JP2003503011A - Methods for detecting microorganisms using immobilized probes - Google Patents

Abstract

(57) Abstract: The present invention discloses a method for detecting the presence or absence of a microorganism, comprising the step of detecting the presence or absence of a microbiological target molecule in a test sample using electrophoresis including an immobilized capture probe. . The present invention also discloses a method for detecting a mutation site in a putative mutation target molecule.

Description

Detailed Description of the Invention

Related Application This application is related to US Provisional Patent Application No. 60 / 046,70 filed on May 16, 1997.
No. 08/971, filed Aug. 8, 1997, claiming benefit of No. 8
, 845, Partial Continuation Application, April 2, 1999 (02.04.999)
It is a continuation application of the application, US patent application Ser. No. 09 / 286,091, and claims priority thereto. These are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION Whole blood and blood products such as red blood cells, platelets, plasma have been transfused to recipient patients throughout the last century. Microbial contamination of these potential transfusion components has been infrequent, yet cases of transfusion-related deaths have been reported, and not only for patients who have to undergo such transfusions, but also in the community of healthcare professionals. Is also a source of concern on both sides. Clinical
As pointed out in Microbiology Reviews, whole blood or its products may initially contain small amounts of bacteria at the time of collection, and this does not mean that the threat of sepsis is present. However, in storing these blood products, bacteria may grow to levels that can cause sepsis in recipient patients. (Wagner, S. J. et al., Cli
medical Microbiology Reviews, 7: 290 (199
4)).

There is clearly a need to detect contaminating microorganisms prior to transfusing a patient with blood or blood products. Currently, there are several methods used to screen for contaminating microorganisms, especially bacterial microorganisms. Visual inspection is used to determine if the color of the contents of the blood bag has changed. If the color changes from a suitable red to a dark red, it may indicate bacterial contamination. However, this method is not without failure. This method relies on qualitative assessments that can be inaccurate and can miss contaminated blood bags.

Staining can also be used to detect microbial contamination. In addition to Wright's stain, Gram stain or acridine orange is often used to detect bacterial contamination. However, such stains may not be sensitive enough to give a positive signal for the bacterial contaminant contained in the blood bag.

A blood sample, or one of its products, can be cultured to determine if it is contaminated. The first drawback of using a culture system is that it typically takes 24-48 hours to obtain a single result. As a result, the blood cannot be tested in a time-effective manner. Another drawback to this technique is the lack of endpoints at which negative findings can be detected.

While there are other useful assay methods, they also have significant shortcomings.
(Rorer, ML, Advance / Laboratory, June Issue: 4
1-46 (1997)). For example, some methods rely on hybridization assays to detect the presence of nucleic acids in contaminating microorganisms in a test sample. In these methods, firstly, a solution phase detection protocol is used to detect the nucleic acid of the microorganism. For example, a labeled nucleic acid probe is hybridized in solution to the nucleotide sequence of a microorganism. The unhybridized nucleic acid obtained from the test sample must be treated, for example by hydrolysis, or the hybridized complex is removed from the unhybridized species of the nucleic acid in solution. There must be. (See below: Brecher, ME et al., Transfusion, 34:
750-755 (1994); Brecher, M .; E. Et al, Transfus
Ion, 33: 450-457 (1993); US Pat. No. 5,541,308 to Hogan et al., US Pat. No. 5,288,6 to Kohne.
11; and U.S. Pat. No. 4,851,330 to Kohne). These solution phase protocols are time consuming and can be unreliable.

[0007] There remains a need for methods of detecting microbial contamination of blood and blood products that are sensitive, rapid and accurate enough to ensure confident monitoring of microbial contamination. The method is sensitive, rapid, and hospital,
It needs to be adaptable to a variety of environments, such as doctors' clinics or areas where complex laboratory equipment is often lacking.

SUMMARY OF THE INVENTION The present invention allows nucleic acids, modified nucleic acids and nucleic acid analogs to be covalently attached (immobilized) to an electrophoretic medium, and electrophoretic methods for immobilizing immobilized nucleic acids, modified nucleic acids or It relates to the discovery that target molecules that specifically bind to (eg, associate with) or are bound by nucleic acid analogs can be used to separate, purify, or analyze. The immobilized nucleic acid, modified nucleic acid or nucleic acid analog is referred to herein as a capture probe. These fixed capture probes can
For example, it can be used for the analysis of microbiological target molecules. One specific binding reaction encompassed by the present invention is hybridization. By hybridization, the capture probe is typically a target nucleic acid, eg, a nucleic acid consisting of a nucleotide sequence that is substantially complementary to the nucleotide sequence of a microbiological nucleic acid molecule, thus resulting in specific hybridization. It happens in a sudden way.
In addition, nucleic acid analogs such as peptide nucleic acids (PNA) can be covalently attached to electrophoretic media for use as capture probes. The capture probe, which is immobilized in the medium used for electrophoretic separation, hybridizes specifically with the capture probe, also resulting in the target nucleic acid being immobilized on its substrate. As used herein, the term "substrate" refers to the immobilized polymeric component of the electrophoretic medium that provides the molecular sieving properties of the medium and also provides the means for immobilization of the capture probe. Examples of suitable matrix materials include cross-linked polyacrylamides, gel-forming polymers such as agarose and starch. Commonly used in capillary electrophoresis applications include linear polyacrylamide, poly (N, N-dimethylacrylamide), poly (hydroxyethyl cellulose).
Non-gel forming polymers such as, poly (ethylene oxide) and poly (vinyl alcohol) also serve as suitable substrates.

