant temperature with strand displacement activity to synthesize, thereby target sequence is efficiently increased.Article 4, among primer 2 be inner primer, i.e. FIP (ForWard inner primer, FIP) and BIP (Backward inner primer, BIP).FTP comprises Flc and F2 (complementary sequence in F2c region), i.e. 5 '-Flc-F2; BIP comprises B1c (complementary sequence in B1 region) and B2, i.e. 5 '-Blc-B2.Outer primer is F3 and B3.
LAMP reaction process comprises dumbbell shaped template synthesis phase, cyclic amplification stage.In LAMP reaction, 60～65 DEG C is the medium temperature of double-stranded DNA renaturation and extension, and DNA is 65 DEG C of left and right in dynamic balance state, and when any one primer carries out base pairing extension to the complementary portions of double-stranded DNA, another chain will dissociate, and becomes strand.Under the effect of strand displacement type archaeal dna polymerase, taking 3 ' end of FIP primers F 2 sections as starting point, with the pairing of template DNA complementary sequence, start strand displacement DNA synthetic.F3 primer and the complementation of F2c front end F3c sequence, taking 3 ' end as starting point, by the effect of strand displacement active dna polysaccharase, replace the synthetic DNA chain of FIP primer ahead on one side.Synthesize self DNA on one side, extend so forward.DNA chain and template DNA that final F3 primer is synthesized into form two strands.The DNA chain first being synthesized by FIP primer is carried out strand displacement by F3 primer and produces a strand, and this strand exists complementary F1c and F1 section at 5 ' end, so there is self-base pairing, forms loop-stem structure.Meanwhile, BIP primer is with this strand hybridization combination, and taking 3 of BIP primer ' hold as starting point, synthetic complementary strand, in this process, loop-stem structure is opened.Then, be similar to F3, B3 primer inserts from BIP primer outside, carries out base pairing, taking 3 ' hold as starting point, under the effect of polysaccharase, synthetic new complementary strand.By above-mentioned 2 processes, form double-stranded DNA.There is complementary sequence in replaced single stranded DNA two ends, naturally-occurring oneself base pairing, forms ring texture, so whole piece chain presents dumbbell shaped structure, this structure is the initial structure of LAMP method gene amplification circulation.The LAMP method gene amplification cycle stage: first in dumbbell shaped structure, taking the F1 section of 3 ' end as starting point, with from as template, carry out that DNA is synthetic to be extended.Meanwhile, FIP primers F 2 and the upper strand F2c hybridization of ring, start new round strand replacement reaction, the double-strandednucleic acid that dissociates and synthesized by F1 section.Equally, on the single-chain nucleic acid dissociateing, also can form loop-stem structure.On loop-stem structure, have single stranded form B2c, the B2 on BIP primer is hybrid with it, and starts new round amplification, through identical process, forms again loop-stem structure.By this process, result complementary sequence on same chain goes round and begins again and forms structure not of uniform size.In order further to shorten the reaction times of LAMP, can also between F1c-F2c/B1c-B2c, design a pair of ring primer LF/LB, ring primer, by being combined on the annular section complementary sequence of loop-stem structure, greatly having accelerated whole strand replacement reaction process, thereby has improved reaction efficiency.LAMP can be special, efficiently, DNA amplification fast, make the amount of product reach 10 within an hour
9individual copy.
The shortcoming of LAMP method maximum is exactly easily to occur false positive, because LAMP has adopted many primers, and is to increase under same temperature condition, likely complementary and amplify non-specific band between primer, causes false positive.Therefore LAMP method is very high to the specificity requirement of primer, designs also more complicated.Because LAMP has omitted 94 DEG C of denaturing steps, anneal simultaneously and extend step and carry out under same temperature (isothermal) condition, the composition of reaction system is also compared with normal PCR complexity, in reaction system the composition such as necessary damping fluid, dNTP primer, enzyme, also need to add the essential composition such as trimethyl-glycine (betaine), magnesium sulfate, reaction system is comparatively complicated, the ratio optimization liquid of main component in system is had relatively high expectations, otherwise can reduce specificity and the susceptibility of reaction.
