CN101845486A - Detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization - Google Patents
Detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization Download PDFInfo
- Publication number
- CN101845486A CN101845486A CN200910227169A CN200910227169A CN101845486A CN 101845486 A CN101845486 A CN 101845486A CN 200910227169 A CN200910227169 A CN 200910227169A CN 200910227169 A CN200910227169 A CN 200910227169A CN 101845486 A CN101845486 A CN 101845486A
- Authority
- CN
- China
- Prior art keywords
- dna
- hybridization
- time
- probe
- rare
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization. A capture probe DNA1 and a recognition probe DNA2 are designed according to the DNA target sequence of the staphylococcus aureus, and a DNA detection model in serial connection based on two probes on the surface of an ordinary slide is constructed. Luminous rare earth europium coordination compounds with long service life are used as markers for recognizing the probe DNA2, and a time-resolved fluorescence method can effectively eliminate the interference of the biosystem intrinsic fluorescence and the solid substrate background light. Through the strict control on the measures of hybridization experiment factors, washing conditions and the like, the optimum detection conditions of the hybridization system is obtained through a large amount of repeated experiment research, satisfactory specificity and sensitivity are obtained, and the invention provides scientific and systemic reference conditions for the detection methods of the staphylococcus aureus. The invention has the characteristics of short time consumption, visualized reaction results, sensitivity and accuracy, and has very obvious advantages.
Description
Technical field
The invention belongs to the microorganism detection field, relate to a kind of method that detects golden yellow staphylococcus based on two probes series connection DNA hybridization models and time-resolved fluorescence method bonded.
Background technology
Streptococcus aureus is widespread in nature, and can find in the movement of air, water body, dust and humans and animals.Streptococcus aureus is a kind of important pathogenic microorganism, it can produce virulence factors such as more toxin and enzyme, and virulence is strong, can cause multiple disease by infection of staphylococcus aureus, comprise food poisoning, pseudomembranous enterocolitis, scalded skin syndromes, toxin shock syndromes, suppurative inflammation and septicemia etc., cause great threat and loss to the mankind, caused people's extensive concern.In recent years, the Center for Disease Control report, the infection that is caused by streptococcus aureus accounts for second, be only second to intestinal bacteria, Staphylococcus aureus enterotoxin is a global health difficult problem, by the food poisoning that Staphylococcus aureus enterotoxin causes, accounts for 33% of whole bacterial food poisoning in the U.S., Canada reaches 45%, and etesian this type of poisoning of China is also very many.
At present, the detection method of streptococcus aureus roughly comprises traditional method for cultivation of bacteria and biochemical identification, immunological method, fluorescence quantitative PCR method, fluorescence in situ hybridization method (FISH, Fluorescent in situ hybridization) and gene chips etc., these detection methods all have advantage separately but also have simultaneously deficiency on the different levels, can directly apply in the detection of complex sample such as the FISH method, and need not to extract the DNA of sample, directly kept the integrity of sample DNA; But factors such as the decay of the autofluorescence of some thalline itself, fluorescent probe signal in the sample, fixed microorganism cells are very few all may cause experiment to produce false negative result; Advantages such as gene chips is the nucleic acid detection technique that develops rapidly in recent years, and it is quick, convenient and simple become the bright spot of this technology, but because the advanced test set and the running cost of this Technology Need costliness make it be difficult to apply.
The fluorescent substance that will have long-term durability luminous performance combines with the bioprobe technology, utilizes time-resolved method can effectively eliminate fluorescence background and the fixedly interference of matrix bias light in the sample, can effectively improve the sensitivity of detection.The present invention adopts the research synthetic long lifetime fluorescent rare earth europium complex Eu (TTA) of this team
3Phen is as the fluorescent substance of sign dna probe, series connection of two double function probes and target DNA fragment specific are formed the detection technique of hybridization system, set up tentatively that a kind of versatility that streptococcus aureus is detected is good, the method for inspection and the testing conditions thereof of highly sensitive and tolerance range.
Summary of the invention
The detection method that the purpose of this invention is to provide a kind of streptococcus aureus based on time-resolved fluorescence method and DNA hybridization.This detection method 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.
