CN101665838A - Escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization - Google Patents
Escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization Download PDFInfo
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- CN101665838A CN101665838A CN200910308402A CN200910308402A CN101665838A CN 101665838 A CN101665838 A CN 101665838A CN 200910308402 A CN200910308402 A CN 200910308402A CN 200910308402 A CN200910308402 A CN 200910308402A CN 101665838 A CN101665838 A CN 101665838A
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Abstract
The invention discloses an escherichia coli detection method based on a time-resolved fluorescence method and DNA hybridization. A capture probe DNA1 and an identifying probe DNA 2 are designed according to a DNA target sequence of escherichia coli; the capture probe DNA1 is fixedly marked on a glass sheet; and the identifying probe DNA 2 is jointed with long-life luminescent rare-earth europium complex. When the capture probe DNA1 and the identifying probe DNA 2 are connected in series and can generate base-pair complementary hybridization reaction with the target DNA (DNA of biology to be detected); and a DNA detection model based on two serial-connected probes on the surface of the glass sheet is established. The long-life luminescent rare-earth europium complex is used as a marker distinguishing the probe DNA 2; and the time-resolved fluorescence method can effectively eliminate the disturbance of fluorescent sources in a biological system and background light of a solid matrix. The detection method takes little time, is visual, flexible, accurate in reaction results and has very obvious advantages.
Description
Technical field
The invention belongs to the microorganism detection field, relate to a kind of detection method of the colon bacillus based on time-resolved fluorescence method and DNA hybridization.
Background technology
Colon bacillus (E.coli) is commonly referred to intestinal bacteria, be that Escherich found in 1885, be considered to a kind of common prokaryotic micro-organisms in the quite a long time, be that the normal microflora in human and the most of warm-blooded animal enteron aisles is a non-pathogenic bacteria always.Up to 20 middle of century, the intestinal bacteria of just recognizing some special serotypes have pathogenic to humans and animals, often cause serious diarrhoea and septicemia.From nineteen eighty-two since the U.S. at first finds, comprise that all there is report in many countries such as China, and day by day increase.To break out incident greatly especially noticeable because of this bacterium of food pollution causes several in 1996 in Japan.In the common isolating pathogen enterobacteria of the U.S. and Canada, it has come front three.At present, colon bacillus is decided to be the indication water body by an index of fecal pollution, is the indirect index of pathogenic bacterium polluted-water.Therefore, be the important means of control bacteriosis generation and medical diagnosis on disease and treatment to fast, accurately checking of colon bacillus with evaluation.
Existing escherichia coli detection method comprises traditional method for cultivation of bacteria and biochemical identification, immunological method, shortcoming such as there is complex operation in these methods, wastes time and energy, detection efficiency is low; Fluorescence in situ hybridization method (FISH, fluorescencein site hybridization) for the detection of pathogenic micro-organism common in the environment, be be widely used, one of otherwise effective technique the most, but fluorescent probe is subject to the interference of endogenous fluorescence background of living things system and fixation of microbe matrix background in the FISH method, and sensitivity is restricted; Biochip technology is the new technology that develops rapidly in recent years, and the detection of bacterium is had advantages such as quick, accurate, efficient, but its high cost of equipment and running cost is difficult to bear for most feeler mechanisies, and its versatility is subjected to very big restriction.Therefore, how adopting better method or comparatively simple instrument to carry out high-sensitivity detection is at present colon bacillus to be detected the heat subject of research.
The fluorescent substance that will have long-term durability luminous performance combines with the bioprobe technology, utilizes time-resolved method can effectively eliminate the interference of fluorescence background and solid substrate background fluorescence in the sample, can effectively improve the sensitivity of detection.The present invention will be with thenoyltrifluoroacetone (TTA), 5-amino-1,10-phenanthroline (NH
2-phen) be the long lifetime fluorescent rare earth europium complex Eu (TTA) of part
3Phen is as the fluorescent substance of sign dna probe, adopt capture probe and the series connection of identification probe and target DNA fragment specific to form the detection technique of hybridization system, set up tentatively that a kind of versatility that colon bacillus is detected is good, the method for inspection of highly sensitive and tolerance range, for developing novel long lifetime biological sensor, make up the long lifetime biological sensing new technology of highly sensitive, highly selective, being applied in the environment detection such as microorganism has important significance for theories and using value.
