CN101573621A - Capture of mycobacteria like micro-organisms - Google Patents
Capture of mycobacteria like micro-organisms Download PDFInfo
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- CN101573621A CN101573621A CN200780048444.0A CN200780048444A CN101573621A CN 101573621 A CN101573621 A CN 101573621A CN 200780048444 A CN200780048444 A CN 200780048444A CN 101573621 A CN101573621 A CN 101573621A
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Abstract
A method for the capture from a sample of micro-organisms having a hydrophobic surface such as mycobacteria, including M. tuberculosis, comprises contacting the micro-organisms with a capture reagent such as pDADMAC which has both a hydrophobic character to bind the micro-organisms by hydrophobic interaction and a polar character, so as to capture the micro-organisms to an acidic surface.
Description
The present invention relates to for example mycobacterium (mycobacteria) catching and relate to subsequently processing of hydrophobicity microorganism to the surface for example with regard to their existence or the analysis carried out of evaluation.
Pathogenic mycobacteria is the reason that forms several serious communicable diseases in the humans and animals.Mycobacterium is characterised in that the hydrophobicity wax adventitia that comprises mycolic acid (mycolic acid) or related compound.Mycolic acid is complicated hydroxylation branched chain fatty acid, has chain length usually at C
77-80Scope in hydrocarbon chain, it produces serious problem in sample preparation, form pallial siphuncle and impel bacterium to swim in the surface of liquid and resist centrifugal thereby cause the bacterium cluster.Hydrocarbon chain can comprise or can not comprise fragmentary oxy radical (oxygenated group) for example hydroxyl, methoxyl, ketone group or carboxyl.Pathogenic mycobacteria is included as the Much's bacillus (Mycobacterium tuberculosis) of the virulence factor of TB, mycobacterium (mainly mycobacterium avium (M.avium) and Mycobacterium intracellulare (M.intracellulare)) for the MAC complex of the conditioned pathogen among the AIDS patient, cause the mycobacterium paratuberculosis (M.Paratuberculosis) of enteritis, cause the Mycobacterium leprae (M.leprae) of leprosy, mycobacterium kansasii (M.kansasii), Mycobacterium marinum (M.marinum), mycobacterium fortutitum (M.fortuitum) complex and many other kinds.Also there are many other avirulence mycobacteriums, comprise mycobacterium smegmatis (M.smegmatis).Equally, other members that contain mycolic acid actinomyces (mycolata) family have the outer membrane component of similar hydrophobicity.In some cases, the chain length of its hydrophobic fat acid is about 50 carbon atoms than the weak point in the branch bacillus, is about 30 carbon atoms in other cases.
For diagnosis of mycobacterial infections tuberculosis for example, must by microscopy, cultivation or molecular method for example PCR prove the existence of described biology.Though can directly carry out microscopy, more commonly before analyzing, at first separate and concentrate mycobacterium from biological sample from biological sample.Biological sample can comprise phlegm, urine, blood, bronchial lavage thing (bronchiallavage) etc.A kind of modal sample type of sending to diagnosis is a phlegm.There is bacteriological oddity problem in phlegm.Phlegm is heterogeneous (heterogenous) in nature, and it can be (purulent) and viscosity band blood, that suppurate.It also can be by for example pseudomonad (Pseudomonas) pollution of other microorganisms.
Usually, make phlegm thinning, simultaneously by using various pre-service to remove pollution.These processing are included in the NaOH that uses 0.25-0.5M under N-acetyl L-halfcystine, lauryl sodium sulfate, oxalic acid or tertiary sodium phosphate existence or the non-existent situation.Processing time can be 20 to 120 minutes.These are handled through being designed for and make phlegm thinning and kill most of contaminative microorganism.Mycobacterium has thick wax adventitia and more can resist this type of processing.Even so, reaching 60% Much's bacillus is according to estimates killed or becomes by this processing and can not survive.In addition, because other members of Much's bacillus and this family grow so slowly, to such an extent as to the growth of the contaminative biology that is killed by this processing does not remain a problem, i.e. the culture of the high number percent pollutant undue growth of being grown fast.
After handling with strong decontaminant, sample is carried out centrifugal to concentrate mycobacterium, it is analyzed by microscopy, cultivation or molecular cloning then.This centrifugation step has been introduced infection experiment chamber staff's risk because any in centrifugal process the content in explosion or the test tube that breaks can nebulize and scatter and contaminated environment.The centrifugal bottleneck of also introducing sample preparation once can only centrifugal limited amount sample because any.In addition, the centrifugal all material (described material is by strong depollution sex change and become and can not dissolve) that makes becomes bulk, and the very large dough that throws into question of can obtain to adjust the telescope to one's eyes inspection technique or molecular method.
Because top listed depollute and the problem of method for concentration about existing, if thereby can directly catch mycobacterium from biological sample, then will be very useful.Thereby some or all of contaminatives are biological to be need not chemical decontamination and dyes and maybe can use not stronger condition to carry out chemical decontamination to dye, then be very useful if this method is removed.This also can increase survival and the increase sensitivity of detection subsequently of the mycobacterium of purifying.
In other different with sample preparation were used, it also may be very useful combine mycobacterium to allow easily to concentrate or operate biology (for example catching and clean mycobacterium to remove external source non-infection bacteriophage or catch mycobacterium and it is transferred to another kind of solution from a kind of solution from phage solution) with solid surface.
Proposed mycobacterium is captured to the method for solid surface before, it comprise the bacteriophage of using combination or be fixed on the bead and as the binding peptide in the bacteriophage of trapping agent source (people such as Stratmann; J Clin Microbiol.2002 November; 40 (11): 4244-4250) from milk, separate mycobacterium paratuberculosis (people such as Grant I.R. with comprising by the bead that uses antibody to apply; Appl Environ Microbiol.1998Sep; 64 (9): 3153-8).Yet, such method is too expensive (especially in not too flourishing country) for being extensive use of, and possible specificity is too high, because be not that the bacterium of all expectations will be hunted down, and relates to the molecule based on protein to proteinase, sex change and strong chemicals sensitivity.
According to people such as Hetland G., Immunology 1994,82, and 445-449 can be by coming bead with the BCG bag by latex beads (microbead) with cultivation and isolated bacterial incubation.Yet this can not be effective for catch this bacterioid effectively from the biological sample that comprises other hydrophobicity biologies or material.
We have observed diallyl dimethyl ammoniumchloride (p-DADMAC) mycobacterium have been bonded to the carboxylic acid microballon.Be not subjected to following theory, we think that the positive charge in the main chain of the cured matter adventitia hydrophobic interaction of the main chain of p-DADMAC and mycobacterium and p-DADMAC also can interact with the lip-deep negative charge of mycobacterium, and p-DADMAC is by the carboxylic acid generation ionic interaction of its quaternary ammonium group that dangles and microballon then.We also observe, and for example plastic and glass can be directly in conjunction with mycobacterium on the surface that p-DADMAC applies.
Therefore, mycobacterium can directly be captured to the surface of p-DADMAC coating or can be captured to the surface indirectly.
Thereby existing the providing in first aspect of the present invention is used for catching the method for microorganism with hydrophobic surface from sample, this method comprises microorganism is contacted with trapping agent, described trapping agent has hydrophobic property, trapping agent is by combining with it with the hydrophobic interaction of described microorganism thus, with have polar character, described trapping agent is present in the surface and goes up and described microorganism is captured on it, or exist in solution described method thereby also comprise by utilizing the polar interaction between described surface and the described trapping agent that described capture reagent bind is captured to the surface with described microorganism to described surface.
