CN102559877B - Method for preparing nucleic acid probe-coated porous solid support strip - Google Patents
Method for preparing nucleic acid probe-coated porous solid support strip Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/60—Detection means characterised by use of a special device
- C12Q2565/625—Detection means characterised by use of a special device being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
Abstract
Disclosed is a method for preparing a nucleic acid probe-coated porous solid support strip. The method includes linking a nucleic acid probe to a carrier to prepare a conjugate and immobilizing the conjugate on a porous solid support by vacuum transfer.
Description
Technical field
The present invention relates to a kind of method of the porosu solid support bar (strip) for preparing nucleotide probe coating.More specifically
For, the present invention relates to it is a kind of prepare nucleotide probe coating porosu solid support test strips method, the method include by
Nucleotide probe is connected to prepare conjugate and the conjugate is fixed in porosu solid support by vacuum transfer with carrier.
Background technology
It is determined that using nucleotide diagnosis in the inherited genetic factorss of disease, the appropriateness for the treatment of, bioassay etc..
It is commonly used for analyzing from the nucleotide sample of antibacterial or cell separation or the sample is expanded by nucleotide polymerization
And obtain product in specific nucleotide sequence technology be used for confirm nucleotide presence, the technology using with will
The nucleotide fragments (that is, nucleotide probe) of the complementary base sequence of analyzed partial nucleotide specificity connection, this is based on
Really there is connection between them.
The example for analyzing the method for specific nucleotide sequence for using at present includes：By probe is adhered to micropore
The specific nucleotide probe of plate surface and the hybridization for wanting analyzed nucleotide, are included on film and fix probe and expand to its addition
The specific nucleotide probe of the nucleotide of increasing and the hybridization (reverse point/line blot hybridization) of nucleotide, and be included in by such as glass
Various probes the nucleotide hybridization (DNA chip) of nucleotide to its addition amplification etc. are fixed on plate made by the material of glass
Deng.
In order to prepare the diagnosis bar for nucleotide hybridization, need a kind of for fixed nucleotide probe on a solid support
Method.
Specifically, physical absorption, ultraviolet (UV) irradiation and including be covalently attached to be connected chemically be by nucleoside acid adhesion
Most common method (reference in solid support：R Lutgarde et al., Applied Environmental
Microbiology, 62,300-303,1996).
Physical absorption is the method in Physical Adsorption on Solid Surfaces biomolecule, and which is caused in diagnosing in vitro greatly
Concern.
The mechanism that biomolecule is adsorbed to solid phase is not known.In fact, may relate to biomolecule and surface
Between interaction.This interaction may occur by 5 steps, i.e. 1) molecule is moved to surface, and 2) its absorption exists
Surface, the 3) rearrangement of binding molecule, 4) the potential desorption (latent desporption) of binding molecule, and 5) inhale
Attached molecule leaves the motion (reference on surface：W Norde, Advances in Colloid and Interface Science,
25,267-340,1986).
Summary prompting desorption be it is potentially intrinsic, but, in fact, when molecule is sufficiently large, with reference to not
Reversibly there is (reference：AW Adamson, " The Solid-liquid Interface：Adsorption From
Solution ", in Physical chemistry of surfaces, 5th ed. (Chichester, UK：Wiley,
1990), 421-459).
The reason for behavior, is, as the quantity of binding site increases with the increase of molecular weight, although therefore one
Binding site is from surface dissociation, but remaining multiple site remains in that bonding state.Thus, it has been reported that nucleotide table
Reveal and act on protein identical.
Although the adhesion of nucleotide is lower than protein, big nucleotide is good with film combination.
But, the serious problems that physical absorption is present are integrally fixed at the nucleotide on surface and are rearranged and therefore may have not
It is suitable to the construction interacted with the base sequence of any introducing.
Nucleotide with long base sequence will not encounter problems, but when the base sequence of nucleotide becomes in short-term, it is potential
The reduction of activity can become serious problem.When base does not keep the free form that can be interacted with solution and is fixed on
When on surface, due to the structure for deforming, hybridization normally can not occur.
Additionally, the molecular size that nucleotide probe has is little for the transfer of direct application of vacuum, and nucleoside
The physical force (electrostatic force, hydrophobic interaction etc.) that acid probe has is for be adsorbed onto
And it is relatively weak, therefore, nucleotide probe is fixed to extremely difficult when porosu solid is supported.
For this reason that, unless carried out other fixation, otherwise nucleotide probe may not be uniformly coated, and
In checkout procedure can easily desorption, therefore nucleotide probe is being applied to excellent repeatability and hypersensitivity
It is adversely invalid in the hybridization assays of molecular diagnosis.In view of these problems, the method that widely application adopts UV.
UV crosslinkings are by one of nucleotide probe and the covalently bound simplest method of nylon membrane.Connection mainly passes through breast
Gland pyrimidine residue (other nucleoside occur not being so frequent) is carrying out.When thymine residue is activated, they with deposit
Be on nylon membrane amine reaction (GM Church and W Gilbert, PNAS, 81,1991-1995,1984；I Saito etc.
People, Tetrahedron Letters, 22,3265-3268,1981).
A part in these bases is covalently attached with film surface, therefore reaction is not involved in crossover process.When film mistake
When degree is exposed to UV, UV connections will necessarily disturb hybridization.It is therefore important that correctly determining the irradiation dose of UV.
In other words, when a small amount of UV is exposed the film to, effectively it is crosslinked, and works as and expose the film to excessive UV
When, the efficiency of hybridization declines.
Meanwhile, an importance related to the UV of film irradiations is membrane water content.Water is absorbed in UV, and drying steps
Deviation cause be crosslinked occur level higher or lower than be expected.Thus, the problem that the UV irradiations of film are present is, by breast
Gland pyrimidine bases control cross-linking level has suitable difficulty.
Meanwhile, by can photoactivated compound be used for film or probe technology be improvement cross-linking method (reference：JK
Veilleux and LW Duran, IVD Technology 2, no.2,26-31,1996).When can photoactivated compound (example
Such as, photobiotin) for probe when, they make probe have correct orientation (directivity) and degree of freedom, but seldom apply it
, because uncontrollable reaction can usually occur being exposed to when UV irradiates.
Covalent attachment is taken as conventional chemical processes and obtains the method that nucleotide probe is fixed to solid support surface
Concern.Amine, carbonyl, carboxyl and sulfydryl are most-often used in various chemical connection process.
Covalent attachment can control orientation of probes, controlled while being chemically attached to control nucleotide by terminal residue
Make nucleotide that adhesion to occur, and there is the interval of suitable size can enter to exercise fixed nucleotide by introducing to add with any
The probe for entering freely hybridizes (free hybridization).
Covalent attachment between probe and solid support etc. is commonly used to microdot array 1 system, i.e. such as sheet glass, bead etc.
Form, and can also be applied to as film porosu solid support.
But, the probe and quilt of amine labelling are normally limited to using the routine techniquess of the direct covalent attachment between probe and film
Activated carboxylic (using carbodiimides (as 1- ethyl -3- [3- dimethylaminopropyls] carbodiimide hydrochloride (EDC or
EDAC) reagent)) film between covalent attachment.
Additionally, method nucleotide probe being fixed on film in the form of the connection controlled on film is substantially in batches
Process, which is difficult to use in large-scale production and automatic business processing.Additionally, these methods need long time period and high cost.