The present invention is particularly directed to the use of microbiological target molecules, which are used in electrophoretic methods to transfer solution phase target molecules in contact with capture probes immobilized on a suitable electrophoretic substrate. The present invention relates to a method for detecting the presence or absence of microorganisms in a test sample by detecting the presence or absence.

The methods of the present invention can be subjected to electrophoretic methods and are bound to or bound by nucleic acids, any chemical entity of microbial origin (eg, when placed in an electrophoretic field). Can be applied to the analysis of charged molecules having detectable mobility. Such entities include, for example, DNA or RNA molecules, nucleic acid binding proteins, and aptamer binding partners (aptamers are nucleic acids selected to bind to specific binding partners such as peptides, proteins and polysaccharides; Jenison et al. , Science, 263: 1425-
1429 (1994)). For example, the methods described herein provide for the analysis of target nucleic acids obtained from microbial organisms using immobilized capture probes, where specific binding involves interaction between the target nucleic acid and the capture probe. Can be used for

The test sample can be from any source and can include any molecule capable of forming a binding complex with the capture probe. Included specifically according to the invention are body tissues (eg skin, hair, internal organs), body fluids such as whole blood, platelets, red blood cells, plasma, urine, semen, sweat or cells or cultures of tissues and organs. A sample obtained from a biological source, including microorganisms, obtained from the system using known techniques.

The test sample is treated in such a manner as is known to those of skill in the art to be the target molecule contained in the test sample useful for binding. For example, if the target molecule in the test sample is a bacteria-specific nucleotide sequence,
Bacterial cell lysates can be prepared and their cell lysates (eg, containing not only other cellular components such as proteins and lipids, but also target nucleic acids) analyzed. Alternatively, the target nucleic acid can be isolated (substantially freeing the target nucleic acid from other cellular components) prior to analysis. Isolation can be accomplished using known lab techniques. The target nucleic acid is also amplified (eg, by polymerase chain reaction or ligase chain reaction techniques) prior to analysis.
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The test sample containing the putative microbial target molecule is then introduced into a suitable electrophoretic medium. The capture probe is immobilized within the electrophoretic substrate by being directly attached to the medium or by being attached to particles that are suspended and collected within the substrate. In either case, the capture probe is fixed, ie it does not move under the influence of its applied electric field.

The test sample containing the microbial target molecule can be detectably labeled before, during, or after the electrophoretic step. Detecting the presence of the microbial target molecule / capture probe complex immobilized on the substrate is an indication of the presence of the microorganism in the test sample from which the target molecule is generated.

Once the test sample is introduced into the electrophoretic medium, the target molecules of the test sample are subjected to an electric field and, if present, are sufficient to bind to one or more capture probes. Under various conditions and times, the test sample undergoes electrophoretic migration in the substrate, resulting in the target molecule / capture probe complex immobilized in the substrate. A typical voltage gradient used in nucleic acid electrophoresis is approximately 1 V / cm.
-100 to 100 V / cm. Other electric field strengths may be useful for certain highly specialized applications.

The electrophoretic substrates useful in the methods described herein can be provided in a number of different formats. For example, the substrate can be provided in a form whose physical length is significantly beyond its width or depth, eg, contained within a tube or formatted as a narrow strip. Alternatively, the substrate may have its length and width significantly beyond its depth, and may be provided, for example, in a format that is formatted as a relatively thin layer or as a slab on the surface. Alternatively, the substrate may be exact or approximately straight, polygonal, spherical,
As an elliptical solid or a similar physical shape, when the length, width and depth are of the same order, they can be basically provided as one solid body. In particular, the substrate can be provided in a disposable container (eg a cassette).

The location of the immobilized capture probes useful in the methods described herein can also be provided in a number of different formats. For example, for the substrate, uniformly over the substrate,
Alternatively, one distributed over one or more discrete regions of the substrate
One or more capture probes can be included. Also, two or more regions of similar (or different) immobilized capture probes or a combination of probes may be combined so that the sample migrates within the sequence of the capture probe region as the sample migrates through its substrate. It is possible to position things.

Alternatively, multiple regions of the fixed capture probe can be positioned to form two or more paths of travel, each passing through one or a sequence of the fixed capture probe regions. it can.

Detection of microbial mutations is also disclosed in the present invention. Nucleic acid capture probes that are complementary to one or more putative mutant target molecules contained in the test sample are immobilized in an electrophoretic medium that forms a capture layer. The test sample is applied to the electrophoretic medium containing the capture layer and subjected to electrophoresis. Complementary single-stranded nucleic acid
Once the (ie putative mutant target molecules) enter the capture layer, they are hybridized and immobilized by the probe. Non-complementary nucleic acids pass unimpeded through the capture layer. The capture probe can be specifically designed to detect sequence variations within the target molecule. In one embodiment, upon entering the capture layer, the putative mutant target molecule hybridizes to an immobilized probe designed to be complementary to the mutant target molecule. As a result of this hybridization, the mutant target is immobilized on the gel.
Wild-type target molecules or target molecules that have not been processed for specific mutations that are not desired to be detected pass through the capture layer. It can be considered possible to detect one or more mutation sites. In this embodiment, multiple capture layers containing immobilized probes specific for specific mutation sites can be formed in the electrophoretic medium.