The present invention is based on the detection method of the streptococcus aureus of time-resolved fluorescence method and DNA hybridization, may further comprise the steps:
A, capture probe DNA
1Fixing
Earlier with the slide aldehyde radicalization, again with base sequence: 5 '-CAC TTT TTC TTA AAT GTT GTT CTTTTTTTTTT-3 '-NH
2Capture probe DNA
1Be diluted to 1 * 10
-7Mol/L gets 40 μ L DNA
1Be 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) with 2mg rare-earth europium title complex Eu (TTA)
3Phen is distributed in 5mL 2.5% glutaraldehyde solution, concuss 5h under the room temperature, and centrifugal back obtains aldehyde group modified Eu (TTA) with second distillation washing 3 times
3It is standby 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 ATT TTC TCT TTT TTC GCT T-3 '
2Be made into working concentration 15 μ g/mL, the identification dna probe
2Join 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 the 2mLPBS damping fluid.
C, streptococcus aureus DNA extraction
D, hybridization
(1) before the hybridization, the staphylococcus aureus gene group 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
1Sheet glass on hybridize 9h.Component in the hybridization system is the single stranded DNA that 10 μ L extract, 10 μ L marks the DNA of rare-earth europium title complex
2, 10 μ L, 12 * SSC solution; Keeping hybridization temperature is 53 ℃;
(3) after the hybridization, slide is cleaned in following 3 kinds of washingss successively, washings I (1 * SSC/0.03%SDS), washing lotion II (0.2 * SSC) and washing lotion III (0.05 * SSC), total washing time is 4.5 minutes, and each 1.5 minutes, wash temperature was 53 ℃;
E, hybridization back signal detection
The identification DNA that has rare-earth europium title complex mark after hybridization is cleaned
2, capture dna
1The crossbred that forms with the target dna probe is 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 612nm optical signal, detects the optical signal of the long-term durability luminous title complex of rare earth on the slide, thereby according to whether having streptococcus aureus DNA in the optical signal detecting sample that detects.
Beneficial effect of the present invention:
1, the probe that uses among the present invention is to screen from the conservative region of the 16S rRNA of streptococcus aureus, 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, streptococcus aureus 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 streptococcus aureus hybridization detects, especially at aspects such as capture probe concentration, hybridization temperature, time, washing times, overcome the many difficulties during practice detects, found optimal condition.
5, first with capture probe DNA
1With the identification dna probe
2The series connection form, be applied to the detection of streptococcus aureus 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, 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.
The present invention is based on the time resolved fluorescence detection and hybridizes the detection method of setting up on the model based with the placed in-line DNA of two probes.For a specific hybridization system, the quality of hybridization and efficient are directly connected to the accuracy of detected result.The present invention is by measures such as strict control hybrid experiment factor and wash conditions, through in a large number, repeatedly experimental study, obtained should the hybridization system optimal detection condition, and obtained satisfied specificity and sensitivity, the reference conditions of scientific system are provided for the method for inspection of streptococcus aureus.
Description of drawings
Fig. 1 is the influences of different capture probe concentration to results of hybridization;
Fig. 2 is the influences of different hybridization time to results of hybridization;
Fig. 3 is the influences of different washing times to results of hybridization;
Fig. 4 is the specific detection result of this hybridization system under the top condition;
Wherein NO.1 is a staphylococcus epidermidis; NO.2 is intestinal bacteria; NO.3 is a Paenibacillus polymyxa; NO.4 is a streptococcus aureus; The positive control of NO.5; NO.6 is blank (sterilized water).
Fig. 5 is the sensitivity detected result of this hybridization system under the top condition.
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, prepares before the hybridization
1, the aldehyde radicalization of slide
Square slide (length of side 13mm) immerses soaked overnight in the chromic acid lotion, the distillation washing, immerse in 25% ammoniacal liquor and spend the night, after the redistilled water washing, in aminosilane 95% ethanolic soln of immersion 2%, regulate pH to 4.5 with Glacial acetic acid, room temperature effect 30min, use the ethanol ultrasonic cleaning, use the redistilled water ultrasonic cleaning, room temperature is dried.2.5% glutaraldehyde under the room temperature (pH is 7.2 PBS solution preparation) and amino slide effect 2h, the PBS flushing, redistilled water washing (1min/ time) is dried.