Summary of the invention
The purpose of this invention is to provide a kind of detection novel method that is used for the environment colon bacillus.Its time spent is short, and reaction result is directly perceived, sensitive, accurate.
The objective of the invention is to be achieved through the following technical solutions.
Escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization is as follows:
A, capture probe DNA1's is fixing
With the slide aldehyde radicalization, be 5 '-ACA TTG ACG CAG GTG ATC GGA CGTTTTTTTTTT-3 '-NH again with base sequence earlier
2Capture probe DNA
1Be made into working concentration 5 μ g/mL, get 40 μ LDNA
1Be added drop-wise to the surface of glass slide of aldehyde radicalization, room temperature reaction 3h; Wash and seal remaining aldehyde radical at last;
B, rare-earth europium title complex mark identification dna probe
2
(1) with 2mg rare-earth europium title complex Eu (TTA)
3(NH
2-phen) be distributed in 5mL 2.5% glutaraldehyde solution, concuss 5h under the room temperature, centrifugal back obtains aldehyde group modified Eu (TTA) with second distillation washing 3 times
3It is in 7.2 the PBS solution that the phen particle is suspended from 4mL pH again;
(2) be NH with base sequence again
2The identification dna probe of-5 '-TTTTTTTTTT GTA TCG GTG TGA GCG TCG CAG AA-3 '
2Be made into working concentration 15 μ g/mL, join in the PBS solution of rare-earth europium title complex particle suspension, in the concussion incubator, react 5h under 30 ℃, centrifuge washing after reaction is finished; Redispersion is standby in 2mL PBS damping fluid;
C, extraction colon bacillus DNA
D, hybridization
(1) before hybridization, the colon bacillus genomic dna that extracts unwind by high temperature makes its sex change become strand;
(2) with the identification dna probe of rare-earth europium title complex on the mark
2Fixing DNA with the single stranded DNA to be measured that extracts
1Slide on hybridize 10h; Crossbred is: the sex change single stranded DNA solution that 10 μ L extract, 10 μ L marks the DNA of rare-earth europium title complex
2Solution, 10 μ L12 * SSC solution; Keeping hybridization temperature is 43 ℃;
(3) after the hybridization, slide is cleaned in following three kinds of washingss successively, washings I 1 * SSC/0.03%SDS, washing lotion II 0.2 * SSC and washing lotion III 0.05 * SSC, washing time is 2min, and wash temperature is 43 ℃;
E, hybridization back signal detection
The identification DNA that has rare-earth europium title complex mark after hybridization is cleaned
2, capture dna
1Crossbred with the ssDNA probe of extracting to be measured forms produces an optical signal under laser excitation, detect the optical signal of the long-term durability luminous title complex of rare earth on the slide, excite and launch slit to be respectively 8.0nm, time of lag 0.1ms, door time 1.0ms; According to whether having colon bacillus DNA in the optical signal detecting sample that detects.
It is as follows to wash and seal remaining aldehyde radical process during described A goes on foot:
(1) with 2 slides of 0.2% SDS washing, second distillation water washing 2 times, room temperature is dried;
(2) with remaining aldehyde radical on the 0.1M glycine sealing slide, be 1h action time;
2 slides of SDS washing of (3) 0.2%, second distillation water washing 2 times, room temperature is dried standby.