In another embodiment of the invention, a method of detecting the presence or absence of a microorganism in a test sample by detecting the presence or absence of one or more microbiological target molecules using an adapter molecule. Disclosed. The microbial target molecule is mixed with an adapter molecule having at least one nucleotide sequence region specific for the target molecule. The adapter / target complex is then subjected to electrophoresis. The complex continues to migrate in the electrophoretic medium until it comes into contact with the immobilized capture probe that is specific to the adapter molecule. The adapter molecule has a second nucleotide sequence region that is specific to a particular class of capture probe. A higher degree complex is formed between the adapter / target complex and a suitable immobilized capture probe. Detection of this tripartite complex is indicative of the presence of the microbial target molecule in the test sample and thus the presence of the microorganism in the test sample.

In another embodiment, the invention employs a signal polynucleotide substitution reaction to detect the presence or absence of one or more microbiological target molecules,
A method of detecting the presence or absence of microorganisms in a test sample is disclosed. The complex is formed between the capture probe and the signal polynucleotide. The capture probe can be immobilized in the electrophoretic medium or in one or more separate areas of the medium. The target molecule is introduced into the electrophoretic medium and also electrophoresed. In the presence of the target molecule, the signal polynucleotide is either replaced by the concentration of the target molecule or it can be replaced by a capture probe that has a higher affinity for the target than the signal polynucleotide. Detecting the presence of the released signal polynucleotide indicates the presence of the microbial target molecule in the test sample and thus the presence of the microorganism in the test sample.

In another embodiment of the invention, a method for detecting the presence or absence of a target molecule in a test sample using a displacement assay is disclosed. A test sample containing one or more target molecules is subjected to electrophoresis in a medium containing immobilized capture probes. These capture probes consist of two components. The first component is a detectably labeled single-stranded nucleic acid molecule. This signal molecule is typically shorter than the second component. The second component is a tether nucleic acid molecule. The tethered molecule includes a nucleotide sequence region that is complementary to the nucleotide sequence region contained within the target molecule. This signal molecule hybridizes to part of this complementary region of the tether molecule. This signal and the tethered form together form one probe complex. The tether molecule is immobilized in the electrophoretic medium. The target molecule can come into contact with its probe complex as it migrates under electrophoresis. The target molecule displaces the signal obtained from the tether and also hybridizes to the tether molecule. The signal molecule continues to move until the electric field is terminated. Detection of the single-stranded signal molecule indicates the presence of one or more target molecules in the test sample.

In another embodiment, a method for detecting the presence or absence of a target molecule in a test sample using a reverse displacement assay is disclosed. A test sample containing one or more target molecules is subjected to electrophoresis. The electrophoretic medium includes a fixed capture probe. These capture probes consist of two components. The first component is a signal nucleic acid molecule that is detectably labeled. The second component is a tethered nucleic acid molecule. The signal molecule is complementary to the nucleotide sequence contained within the target molecule. The tether molecule and the signal molecule form a hybrid, which is called the probe complex. The target molecule contacts the probe complex and the signal molecule displaces from the complex and also hybridizes to the target molecule. This new hybrid will continue to move until the electric field is terminated. Detection of this hybrid is indicative of one or more target molecules in the test sample.

To use an additional enzymatic treatment to hydrolyze unhybridized nucleic acids, or to separate hybridized complexes from unhybridized nucleic acid species by using solid-phase methodologies. The difficulties associated with solution phase detection, such as the need to use size-exclusion chromatography, for example, can be avoided. These methods require additional steps that can result in undesirable effects such as increased assay time.

Furthermore, the sensitivity of any method used to detect contaminating microorganisms should be sufficient to detect the level of microorganisms that are considered clinically relevant. The test needs to be sensitive and accurate, but for example, the actual concentration of bacteria whose clinical significance remains incomprehensible is only 10 ⁵ CFU / mL or more. (Krishnan, LA et al., Trans
fusion Medicine II, 9 (1): 176 (1995)).

Thus, as a result of the studies described herein, the current methods show that potential methods in test samples using immobilized capture probes that specifically bind to microbial target molecules that are indicative of the presence of microorganisms. It is available for rapid, sensitive, efficient and accurate electrophoretic analysis of contaminating microorganisms. The sensitivity of detection described herein by the method allows the detection of microorganisms using non-radioactive means such as fluorescence. This provides a safer and more convenient method of detecting contaminating microorganisms.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an electrophoretic method for analyzing specific binding reactions in which a capture probe is immobilized (eg, covalently attached) in an electrophoretic substrate. In particular, the invention provides a method for detecting the presence or absence of a microorganism in a test sample by detecting the presence or absence of one or more microbial target molecules, including bacterial, viral, fungal, and parasite target molecules. Regarding The capture probe is an acid, modified nucleic acid, or nucleic acid analog (such as PNA) that specifically binds to or hybridizes with the target molecule present in the test sample. The test sample is introduced into the electrophoretic substrate and subjected to an electric field under conditions suitable for the specific binding of its target molecule to the capture probe. The immobilized probe is attached to the interior of the electrophoretic substrate and binding occurs within the substrate. The capture probes can be immobilized throughout the substrate, or alternatively they can be immobilized within one or more discrete areas of the substrate. FIG. 1 is a schematic illustration illustrating some of the steps involved in detecting the presence or absence of microorganisms by detecting the presence or absence of one or more microbial target molecules in a test sample.