2, capture probe DNA
1Fixing
Capture probe DNA
1Base sequence: 5 '-CAC TTT TTC TTA AAT GTT GTT CTTTTTTTTTT-3 '-NH
2(synthetic) by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With dna probe
1Be diluted to the concentration of setting with redistilled water, get 40 μ L DNA
1Be added drop-wise to the surface of glass slide of hexanal baseization, room temperature reaction 3h; Wash and seal remaining aldehyde radical at last;
3, identification dna probe
2Be connected with the rare-earth europium title complex
(1) with 2mg rare-earth europium title complex Eu (TTA)
3Phen is distributed in 5mL 2.5% glutaraldehyde (pH is 7.2 the PBS buffer preparation) solution, concuss 5h under the room temperature, and centrifugal back obtains aldehyde group modified Eu (TTA) with second distillation washing 3 times
3It is standby in 7.2 the PBS solution that the phen particle is suspended from pH again;
(2) identification dna probe
2Base sequence is NH
2-5 '-TTTTTTTTTT ATT TTC TCT TTT TTCGCT T-3 ' (synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) is 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 PBS damping fluid standby.
4, streptococcus aureus DNA extraction
(1) staphylococcus aureus strains is inoculated into concussion cultivation in the NB nutrient solution, 37 ℃ of culture temperature, and the logarithmic phase that arrives cell behind the incubation time 18h 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 staphylococcus aureus gene group DNA according to reagent and scheme that test kit provided, obtains original DNA solution.
(2) genomic dna that extracts used U.S. Bechman company
The nucleic acid protein analyser detects.With 10 times of original DNA solution dilutions, record A
260/ A
280Ratio be 1.9, OD
260Value be 0.35, show that tentatively the DNA purity of extraction is higher, can be directly used in subsequent experimental.
Two, hybridization system
Before the 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 streptococcus aureus DNA to be measured.
Identification dna probe with rare-earth europium title complex on the mark
2With the single stranded DNA to be measured that extracts at mark DNA
1Sheet glass on carry out hybridization.Component in the hybridization system is the single stranded DNA that 10 μ L extract, 10 μ L DNA
2, 10 μ L, 12 * SSC.Wherein each sample is all done in triplicate parallel control, and the target dna of blank substitutes with sterilized water.The slide that will carry out hybridization places the place mat cotton and keeps in the wet box of hybridization of humidity the hybridization temperature constant temperature hybridization to design in constant incubator with 3 * SSC solution.
Three, post-hybridization washing and signal detection
Preparation washings I (1 * SSC, 0.03%SDS), washing lotion II (0.2 * SSC) and washing lotion III (0.05 * SSC).Hybridization is immersed in first slide in the washing lotion after finishing, and slowly mentions slide then, puts into, and mentions again, and washing is up and down dried under the room temperature back and forth.The identification DNA that has rare-earth europium title complex mark after hybridization is cleaned
2, capture dna
1The crossbred that forms with the target dna probe is 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 612nm 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 streptococcus aureus DNA in the optical signal detecting sample that detects.
Five, capture probe DNA
1The influence of concentration
With base sequence is 5 '-CAC TTT TTC TTA AAT GTT GTT C TTTTTTTTTT-3 '-NH
2Capture probe DNA
1Method by 10 times of dilutions is made into working concentration and is respectively 1 * 10
-5, 1 * 10
-6, 1 * 10
-7, 1 * 10
-8, 1 * 10
-9, 1 * 10
-10Mol/L.Then, pipette the DNA of 5 kinds of concentration respectively
140 μ L, and be added drop-wise to the surface of glass slide of hexanal baseization respectively, room temperature reaction 5h; Wash and seal remaining aldehyde radical at last; Room temperature is dried standby.
Experimental result shows that capture probe concentration is bigger to the influence of hybridization efficiency.As can be seen from Figure 1, when capture probe concentration be 1 * 10
-7Mol/L detected more intense and stable signal, and the signal that other concentration obtain is all not as good as this concentration.Show to reach capacity this concentration and probe concentration and being connected of the aldehyde groups that is fixed on surface of glass slide that too high or low excessively concentration all is unfavorable for the efficient of hybridizing.Therefore, Zui Jia immobilized capture probes concentration is 1 * 10
-7Mol/L.