Object of the present invention is exactly by design of primers and constituent optimization, set up a kind of Plesiomonas shigelloides easily and fast, special, sensitive LAMP detection method, promoting the use of on a large scale at laboratories.
Summary of the invention
The invention provides a kind of method that detects Plesiomonas shigelloides, described method comprises the steps:
(1) a pair of outer primer as shown in SEQ ID NO 1-2, sequence a pair of inner primer, a pair of ring primer, dNTP, Bst archaeal dna polymerase, the MgSO of sequence as shown in SEQ ID NO 5-6 as shown in SEQ ID NO 3-4 by sample to be detected, sequence
4, trimethyl-glycine and ring mediated isothermal amplification damping fluid join in reaction vessel;
(2) mixed solution of step (1) gained is positioned in 65 DEG C of isoperibols and carries out DNA amplification reaction;
(3) product) step (2) being obtained is positioned in 80 DEG C of isoperibols, to stop the described DNA amplification reaction of step (2);
(4) product that detecting step (3) obtains.
Wherein, SEQ ID NO 1 is primers F 3, and SEQ ID NO 2 is primer B3, and SEQ ID NO3 is primers F IP, and SEQ ID NO 4 is primer BIP, and SEQ ID NO 5 is primer LF, and SEQ IDNO 6 is primer LB.
The hugA gene that the goal gene that present method detects is Plesiomonas shigelloides, the gene order of design of primers institute foundation is (No. ID, Gene Bank: AY008342.1), the position of SEQ ID NO 1-6 in hugA gene order is as shown in table 1.
LAMP primer sequence used in table 1 experiment
In a preferred technical scheme, MgSO described in step (1)
4concentration be 6mM, the concentration of described trimethyl-glycine is 10mM.
Described in step (1), sample to be detected can be clinical censorship sample, is preferably DNA extraction thing.
In an optimal technical scheme of the present invention, the DNA amplification reaction time described in step (2) is 1 hour.
In an optimal technical scheme of the present invention, the termination reaction time described in step (3) is 5 minutes.
In another optimal technical scheme of the present invention, the described detection method of step (4) can be electrophoresis detection method, turbidity detection method or staining examine method.
Electrophoresis detection method: the DNA fragmentation mixture of the loop-stem structure that the target sequence that is a series of inverted repeats due to amplified production forms and many ring Cauliflower spline structures, after electrophoresis, on gel, manifest the staged collection of illustrative plates of different large communities band composition, can judge according to clip size.
Turbidity detection method: LAMP also produces a large amount of pyrophosphate ions in synthetic DNA, they can be combined with magnesium ion and generate white magnesium pyrophosphate precipitation, can qualitatively judge LAMP reaction and whether occur according to whether forming white precipitate in reaction system, and according to turbidity value, result be carried out to quantitative analysis.
Staining examine method: can add the fluorescence dye that can be incorporated in double-stranded DNA in amplified production, for example SYBY Green I, has positive amplification person's electrophoresis result shown in green, without positive amplification person, does not develop the color.
The present invention also provides a kind of test kit that detects Plesiomonas shigelloides, and described test kit comprises: a pair of outer primer, sequence a pair of inner primer, sequence a pair of ring primer, dNTP, Bst archaeal dna polymerase, the MgSO as SEQ ID NO 5-6 as shown in as SEQ ID NO 3-4 as shown in of sequence as shown in SEQ ID NO 1-2
4, trimethyl-glycine, ring mediated isothermal amplification damping fluid.
In an optimal technical scheme, described MgSO
4concentration be 6mM, the concentration of described trimethyl-glycine is 10mM.
Preferably, described test kit can also comprise DNA extraction test kit.