Beneficial effect of the present invention:
1, the probe that uses among the present invention is to screen from the conservative region of the 16SrRNA of colon bacillus, the specific fragment target dna sequence that obtains by the sequence alignment in the GenBank database again, through Primer Premier 5 software analysis it is cut to two sections with another dna sequence dna of target sequence complementary, sectional is according to the T that is two sections
mBe worth unanimous on the whole.Wherein one section as capture probe DNA
1, another section is as the identification dna probe
2, capture probe DNA
1Fixing mark is on sheet glass; The identification dna probe
2Connect long-term durability luminous rare earth compounding.DNA wherein
13 ' end and DNA
25 ' end connect 10 T, reduce the steric hindrance between probe and connector, make DNA
1With DNA
2The hybridization of base complementrity can more effectively take place with target dna (DNA of biology to be measured) during series connection.Non-specific adsorption in the hybridization is got rid of, obtained believable fluorescent signal, colon bacillus is carried out qualitative and quantitative check.
2, the hybridization that first the original gene group dna direct that extracts is used for model of the present invention, solved the practical problems that runs in the microorganism detection, this DNA is hybridized form for long-chain in crossover process, for the application in practice based on time-resolved fluorescence method and DNA hybridizing method lays a solid foundation.In addition, the DNA of extraction need not steps such as purifying and pcr amplification, has reduced the probability of dna degradation, reduces the pollutent of introducing in the experiment, this some all simplified experimentation greatly.
3, be to use the nanoparticle label DNA that the rare-earth europium title complex is made in the prior art, the present invention directly uses rare-earth europium title complex unit molecule to be marked on the DNA, can satisfy the needs of fluoroscopic examination fully, and can also save the complicated processes for preparing nano particle, save cost, simplified the detection step.
4, hybridization time and washing time are the important factor in order of hybridization, and the quality of reaction and efficient are directly connected to the accuracy of detected result.The present invention has especially done a large amount of work by measures such as strict control hybridization conditions and wash conditions on hybridization and washing time.Through repetition test, finally find out a cover and be suitable for the top condition that colon bacillus hybridization detects.
5, first with capture probe DNA
1With the identification dna probe
2The series connection form, be applied to the detection of colon bacillus with the complementation hybridization mode of target dna base sequence, improved the specificity of hybridization greatly, its detection method time spent is short, traditional total number of bacterial colony measuring method needs just can obtain the result more than 24 hours, and the present invention obtains detected result only needs 12 hours, and be quick on the draw, the result is accurate, can obtain detected result very intuitively, have very significant advantage.
6, the common slide of utilization is as the carrier of hybridization, and long-term durability luminous rare-earth europium title complex has been realized the chemical reaction of solid-liquid-solid system as hybridization result's detection thing, can effectively hybridization signal be separated with non-hybridization signal.
7, with the marker of long-term durability luminous rare-earth europium title complex as hybridization, utilize the method for time resolved fluorescence can effectively eliminate the interference of endogenous fluorescence background of organism and solid substrate background fluorescence, strengthen fluorescence signal intensity, improved the sensitivity that detects.
Description of drawings
Fig. 1 is the influences of different hybridization time to experimental result;
Fig. 2 is the influences of different washing times to experimental result;
Fig. 3 is the spectrogram (time of lag 0.1ms, door time 1.0ms) of colon bacillus sample detected time resolved fluorescence on slide in the landscape water.
1 for detecting the strength of signal of landscape water sample among the figure, and 2 is the strength of signal of blank.
Embodiment
Be intended to below the present invention is described rather than the present invention is further limited that the present invention can implement by the described arbitrary mode of summary of the invention.
One, the design of capture probe and identification probe
Dna probe sequence basis of the present invention is publication (Yao Zhijian, in bravely, the upright people of horse etc., detect gene chip, preparation method, the test kit of multiple common bacteria pathogenic agent, Chinese patent, open day on October 3rd, 2007, publication number CN101045945A) the gene conserved sequence of the 16S rRNA of colon bacillus screens in.