As noted previously, as the thing related to the production of the film bar of the nucleotide probe coating for nucleotide hybridization inspection
Reason and chemical merits and demerits, the large-scale production that can carry out normal hybridisation film bar in easily and rapidly mode are restricted.
Therefore, for the extensive application that the nucleotide realized in diagnostic field based on film is tested, conventional asking is solved to exploitation
Topic and thereby, it is ensured that the film bar of manufacturability need increasingly increase.
The content of the invention
Technical problem
Accordingly, it is considered to the present invention is completed to the problems referred to above, and one object of the present invention is：There is provided one kind to prepare
The method of the porosu solid support bar of nucleotide probe coating, the method can be true by making nucleotide probe be combined with carrier
Protect the orientation of nucleotide probe, it is to avoid the rearrangement of the nucleotide being fixed on solid support surface, also, due to will be with target base
A part for the hybridization base sequence of the probe of the complementary connection of sequence is not fixed, and is occurred because of the activity for reducing so as to prevent
Abnormal hybridization.
Another object of the present invention is：A kind of side of the porosu solid support bar for preparing nucleotide probe coating is provided
Method, the method efficiently avoid the inconvenience of the control UV optimum dosage of irradiation run in conventional method, and due to this
Method does not adopt the batch process required for conventional method (wherein by being connected chemically fixed nucleotide probe) itself, therefore real
Large-scale production and automatic business processing are showed.
A further object of the present invention is：A kind of method for preparing nucleotide probe checking system is provided, the method is adopted
Low cost comes quickly to realize that the method has repeatability, prevents film surface disturbance phase in a straightforward manner by large-scale production
Interaction is so as to be conducive to the hybridization between target base sequence and nucleotide probe, and shows excellent stability and sensitivity
Property.
Technical scheme
Therefore, according to an aspect of the invention, there is provided a kind of porosu solid for preparing nucleotide probe coating is supported
The method of bar, the method include being connected to prepare nucleotide probe conjugate and combine this by vacuum transfer with carrier
Thing is fixed to porosu solid and supports.
For be applied to in-vitro diagnosis nucleotide experiment hybridization, have various methods for by nucleotide probe be fixed to as
On the porosu solid of film is supported, and these methods should be recognized in the case where many factors are considered.Therefore, just with inspection
For the performance joint efficiency relevant with the convenience of preparation method, merits and demerits is certainly existed.With minimum cost with most
Easy way realizes that large-scale production is most important aspect.
Thus, the method for the porosu solid support bar for preparing nucleotide probe coating of the invention is by by core
Thuja acid probe is combined with carrier and is effectively ensured that the orientation of nucleotide probe, it is to avoid fixed nucleotide on a solid support
Reset, also, as a part for the hybridization base sequence of the probe that will be connected with target base sequence complementation is not fixed, so as to
Prevent the abnormal hybridization occurred because of the activity for reducing.
Additionally, methods described efficiently avoid the control UV optimum dosage of irradiation run in conventional method inconvenience it
Place, and as the method does not adopt the batch process being connected chemically needed for itself, it is achieved that large-scale production and automatically
Change is processed.
The nucleotide probe includes just (forward direction) and antisense (reverse) nucleotide, including DNA, RNA, PNA (peptide
Nucleotide) etc. just target-gene sequence.
Visit including the nucleotide of four types of large fragment DNA, short dna (including cDNA), RNA and peptide nucleotide (PNA)
Pin can be fixed in solid phase the purpose for being used for quick inspection.
These methods for being used to fix different molecular have identical property, but actually due to molecular structure and size
It is different and different from each other.For the method for large fragment DNA is fixed on solid from diagnostic test is not used in, typically used in solid
The method of upper fixed shorter dna sequence is replacing.
The preferred chemical bond of connection between nucleotide probe as used herein and carrier, and compound combine include
Covalent bond and the such as Non-covalent binding of ionic interaction and hydrogen bond.Wherein, preferably covalently is combined.
, can show suitable with covalent attachment although which is not to be covalently attached using by the connection of affine combination
Strong adhesion.
Most-often used is that the biotin-avidin/streptavidin related to affine combination is solid
Determine technology, which can be with～10-15The binding constant of M forms strong bond.Additionally, can be using tool between probe and solid support
There are the glutathion-GST (glutathione S-transferase) of strong affine combination power, histidine-metal ion (Co, Ni, Cu), Fructus Hordei Germinatus
The coupling of sugar-MBP (maltose-binding protein), agglutinin (for example, ConA)-mannose/glucose binding protein.
Therefore, in order that whole base sequences of all nucleotide probes participate in hybridization, preferably can be to nucleotide probe
Functional group is introduced so as to by probe and carrier covalent bond and part be introduced to realize between nucleotide probe and carrier
Affine combination.
Can complete to realize covalently connecting between nucleotide probe and solid support using various functional groups as described below
The surface modification for connecing：Carboxyl (- COOH), phosphate-based, amino (- RNH2,-ArNH2；Wherein R represents H or C1-C6Alkyl), chlorine
Acute pyogenic infection of nails base (- ArCH2Cl), amide groups (- CONH2), aldehyde (- CHO), hydroxyl (- OH), epoxy radicals, (the reference such as sulfydryl：2005 &
2010 Integrated DNATechnologies, Strategies for Attaching Oligonucleotides to
Solid Supports).These functional groups being applied in combination individually or with two or more types.
Meanwhile, for realize affinity coupling between nucleotide probe and solid support technology can by as biotin-
It is avidin/streptavidin, glutathion-GST, histidine-metal ion, agglutinin-glycoprotein, anti-
The multiple systems of body-antigen etc. are carrying out.
In a preferred embodiment, in the case of amido modified nucleotide probe, amino is directly connected to nucleoside
On acid probe, or by amino C6 joints (linker) with short carbon chain or amino C12 joints connecting.
It is additionally, in the technique for fixing of nucleotide probe, most effective by the reaction of end group and linking group is excellent
Choosing is incorporated into 5 ' or 3 '-reactive terminal group of nucleotide.
Therefore, the 5 ' of nucleotide probe-end or 3 '-end can be with can modify with the covalently bound functional group of carrier.Core
The modification of thuja acid probe can be entered in the preparation process of nucleotide probe or before film is used it in solution state
OK.
Additionally, nucleotide probe can be, for example including 16 to 22 nucleotide synthesis oligonucleotide.
Specifically, mycobacterium tuberculosis (TB) can detected and is being recognized to rifampicin and the nucleoside of the drug resistance of isoniazid
In the case of acid probe, it is possible to use spacer between the transcription that targeting has specificity to mycobacterium tuberculosis strain (ITS,
Intertranscriptional spacer) gene locis nucleotide probe be used for recognize mycobacterium tuberculosis.
In order to determine the sensitivity and drug resistance of the antituberculotics to such as rifampicin, it is possible to use poly- comprising coding RNA
The nucleotide probe of the wild type-or mutant bases sequence of the gene of synthase β subunits.
Additionally, in order to determine the sensitivity to isoniazid and drug resistance, it is also possible to using containing coding katG (hydrogen peroxide
Enzyme peroxidase) gene locis and inhA (enoyl- carrier protein (ACP) reductase) and ahpC (alkyl peroxides
Reductase) promoter site wild type or the nucleotide probe of mutant bases sequence.