Sample Preparation Suitable sample preparation involves the steps required to lyse the microorganism or microorganisms, thereby releasing their biochemical contents including proteins, peptides and nucleic acids. In the case of a microbial invasion of a host cell, the host cell is invaded by a mammalian host cell, for example by rhinovirus or adenovirus infecting epithelial cells of the respiratory system, in order to release microbial target molecules. , Can be subjected to cell lysis. The microbial target molecule may be specific for a particular microorganism, eg E. coli, or
There may be one class, for example bacteria, which uses the 16S rRNA common to most bacteria to detect bacterial contamination. The presence of one or more microbial target molecules is indicative of the presence of microbial contamination in the test sample. Both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are contemplated as being within the scope of this invention. For bacterial targets, RNA is preferably free of cell wall and cell membrane constraints. However, bacterial DNA, such as plasmid DNA, is also
It is intended to be within the scope of the present invention. If the target molecule is produced from a virus, both DNA and RNA molecules, depending on the class of virus,
Depending on the host cell, or the stage of viral infection, it may be analyzed by first releasing it from the viral coat. Optionally, additional steps can be employed to further purify the desired target molecule from contaminating microorganisms such as cell lysis followed by semi-purification of the lysate. For example, if the desired microbial target molecule is double-stranded D
If NA, then the preparation can be treated to remove single-stranded nucleic acid species from the test sample being analyzed, eg, using S1 nuclease. FIG. 2 is a schematic illustration illustrating the preparation of a microbial target molecule-specific bacterial target molecule obtained from a platelet-containing source.

Electrophoretic Substrate Any substrate suitable for electrophoresis can be used in the methods of the invention.
Suitable substrates include acrylamide and agarose, both commonly used for nucleic acid electrophoresis. However, other materials can also be used. Examples include chemically modified acrylamide, starch, dextran, and cellulosic polymers. Additional examples include modified acrylamides and acrylates (eg, Polysciences, Inc.,
See Polymers and Monomers Catalog, 1996-1997, Warrington, PA,) Starch (Smithies, Biochem. J., 71).
: 585 (1959); product number S5651, Sigma Chemical.
Co. , St. Louis, Missouri, Dextran (eg, Polysci)
See Inc., Polymers and Monomers Catalog, 1996-1997, Warrington, PA,) and cellulosic polymers (eg, Quesada, Current Opin. in Biotechno).
, 8: 82-93 (1997)) and the like. Any of these polymers listed above can be chemically modified to allow specific attachment of the capture probe used in the present invention.

Particularly included by the present invention is a method of using a nucleic acid, modified nucleic acid, or nucleic acid analog as a capture probe. Methods of conjugating nucleic acids to make nucleic acid-containing gels are known to those of skill in the art. Nucleic acids, modified nucleic acids and nucleic acid analogs can be coupled to agarose, dextran, cellulose, and starch polymers using cyanogen bromide or cyanuric chloride activation. Polymers containing carboxyl groups can be attached to synthetic capture probes with primary amine groups using carbodiimide linkages. Polymers carrying primary amines can be attached to amine-containing probes by glutaraldehyde or cyanuric chloride. Many polymers can be modified with thiol reactive groups that can be attached to thiol-containing synthetic probes. Many other suitable methods can be found in the literature. (For a review, see Wong, “Chemistry of Protein Conjugation.”
and Cross-linking ")," CRC Press, 1993, Boca Raton, FL).

Methods for covalently attaching the capture probes described herein to polymerizable chemical groups have also been developed. Substrates containing a high concentration of immobilized nucleic acid can be produced when copolymerized with a suitable mixture of polymerizable monomeric compounds. An example of a method for covalently attaching a nucleic acid to a polymerizable chemical group is described in "Nucleic Acid-Containing Polymerizable Complex (" Nucleic Acid-Contain
No. 08 / 812,105 and Rehman et al., Nucleic Ac, entitled "ing Polymerizable Complex").
id Res. , 27: 649-655 (1999), both references are incorporated herein by reference in their entirety.

For some methods, it may be useful to use a complex substrate containing a mixture of two or more substrate-forming materials, one example including a complex acrylamide-agarose gel. . These gels typically contain 2-5% acrylamide and 0.5% -1% agarose. In such gels, acrylamide gels provide the major sieving function, but in the absence of agarose, such low concentration acrylamide gels lack mechanical strength for convenient operation. Agarose provides mechanical support without significantly modifying the sieve function of acrylamide. In such cases, the components are in intimate contact with the solution phase nucleic acid target so that the nucleic acids can be attached to the components that provide the sieving function of the gel.