Contriver has set up the method that has used LAMP technology to detect Plesiomonas shigelloides.In the process of establishing of LAMP reaction system to MgSO
4, the concentration of reaction additives trimethyl-glycine is screened, and by the aspects such as reaction times, reaction sensitivity and product growing amount are compared, has finally determined MgSO
4, the optimum concn of trimethyl-glycine, thus the LAMP detection kit of Plesiomonas shigelloides set up.By the detection of LAMP detection and real-time fluorescence PCR is compared, we find that LAMP detection is all significantly better than real-time fluorescence PCR in detection sensitivity or on detection speed.Generally, all can be subject to the impact of inhibition in clinical samples taking molecular level as for example PCR of basic detection method and LAMP, some investigators report LAMP use Bst polysaccharase more eager to excel in whatever one does than the Taq enzyme in real-time fluorescence PCR to the tolerance of inhibition in sample, and at least faster more than 20 minutes than the speed of response of real-time fluorescence PCR, can significantly shorten overall detection time.
By contrast, LAMP detection technique has a lot of advantages:
(1) LAMP is high to target sequence amplification efficiency, and in system, other non-target DNAs that coexist are less on the impact of its amplification.Under the detection of LAMP, be limited to several copies, sensitivity, far away higher than regular-PCR technology, has investigator to confirm that the sensitivity of LAMP equals the even detection sensitivity higher than real-time fluorescence PCR.
(2) product is easy to detect, quick.Because LAMP also produces a large amount of by product-pyrophosphate salts in synthetic various stem circular DNAs, and generates white magnesium pyrophosphate throw out.Whether Mori etc. utilize these characteristics to detect LAMP product, can directly qualitatively judge amplified reaction according to the formation of precipitation and occur, and can also carry out real-time quantitative detection by the variation that detects its product turbidity.
(3) LAMP is high to the specificity of target sequence amplification.Because LAMP is accurate in conjunction with producing amplification effect by corresponding 6 special positions on 2 pairs of primers (F3, B3, FIP and BIP) and target sequence, therefore the specificity higher than PCR can be obtained, and inevitable background interference in general nucleic acid amplification technologies can be partly reduced.
(4) equipment is simple, easy to operate.LAMP reaction only needs a common water-bath or thermostat, does not need expensive PCR instrument, is easy to apply at laboratories and Rural areas.
(5) speed of response is fast.LAMP is isothermal amplification reactions, there is no large consuming time of temperature variation in PCR reaction, generally in 30min～60min, can complete, faster than regular-PCR and real-time fluorescence PCR speed of response, can meet better the requirement of clinical rapid detection.In addition, increase by 2 ring primers (LF and LB) and can make to react required time than the original half that shortens in LAMP reaction system, whole reaction can complete in 30min, has greatly improved detection efficiency.These characteristics are more suitable in the time of laboratories, local disease control, vast Rural areas and site disposal epidemic situation LAMP technology to use.
In a word, the LAMP method that the present invention sets up is the detection that first LAMP technology has been applied to Plesiomonas shigelloides, the method just can observations in 1 hour, the needs of clinical quick diagnosis are met, and the method is highly sensitive in real-time fluorescence PCR method to clinical sample, reaction times is faster, and required plant and instrument is simple, can be used as one fast screening method promote the use of on a large scale in Clinical Laboratory quarantine, laboratories and Rural areas
Brief description of the drawings
The turbidity value detected result figure of Fig. 1: LAMP reaction;
Fig. 2: LAMP reaction electrophoresis detection result figure;
The staining examine result figure of Fig. 3: LAMP reaction;
Fig. 4: the detected result figure of 4 parts of Plesiomonas shigelloides samples.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, the defined protection domain of the claims in the present invention is not formed to any restriction.