Meanwhile, the bacterium of non-tuberculous mycobacteria (NTM) and mycobacterium tuberculosis (TB) is distinguished when using nucleotide probe
Kind when, it is also possible to using targeting Mycobacterium specificity transcription between spacer (ITS) gene locis nucleotide probe.
Additionally, the nucleotide probe preferably comprises can be split mirror, photochemistry, biochemistry, immunology or chemistry side
The labelling that formula is directly or indirectly detected, and internally spacer can further be contained to improve base in hybridization site in region
Hybridization efficiency of the sequence (which can be complementally combined with target base sequence) and carrier between.
The example of labelling includes：Enzyme (for example, horseradish peroxidase, alkali phosphatase), radiosiotope are (for example,32P), fluorescence molecule and chemical group (for example, biotin) etc..
The spacer is preferably nonspecific poly- T tails, after which can be added in amido modified site.As spacer
The length of the poly- T tails for using can be 5 to 30 thymine alkali bases.
For example, nucleotide probe can be designed to：5 '-end labelling amino C6 joints, using as spacer including 10
The poly- T tails of individual thymine alkali bases are connected thereto, and the hybridization site that can be connected with the nucleotide sequence complementary of target gene
Base sequence includes 16 to 22 nucleotide.
In the present invention, the carrier can be, such as polysaccharide, protein, synthetic polymer etc..Wherein, preferred polysaccharide, and
And it is special standby be preferably covalently attached with nucleotide probe or affine combination glucosan or derivatives thereof.
In the case of affine combination, for example, antibiotin/streptavidin, maltose-binding protein, sugar
Albumen or metal ion (Co, Ni, Cu etc.) etc. are connected with carrier, and using can be with the biology as part of its affine combination
Element, maltose, agglutinin, histidine or derivatives thereof are marked on nucleotide probe.Wherein, more preferably biotin-antibiont
Element/streptavidin interconnection technique.
As glucosan has various active group, so each dextran molecule can fix multiple DNA probes, therefore
Multiple ligands, such as aspartic acid and oligonucleotide (reference can be fixed：Goss, T.A. et al.,
J.Chromatogr.508,279-287,1990；Schacht E.H et al., Modification of Dextran and
application in prodrug design.In Industrial polysaccharide Engineering：
Genetic Engineering, Structure/Property Relations and Applications Yalpani, M.,
Ed.；Elsevier：Amsterdam, 1987).Additionally, glucosan is the polymer of long and flexibility, thus it is considerably advantageous in rush
Enter the hybridization of fixed DNA molecular, eliminate the sterically hindered (reference caused because of the surface of solid support：Goris J. et al.,
Can J Microbiol, 44,1148-1153,1998；Shepinov M.S et al., Nucleic Acids Res., 25,
1155-1161,1999；Gingeras T.R et al., 15, p.13,1987；Penzol G et al., Biotechnol
Bioeng.60,518-523,1998).
One the most representational example, aldehyde radical-aspartic acid-glucosan is the material with suitable macromolecule,
Which has anion and aldehyde radical, forms very strong covalent attachment therefore, it is possible to the DNA probe with ammonification, wherein, the connection is non-
Often stablize so as to show the resistance to of the extreme pH to hybridization mixture, high temperature or high salt concentration, solvent and nucleopilic reagent
The property of medicine (reference：Fuentes M. et al., Biomacromolecules, 5,883-888,2004).
In a preferred embodiment, the carrier can be the polysaccharide that aldehyde functional group can be formed by oxidized dextran.
For example, polyacetals glucosan can be by aoxidizing the glucosan of 43kDa (compared with the glucosan of 500kDa, with nucleoside
Acid probe has higher joint efficiency) preparing.
This polyacetals glucosan, for example, can be prepared by following steps：By glucosan and KIO4Predetermined etc.
Mix in the glucose of the 62.5mmol units of mole, the mixed solution is rotated with the speed of 40rpm, the solution left standstill 6 is made
Dialyse the solution three times (each circulation 2 hours) more than hour and with aquesterilisa.
As described above, the carrier can be the protein such as albumin or synthetic polymer.In this case, functional group
Can be covalently attached with the amino of protein or synthetic polymer, sulfydryl etc., or can introduce with the part of its affine combination
Nucleotide probe.
For example, polyacetals glucosan carrier reacts to be formed when pH value is 11 at room temperature with the nucleotide probe of amine-labelling
The imine intermediate of schiff base base (Schiff ' s base), gentle reducing agent of the schiff base base with such as sodium borohydride
Reduce as reducing agent, promote irreversible connection to be formed so as to realize stable covalent attachment.
Nucleotide deficient in stability itself, but the reaction work with other groups is obtained when to its applying enough energy
Property.When nucleotide being exposed to visible ray in the presence of the photosensitizer of such as methylene blue, or during no any photosensitizer which is sudden and violent
UV is exposed to, which directly (can be referred to protein cross：R Lalwani et al., J.Photochemistry and
Photobiology 7,57-73,1990；JW Hockensmith et al., Methods in Enzymology, 208,211-
236,1991).
The method for carrying out direct crosslinked nucleic acid by preparing the solid porous support (for example, activating film) of activation has two
Problem.
First problem is loss activity.In other words, the chemically reactive surface of film is with air reaction and therefore loses its work
Property.
When chemism increases, i.e. when the crosslinking rate with target is improved, the speed of loss of activity is improved.To understand
Determine these problems, recently, manufacturer uses more SA group, specifically with target probe reaction itself.Typical example
For the reaction between aldehyde and amine, both of which is metastable.
Due to the built in problem of the active solid related to production, storage and process, active membrane cannot be used for and spy at all
The purpose that pin is covalently attached.
Be usually used with can combine with probe specificity, it is while show compared with low activity and living during storage
Property reduces the film of slower surface chemical property over time.This film generally carries amine or aldehyde radical on its surface.
Advantage with aldehyde activating surface is can just to form important connection by the formation of schiff base base and need not
Add any other reagent.
Schiff base base is metastable, but can be reversed in physiological conditions.For this reason that, it should use boron hydrogen
Change sodium as gentle reducing agent to reduce schiff base base so as to form irreversible connection (MB Meza, " Covalent
Binding in Diagnostic Test Design " in The latex Course, 2000).
Second Problem is that nonspecific connection should be being closed using before and film is activated.If so, sample is not then
Can be connected with film.Closing step should simply, it is quickly and quite effective.It is desirable that as closing result, in the surface shape of film
Into hydrophilic uncharged residue.
When protecting film (passivation) being formed on the surface of film, introduce new chemical functional group and the film can not be with to core
The reactivity of thuja acid.But, as the charge attraction power or hydrophobic captivation of sample connection or detecting system can increase, rather than
Therefore specific signals also can increase.
Great majority in addition to disulfide bond are covalently attached this as batch process, and which includes being connected and then closing target with film
Film, it is therefore desirable to which about 30 minutes to a few hours.
Such deviation makes Automated condtrol and large-scale production become difficult.Photosensitive connection chemistry becomes large-scale production
Simply, but increased cost and impracticable.
On the other hand, the present invention, for example, by specifically (being used as carrier by activating (oxidation) with polyacetals glucosan
Glucosan) covalent bond of amido modified nucleotide probe reacted and solve these problems, therefore make extensive life
Product is possibly realized, and does not additionally need other toxic reagents, and this is from by using 1- ethyl -3- [3- dimethylaminopropyls]
Covalent attachment between the amido and carboxyl of carbonization inferior amine salt hydrochlorate (EDC or EDAC) can be seen that therefore reduce and use
The related cost of organic solvent.