Reach the sequence alignment at American National bioinformation center (NCBI) database more by literature search, special target dna sequence is 5 '-CGT CCG ATC ACC TGC GTC AAT GTA ATG TTC TGC GAC GCTCAC ACC GAT AC-3 ' between guarding, plant in obtaining planting.With target sequence complementary dna sequence dna: 5 '-GTA TCG GTG TGA GCG TCG CAG AACATT ACA TTG ACG CAG GTG ATC GGA CG-3 '.Through Primer Premier 5
Software analysis will be cut to two portions with target sequence complementary dna sequence dna, and wherein one section as capture probe DNA
1, another section is as the identification dna probe
2, capture probe DNA
1Fixing mark is in glass sheet surface; The identification dna probe
2Connect long-term durability luminous rare earth compounding.Capture probe DNA
1With the identification dna probe
2During series connection can with the hybridization of target dna generation base complementrity.
Concrete sequence is as follows:
Capture probe DNA
1: 5 '-AC ATT GAC GCA GGT GAT CGG ACG TTTTTTTTTT, 3 '-NH
2
The identification dna probe
2: NH
2-5 '-TTTTTTTTTT GTA TCG GTG TGA GCG TCG CAG AA-3 '
Two, capture probe DNA
1Fixing
Capture probe DNA
1Base sequence: 5 '-ACA TTG ACG CAG GTG ATC GGA CG TTTTTTTTTT-3 '-NH
2(synthetic) by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
1. the aldehyde radicalization of slide:
(1) slide is cut to square slide (diameter 13mm), puts into the chromic acid soaked overnight, redistilled water cleans 2 times.Then slide is immersed in 25% ammoniacal liquor and soak 5h, redistilled water cleans 2 times.
(2) slide is immersed in the 2% aminosilane reagent (95% ethanolic soln) again, regulate pH to 4.5 with Glacial acetic acid, room temperature effect 30min connects surface of glass slide and goes up amino group.Use the dehydrated alcohol ultrasonic cleaning afterwards 2 times, redistilled water ultrasonic cleaning 2 times.Dry slide under the room temperature.
(3) under the room temperature glutaraldehyde (pH is 7.2 PBS solution preparation) of slide and 2.5% is reacted 3h, make surface of glass slide connect aldehyde groups.After reaction finished, PBS cleaned 2 times, and redistilled water cleans 2 times.Dry slide under the room temperature.
2. surface of glass slide connects capture probe DNA
1
(1) capture probe DNA
1Be made into working concentration 5 μ g/mL, get 40 μ L DNA
1Be added drop-wise to the surface of glass slide of aldehyde radicalization, room temperature reaction 3h makes the amino of capture probe end and the aldehyde radical generation schiff base reaction on the slide, thus with probe stationary on slide.
(2) with 2 slides of 0.2% SDS washing, second distillation water washing 2 times.Room temperature is dried.
(3) with remaining aldehyde radical on the 0.1M glycine sealing slide, be 1h action time.
2 slides of SDS washing of (4) 0.2%, second distillation water washing 2 times.Room temperature is dried standby.
Three, rare-earth europium title complex mark identification dna probe
2
The identification dna probe
2Base sequence: NH
2-5 '-TTTTTTTTTT GTA TCG GTG TGA GCG TCG CAG AA-3 ' (synthetic) by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
1. rare-earth europium title complex aldehyde radicalization:
(1) with 2mg rare-earth europium title complex Eu (TTA)
3(NH
2-phen) be distributed in 5mL 2.5% glutaraldehyde (pH is 7.2 the PBS solution preparation) solution, concuss 5h under the room temperature, centrifugal back obtains aldehyde group modified Eu (TTA) with second distillation washing 3 times
3It is standby in 7.2 the PBS damping fluid that the phen particle is suspended from 4mL pH again;
(2) identification dna probe
2Be made into working concentration 15 μ g/mL, will discern dna probe
2Join in the PBS solution of rare-earth europium title complex particle suspension, in the concussion incubator, react 5h under 30 ℃, redistilled water centrifuge washing after reaction is finished; With DNA on the mark
2The rare-earth europium title complex be scattered in the 2mL PBS damping fluid standby.