Embodiment of the present invention reference culture used is purchased from Nat'l Pharmaceutical & Biological Products Control Institute, and other strains separation are from samples such as food, ight soil and vomituses, all through the qualification of moral spirit WALKAWAY-40SI full automatic microorganism analyser.Be used for the bacterial strain of Evaluation on specificity in table 1.
The detection of embodiment 1:4 part Plesiomonas shigelloides
Compare by the Plesiomonas shigelloides hugA gene order to known in GenBank (No. ID, GeneBank), we select one section of conserved sequence on hugA gene as detecting target.Use primer-design software PrimerExplorer V4 software (
http:// primerexplorer.jp) (Eiken Chemical Co.Ltd., Tokyo, Japan) carry out LAMP design of primers online.Primer is by Shanghai Sheng Gong company limited synthetic (be shown in table 2).The reaction system of LAMP is as follows: the concentration of primer SEQ ID NO 1 and SEQ ID NO 2 is 5pmol, the concentration of primer SEQ ID NO 3 and SEQ ID NO4 is 40pmol, the concentration of primer SEQ ID NO 5 and SEQ ID NO 6 is 20pmol, the Betain of 10mM, the MgSO of 6mM
4, the dNTP of 1mM, 10 × Bst DNA polymerase buffer liquid of 2.5 μ L, the strand displacement archaeal dna polymerase of 8U, the template of 2 μ L, adds deionized water to 25 μ l.Whole reaction is carried out in the real-time turbidimeter of LA320 (Teramecs, Tokyo, Japan), 65 DEG C of 1h of isothermal duplication, 80 DEG C of 5min termination reactions.Result can qualitatively judge according to the formation of precipitation, also can carry out real-time quantitative detection by the variation that detects product turbidity.
Fig. 4 is the detected result of 4 parts of Plesiomonas shigelloides.In Fig. 4, the LAMP detection system that 4 parts of Plesiomonas shigelloides and a colibacillary sample adopt this research to set up detects, all there is positive amplification curve in 4 parts of Plesiomonas shigelloides samples as a result in 60 minutes, intestinal bacteria sample occurs without amplification curve, illustrates that the LAMP detection system that this research is set up can detect fast and effectively to Plesiomonas shigelloides.
The sensitivity evaluation that embodiment 2:LAMP detects:
1. contain structure and the preparation of the plasmid standard of hugA gene
(1) use hugA special primer to carry out obtaining object fragment after pcr amplification;
(2) cut glue and reclaim, purified pcr product;
(3) be connected to T carrier;
(4) transform JM109 competent cell;
(5) screening positive clone, verifies with PCR, and final order-checking is confirmed;
(6) extract plasmid, according to the molecular weight of plasmid, plasmid sample concentration is scaled to copy number concentration: copy number=concentration (ng/ μ L) × Avogadro constant number × 10 of detecting gene in every μ L sample
-9/ (660 × recombinant plasmid base number);
(7) with Easy Dilution, above-mentioned plasmid is diluted to 1.0 × 10 successively
0copy/μ L～1.0 × 10
6copy/μ L, totally 7 concentration gradients.
The sensitivity of 2.LAMP detection system
Get the every reaction system 10 of plasmid standard
0be copied to 10
6copy is template, and each concentration is carried out 3 times and repeated, and taking real-time fluorescence PCR as contrast, evaluates the sensitivity of LAMP detection system.
Evaluation result:
LAMP and real-time fluorescence PCR are respectively 20 copy/reactions (Fig. 1) and 200 copies/reaction (copies/reaction) for the detection lower limit of hugA gene.This presentation of results LAMP is sensitiveer than real-time fluorescence PCR in the time detecting Plesiomonas shigelloides DNA.Fig. 1 is for to carry out Plesiomonas shigelloides plasmid standard after 10 times of serial dilutions, increases by LAMP detection system, evaluates the sensitivity of this system.In figure, the concentration of amplification curve is respectively 2.0 × 10
6～2.0 × 10
1copy/reaction.