Preferably, the trapping agent of use in solution carries out said method, this method comprises microorganism is contacted with trapping agent in the solution like this, described trapping agent has hydrophobic property, trapping agent is by combining with it with the hydrophobic interaction of described microorganism thus, with have for example polyion characteristic of polar character, thereby by utilizing polar interaction between described surface and the described trapping agent that described trapping agent is bonded to described surface described microorganism is captured to the surface.
Sample can be that for example phlegm, urine, blood, bronchial lavage thing etc. maybe can be for example biopsy thing skin samples for example of solid sample (preferably described sample is handled to extract microorganism or microorganism is dispensed into and produced fluid sample in the liquid) to fluid sample.
Randomly, described trapping agent comprises and has for example long hydrocarbon chain of ionic sites (ionicsite) of a plurality of polarity.The part that described a plurality of polarity or ionic sites can be arranged in described chain jointly is end portion or can distribute at interval along described chain (as them the p-DADMAC) for example.
Trapping agent can be anionic but preferably cationic, as in the situation of p-DADMAC, and preferably gathers-diallyldimethylammonium chloride (DADMAC) itself.Because most of bacterial cells are electronegative, so p-DADMAC is that cell is transformed into clean positive charge in conjunction with the effect of the cured matter adventitia of mycobacterium.This is favourable, and is still not electronegative in conjunction with other contaminative microorganisms of p-DADMAC because it is guaranteed, thereby can not combine with microballon.
In addition, directly catching in the embodiment, under the situation that scaling agent exists, the surface that the biology of hydrophobicity deficiency will not apply in conjunction with p-DADMAC, thereby the selectivity of the type of generation captive biology to a certain degree.
Other trapping agents that can consider comprise poly-D-lysine or polyethyleneimine.Selection be hydrophobic amino acid for example tryptophane, leucine, valine, methionine, isoleucine, halfcystine or phenylalanine and polar amino acid for example lysine at random or segmented copolymer.
Trapping agent in nature should be preferably enough hydrophobic to be bonded to plastics (for example being bonded to the polystyrene microtest plate that is generally used for conjugated protein) by hydrophobic interaction, or alternatively can be by polar interaction or by being enough to come in conjunction with glass or hyaloid surface having in nature by the abundant hydrophobicity of hydrophobic interaction mating surface, described surface can be suitably for example can be seen in microslide or cover glass the surface.But it should be fully hydrophilic in nature, like this its can be at least under the organic cosolvent situation that for example DMSO exists of suitable scaling agent system or dosis tolerata in the water-soluble or buffering aqueous medium.Therefore it dissolves in and has in sample and any other employed mixtures of material.
No matter above-mentioned theory, the present invention second independent aspects provide from fluid sample catch microorganism with hydrophobic surface method, this method comprises microbiological specimens is contacted with the solubility trapping agent, described trapping agent comprises poly-DADMAC, trapping agent is in conjunction with described microorganism thus, and by described trapping agent is bonded to described surface described microorganism is captured to the surface.
In either side of the present invention, described surface is suitably provided by bead.These surfaces can be micron or nano-scale.Suitably, they are paramagnetic so that easily separate from liquid medium.They can have the carboxylic acid polyalcohol surface or be characterised in that the surface of sulfuric acid or phosphate group.
The molecular weight of poly-DADMAC can be lower than 100,000 (very low), 100,000-200,000 (low), 200,000-400,000 or 500,000 (medium) or surpass in the scope of 500,000 (height).
Preferably, under the combination situation that optionally the scaling agent system of scaling agent exists of the microorganism that one or more enhancings are expected, sample is contacted with trapping agent.Be desirable to, not in conjunction with some or all of be present in the sample the contaminative hydrophobic material or not in conjunction with the situation of the some or all of microorganisms in the sample (described microorganism is not the microorganism that catches of expectation) under in conjunction with microorganism.
The scaling agent system can comprise the amino acid amide of the fatty acid that is preferably the N-Hamposyl L.The scaling agent system can comprise or also comprise Triton X scaling agent alternatively, preferred Triton X-100.
For most of samples, preferably for example provide trapping agent in phosphate buffer or the Tris damping fluid at capture buffer liquid (suitably having 7 to 10, more preferably 7 to 98 to 9 or 8.2 to 8.6 pH for example).For the very dense mucoid sputum sample product that comprise the mucopolysaccharide that has many hydroxy-acid groups in a large number, lower pH can be favourable for catching.Under enough low pH, carboxylic group is neutralized and does not disturb pDADMAC or other sulfuric acid or phosphate group, thereby surface combination of mycobacterium and catching subsequently of pDADMAC or other surfaces are provided.Such condition (handling height mucoid sample although be designed for) can be used for all samples.The pH of suitable in this case trapping agent is 0 to 4, is that 4 pH still is enough to the protonated carboxylic acid group.Therefore, depend on the selection of solid surface, the pH of trapping agent can be at least 0 to 10.
The processing of sample certainly comprises the stage of depolluting, in this stage, handle sample or have the microorganism that catches the surface so that the microorganism except the purpose microorganism can not survive.This can N-acetylcystein exist or non-existent situation under use the material NaOH or use N-acetylcystein to carry out separately for example that becomes known for this purpose.Yes makes captive purpose microorganism be in the state that can survive for purpose.
Captive microorganism can be mycobacterium especially, and it can be above mentioned any mycobacterium.
The present invention includes and be used to detect method of microorganism, it comprises by said method described microorganism is captured to the surface, clean described captive microorganism and detect described lip-deep described captive microorganism or the described captive microorganism of detection after taking off from described surface.
Employed detection method can be any method that is suitable for described microorganism.Usually for mycobacterium with in particular for Much's bacillus, these class methods will comprise cultivates and microscopic examination (for example being undertaken by dyeing), the PCR-PCR, the amplification of TMA-transcriptive intermediate, the SDA-strand displacement is measured or at biological other amplifications of nucleic acid and detection method and based on the method for bacteriophage itself, comprise that FASTPlaqueTB (wherein adds the mycobacterial infections bacteriophage and allows it enter cell, kill the bacteriophage of staying the cell outside, further afterwards incubation is to discharge bacteriophage from cell, by infecting the existence that other microorganism detects the bacteriophage of release).
The vital material of implementing needed material of mentioned microorganism detection method or selection can be provided with the form of kit.Therefore, the present invention includes the microbioassay kit, described kit comprises solubility trapping agent and matrix and at least a following material, described trapping agent has hydrophobic property, trapping agent can be by with the hydrophobic interaction of microorganism to be detected and combine with it thus, with have the polyion characteristic, described matrix has the surface, described surface is used for by utilizing polar interaction between described surface and the described trapping agent that described trapping agent is bonded to described surface described microorganism being captured to described surface, and described following material is:
-can infect the bacteriophage of described microorganism;
-be used to carry out the primer of amplification of the genomic nucleic acids of described microorganism or described bacteriophage;
-be used to cultivate the nutrient culture media of described microorganism;
-be used to manifest described microorganism to carry out the stain of microexamination;
-be used for antibody (no matter still having the part of selecting binding affinity) as it as complete antibody in conjunction with described microorganism; Or
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces.
According to foregoing invention, sample can also be the gaseous sample (for example air) that wherein carries microorganism.Such sample can be charged into trapping agent solution with the form of bubble, thereby microbes is bonded to trapping agent.