For many applications gel-forming substrates such as agarose and cross-linked polyacrylamide will be preferred. However, for capillary electrophoresis (CE) applications, it is convenient and reproducible to use soluble polymers as electrophoretic substrates. Examples of soluble polymers that have proven useful in CE analysis are Ques
ada (Current Opinion in Biotechnology)
, 8: 82-93 (1997)), such as polyacrylamide, poly (N, N-dimethylacrylamide), poly (hydroxyethylcellulose), poly (ethylene oxide) and poly (vinyl alcohol). It is a polymer. Such soluble substrates can also be used to practice the methods of the invention. "Preparation of Nucleic Acid-Containing Polymerizable Complexes" (Nucleic Acid-Con) for the preparation of soluble polymeric substrates containing immobilized capture probes.
It is particularly convenient to use the method found in US patent application Ser. No. 08 / 812,105, entitled "taining Polymerizable Complex"), which is incorporated by reference herein in its entirety. T
imofeev et al., Nucleic Acids Res. , 24: 3142-
Another approach for attaching oligonucleotide probes to preformed polyacrylamide gels found in 3148 (1996) was also used to attach capture probes to prepolymerized soluble linear polyacrylamide. Can be done, the teachings of which are incorporated herein by reference in their entirety.

The nucleic acid can be attached to particles that can themselves be incorporated into an electrophoretic substrate. The particles are macroscopically visible under a microscope or can be in the form of natural colloids. (See Polysciences, 1995-1996 Particle Catalog, Warrington, PA). Cantor et al., US Pat. No. 5,482,863 describe a method for casting electrophoretic gels containing suspensions or particles. The particles are linked to nucleic acids using methods similar to those described above, which are mixed with a gel-forming compound and also cast as a suspension in the desired substrate form.

Substrate Format and Method of Producing the Substrate The substrate can be configured in various formats. For example, where the gel is formed by polymerization within a support, linear gels are formed by techniques including formation within linear supports such as holes or tubes, or alternatively by forming partitions or grooves. It is formed by subdividing the two-dimensional gel into a number of strips. In the form of holes, strips or grooves, one or more copolymerizable capture probes may be provided prior to or during gel polymerization to provide one or more capture probes within the polymer gel. In any case, it is added to the gel material at any spatially defined location, such as spatial positioning dropper technology. In tube format, sequences of gel monomers, mixtures of gel monomers and polymerizable capture probes are subsequently polymerized in situ to protect the spatial relationships of their components, spatially distinct. It can be introduced continuously into the tube to provide a set and concentration of ingredients. To protect the integrity of the gel during polymerization-induced shrinkage, the tube wall can be made of an elastic material that shrinks laterally as the gel shrinks. Alternatively, a gradual polymerization can be induced from one end of the tube, while additional liquid material can be added to the other end to compensate for shrinkage. Such gradual polymerization may be accomplished by means including diffusion of a polymerization catalyst agent, or by electromagnetic or other means from one end of the tube to the other, such as by moving or gradual exposure to the irradiating source. It can be induced by the progressive application of irradiation-induced polymerization. Alternatively, linear format gels can be made by removing linear slices obtained from two-dimensional gels or linear cores obtained from three-dimensional gels produced as described below.

Two-dimensional gels can be formed by techniques including formation on the surface of a support or formation between two support surfaces. A layer of gel monomer is applied and a large amount of copolymerizable capture probe is applied to protect the optional spatial location in the gel.
It can be polymerized in situ and applied to the layer in any spatially significant manner before or during polymerization. Application of the multi-polymerizable capture probe can be carried out by known means, including positioning programmable dropper technology. The gel shrinkage during polymerization is tuned by means that include allowing the reduction of the gap between the support surfaces and by allowing lateral shrinkage with additional material added laterally to compensate. can do. The two-dimensional gel is further divided into a number of strips by using a partition before gel formation, during gel formation, or after gel formation, by forming into grooves, or by slicing into narrower sections after formation. be able to.

Three-dimensional gels can be formed by a number of techniques. Multiple linear strips or two-dimensional layers can be repeatedly constructed as described above, each containing an optionally localized capture probe, each strip or layer being an underlying layer to produce a three-dimensional volume. Polymerized above. Alternatively, many two-dimensional gels, optionally with capture probes, optionally located in place, can be formed as described above and assembled together to provide a three-dimensional structure. .

Fixed Probes for Analysis of Hybridization Binding Reactions Various capture probes can be used in the methods of the invention.
Typically, the capture probe of the present invention consists of a nucleic acid having a polynucleotide sequence in which the target molecule is substantially complementary to a microbial target molecule that hybridizes to the capture probe. The complementarity of the nucleic acid capture probe is sufficient to specifically bind the target molecule in the test sample and also to indicate the presence or absence of the microbial target molecule in the test sample, and thus the presence or absence of the microorganism in the test sample. Just need one. Suitable probes for use in the present invention include mixed classes of chimeric probes consisting of RNA, DNA, nucleic acid analogs, modified nucleic acids, and nucleic acids having other organic moieties such as, for example, peptide nucleic acids. The capture probe can be a single-stranded or double-stranded nucleic acid. Typically the length of the capture probe is at least 5 nucleotides in length, more typically between 5 and 50 nucleotides in length, and it can also be thousands of bases in length.