Six, the influence of hybridization temperature
Select the suitableeest hybridization temperature of crucial importance in hybridization technique.If the hybridization temperature is low excessively, though also can form stable two strands between the complementary strand, the complementary base pairing reduces, and mispairing is to increasing, and hydrogen bonded is more weak.If two homologys about 50% or lower DNA, are adjusted hybridization temperature and can be made the hybridization between them change 10 times.Therefore, for a specific hybridization system, hybridization temperature is optimized is very important.
Based on designed probe, its melting temperature(Tm) T
mValue is 63 ℃.The effects optimum renaturation temperature T
Or, harsh renaturation temperature T
sAnd non-harsh renaturation temperature T
NsInfluence to hybridization efficiency.
Experimental result shows, when temperature is T
OrPerhaps T
NsThe time, sample and barren signal are all lower, and the difference between them is also very little.Analyzing its reason, may be because under lower temperature of reaction, the sample DNA that molecular weight is bigger is difficult to disperse to cause hybridization not exclusively fully, and base mismatch reduces increasing hybridization efficiency.And work as hybridization temperature is T
sWhen (53 ℃), can well distinguish the signal between sample and the blank, hybridization efficiency is than higher, and the result of this hybridization temperature and bibliographical information is similar.Therefore, the hybridization temperature that detects streptococcus aureus in this experiment is 53 ℃.
Seven, the influence of hybridization time
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.The ideal hybridization time is relevant with several factors, the chain length of probe for example, sequence etc.Therefore, in specific hybridization system, it is extremely necessary that hybridization time is optimized.
Design hybridization time length respectively and be 3,5,7,9,11,13,15, single factor optimization experiment of 17h.After arriving the time of setting, slide is taken out washing from constant temperature hybridization case.Experimental result as shown in Figure 2.The result show when hybridization time when 3h is increased to 9h, the fluorescence intensity that measures surface of glass slide strengthens gradually, and surpasses 9h when hybridization time, fluorescence intensity reduces on the contrary.Too short when the reaction times, hybridization will be carried out not thoroughly, and the long generation that can cause side reaction of hybridization time.Therefore, the Zui Jia hybridization time is 9h.
Eight, the influence of washing time
Adopt the non-specific binding in strict washing method removal of the two steps hybridization.Its objective is that in the low tightness washing stage (low salt concn, high temperature) removal has neither part nor lot in the probe of hybridization.Its objective is the non-specific hybridization of getting rid of homology in the washing stage of high tightness (low salt concn, high temperature).The washings of high tightness is preheating to the temperature of setting earlier.The washings prescription is: and washings I (1 * SSC, 0.03%SDS), washing lotion II (0.2 * SSC) and washing lotion III (0.05 * SSC).During cleaning, allow slide stay in the washing lotion always, slowly from washing lotion, mention slide, put into, mention again, clean several times up and down back and forth.
With T
mBe worth 53 ℃ of temperature as washing, design washing time length is respectively 1.5,3,4.5,6,9, the single factor optimization experiment of 15min.Experimental result as shown in Figure 3, the difference that detects sample and barren fluorescence intensity when washing time is 4.5 minutes is the most obvious.When washing time is less than 4.5 minutes, the value that blank then occurs is very high, but washing time is greater than 4.5 minutes, and the fluorescence intensity of blank remains unchanged substantially, and the fluorescence intensity of sample but weakens.May destroy originally paired base owing to cross long washing, and this destruction is irreversible.Therefore, selecting best washing time for use is 4.5 minutes.
Nine, the specificity of hybridization system
Adopted the genome of other 3 kinds of pathogenic micro-organisms to investigate the specificity of this system.Wherein staphylococcus epidermidis and streptococcus aureus belong to Staphylococcus; Intestinal bacteria are the bacterium that coexist together with streptococcus aureus in a kind of most of pollutent; Paenibacillus polymyxa is any bacterium of selecting at random.Hybridization and wash conditions are all carried out under top condition.
Experimental result shows, streptococcus aureus sample and positive control experiment, and the time resolved fluorescence intensity that obtains is between 163-185, and other 3 kinds of Institute of Micro-biology obtain fluorescent value and blank sample is approaching, as shown in Figure 4.In addition, this detection method does not have false positive results basically, illustrates that this detection methods of staphylococcus aureus has gratifying specificity.