Alternatively, the present invention includes the microbioassay kit, thereby described kit comprises and is coated in trapping agent fixed thereon on the solid surface that described trapping agent has hydrophobic property and polyion characteristic, trapping agent can be in conjunction with microorganism to be detected and at least a following material thus:
-can infect the bacteriophage of described microorganism;
-be used to carry out the primer of amplification of the genomic nucleic acids of described microorganism or described bacteriophage;
-be used to cultivate the nutrient culture media of described microorganism;
-be used to manifest described microorganism to carry out the stain of microexamination;
-be used for antibody (no matter still having the part of selecting binding affinity) as it as complete antibody in conjunction with described microorganism; Or
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces.
Solid surface can be a microslide.
Preferably, captive mycobacterium (directly catch or catch indirectly) is not caught damage by this and still can survive.Therefore the present invention can be used for biological medicament sensitivity test.In one aspect, mycobacterium can be exposed to medicine as the mode that allows the drug influence biology by this way.Subsequently, can catch mycobacterium with any method of describing herein, can use the methods of describing before many with regard to viability it to be checked then, described method can comprise the microscopy that uses survival stain (viability stain), based on the method for bacteriophage, based on the method for cultured method or PCR-based.In yet another aspect, at first can catch mycobacterium herein, be exposed to medicine as the mode that allows the drug influence biology by this way then with any method of describing.Subsequently, can use the method for many descriptions to check mycobacterium with regard to viability then, described method can comprise the microscopy that uses the survival stain, based on the method for bacteriophage, based on the method for cultured method or PCR-based.Employed medicine can comprise and is generally used for treating tuberculosis for example rifampin, streptomysin, isoniazid, ethambutol, pyrazinamide and Ciprofloxacin.
The vital material of implementing mentioned microorganism needed material of drug susceptibility method or selection can be provided with the form of kit.Therefore, the present invention includes microbial medicine sensitivity testing kit, described kit comprises the following material of solubility trapping agent and matrix and one or both, described trapping agent has hydrophobic property, trapping agent can be by with the hydrophobic interaction of microorganism to be detected and combine with it thus, with have the polyion characteristic, described matrix has the surface, described surface is used for by utilizing polar interaction between described surface and the described trapping agent that described trapping agent is bonded to described surface described microorganism being captured to described surface, and described following material is:
-one or more medicines to be detected; With
-being used to measure the instrument whether captive microorganism survives, this instrument can be following one or more:
-be used to manifest described microorganism to carry out the survival indication stain of microexamination;
-can infect the bacteriophage of described microorganism;
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces;
And if not already present, one or more following materials randomly:
-can infect the bacteriophage of described microorganism;
-be used to carry out the primer of amplification of the genomic nucleic acids of described microorganism or described bacteriophage;
-be used to cultivate the nutrient culture media of described microorganism;
-be used to manifest described microorganism to carry out the stain of microexamination;
-be used for antibody (no matter still having the part of selecting binding affinity) as it as complete antibody in conjunction with described microorganism; Or
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces.
Alternatively, the present invention includes microbial medicine sensitivity testing kit, thereby described kit comprises and is coated in solid surface trapping agent fixed thereon, described trapping agent has hydrophobic property and polyion characteristic, trapping agent can be in conjunction with microorganism to be detected thus, and one or both following materials:
-one or more medicines to be detected; With
-being used to measure the instrument whether captive microorganism survives, this instrument can be following one or more:
-be used to manifest described microorganism to carry out the survival indication stain of microexamination;
-can infect the bacteriophage of described microorganism;
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces;
And if not existing, one or more following materials randomly:
-one or more medicines to be detected;
-can infect the bacteriophage of described microorganism;
-be used to carry out the primer of amplification of the genomic nucleic acids of described microorganism or described bacteriophage;
-be used to cultivate the nutrient culture media of described microorganism;
-be used to manifest described microorganism to carry out the stain of microexamination;
-be used for antibody (no matter still having the part of selecting binding affinity) as it as complete antibody in conjunction with described microorganism; Or
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces.
Solid surface can be a microslide.
Carry Much's bacillus in the airborne particle (airborne particle) that when the infected experimenter who suffers from pulmonary tuberculosis or laryngophthisis disease coughs, sneezes or calls out, produces, the droplet nuclel (dropletnuclei).Particle is about 1-5 μ m and can remains on a few hours in the air, thereby guarantees that they can diffuse to whole room or building.When the susceptible person sucks the droplet nuclel (described droplet nuclel is through port or nasal meatus, the upper respiratory tract and bronchus arrival alveolar then) that contains Much's bacillus, infect and take place.The MDR Much's bacillus also is categorized as the C class reagent of bioterrorism by CDC, and delivery mechanism may be to produce gas carrier gas colloidal sol (airborne aerosol).
Near the infected experimenter of expectation protection health worker, other people and army personnel avoids by sucking the danger that generation is infected.In addition, the laboratory worker of using sample, TB culture that TB infects and the sample that contains other pathogenic mycobacterias (for example, the mycobacterium paratuberculosis in the ight soil) to carry out work also is in the danger of infection.Health worker and laboratory worker attempt to use face shield or more advanced particulate filtering respirator to prevent to infect at present.
CDC recommend to use ability with the smallest particles in effective filtering 1-5 mu m range through National Institute for Occupational Safety and Health (National Institute forOccupational Safety and Health) (NIOSH)-the particulate filtering respirator (for example, N95, N99 or N100) of authentication.Face shield is made up of simple knitting or non-knitting material usually; They can have several layers and can have the instructions of indicating definite pore size.Yet most of face shields are not the respirators of NIOSH-authentication, and its user that can not adequately protect avoids the exposure of TB and do not satisfy the OSHA requirement of respiratory protection.Research has shown that the use of respiratory protection reduces health care worker's infection risk according to estimates with following ratio (comparing with non-protection): surgery face shield, 2.4 times; Disposable dust, flue dust, mist or disposable highly effective air micro particle filtering (HEPA) face shield, 17.5 times; Elastomeric HEPA cartridge case respirator (cartridgerespirator), 45.5 times; Or PAPR (powered air-purifyingrespirator) (PAPR), 238 times.(J Occup Environ Med.1997Sep；39(9)：849-54)。
When particulate filtering respirator provided high-caliber protection, it had expensive unfavorable aspect and in use is restricted.Existence is to the needs of the disposable face shield of improvement (described face shield provides the protective effect of avoiding airborne mycobacterial infections of enhancing therein under the respirator situation that can not obtain or be not suitable for using as the user).This is that the situation in the developing country also is simultaneously the situation in the laboratory environment.Needed is face shield and/or filtrator; described face shield and/filtrator provide in conjunction with produce by infected experimenter and in the laboratory the accidental gasoloid that contains mycobacterium that produces, thereby greatly improve the special and effective method of the standard of user's protection.
Therefore the present invention provides and has been used for filtered airflow to remove the filtrator of the microorganism of wherein carrying, described filtrator comprises polar surfaces and on polar surfaces or the trapping agent of its upstream, described trapping agent has hydrophobic property, it can be by the bacterium of hydrophobic interaction in conjunction with hydrophobicity bag quilt thus, with polar character polyion characteristic for example, it is bonded to or is suitable for being bonded to described polar surfaces thus.
It maybe can be the filter element that is connected to the face shield or the helmet that filtrator can be taked to be used to protect the form of wearer's face shield.It can be mounted in the air supply pipe road or be used for installed filters therein.