As defined herein, the term "nucleic acid" includes DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Nucleic acid, as referred to herein as "isolated," refers to a component of its source of origin (eg, when present in a cell, or a mixture of nucleic acids such as a library). A nucleic acid which has been separated from the above) and can be further processed.
Isolated nucleic acids include nucleic acids obtained by methods known to those of skill in the art. Such isolated nucleic acids include substantially pure nucleic acids (ie, nucleic acids released from proteins, carbohydrates, or lipid associations), produced by chemical synthesis, by a combination of biological and chemical methods. Nucleic acids, and isolated recombinant nucleic acids are included.

As used herein, "modified nucleic acid" includes nucleic acids containing modified sugar groups, phosphate groups, or modified bases. Examples of nucleic acids having modified bases include, for example, acetylated, carboxylated, or methylated bases (eg, 4-acetylcytidine, 5-carboxymethylaminomethyluridine, 1-methylinosine,
Norvaline, or allo-isoleucine). Probes containing modified polynucleotides may also be useful. For example, polynucleotides containing deazaguanine bases and uracil bases can be used in place of guanine and thymine containing polynucleotides to reduce the thermal stability of hybridized probes (Wetmur, Critical Reviews).
in Biochemistry and Molecular Biolog
y, 26: 227-259 (1991)). Similarly, 5-methylcytosine can be substituted for cytosine if a hybrid with increased thermostability is desired (Wetmur, Critical Reviews in B.
iochemistry and Molecular Biology, 26
: 227-259 (1991)). Modifications to the ribose sugar group, such as the addition of 2'-O-methyl groups, can reduce the nuclease sensitivity of immobilized RNA probes (Wagner, Nature, 372: 333-335 (1994)). Modifications that remove the negative charge from the phosphodiester backbone can increase the thermal stability of hybrids (Moody et al., Nucleic Acids Res.
, 17: 4769-4782 (1989); Iyer et al., J. Am. Biol. Che
m. , 270: 14712-14717 (1995)).

As used herein, “nucleic acid analog” includes molecules that lack a sugar phosphate backbone but retain the ability to form a complex via base pairs. Be done. Such nucleic acid analogs are known to those of skill in the art. One example of a useful nucleic acid analog is a peptide nucleic acid (PNA) in which standard DNA bases are attached to a modified peptide backbone consisting of repeating N- (2-aminoethyl) -glycine units (Nielse).
n et al., Science, 254: 1497-1500, (1991)). The peptide backbone is capable of retaining bases at appropriate distances to base pairs by standard DNA and RNA single strands. Probably due to the fact that there are no negatively charged phosphodiester bonds in the PNA chain, PNA-D
NA hybrid duplexes are much stronger than equivalent DNA-DNA duplexes. Moreover, because of their unusual structure, PNAs are highly resistant to nuclease degradation. For these reasons, PNA nucleic acid analogs are useful in fixed probe assays. It will be apparent to those skilled in the art that similar design strategies can be used to construct other nucleic acids that will have useful properties for fixed probe assays.

As defined herein, "substantially complementary" does not require that the polynucleotide sequence of the capture probe reflect the exact polynucleotide sequence of the microbial target molecule, but under certain conditions. Means that it must be sufficiently complementary to hybridize to the target molecule below. For example, non-complementary bases or additional polynucleotides can be interspersed in the sequence with sufficient complementary bases to hybridize therewith. Generally, the degree of complementarity required is from about 90% to about 100%.

Specific conditions for hybridization can be empirically determined by those skilled in the art. For example, stringent conditions should be chosen that significantly reduce non-specific hybridization reactions. Stringent conditions for nucleic acid hybridization are described in, for example, Ausubel, F .; M. Et al. "Current Protocols in Molecular Biology (Cu
current Protocols in Molecular Biology
), Vol. 1, Addendum 26, 1991, the teachings of which are incorporated by reference in its entirety in this application. Factors such as probe length, base composition, percent mismatch between hybridizing sequences, temperature and ionic strength affect the stability of nucleic acid hybrids. For example, moderate or highly stringent stringency conditions can be empirically determined depending on some of the chemical properties of the probe and microbial target molecule.

To screen a test sample for microbial contamination, the capture probe used must be directed to the microbial specific molecule. For example, if a test sample is suspected of being infected with a retrovirus, then the probe can be directed against any of the gag, pol, or env viral nucleotide sequences or fragments thereof. . In the example just presented, multiple probes are used when three classes of probes are used, each class directed against one of the viral nucleotide sequences listed or fragments thereof. Can be used. If a test sample, eg, platelets, is analyzed for the presence of bacterial contamination, then the probe can be for a particular bacterial DNA sequence, or bacterial RNA sequence (eg, rRNA sequence). In the latter case, the probe will preferably not react with human RNA, but will react with RNA obtained from the bacterial species contaminating the platelets. (See below for this: Brecher, ME et al., Transfusion, 34: 750-755 (1994); Breche.
r, M. E. Et al., Transfusion, 33: 450-457 (1993).
US Pat. No. 5,541,308 to Hogan et al .; US Pat. No. 5,288,611 to Kohne; US Pat. No. 4,851,330 to Kohne; The entirety is incorporated herein by reference in its entirety. ) Several bacterial species known to contaminate platelets are listed in Table 1. The species listed in that table are examples of suitable target microorganisms for screening using the present invention.