Ten, the sensitivity of hybridization system
Pure culture streptococcus aureus concentration is by carrying out ten grades of stepwise dilutions with aseptic PBS damping fluid, dull and stereotypedly cultivates that counting process detects.By extracting corresponding cell genomic dna in each diluent, be applied to obtain the fluorescence intensity of every kind of diluent correspondence in the hybridization under the optimal conditions.Concrete steps are as follows:
(1) the streptococcus aureus seed liquor is inoculated in the 150mLNB liquid medium, inoculum size is 5mL, and 18h is cultivated in concussion in the constant incubator, and culture condition is: 37 ℃ of temperature, shaking table hunting speed 120rpm, the logarithmic phase that promptly arrives streptococcus aureus stops to cultivate.
(2) PBS (pH=7.2) damping fluid is distributed into the sterilization of 9mL/ pipe, sterilising conditions is 121 ℃, 20min.
(3) the aseptic 1mL bacterium liquid that pipettes is to the aseptic PBS damping fluid of 9mL from the streptococcus aureus nutrient solution that arrives the logarithmic phase growth, and being concentration is 10
-1Bacterium liquid.It is 10 that Using such method obtains a series of concentration
-2, 10
-3, 10
-4, 10
-5Bacterium liquid.
(4) the streptococcus aureus nutrient solution of 1mL logarithmic phase is cultivated in aseptic NB agar culture plate, with enumeration and report manner CFU/mL (colony forming unit) report detected result, this result is as the foundation of sensitivity detected result.Cultivation results is from 10
-1～10
-5The colony number of bacterial concentration is 10
6～10
2CFU/mL.
(5) be centrifugal 10min in the supercentrifuge of 12000rpm in centrifugal speed respectively with the bacterium liquid of above-mentioned serial dilution concentration, abandoning supernatant gets thalline.The DNA extraction of thalline all uses the bacterial genomes DNA extraction test kit (centrifugal column type) of Shanghai Jierui Biology Engineering Co., Ltd to be numbered GK1071, extracts according to reagent and scheme that test kit provided.
(6) hybridize detection with present method under hybridization conditions after the serial DNA that extracts optimizes respectively and the wash conditions, determine the sensitivity of this probe according to the fluorescence signal intensity that detects.
Experimental result as shown in Figure 5, the fluorescence intensity of curve 5 is minimum in all samples.Detection signal is as long as then think positive findings greater than the standard deviation of 2 times of average baselining emissions.According to this standard, the detection that we obtain this detection methods of staphylococcus aureus is limited to 8 * 10
3CFU mL
-1This detectability can be compared with the detection method of the streptococcus aureus of the utilization pcr amplification reported in the document.
Sequence table
＜110〉Hunan University
＜120〉detection method of a kind of streptococcus aureus based on time-resolved fluorescence method and DNA hybridization
<130>GenBank
<160>2
<170>PatentIn?version?3.3
<210>1
<211>32
<212>DNA
<213>Staphylococcus?aureus
<400>1
cactttttct?taaatgttgt?tctttttttt?tt 32
<210>2
<211>29
<212>DNA
<213>Staphylococcus?aureus
<400>2
tttttttttt?attttctctt?ttttcgctt 29
Claims (1)
1. detection method based on the streptococcus aureus of time-resolved fluorescence method and DNA hybridization is characterized in that:
A, capture probe DNA
1Fixing
With the slide aldehyde radicalization, be 5 '-CAC TTT TTC TTA AAT GTT GTT CTTTTTTTTTT-3 '-NH again with base sequence earlier
2Capture probe DNA
1Be diluted to 1 * 10
-7Mol/L gets 40 μ L DNA
1Be 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) with 2mg rare-earth europium title complex Eu (TTA)
3Phen is distributed in 5mL 2.5% glutaraldehyde solution, concuss 5h under the room temperature, and centrifugal back obtains aldehyde group modified Eu (TTA) with second distillation washing 3 times
3It is standby 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 ATT TTC TCT TTT TTC GCT T-3 '
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 ℃, centrifuge washing after reaction is finished; Redispersion is standby in the 2mLPBS damping fluid;
C, streptococcus aureus DNA extraction
D, hybridization
(1) before the hybridization, the staphylococcus aureus gene group 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
1Sheet glass on hybridize 9h.Component in the hybridization system is the single stranded DNA that 10 μ L extract, 10 μ L marks the DNA of rare-earth europium title complex
2With 10 μ L, 12 * SSC solution; Keeping hybridization temperature is 53 ℃;