Of the present invention preferred aspect; for the protective effect of raising is provided; face shield with solubility trapping agent dipping can be provided; described trapping agent has hydrophobic property; thus trapping agent by hydrophobic interaction in conjunction with mycobacterium; with polar character polyion characteristic for example, trapping agent is bonded to ion surface by polar interaction thus.
The solubility trapping agent can be injected into suitable sulidus face cover material for example on the filter material of face shield, before the packing of product, carry out drying then.When using, face shield is because the breath of user's exhalation becomes moistening, thereby trapping agent will be dissolved in the moist layer on the face shield material surface.The gasoloid that contains mycobacterium will cause the solubility trapping agent to combine with the quick of mycobacterium cell to the collision on this surface.The use of polyionic solid phase material will cause mycobacterium fixing to solid phase in the face shield.This will eliminate and further produce infectious aerocolloidal any possibility from the surface in suction process, thereby will provide high-caliber protection for the operator.
In this first example, the solubility trapping agent will be when becoming wet and begin to be bonded to the polyion solid phase material of face shield before the gasoloid collision.This can have the effect that reduces the efficient catch mycobacterium because the surface can be hunted down agent saturated, thereby will be not in conjunction with the mycobacterium cell/solubility trapping agent compound in the collision gasoloid.Alternatively, in conjunction with trapping agent is owing to can have the affinity or the affinity to microorganism of minimizing from the interference of solid surface.
This unfavorable aspect can overcome by using two face mask, and described two face mask have first skin on neutrality, uncharged material with solubility trapping agent dipping.The collision gasoloid will cause the formation of mycobacterium/solubility trapping agent compound, can be combined closely by the polyion material in second internal layer of face mask structure after the described compound.
Therefore, the present invention includes the filter for molten of initial description, wherein on solid surface (it has the binding affinity of low trapping agent to described polar surfaces upstream), provide described trapping agent.
In the accompanying drawings:
Step 5 (figure of the left-hand side) neutralization procedure 6 (dexter figure) that Fig. 1 shows embodiment 10 microscope of the Ziehl Neelson dyeing of Mycobacterium bovis (Mycobacterium bovis) afterwards manifests;
Fig. 2 is presented in the step 6 of embodiment 10 microorganism that separates from bead under the enlargement factor of higher (last figure) and lower (figure below);
Fig. 3 shows the processing on (left side) and uncoated (right side) that apply among the embodiment 11; With
Thereby Fig. 4 shows the mycobacterium that catches among the embodiment 12 and dye the demonstration viability.
The present invention will further describe and illustrate by the following example.In these embodiments, because total many common characteristics of mycobacterium smegmatis and Much's bacillus but do not have infectivity, so used as the representative mode biology of Mycobacterium.
Embodiment
Embodiment 1.pDADMAC part and the titration of catching bead
Ultimate principle.In order to measure the optimal dose of the part and the bead that are used to catch mycobacterium, carry out present embodiment.Amount by the captive mycobacterium of the genomic pcr analysis of mycobacterium.
Method
1. the culture with 1 μ l mycobacterium smegmatis (is supplemented with the 7H9 nutrient culture media of 10%OADC, Difco) duplicates in the nutrient culture media at 1ml 7H9 OADC.
2. add 250 μ l 5x capture buffer liquid (250mM Tris pH8.3,5% (w/v) N-Hamposyl L, 5% (v/v) Triton X-100,5% (w/v) BSA) and mix.
3. add the pDADMAC (Sigma Aldrich, intermediate molecular weight, 400,000 to 500,000) of the Yu Shuizhong dilution of different amounts, mix, incubation is 15 minutes then.
4. the volume ratio with 10: 1 (comparing with the initial volume of pDADMAC) adds MyOne carboxylic acid paramagnetism bead, mixes, and incubation is 15 minutes then.
5. catch bead by magnet base (magnetic stand), in 1ml PBS, clean then.
6. add 20 μ l 100mM NaOH, 0.05% (v/v) Triton X-100, the resuspension bead heated 5 minutes down at 95 ℃ then.
7. add 10 μ l 200mM HCl, pass through eluate then the quantitative PCR analysis 2 μ l of mycobacterium smegmatis.
Pcr analysis
Use MJ Research Inc. (Hercules, California) Chromo 4 machines.(Belgium), this kit can make the PCR product monitored by the fluorescence of dna double chain insertion agent for Eurogentec, Seraing to use Sybr Green kit.Employed PCR parameter comprises, heats 10 seconds down at 95 ℃, anneals 15 seconds down and extends 15 seconds down at 72 ℃ at 65 ℃.Use is specific PCR primer 5 ' TCAGGC CCT CGA AAG CCG ACT GGG 3 ', 5 ' CCA GGA CTC GGT ACA AGA CTCTGC 3 ' for the mycobacterium smegmatis genome.
The result
| The amount of the pDADMAC that uses | The amount of the bead that uses | The positive circulation mycobacterium smegmatis of PCR exists | The shameless dirty mycobacterium contrast of circulation that PCR is positive |
| 5μl 0.01％(v/v) | 50μl | 25.2 | 34.1 |
| 2μl 0.01％(v/v) | 20μl | 27.2 | Keep negative |
| 5μl 0.004％(v/v) | 10μl | 26.5 | 37.1 |
| 2.5μl 0.004％(v/v) | 2.5μl | 28.7 | 35.7 |
Conclusion
5 μ l 0.01%pDADMAC effects are best, compare with the 34th circulation that contrasts (PCR primer dimer background) for no bacillus, produce signal the 25th circulation.The dilution of pDADMAC and bead has produced the recovery of the mycobacterium smegmatis that reduces gradually.
The Study on Efficiency that embodiment 2. catches
Research is compared with the mycobacterium smegmatis with directly detecting by PCR that passes through alkali heating extraction of same amount, catches the efficient of the mycobacterium smegmatis that enters nutrient culture media.
Method
1. the culture of mycobacterium smegmatis is diluted into 1ml 7H9OADC and (be supplemented with the 7H9 nutrient culture media of 10%OADC, Difco) nutrient culture media.
2. add 250 μ l 5x capture buffer liquid (250mM Tris pH 8.3,5% (w/v) N-Hamposyl L, 5% (v/v) Triton X-100,5% (w/v) BSA) and mixing.
3. the pDADMAC (Sigma Aldrich, intermediate molecular weight, 400,000 to 500,000) that adds 10 μ l 0.01% (v/v) mixes, and incubation is 15 minutes then.
4. add 50 μ l MyOne carboxylic acid paramagnetism beads, mix, incubation is 15 minutes then.
5. catch bead by magnet base, in 1ml PBS, clean then.
6. add 20 μ l 100mM NaOH, 0.05% (v/v) Triton X-100, the resuspension bead heated 5 minutes down at 95 ℃ then.
7. add 10 μ l 200mM HCl, and pass through eluate the quantitative PCR analysis 2 μ l of mycobacterium smegmatis.
8. to directly handling the identical mycobacterium smegmatis culture of 1 μ l with alkali described in 7, it is analyzed by PCR with identical method then in addition, as above-mentioned step 6.
Pcr analysis
PCR is described in embodiment 1.