The amplification (Fig. 2) that can detect by the method for electrophoresis LMAP, also can add 1,000 × SYBR green I (Fig. 3) of 1 μ L to detect by the change of visual inspection color.Fig. 1 is that the result of turbidimeter Real-Time Monitoring is passed through in the sensitivity of hugA-LAMP.Under detection, being limited to each reaction 20 copies.
Fig. 2 is the amplified production of analyzing hugA-LAMP with agarose gel electrophoresis.Swimming lane M:DL2000marker; Swimming lane 1:2 × 10
6copy/reaction; Swimming lane 2:2 × 10
5copy/reaction; Swimming lane 3:2 × 10
4copy/reaction; Swimming lane 4:2 × 10
3copy/reaction; Swimming lane 5:2 × 10
2copy/reaction; Swimming lane 6:2 × 10
1copy/reaction; Swimming lane 7:2 × 10
0copy/reaction; Swimming lane 8: without template.
Fig. 3 is for using SYBR green I detection of fluorescent dyes hugA-LAMP amplified production. and in the time there is positive amplification, the tangerine look of SYBR Green I dyestuff becomes green fast, more remarkable under black background; In the time that amplification is negative, the interior color of pipe maintains tangerine look constant.
The Evaluation on specificity that embodiment 3:LAMP detects: the specificity taking common pathogen and conditioned pathogen DNA (in table 2) for template evaluation LAMP system.
The specificity that LAMP detects is used 52 strain common pathogens and conditioned pathogen DNA (in table 2) to evaluate.All there is positive amplification in 20 strain Plesiomonas shigelloides, amplification does not all appear in the non-Plesiomonas shigelloides of 32 strain, illustrates that the specificity of this LAMP detection technique is good in 1 hour.
Bacterial strain used in table 1 experiment
The reproducibility that embodiment 4:LAMP detects:
When experiment in criticizing, in experiment once by the plasmid standard of 3 parts of different concns (10
6, 10
4, 10
2) do replication 10 times, gained detected result is analyzed, obtain CVi; While experiment in criticizing, by the plasmid standard of 3 parts of different concns (10
6, 10
4, 10
2) do 10 replications, in 10 day time, divide and carry out for 10 times, gained detected result is analyzed, obtain CVo.
LAMP detects repeatability by the plasmid standard (10 of 3 parts of different concns
6, 10
4, 10
2) evaluate.Variation within batch coefficient is 1.21%-1.54%, and interassay coefficient of variation is 2.17%-3.23%.Data presentation, LAMP of the present invention has good repeatability.
Embodiment 5: evaluate the application of LAMP technology in ight soil analog sample
(1) Plesiomonas shigelloides (ATCC51903) shakes bacterium to logarithmic phase;
(2) with phosphate buffered saline buffer, this bacterium is carried out to continuous 10 times of dilutions, and carry out plate count;
(3) get Healthy People fresh excreta 0.2g/ pipe, add respectively the Plesiomonas shigelloides dilution suspension of 100 μ l different concns, make 5 × 10
1～5 × 10
7the ight soil simulated samples of CFU/g gradient concentration;
(4) all stool samples of application QIAamp DNA stool mini kit (QIAGEN, Venlo, Netherlands) test kit extracting, each bacterium amount concentration is carried out 3 times and is repeated, and the parallel independent DNA that extracts carries out LAMP detection.
Result: LAMP detects under the detection in ight soil simulated samples and is limited to 5 × 10
3cFU/g, by contrast, real-time fluorescence PCR is limited to 5 × 10 for hugA gene under the detection in ight soil simulated samples
4cFU/g, illustrates that LAMP is sensitiveer to the detection of ight soil simulated samples than real-time fluorescence PCR.