In a preferred embodiment, the porosu solid support can be film and its substrate (substrate) can be fibre
Dimension element or nylon.
The example of the solid support fixed for nucleotide includes：Film (nitrocellulose or nylon membrane), bead are (controllable
Pore glass beads), acrylamide gel, polystyrene matrix, activation glucosan, avidin coating polystyrene
Pearl and glass etc., and these different solid supports are used for into DNA diagnosis (references：Saiki R.K et al, PNAS, 86,
6230-6234,1989；Meinkoth Jet al, Anal.Biochem, 138,267-284,1984；Ghosh S.S et al,
Nucl.Acids Res., 15,5353-5372,1987；Rasmussen S.R et al., Anal.Biochem, 198,138-142,
1991；Gingeras T.R, Nucl.Acids Res., 15,5373-5390,1987；Lund V. et al., Nucl.Acids
Res., 16,10861-10880,1988).
The present invention mainly have studied and for target substances of multiple types be fixed to the mechanism that porosu solid is supported, in these many kinds of solids
Film is have studied in support especially.
The film includes the large-scale material that can be used for nucleotide fixation.Specifically, the example of the film includes：It is conventional
Casting film, such as nitrocellulose and nylon；New membrane, such as ceramics or trace-etching-film；And for the substrate of microarray
Type film (substrate-type membrance).
The Best link state of the film of the various suggestions fixed for nucleotide should be determined.In any interconnection technique
In, nucleotide can be added on film in uncontrollable mode.
In this case, as nucleotide probe will not be arranged in its main direction, so reaction can be potentially
It is limited.Therefore, in most of the cases, the end connection of the fixed nucleotide in oriented adsorption, i.e. the application is preferred.
The major advantage of nitrocellulose for can the quickly suitability, but unless by dry polyester coating nitrocellulose,
Otherwise the performance is weaker, and with relatively low adhesion compared with other films.
Recently, nylon has the adhesion of higher physical strength and Geng Gao, and which has the usable surface of wider range
Property is closed, is capable of the adhesion of optimization nucleotide, and therefore is promoted it as the substrate connected for nucleotide.
It is physical absorption that nucleotide is connected with nitrocellulose most-often used, and the fixation on nylon membrane is by thing
Manage absorption, UV crosslinkings or chemical activation to realize.
As described above, the present invention can solve the problem that the problem of Typical physical absorbing process.But, with regard to production and manufacture efficiency
Speech, physical absorption are comparatively simple and coarse technology (references：TC Tisone, IVDTechnology 6, no.3,43-60,
2000)。
When in dry run being used to connect by material by continuous processing, it is most based on the simple production of the system of film
Readily.Fixable material can be used for continuous processing, and by simple and control good technique (that is, being dried) entering
Row connection.
If solving the particular problem of physical absorption, i.e. strengthen physical absorption so that higher be directly connected to become
May, used as the technology of the large-scale production for nucleotide diagnostic test, physical absorption is beneficial.
Thus, the present invention can be further included between the hole that carrier and porosu solid are supported by being dried what is formed
Physical absorption conjugate.
The physical adsorption techniques that great majority are strengthened not only improve the joint efficiency with very short probe, and make probe
Important base sequence be arranged in and project upwards in the side away from the surface of solids.As a result, in the sample and important base for adding
Free interaction can occur between sequence.Even if particularly using short probe, due to improve selectivity and sensitivity,
Physical adsorption techniques are also quite effective.
Thus, according to the present invention, the important base sequence of nucleotide probe has to be arranged them by covalent attachment
Arrange into the orientation in the direction away from porosu solid stayed surface.
In the present invention, the vacuum transfer is preferably included：In transfer matic blotter (automatic line
Blotter load nucleotide probe-carrier conjugates in nucleotide slit lines (slit line)) and incited somebody to action using vacuum pump
Conjugate is adsorbed onto porosu solid and supports.
Present invention also offers the porosu solid support bar of the nucleotide probe coating prepared by methods described.
The porosu solid support bar of nucleotide probe coating of the invention itself has specific structure, and the structure is led to
Cross specific preparation technology as above and make a distinction with conventional support bar.
Beneficial effect
From the above it is apparent that the present invention is effectively ensured by nucleotide probe combines with carrier
The orientation of nucleotide probe, it is to avoid the rearrangement of the nucleotide being fixed on solid support surface, and due to target base sequence
A part for the hybridization base sequence of the complementary connection of row is not fixed, so preventing the exception caused due to the activity for reducing
Hybridization.
Additionally, present invention effectively avoids the inconvenience of the control UV optimum dosage of irradiation being related in conventional method, and
And as methods described is not adopted required for conventional method (wherein by being covalently attached fixed nucleotide probe) itself in batches
Process, it is achieved that large-scale production and automatic business processing.
In other words, the present invention effectively prepares nucleotide-probe checkout system, and which can be using low cost by extensive
Production comes quickly to realize which has repeatability, prevents film surface disturbance target base sequence and nucleotide probe in a straightforward manner
Between interaction, so as to be conducive between both hybridization and show excellent stability and sensitivity.
Description of the drawings
Connection with figures will be more clearly understood from from being described in detail below the present invention above and other purpose, feature and
Other advantages, in the accompanying drawings：
Fig. 1 is the view of the whole process that diagram prepares bar, including by being covalently attached the spy of nucleotide oligonucleotide
Pin is fixed to the conjugate on film with the glucan binding domian as carrier and by vacuum transfer；
Nucleotide probe is wherein coated in film bar together with patent blueness V dyestuffs (as blue dyess) to show by Fig. 2
On state figure, wherein, the probe on top is to the sensitivity of Rimactazid/resistance to according to an embodiment detection
The probe of the property of medicine, according to the probe of bottom, another embodiment is recognized and detects the probe of Mycobacterium；
Fig. 3 is to show the PCR and reverse hybridized line trace inspection by for rifampicin and isoniazid sensitivity/drug resistance sample
Scheme obtained from testing；And
Fig. 4 is display by the PCR and the inspection of reverse hybridized line trace organized for mycobacteria sample and control (the negative, positive)
Scheme obtained from testing.
Specific embodiment
Now, the present invention will be more fully described with reference to the following examples.These embodiments are only used for illustrating this
Invent and bel not applied to limit the scope of the present invention and essence.
Following experiment is related to the method and card for nucleotide probe being fixed on film to prepare bar by vacuum transfer
The experiment of real following effectiveness：I () is detected mycobacterium tuberculosis and is confirmed to rifampicin and the reality of the drug resistance of isoniazid medicine
Test, (ii) using the reverse hybridized line trace method of inspection of film bar used in two specific embodiments being detected and be recognized branch
The experiment of bacillus, i.e. distinguish tuberculosis point in sputum, Bronchial washing, cerebrospinal fluid, urine, tissue, whole blood and culture bacterial strain
Branch bacillus (TB) and non-tuberculous mycobacteria (NTM).