The result
| The dilution of mycobacterium smegmatis | The roughly number of bacillus | The circulation that PCR is positive |
| 10 -3 | 100,000 | 26.7 |
| 10 -4 | 10,000 | 32.0 |
| 10 -5 | 1000 | 37.3 |
| The contrast of shameless dirty mycobacterium | 0 | 35.6 |
| The mycobacterium smegmatis that 1 μ l directly handles | 100,000 | 26.5 |
Conclusion
The efficient that bacillus catches is very high, because the bacillus that enters with reclaim of same amount produces and the similar signal of directly analyzing of bacillus.The bacillus that enters 1 milliliter of nutrient culture media to 10,000 is lacked in recyclable and detection.
The research of the needs of 3. pairs of capture buffer liquid of embodiment
Ultimate principle.Exist or non-existent situation is taken in mycobacterium smegmatis into nutrient culture media next time at capture buffer liquid.
Method
Except not adding the capture buffer liquid in a sample, method is the same with the method described in the embodiment 1.
The result
| The existence of capture buffer liquid | The circulation that PCR is positive |
| No capture buffer liquid | 25.5 |
| Use capture buffer liquid | 23.0 |
Conclusion
Capture buffer liquid increases the recovery of bacillus or calculates about 6 times of increase according to bacillus gene group and bacillus with 2.5 circulation.This may owing to scaling agent to the effect of nutrient culture media and minimizing by suppressing the interference of mycobacterium smegmatis with the inhibition composition that the combines generation of trapping agent.
The function that embodiment 4. catches based on the part of mycobacterium smegmatis in the mensuration of bacteriophage
Proof
Ultimate principle.Can detect mycobacterium to the ability of host's phage-infect with regard to viability by bacterium.A problem of this method is that the non-infection of infected bacillus and external source bacteriophage is separated.In case separate with the external source bacteriophage, come it is studied with regard to the cleavable bacillus and with regard to the endogenous infection bacteriophage.
Method.
In 10ml 7H 9OADC nutrient culture media, add 100 μ l mycobacterium smegmatis, and 37 ℃ of following incubations 3 hours.The negative control that also prepares no bacillus simultaneously.
Add 100 μ l (about 10 in test tube in putting back incubator and the sample
10Pfu) D29 mycobacteriophage.Time points different after infection take out the 1mI five equilibrium, and (except carry out 3 extra cleanings in PBS) catches mycobacterium smegmatis from nutrient culture media as described in example 1 above.
The bacillus that catches of cracking and with regard to the existence of endogenous infection phage genome it is studied as described by PCR.Except using phage gene group-specific primers 5 ' CCTCGG GCT AAA AAC CAC CTC TGA CC 3 ', 5 ' CTG GGA GAA TGT GAC ACGCCG ACC 3 ', PCR is with described PCR is the same before.
The result
| Infect the back time | The existence of mycobacterium smegmatis | The circulation that PCR is positive |
| 15 minutes | Exist | Keep negative |
| 15 minutes | Do not exist | 39.8 |
| 30 minutes | Exist | 27 |
| 30 minutes | Do not exist | Keep negative |
| 60 minutes | Exist | 30 |
| 60 minutes | Do not exist | Keep negative |
| 90 minutes | Exist | Keep negative |
| 90 minutes | Do not exist | Keep negative |
| 120 minutes | Exist | 31 |
| 120 minutes | Do not exist | Keep negative |
Conclusion
The ability of catching mycobacterium smegmatis from nutrient culture media allows bacillus to be cleaned with the external source bacteriophage to be removed.The bacteriophage that is detected has subsequently just infected the bacteriophage of bacillus.In this embodiment, using method is monitored course of infection.After adding bacteriophage 15 minutes, do not detect signal from bacillus.Bacteriophage does not also infect, thereby phage genome is not replicated.Endogenous phage genome appeared in the bacillus at the 30th minute, but descended time point the 60th minute, in time point complete obiteration in the 90th minute because phage replication and cracking bacillus.Signal reappeared at the 120th minute, because the bacteriophage that the second generation that discharges is duplicated experiences another infection of taking turns and duplicates.
Embodiment 5. catches the proof of mycobacterium from phlegm
Ultimate principle.Phlegm is the matrix of complicated and thickness.Carry out this experiment and do not disturb catching of mycobacterium to show this matrix.
Method.
Prepare the potpourri of 5 sputum sample product and it is divided into the 1ml volume.
In half five equilibrium, add 10 μ l mycobacterium smegmatis cultures.
Add 100 μ l 5M NaOH, 2.5%N-acetylcysteine and incubation 15 minutes are to dilute and decontamination phlegm.
Add 100 μ l 5M HCl, add 250 μ l 5x capture buffer liquid (as previously described) then.
Then as the step 3 of embodiment 2 to from phlegm, catching mycobacterium smegmatis described in 8, and quantitative to it by PCR.
The result
Sample with mycobacterium smegmatis shows positive the 20th PCR circulation.
The sample of no bacillus (being background) shows positive the 36.3rd PCR circulation.
Conclusion
The extracting method that uses pDADMAC and bead to catch is not suppressed by sample substrate (phlegm), and has successfully reclaimed mycobacterium smegmatis from this sample.
Embodiment 6. catches the branch bar from phlegm under situation about need not with alkali dilution and decontamination
The proof of bacterium.
Ultimate principle.Phlegm is the matrix of complicated and thickness.Use the processing of alkali that phlegm is diluted and decontamination, but also can damage mycobacterium.Carry out this experiment and can under the situation of not having alkali treatment in advance, use extracting method to show.Once more with the model organism of mycobacterium smegmatis as Mycobacterium.
Method
Prepare the potpourri of 5 sputum sample product and it is divided into the 1ml volume.
In half five equilibrium, add 10 μ l mycobacterium smegmatis cultures.
Add 250 μ l 5x capture buffer liquid (as previously described) and mixing.
Then as the step 3 of embodiment 2 to from phlegm, catching mycobacterium smegmatis described in 8, and quantitative to it by PCR.
The result
Sample with mycobacterium smegmatis shows positive the 24.7th PCR circulation.
It is negative that the sample of no bacillus (being background) keeps.
Conclusion
The extracting method that uses pDADMAC and bead to catch does not need to use alkali anticipating phlegm.The adding of observing capture buffer liquid is enough to cause the decomposition and the dilution of phlegm, and this allows to reclaim mycobacterium smegmatis from this sample subsequently.
In embodiment 7. capture systems to the proof of the needs of pDADMAC part.
Ultimate principle.For prove trapping agent (this sentences pDADMAC is example) be vital for catching of mycobacterium and mycobacterium under the non-existent situation of trapping agent not in conjunction with the carboxyl bead, carry out this experiment.
Method
Culture repose period of the mycobacterium smegmatis of 0.5ml five equilibrium is centrifugal to precipitate biology, then biology is resuspended to capture buffer liquid (50mM Tris pH 8.3,1% (w/v) N-Hamposyl L, 1% (v/v) Triton X-100,1% (w/v) BSA).
Add 10 μ l 0.01%pDADMAC in 1 five equilibrium, mix, incubation is 15 minutes then.Identical five equilibrium does not add pDADMAC and carried out 15 minutes.
Add 50 μ l MyOne carboxylic acid paramagnetism beads (before use at dH to each five equilibrium then
2Clean 3 times and be resuspended to the dH of initial volume among the O
2Among the O), incubation 15 minutes.Then grade is placed on the magnet, estimates any clarification of biological muddy suspending liquid by eyes.
Take out and preserve supernatant then.
Then bead is cleaned 1 time in capture buffer liquid and in 7H 9OADC nutrient culture media, clean 2 times, be resuspended to then in this nutrient culture media of 1ml.