(3) after the hybridization, slide is cleaned in following 3 kinds of washingss successively, washings I (1 * SSC/0.03%SDS), washing lotion II (0.2 * SSC) and washing lotion III (0.05 * SSC), total washing time is 4.5 minutes, and each 1.5 minutes, wash temperature was 53 ℃;
E, hybridization back signal detection
The identification DNA that has rare-earth europium title complex mark after hybridization is cleaned
2, capture dna
1The crossbred that forms with the target dna probe is 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 612nm optical signal, detects the optical signal of the long-term durability luminous title complex of rare earth on the slide, thereby according to whether having streptococcus aureus DNA in the optical signal detecting sample that detects.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910227169A CN101845486A (en) | 2009-12-10 | 2009-12-10 | Detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910227169A CN101845486A (en) | 2009-12-10 | 2009-12-10 | Detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101845486A true CN101845486A (en) | 2010-09-29 |
Family
ID=42770305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910227169A Pending CN101845486A (en) | 2009-12-10 | 2009-12-10 | Detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101845486A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164100A (en) * | 2021-12-31 | 2022-03-11 | 上海山恒生态科技股份有限公司 | Environmental protection aerobic bacteria cultivates and uses hybridization appearance |
-
2009
- 2009-12-10 CN CN200910227169A patent/CN101845486A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114164100A (en) * | 2021-12-31 | 2022-03-11 | 上海山恒生态科技股份有限公司 | Environmental protection aerobic bacteria cultivates and uses hybridization appearance |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Rompre et al. | Detection and enumeration of coliforms in drinking water: current methods and emerging approaches | |
| CN1957089B (en) | Measuring contamination | |
| AU2002327163B2 (en) | Rapid methods for microbial typing and enumeration | |
| AU2002327163A1 (en) | Rapid methods for microbial typing and enumeration | |
| CN109913565B (en) | Kit, primer pair, probe and method for detecting vibrio parahaemolyticus | |
| Feng et al. | Advances in detection of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits | |
| CN101113473A (en) | Method for detecting food-derived pathogenic vibrio bacteria by composite fluorescence PCR technique | |
| CN101724690A (en) | Method for detecting polymorphism of flora of prawn culture water body | |
| Pereira et al. | When FLOW-FISH met FACS: Combining multiparametric, dynamic approaches for microbial single-cell research in the total environment | |
| Bidartondo et al. | Fungal diversity in molecular terms: profiling, identification, and quantification in the environment | |
| CN103451310B (en) | Gene chip capable of simultaneously detecting various vibrios and method for detecting vibrios | |
| JP5349579B2 (en) | Microbe detection and counting | |
| CN103233078A (en) | Multi-gene detection method of Listeria monocytogenes based on quantum dot/graphene oxide nanometer platform | |
| CN101665838A (en) | Escherichia coli detection method based on time-resolved fluorescence method and DNA hybridization | |
| CN102260738B (en) | Oligonucleotide gene chip and application of oligonucleotide gene chip to detection of various bacteria | |
| JP2003061675A (en) | Nucleic acid probe for detecting genus selenomonas bacterium, and method for detecting beer-contaminating bacterium | |
| Girard et al. | Spatiotemporal dynamics of total viable Vibrio spp. in a NW Mediterranean coastal area | |
| CN101845486A (en) | Detection method of staphylococcus aureus based on time-resolved fluorescence method and DNA hybridization | |
| JP2009535052A (en) | Microbial detection and counting method | |
| Ruan et al. | Rapid detection of Staphylococcus aureus via a sensitive DNA hybridization assay based on a long-lifetime luminescent europium marker | |
| EP1490507B1 (en) | Method for the identification of microorganisms by means of in situ hybridization and flow cytometry | |
| Lepeuple et al. | Laser scanning detection of FISH-labelled Escherichia coli from water samples | |
| Dazzo et al. | Immunofluorescence Microscopy and Fluorescence In Situ Hybridization Combined with CMEIAS and Other Image Analysis Tools for Soil‐and Plant‐Associated Microbial Autecology | |
| CN1261590C (en) | Process for testing gene chip used to detect bacteremia | |
| JP3607327B2 (en) | Rapid detection of single microbial cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100929 |