[0045]

[Table 1]

Single-Stranded and Double-Stranded Target Molecules In one embodiment of the invention, single-stranded microbial target molecules and single-stranded immobilized capture probes are used. This embodiment is particularly useful for the analysis of microbial RNA target molecules. It is also useful for capturing specific targets obtained from complex samples, where target recovery is not rapid. Highly concentrated microbial targets such as PCR products can require denaturation just prior to electrophoresis due to their rapid recovery.
For example, for the analysis of PCR products that are 100 to 250 base pairs long, 75
It is convenient to pull the sample up to% formamide (vol / vol) and heat above 70 ° C. for 5 minutes just prior to electrophoresis.

In another embodiment of the invention, the double-stranded microbial target is captured by a single-stranded immobilized capture probe. For example, the capture probe is designed to associate with double-stranded nucleic acid to form a triple-stranded structure. The third strand is located in the major groove of the double-stranded molecule and forms a Hoogsteen base pair interaction with the base of the double-stranded molecule
(Hogan and Kessler, US Pat. No. 5,176,966 and Cant.
or et al., U.S. Pat. No. 5,482,836). The design of the probe is therefore subject to the constraints that govern these chemical interactions. However, the frequency of sequences capable of forming triple structures in naturally occurring nucleic acids is so high that many microbial target nucleic acids can be specifically captured using this probe design strategy.

Alternatively, the capture probe can be designed to associate with double-stranded nucleic acid by forming a displacement loop structure. Such a probe binds only to the single strand of the double-stranded molecule nucleic acid and also locally displaces the probe homologous duplex of the double-stranded molecule. This displacement can only be achieved if the probe-target strand interaction is much more favorable than the target strand interaction. These probes have modified bases and W
etmur, Critical Reviews in Biochemist
ry and Molecular Biology, 26: 227-259.
(1991), backbone modification (Moody et al., Nucleic Acids Res.
． , 17: 4769-4782 (1989)) and nucleic acid analogs (Niel.
Son et al., Science, 254: 1497-1500 (1991)). The use of peptide nucleic acid (PNA) probes whose base pairs are very tightly and specifically bound to naturally occurring nucleic acids is particularly useful in this embodiment.

Homogeneously Modified Electrophoresis Media for the Analysis of Target Molecules In this embodiment of the invention, substantially all media is modified with a capture probe or multiple probes. The choice of capture probe and electrophoretic conditions is such that the binding between the capture probe and the target molecule depends on the time scale of the electrophoretic analysis.
It is transient and is done so as to be rapid and reversible. Under these conditions, the target molecule undergoes many cycles of binding, releasing and rebinding to the capture probe during the electrophoretic run. This reversible binding has the effect of reducing the electrophoretic mobility of the measured target relative to its mobility in the absence of the capture probe. The mobility of the target is substantially reduced when the binding to the capture probe is strong.
If the binding to the capture probe is weak, the mobility of the target will be reduced only slightly.
Thus, structurally related targets that have similar electrophoretic mobilities in the absence of capture probe or multiple probes can be identified based on their affinity for a specific capture probe. . The method is particularly useful for analyzing microbial target nucleic acid sequence changes, as described below. This method of analysis can be used to screen for multiple contaminants in test samples, such as platelets, where there is a need to identify multiple pathogens suspected of contaminating the sample.

One-Dimensional Array for Target Molecule Analysis In this embodiment of the invention, a sample containing target molecules is electrophoresed through a series of discrete substrate layers, each containing at least one class of capture probe. Can be electrophoresed. For example, in a hybridization binding reaction, a microbial target nucleic acid that is complementary to the capture probe hybridizes to the capture probe contained within the gel layer and is thus retained on that gel layer, ie the capture layer. . Non-complementary sample nucleic acid passes through the capture layer. The presence of hybrids between the capture probe and the complementary sample nucleic acid is detected in the capture layer by the appropriate labeling strategy described herein.

This one-dimensional format has some important advantages. First, all of the sample passes through the capture layer and is therefore useful for hybridization. This is a major advantage over most other solid phase hybridization methods. A high concentration of immobilized probe can be used to capture all hybridizable sample nucleic acid strands in the small gel band.

Second, complete nucleic acid species with distinct electrophoretic mobilities are not required for analysis by this method. Hybridization and detection only require short sequence homologies, and partially degraded microbial nucleic acids will still give a signal. This attribute also increases the sensitivity of detection, since all microbial target nucleic acids are concentrated at specific points in their substrate, whether they are degraded or not. In traditional zone electrophoresis, all sample nucleic acids must migrate as individual bands for detection.

Third, sample size is not critical. In the present invention, all sample nucleic acids pass through the capture layer, even if large sample volumes are used. This is a significant advantage over traditional zone electrophoresis, where the sample volume needs to be as small as possible for maximum detection sensitivity and resolution.