0.1 μ l supernatant of equivalent and bead suspending liquid is coated on the 7H9OADC agar plate and at 37 ℃ of following incubations 2 days, the number of colony on each flat board of counting after this time.
The result
Adding magnetic bead and with after waiting on the magnet that is placed in, existence is with the remarkable clarification of the five equilibrium of pDADMAC trapping agent incubation.This causes owing to most of biologies in the suspending liquid are caught into magnetic-particle.The five equilibrium that does not add trapping agent keeps muddy, because biology is retained in the suspending liquid.From agar plate culture counting trapping agent exist and non-existent situation under captive and bacillus at large number and with its tabulation (seeing table).
| There is not the pDADMAC trapping agent in the number of the colony of counting | The number of the colony of counting has added the pDADMAC trapping agent | |
| Supernatant | Greater than 1000 | 490 |
| The bead of catching | 359 | Greater than 1000 |
Conclusion
The catching of the mycobacterium that is undertaken by the carboxylic acid bead (as measuring by visual turbidimetry (visual turbidity)) depends on the existence of pDADMAC.This visualization is quantitatively verified by microorganism.Under the situation that trapping agent exists, most mycobacteriums are hunted down, yet under the non-existent situation of trapping agent, have only the minimum bead that is adsorbed to.
The optionally proof that the part of embodiment 8. mycobacteriums is caught.
Ultimate principle.In order to prove that pDADMAC trapping agent specificity in conjunction with mycobacterium and not in conjunction with other biological subjects, comprises the representative Gram-negative and the Gram-positive biology that also can pollute the associated biomolecule sample, carry out this experiment.
Method.
The mycobacterium smegmatis of centrifugal 0.5ml five equilibrium, Pseudomonas aeruginosa (Pseudomonasaeruginosa), staphylococcus aureus (Staphylococcus aureus) and Escherichia coli (Escherichia coli) repose period culture to precipitate biology, then biology is resuspended to capture buffer liquid (50mM Tris pH 8.3,1% (w/v) N-Hamposyl L, 1% (v/v) Triton X-100,1% (w/v) BSA) in.
Each five equilibrium to each biology adds 10 μ l 0.01%pDADMAC, mixes, and incubation is 15 minutes then.
Add 50 μ l MyOne carboxylic acid paramagnetism beads (before use at dH to each five equilibrium then
2Clean 3 times among the O, be resuspended to the dH of initial volume then
2Among the O), incubation 15 minutes.Then grade is placed on the magnet, estimates any clarification of biological muddy suspending liquid by eyes.
Take out and preserve supernatant then.
In capture buffer liquid, clean bead 1 time then.In 7H9 OADC nutrient culture media, clean mycobacterium smegmatis etc. then and divide 2 times, be resuspended to afterwards in this nutrient culture media of 1ml.In Mueller Hinton nutrient culture media, clean other biological with identical method, be resuspended to then in this nutrient culture media of 1ml.
The 0.1 μ l supernatant and the bead suspending liquid of equivalent are coated on 7H9OADC agar plate (for mycobacterium smegmatis) or the Mueller Hinton agar plate (for other biological) and at 37 ℃ of following incubations, the number of colony on each flat board of counting after this time appears until the bacterium colony.
The result
As before, in the time of on placing magnet, it is clear that the suspending liquid of mycobacterium smegmatis becomes, and proves and caught biology from solution.The suspending liquid of every other biological subject is still very muddy, shows biologically at largely to obtain and be retained in the suspending liquid.The number of and at large bacillus captive and with its tabulation from agar plate culture counting.
| Biological | Number from the colony of supernatant | Number from the colony of bead |
| Mycobacterium smegmatis | 221 | Greater than 1000 |
| Pseudomonas aeruginosa | Greater than 10000 | 12 |
| Staphylococcus aureus | Greater than 1000 | 8 |
| Escherichia coli | Greater than 10000 | 22 |
Conclusion
The pDADMAC trapping agent allows the specificity of mycobacterium mycobacterium smegmatis to catch.Other biological subjects are in conjunction with this trapping agent, thereby are not hunted down.
Embodiment 9. specificitys are caught the research that required capture buffer liquid is formed.
Ultimate principle.For study pDADMAC to the specificity of mycobacterium in conjunction with in be the component of important capture buffer liquid, carry out this experiment.
Method
The mycobacterium smegmatis of centrifugal 0.5ml five equilibrium, Pseudomonas aeruginosa, staphylococcus aureus and colibacillary repose period culture to precipitate biology, then biology is resuspended to the different component part of capture buffer liquid.
Each five equilibrium to each biology adds 10 μ l 0.01%pDADMAC, mixes, and incubation is 15 minutes then.Another five equilibrium that does not add each biology of trapping agent is observed the contrast of catching that trapping agent mediates with acting on.
In each five equilibrium, add 50 μ l MyOne carboxylic acid paramagnetism beads then (before use at dH
2Clean 3 times among the O, be resuspended to the dH of initial volume then
2Among the O), incubation 15 minutes.Then grade is placed on the magnet, estimates any clarification of biological muddy suspending liquid by eyes.
The result
Record result and being shown in the following table.Under any buffer conditions, under the situation that trapping agent does not exist or exists, non-branch bacillus biology is not caught by bead.Mycobacterium catch the existence that depends on the pDADMAC trapping agent, and under all buffer conditions that are studied, observe and catch.As if the use that only contains the damping fluid of N-Hamposyl L partly suppress to catch.Yet when methyl amimoacetic acid used with Triton-X100, the efficient of catching was recovered.If the N-Hamposyl L is not present in the capture buffer liquid, after mycobacterium caught, bead was very easy to caking behind resuspension.Prevented from the comprising of N-Hamposyl L to lump after this from catching and helped and cleaned and caught back processing and analysis is very important bead operation for subsequently bead.
Conclusion
As indicated in before, be subjected under the strip spare any, under trapping agent existence or non-existent situation, biological at large the obtaining of non-branch bacillus to bead.The catching the existence that depends on trapping agent and can be subjected to be proved to be under the strip spare of mycobacterium at all.Comprising the N-Hamposyl L in the capture buffer liquid is vital for back operation of catching of captive material and analysis.
Embodiment 9: the result
Embodiment 10. catches the mycobacterium that carries out with resistance to acid microexamination by part
Detection
From solution, catch Mycobacterium bovis and by resistance to acid Ziehl Neelsen decoration method and auramine-phenol fluorescence colour directly on the bead or behind wash-out to its dyeing.
Method
1. in comprising the 7H9 nutrient culture media of suspending liquid of Mycobacterium bovis, 1ml adds 250 μ l 5x capture buffer liquid (250mM Tris pH 8.3,5% (v/v) Triton X-100,5% hybrid ion [3-(n, N-dimethyl myristyl ammonia)-propane sulfonic acid]).
2. add 10 μ l 0.01% (v/v) pDADMAC, mix, incubation is 15 minutes then.
3. add 50 μ l MyOne carboxylic acid paramagnetism beads, mix, incubation is 15 minutes then.
4. catch bead by magnet base, in 1ml PBS, clean then.
With the bead point of half volume on microslide, allow its drying, and before ZiehlNeelsen dyeing, carry out heat fixation.In the figure of Fig. 1 left-hand side, before part/bead wash-out, seeing captive mycobacterium.Magnetic beads is indicated by the below arrow.Indicated by upper arrow by resistance to acid mycobacterium magnetic capture, that highly assemble and as seen it is surrounded by bead.