Claims (7)
1. for a method for the detection Plesiomonas shigelloides of non-diagnostic purpose, described method comprises the steps:
(1) a pair of outer primer as shown in SEQ ID NO1-2, sequence a pair of inner primer, a pair of ring primer, dNTP, Bst archaeal dna polymerase, the MgSO of sequence as shown in SEQ IDNO5-6 as shown in SEQ ID NO3-4 by DNA extraction thing, sequence
4, trimethyl-glycine and Bst DNA polymerase buffer liquid joins in reaction vessel; Wherein, described MgSO
4concentration be 6mM, the concentration of described trimethyl-glycine is 10mM;
(2) mixed solution of step (1) gained is positioned in 65 DEG C of isoperibols and carries out DNA amplification reaction;
(3) product) step (2) being obtained is positioned in 80 DEG C of isoperibols, to stop the described DNA amplification reaction of step (2);
(4) product that detecting step (3) obtains.
2. method according to claim 1, is characterized in that, the DNA amplification reaction time described in step (2) is 1 hour.
3. method according to claim 1, is characterized in that, the termination reaction time described in step (3) is 5 minutes.
4. method according to claim 1, is characterized in that, the described detection method of step (4) can be electrophoresis detection method, turbidity detection method or staining examine method.
5. method according to claim 4, is characterized in that, the dyestuff that described staining examine uses is SYBR green I fluorescence dye.
6. detect a test kit for Plesiomonas shigelloides, described test kit comprises: a pair of outer primer, sequence a pair of inner primer, sequence a pair of ring primer, dNTP, Bst DNA polysaccharase, the MgSO as SEQ ID NO5-6 as shown in as SEQ ID NO3-4 as shown in of sequence as shown in SEQ ID NO1-2
4, trimethyl-glycine, Bst DNA polymerase buffer liquid.
7. test kit according to claim 6 is characterized in that described test kit also comprises DNA extraction test kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210262462.1A CN102827928B (en) | 2012-07-27 | 2012-07-27 | Rapid diagnosis method for plesimonas shigelloides |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210262462.1A CN102827928B (en) | 2012-07-27 | 2012-07-27 | Rapid diagnosis method for plesimonas shigelloides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102827928A CN102827928A (en) | 2012-12-19 |
| CN102827928B true CN102827928B (en) | 2014-07-30 |
Family
ID=47331235
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210262462.1A Expired - Fee Related CN102827928B (en) | 2012-07-27 | 2012-07-27 | Rapid diagnosis method for plesimonas shigelloides |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102827928B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105050720A (en) | 2013-01-22 | 2015-11-11 | 华盛顿大学商业化中心 | Sequential delivery of fluid volumes and associated devices, systems and methods |
| CN106868160A (en) * | 2017-03-21 | 2017-06-20 | 杭州迪安生物技术有限公司 | Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously |
| CN107988399A (en) * | 2017-12-06 | 2018-05-04 | 佛山科学技术学院 | Loop-mediated isothermal amplification (LAMP) primer group, nucleic acid test strip kit and its detection method of a kind of Plesiomonas shigelloides |
| CN110724752B (en) * | 2019-11-06 | 2023-03-07 | 广东海洋大学深圳研究院 | Method for detecting zoonosis pathogenic bacteria of human fish by multiple PCR |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101082581A (en) * | 2006-05-30 | 2007-12-05 | 广州华峰生物科技有限公司 | Colon bacillus 0157 gene rapid diagnosis reagent kit of annular mediated isothermal amplification technology |
| CN101659992A (en) * | 2009-03-20 | 2010-03-03 | 曹际娟 | Detection kit and detection method for 5 new pathogens in foods |
| CN102304573A (en) * | 2011-08-24 | 2012-01-04 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence for bacterium diagnosis and application |
-
2012