Embodiment 1：Prepare polyacetals glucosan (PAD)
(1) preparation of reagent needed for and instrument
Using glucosan (Sigma, D-4133, Mr～43kDa), KIO4It is (Aldrich, 32242-3, F.W230.00), fine
The plain dialyzer of dimension retains 12000：Spectra/Por, MWCO 12-14000), oxammonium hydrochloride. (NH2OH-HCl=69.49), first
Base orange powder, 10N hydrochloric acid, acidometer, 0.1N sodium hydroxide (NaOH), freezer dryer and SpeedVac etc..
(2) preparation of oxidized dextran
The glucosan (glucoses of Sigma, D-4133, the Mr～43kDa/ equivalent to 62.5mmol) of 0.5g is completely dissolved
So as to cumulative volume is adjusted to 10ml in the D.W that hot-pressing processing is crossed.It is added thereto to the KIO of 0.72g4(equivalent to
The identical equivalent of 62.5mmol) (reference：Azzam T et al., J.Med.Chem., 45,1817-1824,2002；Azzam T etc.
People, Macromolecules, 35,9947-9953,2002；Azzam T et al., J.Controlled Release, 96,309-
323,2004；Hosseinkhani H.et al .Gene Therapy, 11,194-203,2004).
The mixture for obtaining is kept for 6 to 12 hours while rotating under the speed of 40rpm.Oxidized dextran is added to into fibre
On the plain dialyzer of dimension, the O/N dialysis in 2L sterilizing tri-distilled waters once and continues dialysis, three times altogether, every time dialysis 2 hours more
Change sterile purified water.After dialysis, the volume of polyacetals glucosan is accurately measured.
(3) in oxidized dextran the amount of aldehyde radical measurement
Reported in periodical according to Huiru Zhao et al. open method (Pharmaceutical Research, 8,
400-402,1991) measuring oxidationGlucosanThe amount of middle aldehyde radical.First, by the oxammonium hydrochloride. that is dried of 1.75g is dissolved in
0.25N oxammonium hydrochloride .s-methyl orange (pH4.0) reagent is prepared in the sterile purified water of 15ml.Individually, prepare the end of 0.6ml
Concentration is 0.05% methyl orange reagent and is added into hydroxylamine hydrochloride solution.
10N HCl are slowly added in obtained mixture pH value is adjusted to 4.0, and it is molten to dilute this with sterile purified water
Liquid is preparing 0.25N oxammonium hydrochloride .s-methyl orange (pH 4.0) reagent of 100ml.
Oxidation-reduction titration is carried out to polyacetals glucosan with 0.1N NaOH standard solution.After dialysis, by 1.25ml's
0.25N oxammonium hydrochloride .s-methyl orange (pH 4.0) solution is added in polyacetals glucosan obtained in 100 μ L, is then entered at room temperature
Row reaction 2 hours, while rotating under 40rpm.
Then, titrated as standard solution with 0.1N NaOH.Accurately measurement is added in oxidation-reduction titration
The volume (μ L) of NaOH and titration end-point can be found out from the change (color is changed into yellow from redness) of color.
The calculating of the substitution level of the oxidation-reduction reaction and aldehyde radical of polyacetals glucosan is displayed in equation below, and
And calculate the total mole number of aldehyde radical present in the cumulative volume of polyacetals glucosan (PAD) measured after dialysis and including
In table 1 below.
Glucosan-(CHO)n+H2N-OHHCl=glucosans-(CH=N-OH)n+H2O+HCl (1)
HCl+NaOH=NaCl+H2O (2)
The substitution level of aldehyde radical
[table 1]
(4) lyophilizing of polyacetals glucosan and the preparation of stock solution
The a small amount of polyacetals glucosan of 10.0 μ L is loaded in screw-cap test tube and with SpeedVac or freezing
Drying machine carries out O/N lyophilizing.By in sterile purified water add lyophilizing polyacetals glucosan be then completely dissolved so as to
Final concentration is adjusted 100nmol/ μ L to prepare stock solution.
From table 2 it can be seen that being then completely dissolved by adding the sterile purified water of 468.3 μ L, 1.0 μ can be prepared
The stock solution of mol/ μ L, because the total mole number of aldehyde radical is 468.3 μm of ol in the polyacetals glucosan of lyophilizing.
[table 2]
Obtained product is diluted with 10 × sterile purified water and fully dissolve to prepare the original of final concentration of 100nmol/ μ L
Liquid, and will preserve under -20 degrees Celsius.
(5) preparation of the polyacetals glucosan of 1pmol/ μ L
The polyacetals stock solution of 100nmol/ μ L is diluted into 10 times of polyacetals stock solutions so that 1pmol/ μ L are obtained progressively, i.e. with nucleoside
Acid probe is covalently attached the concentration of required polyacetals glucosan.
Embodiment 2：The preparation of nucleotide probe
The probe for using in this experiment is designed to into detection：I () mycobacterium tuberculosis are simultaneously recognized to rifampicin and different cigarette
The drug resistance of hydrazine medicine, (ii) detection branches bacillus, i.e. differentiation non-tuberculous mycobacteria (NTM) and mycobacterium tuberculosis (TB)
Strain.
Firstly, for aspect (i), when probe go to detect mycobacterium tuberculosis and recognize mycobacterium tuberculosis to rifampicin and
During the drug resistance of isoniazid, by targeting spacer (ITS) gene locis between the transcription of mycobacterium tuberculosis complex specificity
Probe be used for mycobacterium tuberculosis identification.
When going to recognize the sensitivity and drug resistance to rifampicin (antituberculotics) with probe, using antituberculotics,
The wild type or the oligonucleotide of mutant bases sequence of the gene i.e. containing coding rpoB (that is, RNA polymerization beta subunits).
Additionally, when probe goes to detect the sensitivity and drug resistance to isoniazid, using containing coding katG (hydrogen peroxide
Enzyme peroxidase) gene locis and inhA (enoyl- carrier protein (ACP) reductase) and ahpC (alkyl peroxides
Reductase) promoter site wild type or the nucleotide probe of mutant bases sequence.
These probes are designed with into such structure：Wherein modify in 5 '-position of corresponding gene base sequence
Amino-C6- the joints and the poly- T tails comprising 10 thymine alkali bases of labelling at 5 '-end.
Simultaneously for aspect (ii), when probe removes to detect and recognize mycobacteria (that is, mycobacterium tuberculosis (TB) and non-
The strain of mycobacterium tuberculosis (NTM)) when, there is to various Mycobacteriums the probe of the ITS genes of specificity using targeting.
It is similar with the probe of aspect (i), these probes are designed with into such structure：Wherein in corresponding gene base sequence
Amino-C6- joint and poly- T tail comprising 10 thymine alkali bases of the modification of 5 '-position in 5 '-end labelling.
In the following embodiments, will be described in for binding nucleotide probe and the glucan derivative as carrier
The method of (polyacetals glucosan) and the probe base sequence used by non-specific design, wherein, the nucleotide probe by using 5 '-
Amino linker and the modification of poly- T tails spacer are obtained according to the probe of required purpose preparation.Have with the probe for (i) and (ii)
The brief information of pass is displayed in (table 3 in mark 3 and 4：For detection to rifampicin or the probe of the sensitivity/drug resistance of isoniazid
Brief information；
Table 4：For identification and the brief information of the probe of detection branches bacillus).