6. remaining bead is resuspended to 100 μ l dH
2Among the O, add 10 μ l chloroforms then.Vortex with chloroform with after aqueous layer is mixed, utilize magnet that bead is drawn side to test tube, then with supernatant point on microslide, drying, heat fixation dyes by ZiehlNeelsen decoration method and auramine-phenol method then.As Medical Microbiology, aPractical Approach, Eds., Peter Hawkey and Deidre Lewis described in the OxfordUniversity Press, carry out all dyeing.In the dexter figure of Fig. 1, behind part/bead wash-out, seeing captive mycobacterium.Wash-out has disperseed resistance to acid mycobacterium (below arrow).Still there be (upper arrow) in some beads.Fig. 2 shows the microorganism that catches from bead.Under high-amplification-factor, the caking of the mycobacterium fluorescens that visible part is caught under lower enlargement factor, can be seen typical ' an array of stars night sky ' of the mycobacterium fluorescence of dispersion in figure below in last figure.
The result
Mycobacterium caught by part/paramagnetism bead and, under the situation of wash-out not, can see that after Ziehl Neelsen dyeing it is the pink material of being assembled by the height that bead centers on.Behind wash-out, mycobacterium separates with bead and is disperseed (referring to Fig. 1 and 2).
Conclusion
Mycobacterium can be caught by the TB-part and can observe by acid-fast staining and microexamination.Behind the wash-out, mycobacterium separates and is disperseed from bead.Experiment in addition shown that part is caught and the dyeing scheme for the clinical TB sample in the phlegm very effectively and fluorescence microscopy can be used for sensitiveer detection.
Embodiment 11. mycobacteriums directly catching on the solid surface that part applies used
The detection of original position dyeing and the captive biology that undertaken by microscopy.
Ultimate principle.The catching of the microslide that applies to p-DADMAC for the proof mycobacterium (it is observed by original position dyeing and microexamination) carry out this experiment.
Method.
By with 2% (v/v) p-DADMAC (in distilled water, coming) covering microslide, allow it be evaporated to drying then and come to apply microslide with p-DADMAC from the dilution of 20% stoste.Uncoated microslide is with comparing.In a large amount of distilled water, clean microslide then and carry out drying.100 μ l mycobacterium smegmatis cultures are added to 800 μ l dH
2In O and the 100 μ l capture buffer liquid (10% (w/v) hybrid ion [3-(n, N-dimethyl myristyl ammonia)-propane sulfonic acid, 10% (v/v) Triton X-100,500mM Tris pH 8.3) and put to microslide.Behind the incubation 10 minutes, in distilled water, clean microslide, as MedicalMicrobiology, a Practical Approach, Eds., Peter Hawkey and DeidreLewis carry out Gram described in the Oxford University Press.
The result
Can observe the Gram-positive mycobacterium (Fig. 3) that is captured in a large number on the microslide that p-DADMAC applies by microscopy, yet the mycobacterium of considerably less (if any) is caught on the uncoated microslide.
Conclusion
This shows that mycobacterium can be caught by the microslide that p-DADMAC applies and can carry out original position dyeing and observe by microexamination these mycobacteriums.Similar result also can obtain from the culture (dyeing by resistance to acid Ziehl Neelsen decoration method and fluorescence auramine-phenol method) of BCG.
Embodiment 12. mycobacteriums directly catching on the solid surface that part applies used
Original position viability dyeing and the detection by microscopy.
Ultimate principle.In order to prove that the mycobacterium that is caught into the wave carrier piece that p-DADMAC applies still can survive and can manifest by microscopy then by the dyeing of original position viability, carries out the present invention.
Method.
By with 2% (v/v) p-DADMAC (in distilled water, coming) covering microslide, allow it be evaporated to drying then and come to apply microslide with p-DADMAC from the dilution of 20% stoste.Uncoated microslide is with comparing.In a large amount of distilled water, clean microslide then and carry out drying.100 μ l mycobacterium smegmatis cultures are added to 800 μ l dH
2In O and the 100 μ l capture buffer liquid (10% (w/v) hybrid ion [3-(n, N-dimethyl myristyl ammonia)-propane sulfonic acid], 10% (v/v) Triton X-100,500mM Tris pH 8.3) and put to microslide.Behind the incubation 10 minutes, in distilled water, clean microslide, add the 7H9 of 1mg/ml, the thiazole bromide blue tetrazolium in the OADC nutrient culture media (Thiazolyl Blue TetrazoliumBromide) (MTT), incubation 30 minutes at room temperature then.After being in the distilled water to clean, the mycobacterium that can survive by microscopic examination.
The result
It is painted to be deposited as insoluble blue/black in the biology that can survive of MTT stain on the microslide that is caught into the p-DADMAC coating, thereby the biology that allows to survive detects (referring to Fig. 4) by microscopy.
Conclusion
This proof mycobacterium can be caught by the microslide that p-DADMAC applies and these captive mycobacteriums still can survive, and can be by for example MTT dyeing of viability stain.
Embodiment 13. is used for the method for mucoid phlegm
Ultimate principle.Some sputum sample product can be very dense with myxoid, its have high concentration pass through covalency thioether bridge (sulphide bridge) highly cross-linked and because of having the mucopolysaccharide of many carboxyl altitudinal belt electric charges.Described the use reductive agent for example dithiothreitol (DTT) and N-acetylcystein interrupt disulfide bond, but the mucopolysaccharide of high concentration still can disturb catching of mycobacterium by the interaction of electronegative carboxyl and positively charged pDADMAC.In order to reduce this inhibition, can be desirably under the low pH-be neutralized but catch under the still charged pH of pDADMAC at carboxyl.Under so low pH, pDADMAC can not be captured on the carboxyl bead, therefore because bead has also lost their electric charge, under low pH, the carboxyl bead must be used in the carboxyl bead and become the sulfate bead that still has negative charge under the neutral condition and substitute.With regard to Much's bacillus these conditions of Acquisition Detection from the phlegm that provides by World Health Organization (WHO) phlegm storehouse.
Method
1. at first at the dH of 5% (v/v)
2(BM546, Bangs Laborator ies Inc. US), carried out 1 hour, then, was dH to apply BioMag amine bead among the pDADMAC among the O (high molecular)
2After cleaning among the O, at dH
2Coat with 10mg/ml dextran sulfate (500 000mwt) among the O, carried out 1 hour.Be dH
2After cleaning among the O, bead is resuspended to the dH of initial volume
2Among the O, prepare then to use.
With the dithiothreitol (DTT) of 2% (w/v) final concentration handle 0.5ml phlegm sample (for mycobacterium, microexamination positive or negative) 20 minutes.As positive control, some sputum sample product also added the BCG that cultivates before handling.
3. after this is handled, add 50 μ l 10% (v/v) Triton X-100,10mMEDTA, 20 μ l 0.004% (v/v) pDADMAC (500000mwt) and 50 μ l 2.5MHCl, incubation is 10 minutes then.The pH in this stage is contemplated to about 0.6.
4. add the paramagnetism bead that 20 μ l dextran sulfates apply then, incubation is 10 minutes then.
5. collect bead by magnet, at 1ml dH
2Clean (do not need this cleaning step usually, but carry out this cleaning step for active the catching that proves mycobacterium) among the O, and be resuspended to 10 μ l dH
2O, then with its point on microslide.