- 2012-07-27 CN CN201210262462.1A patent/CN102827928B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101082581A (en) * | 2006-05-30 | 2007-12-05 | 广州华峰生物科技有限公司 | Colon bacillus 0157 gene rapid diagnosis reagent kit of annular mediated isothermal amplification technology |
| CN101659992A (en) * | 2009-03-20 | 2010-03-03 | 曹际娟 | Detection kit and detection method for 5 new pathogens in foods |
| CN102304573A (en) * | 2011-08-24 | 2012-01-04 | 中国疾病预防控制中心传染病预防控制所 | Nucleotide sequence for bacterium diagnosis and application |
Non-Patent Citations (4)
| Title |
|---|
| 孟双等.类志贺邻单胞菌实时荧光TaqMan PCR快速检测体系的建立.《疾病监测》.2011,第26卷(第11期),第906-910页. |
| 王素秋等.类志贺邻单胞菌检测的研究.《中国卫生检验杂志》.1993,第3卷(第5期),第291-293页. |
| 类志贺邻单胞菌实时荧光TaqMan PCR快速检测体系的建立;孟双等;《疾病监测》;20111130;第26卷(第11期);第906-910页 * |
| 类志贺邻单胞菌检测的研究;王素秋等;《中国卫生检验杂志》;19931231;第3卷(第5期);第291-293页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102827928A (en) | 2012-12-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Boughner et al. | Microbial ecology: where are we now? | |
| CN101831493A (en) | Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method | |
| CN102816847B (en) | LAMP primer for detecting Brucella and kit containing the same | |
| CN102747165B (en) | Reagent kit and method for quickly detecting tilapia streptococcus agalactiae by using loop-mediated isothermal amplification technology | |
| Lu et al. | LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila | |
| CN102827928B (en) | Rapid diagnosis method for plesimonas shigelloides | |
| CN102864228A (en) | Loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus | |
| CN102094090B (en) | Cholera toxin virulence gene detection kit and detection method thereof | |
| CN101880709B (en) | Salmonella enteritidis detection reagent kit and method based on loop-mediated isothermal amplification technology | |
| CN103468811A (en) | Yersinia enterocolitica virulence gene multiplex-PCR (Polymerase Chain Reaction) detection primer group and kit | |
| Lee et al. | Phylogenetic analysis of microalgae based on highly abundant proteins using mass spectrometry | |
| CN111378774B (en) | Primer group, kit and method for rapidly detecting listeria monocytogenes | |
| CN102367475A (en) | M-PCR (multiplex-polymerase chain reaction) detection kit for different serotype vibrio cholerae and detection method thereof | |
| CN116042902A (en) | Real-time fluorescent nucleic acid isothermal amplification detection kit for simultaneously detecting six candida species and special primer and probe thereof | |
| CN109439781A (en) | For detecting the application of the Primer composition, kit and kit of clostridium difficile gene | |
| CN104372099B (en) | A kind of LAMP detection primer compositionss of Phytophthora cactorum bacterium and its LAMP detection kit and LAMP detection method | |
| CN101736081A (en) | Kit for rapidly detecting isothermal gene amplification of Mycobacterium tuberculosis and detection method | |
| CN104152546A (en) | Kit and method for simultaneously detecting salmonella, listeria monocytogenes and staphylococcus aureus | |
| CN112725480B (en) | Primer group, detection method and kit for rapidly detecting salmonella typhi by LAMP (loop-mediated isothermal amplification) technology | |
| CN102851383A (en) | LAMP kit for rapid detection of Salmonella | |
| CN101565753A (en) | Rapid diagnostic kit for staphylococcus aureus gene based on loop-mediated isothermal amplification technology and detecting method thereof | |
| CN102851382A (en) | LAMP kit for rapid detection of Shigella | |
| CN101368203A (en) | Primer, reagent kit and detection method for monotonic increasing Listeria hymenial veil mediated isothermality amplification technique fast detection | |
| CN101586157A (en) | Diagnostic kit for Streptococcus iniae molecule and detection method | |
| CN101555529A (en) | Loop-mediated isothermal amplification technology-based Listeria monocytogenes rapid diagnostic kit and testing 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 | ||
| 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: 20140730 Termination date: 20160727 |