Four, colon bacillus DNA extraction
1. the colon bacillus strain is seeded in concussion cultivation in the LB nutrient solution, 37 ℃ of culture temperature, and the logarithmic phase that arrives cell behind the incubation time 16h stops to cultivate.DNA extraction adopts the bacterial genomes DNA extraction test kit (centrifugal column type) of Shanghai Jierui Biology Engineering Co., Ltd to be numbered GK1071, extracts the colon bacillus genomic dna according to reagent and scheme that test kit provided, obtains original DNA solution.
2. the nucleic acid protein analyser that uses U.S. Bechman company to produce the genomic dna that extracts detects, and with 15 times of original DNA solution dilutions, records A
260/ A
280Ratio be 1.8, OD
260Value be 0.42, show that tentatively the DNA purity of extraction is higher, can be directly used in subsequent experimental.
The optimization of five, hybridization system and hybridization time
1. hybridization system
Before hybridization, the genomic dna that extracts unwind by high temperature makes its sex change become strand, and wherein melting temperature(Tm) is 95 ℃, and time 5min unwinds.For the DNA that prevents to have unwind renaturation in the temperature that slowly falls after rise, separate in the mixture of ice and water that rapidly it is changed over to 4 ℃ after the end of chain (EOC), place 5min, obtain strand target colon bacillus DNA to be measured.
Identification dna probe with rare-earth europium title complex on the mark
2Fixing DNA with the single stranded DNA to be measured that extracts
1Slide on carry out hybridization.Crossbred is: the single stranded DNA solution that 10 μ L extract, 10 μ L marks the DNA of rare-earth europium title complex
2Solution, 10 μ L12 * SSC solution.The slide that will carry out hybridization places the place mat cotton also to keep in the wet box of hybridization of humidity with 3 * SSC solution, and keeping hybridization temperature in constant incubator is 43 ℃.
2. hybridization time gropes
The hybridization time is making nucleic acid molecular hybridization experiment significant effects factor.Under the situation that other conditions all satisfy, the time of reaction is depended in the success or failure of hybridization.Time is too short, and hybridization is not finished; Overlong time is also unhelpful, can cause that non-specific binding increases.
Grope by a large amount of experiments, hybridization time has been carried out comprehensive research, investigation scope is the most at last dwindled between 2h to 18h, the hybridization time of investigate setting is 3,5,7,9,10,12,16,18h, after arriving the time of setting, slide is taken out washing from constant temperature hybridization case.Different hybridization time are seen accompanying drawing 1 to result's influence.Determine that from detected result hybridization time is 10h.
Six, washing time gropes
In the elution process, if probe and its complementary strand unwind, this result that unwinds is irreversible mostly.Because free concentration and probe concentration in washings was hanged down excessively and was difficult to restart hybridization this moment, and along with further wash-out, concentration and probe concentration can be more and more lower.For dissociating of the base that prevents to hybridize the back complementary pairing, guarantee again nonspecific adsorbent is removed in washing process, must be optimized wash conditions.
Set different wash temperatures, the kind of washing composition, and factor such as washing time are groped by a large amount of tests, and the washings composition that obtains is: and washings I (1 * SSC, 0.03%SDS), washing lotion II (0.2 * SSC) and washing lotion III (0.05 * SSC); Wash temperature is 43 ℃; Especially washing time is defined as 2min/ washing lotion kind.Different washing times are seen accompanying drawing 2 to the influence of detected result.
Seven, hybridization back signal detection
The identification DNA that has rare-earth europium title complex mark after hybridization is cleaned
2, capture dna
1Form crossbred with the ssDNA probe of extracting in surface of glass slide, be 8.0nm exciting and launch slit, time of lag 0.1ms, door time 1.0ms; Excitation wavelength is that emission wavelength of the following generation of the testing conditions of 368nm is the 610nm optical signal, has detected the optical signal of the long-term durability luminous title complex of rare earth on the slide, thereby according to whether having colon bacillus DNA in the optical signal detecting sample that detects.