[table 3]
[table 4]
| Line (line) | Band (cord) | Detecting probe information |
| 1 | CC | With reference to control |
| 2 | AC | Amplification control |
| 3 | GPC | Mycobacteria Pseudomonas |
| 4 | GNC | Mycobacteria negative control |
| 5 | TUB | 1) mycobacterium tuberculosis complex |
| 6 | AVI | Mycobacterium avium (M.avium) |
| 7 | INT | Mycobacterium intracellulare (M.intracellulare) mycobacteria chimera (M.chimaera) |
| 8 | SCO | Mycobacterium scrofulaceum (M.scrofulaceum) |
| 9 | ABS | Mycobacterium abscessuses (M.abscessus) |
| 10 | CHE | Mycobacterium chelonei (M.chelonae) |
| l1 | KAN | Mycobacterium kansasii (M.kansasii) |
| 12 | SZU | Si Shi mycobacterias (M.szulgai) |
| 13 | GOR | Ge Dengshi mycobacterias (M.gordonae) |
| 14 | CEL | Hiding mycobacteria (M.celatum) |
| 15 | M-U | Mycobacterium marinum (M.marinum) mycobacterium buruli (M.ulcerans) |
| 16 | SIM | Mycobacterium habana (M.simiae) |
| 17 | L-G | Slow SHENGHUANG mycobacteria (M.lentiflavum) Geneva mycobacteria (M.genavense) |
| 18 | XEN | Mycobacterium littorale (M.xenopi) |
| 19 | SME | Mycobacterium smegmatis (M.smegmatis) |
| 20 | MAL | Ma Ermo mycobacterias (M.malmoense) |
| 21 | GAS | Mycobacterium gastri (M.gastri) |
| 22 | FLA | Micro- yellow mycobacteria (M.flavescens) |
| 23 | VAC | Mycobacterium vaccae (M.vaccae) |
| 24 | FOR | 2) Mycobacterium fortuitum complex (M.fortuitum) |
| 25 | TERC | Mycobacterium terrae complex (M.terrae) |
1) mycobacterium tuberculosis complex：Mycobacterium tuberculosis, Mycobacterium bovis (M.bovis), mycobacterium africanum
(M.africanum), mycobacterium microti (M.microti)
2) Mycobacterium fortuitum complex：It is Mycobacterium fortuitum, Senegal mycobacteria (M.Senegalense), outer
Carry out mycobacteria (M.peregrinum)
Embodiment 3：The preparation of nucleotide probe-polyacetals glucan binding domian thing
(1) preparation of reagent needed for
The buffer for preparing the sodium borate buffer liquid (pH 11.0) of 0.5M and being used as association reaction, prepares
The NaBH of 0.1mM4Gentle reducing agent of the solution for use as conversion schiff base base, the schiff base base is by as carrier
The Jing stable covalent attachment of reaction between the amino at the aldehyde radical of polyacetals glucosan and nucleotide probe 5 '-end is formed.
(2) for association reaction main mixed solution preparation
Main mixed solution is by polyacetals glucosan (1pmol/ μ L), the sodium borate buffer liquid (pH11.0) of 0.5M and sterilizing
Distilled water is constituted.The proportioning listed in these component accordings to the form below 5 is mixed.
[table 5]
(3) association reaction
40 sizes of preparation are 16.7cm × 5.5cm and the thickness of each line is the film of 0.8mm.By 48.00 μ L, 100 μ
The probe solution of M is mixed with the sterile purified water of 4752.00 μ L so that its final volume is adjusted to 4800.00 μ L.Add and mix
Close the 11200.00 μ L of main mixed solution for combining as shown in table 5, then carry out O/N reactions, at the same at room temperature with
The speed rotation of 40rpm.
Specifically, based on sheet of membrane (size：16.7cm × 5.5cm), when transfer matic blotter examination slit it is a width of about
During 0.8mm, in online coating procedure, by the Master Mix of 280.00 μ L with the water-reducible 120.00 μ L amine of aseptic distillation-
Probe solution (final concentration：1 μM) mixing.As a result, in final cohesive process, liquor capacity is 400.00 μ L.
Reduce polyacetals glucosan-probe imines conjugate to prepare stable amine conjugate with reducing agent.Specifically, will make
For the 0.1mM NaBH of 1.0 μ L of reducing agent4Solution adds polyacetals glucosan-probe imines conjugate that cumulative volume is 100 μ L,
Add altogether twice, midfeather 1 hour, 2 hours when sharing.After adding reducing agent, in room while conjugate reacts
Rotated with the speed of 40rpm under temperature.
Embodiment 4：The preparation of the film bar of nucleotide probe coating
(1) setting of transfer matic blotter
The line blotter with about 8mm slits that preparation is matched with a film size (16.7cm × 5.5cm), and to upper
Portion's disk and lower disc carry out ultrasonic Treatment as using front cleaning step.
The line blotter is arranged to into upper disc with lower disc (sterilized distilled water and 1 × PBS are several times) fully
With reference to being automatically loaded with preparation conjugate.
(2) using transfer matic blotter nucleotide probe coat film
The patent blue V dyestuff (5000 ×=3g/500ml) of suitable concentration is mixed as blue dyess with 1 × PBS.Plus
Enter the dyestuff to detect uniform drafting that film is reached the standard grade.For every line (each probe), the PAD- nucleotide probes of 400 μ L are tied
Compound is mixed with the 1 × PBS (containing patent blue V dyestuff) of 600 μ L to prepare solution of the cumulative volume as 1mL.
The film for using in the present embodiment is the Biodyne B films buied from Pall company limiteies and is cut into 3mm paper suitable
Close the size of slit imprinting device size.A piece of 3MM paper is placed in the lower disc of slit imprinting device, by film to thereon,
And upper disc is adhered to film, then load 1 × PBS solution containing patent blue V dyestuff first and load in slit to detect
The seepage and speckle of the appearance of solution.
In the same manner described above, 3MM paper is placed in the lower disc of line blotter, places 1 × PBS of immersion thereon
Biodyne B films, by upper disc with its adhere to prepare nucleotide probe is coated on film.
PAD- nucleotide probe conjugate solution is automatically loaded in the slit of line with the sample aliquot of 1mL.Using vacuum
Pump is with the speed of 150rpm by its applying vacuum 5 minutes, making PAD- nucleotide probe conjugates be absorbed into Biodyne B
On film, and before pretreatment is carried out with pre--hybridization buffer, film is air-dried to strengthen physical absorption and doing
It is dried in dry device.
(3) with pre--hybridization buffer pretreatment film
In the present embodiment, with pre--hybridization buffer pretreatment film to prepare film bar so that it is guaranteed that nucleotide hybridization inspection
The rapidity of experiment simultaneously improves the ratio (S/N ratios) of signal and noise.
Pre--the hybridization buffer by 5 × denhardt solution (Denhardt ' s solution), 5 × SSC, 0.01～
The salmon sperm DNA composition of the degeneration of 1%SDS and 0.01～1mg/ml.The film in advance-hybridization buffer pretreatment 30 at 42 DEG C
Minute, then in exsiccator through one day time be dried.
(4) preparation of the shell of adhesive film bar
By the film of drying and the viscosity holder (size being arranged on the surface relative with the surface of coating probe：It is long
Spend for 6cm, hyalomere is divided into 0.5cm) adhere to and be cut into the width of 3.4mm.The holder is biadhesive film.By the film
With shell adhere to, so as to using the holder surface relative with film adhesive surface come storage films bar.