Handle the auramine-phenol fluorescence microscopy of microslide to carry out the mycobacterium described in the embodiment 10.
The result
10 sputum sample product that are reported to the Much's bacillus feminine gender by WHO phlegm storehouse (WHO sputum bank) catch with microexamination after be negative.Being reported to 10 positive sputum sample product by WHO phlegm storehouse is tangible positives, and the contrast that adds BCG also is like this.In addition, there be the recovery of mycobacterium from phlegm efficiently in the control sample demonstration, as estimating that by the comparator microscope inspection technique mycobacterium of the adding of 90-95% is recovered.
Conclusion
For dense mucoid sample, being captured in of mycobacterium be low to moderate be enough to make under the pH that the hydroxy-acid group on the mucopolysaccharide is neutralized very effective.
In this manual, unless point out clearly in addition, word ' or ' uses with the meaning of sign of operation, and described sign of operation returns true value when satisfying any or two of described condition, and this sign of operation ' XOR ' with a condition during requirement only satisfies condition is opposite.Word ' comprises ' and means ' comprising ', but not ' by ... form '.The prior art of generally acknowledging above all is integrated with this paper by reference.The document of announcing before any herein admit not will be understood that be its to be taught in that its date rises be admitting or describing of Australia or other local common general knowledges.
Claims (19)
1. be used for catching method of microorganism with hydrophobic surface from sample, this method comprises microorganism is contacted with trapping agent, described trapping agent has hydrophobic property, trapping agent is by combining with it with the hydrophobic interaction of described microorganism thus, with have polar character, described trapping agent is present in the surface and goes up and described microorganism is captured on it, or exist in solution described method thereby also comprise by described trapping agent being bonded to described surface described microorganism is captured to the surface by means of the polar interaction between described surface and the described trapping agent.
2. the method described in the claim 1, wherein said trapping agent comprises the long hydrocarbon chain with a plurality of polarity site.
3. the method described in the claim 2, arrange at interval along described chain in wherein said a plurality of polarity site.
4. the method described in any aforementioned claim, wherein said trapping agent is cationic.
5. the method described in the claim 4, wherein said trapping agent is diallyl dimethyl ammoniumchloride (DADMAC).
6. be used for catching method of microorganism with hydrophobic surface from fluid sample, this method comprises microorganism is contacted with the solubility trapping agent, described trapping agent comprises poly-DADMAC, trapping agent is in conjunction with described microorganism thus, and by described trapping agent is bonded to the surface described microorganism is captured to described surface.
7. the method described in any aforementioned claim, wherein said surface is provided by bead.
8. the method described in any aforementioned claim wherein contacts sample under the situation that scaling agent exists with trapping agent, described scaling agent increases the selectivity of the combination of the microorganism of expecting.
9. the method described in the claim 8, wherein scaling agent comprises the amino acid amide of fatty acid.
10. the method described in the claim 8, wherein scaling agent comprises the N-Hamposyl L.
11. the method described in each of claim 8 to 10, wherein scaling agent comprises Triton X scaling agent.
12. be used to detect method of microorganism, this method comprises by the method described in any aforementioned claim described microorganism is captured to the surface, clean described captive microorganism and detect described lip-deep described captive microorganism or the described captive microorganism of detection after taking off from described surface.
13. the method described in the claim 12 is wherein measured the viability of captive microorganism.
14. the method described in the claim 13, wherein captive microorganism of treated with medicaments and the viability of measuring described microorganism are to determine whether medicine influences the viability of microorganism.
15. microbioassay kit, this kit comprises (a) solubility trapping agent and matrix, described trapping agent has hydrophobic property, trapping agent can be by with the hydrophobic interaction of microorganism to be detected and combine with it thus, with have the polyion characteristic, described matrix has the surface, described surface is used for by utilizing polar interaction between described surface and the described trapping agent that described trapping agent is bonded to described surface described microorganism being captured to described surface, thereby or (b) be coated in trapping agent fixed thereon on the solid surface, described trapping agent has hydrophobic property and polyion characteristic, trapping agent can be in conjunction with microorganism to be detected thus
With at least a following material:
-can infect the bacteriophage of described microorganism;
-be used to carry out the primer of amplification of the genomic nucleic acids of described microorganism or described bacteriophage;
-be used to cultivate the nutrient culture media of described microorganism;
-be used to manifest described microorganism to carry out the stain of microexamination;
-be used for antibody in conjunction with described microorganism; Or
-be used to detect the detectable of the metabolic product that when cultivating described microorganism, produces.
16. the kit described in the claim 15, wherein said trapping agent are poly-DADMAC.
17. the kit described in claim 15 or the claim 16, wherein said bacteriophage, described primer, described antibody or described detectable are specific for the evaluation of Much's bacillus, mycobacterium avium, Mycobacterium intracellulare, mycobacterium paratuberculosis, Mycobacterium leprae, mycobacterium kansasii, Mycobacterium marinum or mycobacterium fortutitum complex.
18. the kit described in each of claim 15 to 17, wherein to comprise for the microorganism that can survive be specific detectable to this kit.
19. the kit described in each of claim 15 to 18, wherein this kit comprises one or more medicines that can influence the viability of described microorganism potentially.
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| GB0623866A GB0623866D0 (en) | 2006-11-29 | 2006-11-29 | Capture of mycobacteria like micro-organisms |
| GB0623866.1 | 2006-11-29 | ||
| GB0710977A GB0710977D0 (en) | 2007-06-07 | 2007-06-07 | Capture of Mycobacteria like micro-organisms |
| GB0710977.0 | 2007-06-07 | ||
| PCT/EP2007/062732 WO2008065047A1 (en) | 2006-11-29 | 2007-11-23 | Capture of mycobacteria like micro-organisms |
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| CN103189747A (en) * | 2010-07-02 | 2013-07-03 | 米克罗森斯医疗技术有限公司 | Capture of micro-organisms |
| CN112574885A (en) * | 2019-09-29 | 2021-03-30 | 广东体必康生物科技有限公司 | Mycobacterium tuberculosis detection method based on magnetic bead enrichment sputum sample and special kit thereof |
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|---|---|---|---|---|
| US6673629B2 (en) * | 1998-01-15 | 2004-01-06 | Abbott Laboratories | Neutralization of polycations in a chromatographic device for whole blood use |
| SE0302652D0 (en) * | 2003-10-06 | 2003-10-06 | Amersham Biosciences Ab | Attachment of cells to surfaces |
| BRPI0517325B1 (en) * | 2004-09-23 | 2021-02-02 | Tripath Imaging, Inc | method for the preparation of a coated substrate adapted for immobilization of a biological sample |
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2006
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2007
- 2007-11-23 CN CN200780048444.0A patent/CN101573621B/en not_active Expired - Fee Related
- 2007-11-23 ZA ZA200903650A patent/ZA200903650B/en unknown
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103189747A (en) * | 2010-07-02 | 2013-07-03 | 米克罗森斯医疗技术有限公司 | Capture of micro-organisms |
| CN103189747B (en) * | 2010-07-02 | 2015-03-11 | 米克罗森斯诊断有限公司 | Capture of micro-organisms |
| CN112574885A (en) * | 2019-09-29 | 2021-03-30 | 广东体必康生物科技有限公司 | Mycobacterium tuberculosis detection method based on magnetic bead enrichment sputum sample and special kit thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101573621B (en) | 2013-05-29 |
| GB0623866D0 (en) | 2007-01-10 |
| ZA200903650B (en) | 2010-09-29 |
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