Eight, detect example---the detection of colon bacillus in the landscape water
Water sampling 100mL in certain landscape lake from mountain scene district, the foot of a hill or mountain, high mountain takes back the laboratory rapidly and preserves analysis after the sealing.Get 10mL view water sample high-speed refrigerated centrifuge (Eppendorf5415R, Germany) in temperature be that 4 ℃, centrifugal speed are centrifugal 10min under the 12000rpm condition, obtain the microorganism cells in the water sample after the abandoning supernatant.The microorganism cells that this 10mL water sample is obtained carries out DNA extraction, concrete grammar adopts the bacterial genomes DNA extraction test kit (centrifugal column type) of Shanghai Jierui Biology Engineering Co., Ltd to be numbered GK1071, the mixing genomic dna that extracts the view water sample according to reagent that test kit provided and scheme.At last the DNA that extracts is hybridized detection with the inventive method under hybridization conditions after the optimization and wash conditions, determine whether there is colon bacillus in the landscape water.Detected result is seen accompanying drawing 3.
Experiment has used traditional microbe colony detection method to carry out with reference to the contrast experiment when adopting the inventive method to detect.Promptly adopting the special substratum of differentiating colon bacillus---the U.S. blue substratum in Yihong (EMB) carries out the dilution plate experiment to the view water sample.The former view water sample of 10mL is got in experiment, and to obtain concentration by 10 times of concentration dilution methods be 10
-1With 10
-2Serial water sample.2 dilutions water samples and former water samples are got 1mL respectively be coated on and sterilize and be frozen on the U.S. blue substratum in dull and stereotyped Yihong, after the coating evenly culture dish is inverted, in 37 ℃ constant incubator, leave standstill cultivation 24h.According to the total number of bacterial colonies assay method, with enumeration and report manner CFUmL
-1(colony forming unit) reports detected result, records that the colon bacillus colony number is 2 * 10 in the view water sample
4CFU mL
-1
Sequence table
＜110〉Hunan University
＜120〉a kind of escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization
<130>CN?101045945?A
<160>2
<170>PatentIn?version?3.3
<210>1
<211>33
<212>DNA
<213>Escherichia?coli
<400>1
acattgacgc?aggtgatcgg?acgttttttt?ttt 33
<210>2
<211>33
<212>DNA
<213>Escherichia?coli
<400>2
tttttttttt?gtatcggtgt?gagcgtcgca?gaa 33
Claims (2)
1. the escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization is characterized in that,
A, capture probe DNA1's is fixing
Earlier with the slide aldehyde radicalization, the capture probe DNA1 that with base sequence is 5 '-ACA TTG ACG CAG GTG ATC GGA CGTTTTTTTTTT-3 '-NH2 again is made into working concentration 5 μ g/mL, get 40 μ LDNA1 and be added drop-wise to the surface of glass slide of hexanal baseization, room temperature reaction 3h; Wash and seal remaining aldehyde radical at last;
B, rare-earth europium title complex mark identification dna probe 2
(1) 2mg rare-earth europium title complex Eu (TTA) 3 (NH2-phen) is distributed in 5mL 2.5% glutaraldehyde solution, concuss 5h under the room temperature, centrifugal back is with second distillation washing 3 times, and obtaining aldehyde group modified Eu (TTA) 3phen particle, to be suspended from 4mL pH again be in 7.2 the PBS damping fluid;
(2) be that the identification dna probe 2 of NH2-5 '-TTTTTTTTTT GTA TCG GTG TGA GCG TCG CAG AA-3 ' is made into working concentration 15 μ g/mL again with base sequence, join in the PBS solution of rare-earth europium title complex particle suspension, in the concussion incubator, react 5h under 30 ℃, centrifuge washing after reaction is finished; Redispersion is standby in 2mL PBS damping fluid;