When film bar is adhered to shell, suppress by with roller made by rubber, the probe of coating can be separated therefrom.Cause
This, suppresses film by with soft 3MM paper or sponge, and the film bar can be with shell tight adhesion.
The film bar for preparing in the present embodiment is as shown in Figure 2.Fig. 2 show in more detail the film bar of nucleotide probe coating
Size and the adhesion of the film bar and shell state.
Test example 1：For detecting mycobacterium tuberculosis to the sensitivity/drug resistance of rifampicin/isoniazid medicine
Reverse hybridized line trace inspection
The detailed schematic of this test example is as described below.Using using the multiple asymmetric PCR (one-tube of single tube nest bed
Nested multiplex asymmetric PCR) the inspection of reverse hybridized line trace.
In PCR courses of reaction, expanded by the primer Jing asymmetric PCRs method of the biotin labeling of target gene specific
The gene code site and inhA (alkene of rpoB (RNA polymerase beta subunit) and katG (catalase peroxidase)
Acyl carrier protein (ACP) reductase) and ahpC (alkyl peroxide reductase) promoter site, it is final so as to prepare
Biotin labeling, single-stranded target DNA.
Meanwhile, also prepare the amplification of PCR amplification matched groups and the ITS (spacer between transcription) from mycobacterium tuberculosis
Son.
During reverse hybridized, biotin-labelling, single-stranded target DNA respectively with adhesion nylon membrane probe (rpoB,
The wild type or saltant type probe of katG, inhA and ahpC) hybridization.
To strict cleaning being carried out for the hybridization bar for detecting the mycobacterium tuberculosis of multidrug resistant, then carry out enzyme combination
React so as to Avidin-alkaline phosphatase (AP) conjugate is combined with the target of biotin labeling, the biotin mark
Oligonucleotide probes and amplicon hybridization as a control group that the target of note has been absorbed with film.
Not connected product is removed by cleaning step, will be containing chlorination nitro nitroblue tetrazolium (4-nitro blue
Tetrazolium chloride, NBT) and the substrate solution of 5- bromos -4- chloros -3- indolyl phosphates (BCIP) be added to bar
On developed the color.
Blue precipitate is formed in hybridization probe area by oxidation-reduction reaction.That is, the alkaline phosphatase enzyme catalysiss BCIP of connection
Dephosphorylation being produced as the navy blue indigo dye of oxide.
Now, the NBT as oxidant also form dark blue dyes, therefore make blue deeper and improve detection sensitivity
Property.
Can be by explaining the graphic result of Blue color bands with laboratory reference guide/reading card, or using automatic
Analytical tool (AdvanSureTMGenoLine Scan are raw by LG Life Science Ltd (Life Science Ltd.)
Produce) can just detect mycobacterium tuberculosis to rifampicin and the drug resistance of isoniazid medicine.
(1) preparation of solution needed for
Prepare what is listed in table 6 to carry out sample DNA extraction, PCR reactions and reverse hybridized line trace inspection experiment
Reagent.
[table 6]
(2) preparation of sample and DNA extraction thing
By the sample pretreatment solution 1 of the sputum of the lunger of 1 to 2ml or reference culture culture solution and equivalent
(that is, 1N NaOH) is added in 15mL test tubes, then fully shaking and under 4,000rpm be centrifuged 20 minutes.
Gained supernatant is removed, and 10ml is added as the PBS (0.15M of sample pretreatment solution to precipitate
NaCl, 0.01M NaH2PO4), then equably shake and be centrifuged 20 minutes under 4,000rpm.Then, supernatant is removed, will
Precipitation adds 1.5ml test tubes, is added thereto to the PBS of 1ml, is then centrifuged 5 minutes under 13,000rpm.
Additionally, removing supernatant, 50-100 μ L are added as 5% (w/v's) of extract buffer soln to precipitate
100 resins of Chelex (are prepared by Bio-Rad laboratorys), and gained mixture is heated 20 minutes and 13 at 100 DEG C,
It is centrifuged 3 minutes under 000rpm.The DNA supernatant isolated is used as into pcr template DNA.
(3) PCR reactions
Under conditions of table 7 shows, mixed by adding the primer of 12.5 μ L, 2 × PCR mixed solutions that for example table 6 is listed, 5 μ L
Close the sample DNA of solution and 7.5 μ L to enter performing PCR reaction.
[table 7]
*The sample (solid-culture, the M. tuberculosis strains of liquid-culture) of culture
**The direct PATIENTS MATERIALS's (sputum, Bronchial washing, cerebrospinal fluid, urine, tissue and whole blood etc.) of smear-positive
(4) reverse hybridized line trace inspection
Reverse hybridized line trace inspection is carried out using the reagent listed in upper table 6.Add 20 μ L PCR primer and
The solution is added on the film bar for adsorbed probe and is hybridized, together to prepare hybridization solution by the hybridization buffer of the preheating of 250 μ L
When in 55 DEG C of hybridization chamber/casees concussion 30 minutes it is (vertical to shake：9rpm, level concussion：80rpm).
After hybridization, the reaction solution of the bar for absorbing probe is removed by vacuum suction (suction), by the tight of 250 μ L preheatings
Lattice cleaning solution is added on bar, and at 55 DEG C with 9rpm by vertical concussion 15 minutes rinsing bar.After flushing, remove from bar
Dereaction solution, the rinse solution of 250 μ L is added on bar, and at room temperature with 9rpm by vertical concussion 1 minute cleaning
Test strips.
After cleaning reaction, the reaction solution of bar is removed.Enzyme binding soln is used after standing at room temperature more than 10 minutes, will
The enzyme binding soln of 250 μ L is added in bar, at room temperature with 9rpm carrying out vertical concussion 30 minutes and after enzyme association reaction
Remove the reaction solution of bar.
The rinse solution of 250 μ L is added on bar and carries out vertical concussion 1 minute to be rinsed with 9rpm at room temperature.
After flushing, reaction solution is removed from bar.Hung down by the rinse solution of 250 μ L is added on bar and at room temperature with 9rpm
It is straight to shake 1 minute to be rinsed again.
After cleaning reaction, remove the reaction solution of bar and carry out chromogenic reaction.Substrate solution stands 10 minutes at room temperature
Use after above.The substrate solution of 250 μ L is added on bar and vertical concussion 10 is carried out with 9rpm in room temperature in the case where there is optical condition
Minute.
The reaction solution of bar after chromogenic reaction, is removed, rinses bar to remove remaining substrate with the sterile purified water of 250 μ L
Solution and precipitation.With hair-dryer rapid draing or natural air desciccator diaphragm bar.Using what is produced by LG Life Science Ltd
AdvanSureTMGenoLine Scan programs carry out numerical analyses, or adopt laboratory reference guide (reading card by naked eyes
Piece) or data explanation table carry out explanation results.
From the concrete outcome of test example 1, the detecting probe information based on table 3 can analyze mycobacterium tuberculosis to rifampicin
Or sensitivity/the drug resistance of isoniazid.By comparing the Li Fu between wild type and saltant type band intensity in each locus
Put down the sensitivity/drug resistance of (rpoB) and isoniazid (katG, inhA, ahpC) to carry out interpretation of result.When wild type bands of a spectrum
When intensity is better than saltant type bands of a spectrum, it is believed that mycobacterium tuberculosis are sensitive, and the intensity for working as wild type bands of a spectrum is weaker than saltant type
When, it is believed that mycobacterium tuberculosis are drug resistances.