C, extraction colon bacillus DNA
D, hybridization
(1) before hybridization, the colon bacillus genomic dna that extracts unwind by high temperature makes its sex change become strand;
(2) the identification dna probe 2 of rare-earth europium title complex on the mark is hybridized 10h with the single stranded DNA to be measured that extracts on the slide of having fixed DNA1; Crossbred is: the sex change single stranded DNA solution that 10 μ L extract, 10 μ L marks the DNA2 solution of rare-earth europium title complex, 10 μ L12 * SSC solution; Keeping hybridization temperature is 43 ℃;
(3) after the hybridization, slide is cleaned in following three kinds of washingss successively, washings I 1 * SSC/0.03%SDS, washing lotion II 0.2 * SSC and washing lotion III 0.05 * SSC, washing time is 2min, and wash temperature is 43 ℃;
E, hybridization back signal detection
The crossbred that the ssDNA probe to be measured that has identification DNA2, the capture dna 1 of rare-earth europium title complex mark after hybridization is cleaned and extract forms, under laser excitation, produce an optical signal, detect the optical signal of the long-term durability luminous title complex of rare earth on the slide, excite and launch slit and be respectively 8.0nm, time of lag 0.1ms, the door time 1.0ms; According to whether having colon bacillus DNA in the optical signal detecting sample that detects.
2. method according to claim 1 is characterized in that, described A washing and to seal remaining aldehyde radical process as follows in the step:
(1) with 2 slides of 0.2% SDS washing, second distillation water washing 2 times, room temperature is dried;
(2) with remaining aldehyde radical on the 0.1M glycine sealing slide, be 1h action time;
2 slides of SDS washing of (3) 0.2%, second distillation water washing 2 times, room temperature is dried standby.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507934A (en) * | 2011-11-02 | 2012-06-20 | 集美大学 | Time-resolved fluoroimmunoassay (TRFIA) of Klebsiella oxytoca |
| CN103966344A (en) * | 2014-05-29 | 2014-08-06 | 福建省中医药研究院 | DNA (deoxyribonucleic acid) bioluminescence sensor for identifying radix psuedostellariae species |
| WO2022032447A1 (en) * | 2020-08-10 | 2022-02-17 | 深圳先进技术研究院 | Detection method for enhancing rare earth complex fluorescence based on nucleic acid hybridization |
| CN114062328A (en) * | 2020-08-10 | 2022-02-18 | 深圳先进技术研究院 | Detection method for enhancing fluorescence of rare earth complex based on nucleic acid hybridization |
-
2009
- 2009-10-16 CN CN200910308402A patent/CN101665838A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507934A (en) * | 2011-11-02 | 2012-06-20 | 集美大学 | Time-resolved fluoroimmunoassay (TRFIA) of Klebsiella oxytoca |
| CN102507934B (en) * | 2011-11-02 | 2014-02-26 | 集美大学 | Time-resolved fluoroimmunoassay (TRFIA) of Klebsiella oxytoca |
| CN103966344A (en) * | 2014-05-29 | 2014-08-06 | 福建省中医药研究院 | DNA (deoxyribonucleic acid) bioluminescence sensor for identifying radix psuedostellariae species |
| CN103966344B (en) * | 2014-05-29 | 2015-11-25 | 福建省中医药研究院 | A kind of DNA bioluminescence sensor identifying Root of Heterophylly Faalsestarwort kind |
| WO2022032447A1 (en) * | 2020-08-10 | 2022-02-17 | 深圳先进技术研究院 | Detection method for enhancing rare earth complex fluorescence based on nucleic acid hybridization |
| CN114062328A (en) * | 2020-08-10 | 2022-02-18 | 深圳先进技术研究院 | Detection method for enhancing fluorescence of rare earth complex based on nucleic acid hybridization |
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