Test example 2：For recognizing and detecting the reverse hybridized line trace inspection of mycobacteria
The principle of test example 2 is identical with test example 1.Using using the multiple asymmetric PCR of single tube nest bed
Reversely-hybridization line marking inspection.
In PCR courses of reaction, by asymmetric PCR method with the mycobacteria Pseudomonas primer of biotin labeling expanding
The ITS sites of mycobacterium tuberculosis (M.TB) and non-tuberculous mycobacteria (NTM) so as to prepare it is final it is biotin-labelling,
Single-stranded target DNA.Meanwhile, also prepare PCR amplification matched groups.
During reverse hybridized, biotin-labelling, single-stranded target DNA respectively with nylon membrane-absorption probe (rpoB,
The wild type or saltant type probe of katG, inhA and ahpC) hybridization.
Then, with identical mode in test example 1, by hybridization, cleaning and development step by obtained film
Bar is for the detection from sample and recognizes mycobacteria (mycobacterium tuberculosis (MTB) or non-tuberculous mycobacteria (NTM)).
(1) preparation of solution needed for
Prepare such as the examination listed in table 8 below to carry out DNA extraction, PCR reactions and the inspection experiment of reverse hybridized line trace
Agent.
[table 8]
(2) preparation of sample and DNA extraction thing
The preparation of sample and DNA extraction thing with it is identical in test example 1, therefore no longer provide detail explanation.
(3) PCR reactions
Under conditions of table 9 shows, by adding the primer of 12.5 μ L, 2 × PCR mixed solutions that for example table 8 is listed, 5 μ L molten
The sample DNA of liquid and 7.5 μ L come enter performing PCR reaction.
[table 9]
(4) reverse hybridized line trace inspection
The inspection of reverse hybridized line trace with it is identical in test example 2, therefore which be no longer provided describe in detail.
According to the concrete outcome of test example 2, can detect in the embodiment that Fig. 4 shows and recognize branch bar
Bacterium.
Although based on purpose of explanation disclose the present invention be preferable to carry out method, those skilled in the art can manage
Solution may be many modifications under conditions of without departing from the such as scope and spirit of following claims present invention disclosed,
Addition and replacement.
Claims (22)
1. it is a kind of prepare nucleotide probe coating porosu solid support bar method, which includes：
Nucleotide probe is connected to prepare conjugate with carrier；And
Shifted by vacuum and the conjugate be fixed in porosu solid support,
Wherein, the 5 ' of the nucleotide probe-end or 3 '-end with can be covalently attached with carrier or affine combination functional group or match somebody with somebody
Body is modified,
Wherein, the nucleotide probe includes spacer to improve the hybridization efficiency in region internally,
Wherein, the spacer is non-specific poly- T tails, and
Wherein, the vacuum transfer is included using transfer matic blotter.
2. method according to claim 1, wherein, the nucleotide probe is at least in DNA, RNA and PNA
Kind.
3. method according to claim 1, wherein, the connection between the nucleotide probe and the carrier is covalently to connect
Connect, or be the affine combination with the adhesion suitable with covalent attachment.
4. method according to claim 1, wherein, the functional group is selected from carboxyl (- COOH), phosphate-based ,-NH2、-
ArNH2、-CH2Cl, amide groups (- CONH2), aldehyde radical (- CHO), hydroxyl (- OH), at least one in epoxy radicals and sulfydryl.
5. method according to claim 1, wherein, the part is selected from biotin, glutathion, maltose, group ammonia
At least one in acid, agglutinin and its derivant.
6. method according to claim 4, wherein, the amino is directly connected with the nucleotide probe.
7. method according to claim 5, wherein, the part is directly connected with the nucleotide probe.
8. method according to claim 4, wherein, the amino is the amino C6 joints with short carbon chain or amino C12
Joint.
9. method according to claim 1, wherein, the nucleotide probe includes mirror, photochemistry, the bioid of being split
The labelling that the mode of, immunology or chemistry is directly or indirectly detected.
10. method according to claim 1, wherein, the nucleotide probe is the oligonucleotide of synthesis.
11. methods according to claim 10, wherein, the oligonucleotide of the synthesis includes 16 to 22 nucleotide.
12. methods according to claim 1, wherein, the carrier is in polysaccharide, protein and synthetic polymer
It is at least one.
13. methods according to claim 12, wherein, the carrier is glucosan or glucan derivative.
14. methods according to claim 13, wherein, the glucan derivative is oxidized dextran.
15. methods according to claim 14, wherein, the oxidized dextran is polyacetals glucosan.
16. methods according to claim 1, wherein, the conjugate is by the nucleotide probe of amine-labelling and is used as load
Body polyacetals glucosan reaction with formed schiff base base imine intermediate and adopt reducing agent to reduce the schiff base base with
Cause irreversible connection to prepare.
17. methods according to claim 1, wherein, the porosu solid is supported as film.
18. methods according to claim 17, wherein, the film is made up of nitrocellulose or nylon.
19. methods according to claim 1, which further includes the thing between the carrier and the porosu solid are supported
The reason absorption conjugate.
20. methods according to claim 1, wherein, the vacuum transfer includes：In the nucleotide of transfer matic blotter
Load the conjugate of the nucleotide probe and carrier in slit lines；And the conjugate is adsorbed onto admittedly by porous using vacuum pump
On body is supported.
21. methods according to claim 1, which is further included with solid support described in pre-hybridization buffer pretreatment.
22. methods according to claim 21, wherein, the pre-hybridization buffer is containing denhardt solution, SSC, SDS
With the compositionss of the salmon sperm DNA of degeneration.
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|---|---|---|---|---|
| CN1270229A (en) * | 2000-03-28 | 2000-10-18 | 陈高明 | Polygenic amplification chip and preparing process thereof |
| CN1402796A (en) * | 1999-11-02 | 2003-03-12 | 胡元徽 | Molecular microarrays and methods for production and use thereof |
| CN1524180A (en) * | 2001-04-10 | 2004-08-25 | 纽约市哥伦比亚大学信托人 | Novel microarrays and methods of use thereof |
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| GB8523801D0 (en) * | 1985-09-26 | 1985-10-30 | Jones K W | Vacuum molecular transfer(blotting)apparatus |
| US4818701A (en) * | 1987-05-13 | 1989-04-04 | American Bionetics, Inc. | Apparatus for transferring biological specimens from a gel to a transfer membrane and method |
| US7501250B2 (en) * | 2006-09-26 | 2009-03-10 | Chung-Cheng Chang | Blotting method for rapidly analyzing nucleic acid |
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| CN1402796A (en) * | 1999-11-02 | 2003-03-12 | 胡元徽 | Molecular microarrays and methods for production and use thereof |
| CN1270229A (en) * | 2000-03-28 | 2000-10-18 | 陈高明 | Polygenic amplification chip and preparing process thereof |
| CN1524180A (en) * | 2001-04-10 | 2004-08-25 | 纽约市哥伦比亚大学信托人 | Novel microarrays and methods of use thereof |
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| WO2012081856A2 (en) | 2012-06-21 |
| WO2012081856A3 (en) | 2012-11-29 |
| KR20120067401A (en) | 2012-06-26 |
| CN102559877A (en) | 2012-07-11 |
| WO2012081856A9 (en) | 2012-09-27 |
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