In this embodiment, the capture layer may contain single or multiple classes of capture probes. The use of multiple capture probes in a monolayer is useful in assays where any one of a number of different microorganisms needs to be detected. For example, a blood sample (eg, platelets) in the medium or used for transfusion
This embodiment can be used for the presence and / or identification of any bacteria therein. Therefore, general testing on bacteria may use as a capture probe a collection of conserved bacterial gene sequences common to most, if not all, bacteria. Since the identification of a particular bacterium is not always important, a collection of probes will allow multiple probes specific for different bacteria to ensure that any and all bacteria are detected in the same capture layer. Could consist of a broad spectrum of.

Multiple acquisition layers can also be used in this embodiment. This is a simple one that sequentially molds multiple capture layers on the same gel device to create a multiplex hybridization assay. During the assay, the test sample is electrophoresed through all its layers and a complementary sample target nucleic acid is captured on each layer. The amount of hybrids in each layer directly reflects the sample composition with respect to the capture probe used. For example, if there are different, distinct layers of immobilized capture probes, each layer being specific to a particular bacterial species, when a test sample is electrophoresed, that complementary species of bacteria is It will be detected by a particular class of immobilized capture probe that is specific to the particular species.

For example, the methods described herein can be used in three bacterial species, E. coli (E.co.
li), S.I. S. aureus and S. aureus. Pyogenes (S.py
If used to screen a test sample for genes), the electrophoretic medium is constructed so that there are three separate layers containing immobilized capture probes. Each layer is specific for one of three bacteria, for example layer 1 contains capture probes specific for E. coli and layer 2 contains S. Aureus-specific capture probes are included, and layer 3 is S. aureus. It contains a capture probe specific for pyogenes. The test sample is then subjected to electrophoresis. Test samples were E. coli and S. If only contaminated with Piogenes, then
The capture layers 1 and 3 were tested with E. coli and S. Contaminated by P. genogens, S. It contains a hybridization complex formed between the capture probe and the target molecule that is not Aureus.

Conditions can be identified that ensure that only properly hybridized nucleic acids are retained in each layer. Electrophoretic hybridization with a 20 base long capture probe can be performed at room temperature using traditional non-denaturing gel and buffer systems. A perfectly complementary hybrid of this size
It seems to be stable for a long time. However, additional stringency can be achieved by adding denaturing agents such as urea or formamide to the gel, or by running the gel at elevated temperatures.

Two-Dimensional Probe Arrays One-dimensional probe arrays can be used for analysis with a limited number of capture probes. Two-dimensional arrays of fixed probes can be used for analysis of large numbers of sequences. The array can be formed in many ways. Simple 2
The dimensional array can be cast on a conventional slab gel machine, for example, using multiple vertically aligned spacers, resulting in the creation of one array of one dimensional arrays.

A more complex two-dimensional array involves two steps, first polymerizing the capture probe regions as an array of substrate (eg, polyacrylamide gel) spots on a plate, and then the array. The set of points can be "sandwiched" by placing an upper gel plate on top of it and created by the step of filling with unmodified gel in the void spaces between the set of probe points.

In each case, the test sample is loaded as a band over the length of the substrate, eg at the top edge of the substrate. In this embodiment, the entire test sample does not contact all of the capture probes. However, in most applications where two-dimensional analysis is desired, the sample nucleic acid is present in high copy number, so this problem does not lead to high impact obstacles.

Three-Dimensional Probe Arrays The hybridization methods described in this application also include, for example:
Such three-dimensional arrays can also be included, which can be particularly useful in multiplex parallel assays to achieve high yields and / or price effects. Such an assay can be provided in the form of a three-dimensional solid when multiple samples can be applied to the surface or surface, and the solid Made to move the entire capacity. The array can be produced such that each sample encounters the same sequence of capture probes as it travels through the array, alternatively to analyze different sample mixtures, or one or more sample mixtures. Different sequences of capture probes can be positioned for this purpose so as to analyze different sets of internal components.

Mutation Detection In this embodiment, the invention relates to a method of detecting a mutation within the nucleotide sequence of a target molecule. For example, it may be desirable to determine if a bacterium, virus, parasite, etc. has been mutated. Detecting mutations in the genome of a microorganism can lead to significant information about that microorganism. For example, bacteria can become resistant to antibiotics by receiving genetic material that transmits resistance. Certain viruses, such as retroviruses, are notorious for their rapid mutation rate. Viruses that cause common colds
(For example, adenovirus) can very easily mutate between hosts. In order to predict what therapeutic regimen should be adopted for a particular microbial contamination, the virus will be mutated in such a way, for example, to remove previous antigens that are susceptible to antibody attack. It can be said that it is very important to confirm the gene status of whether or not to do. In this embodiment, the test sample comprises one or more putative mutant target molecules, ie a target nucleic acid having at least one mutation site contained within its nucleotide sequence. The mutation site may contain one or more base mutations in the nucleotide sequence. In this embodiment, the capture probe immobilized in the electrophoretic medium contains a nucleotide sequence region that is complementary to a particular mutation site contained within the mutant target molecule. This complementary region is used to hybridize to that particular mutant target molecule if that mutation is indeed present in any test sample. In one embodiment, the capture probe can be found in the substrate at a uniform concentration. In one preferred embodiment, the capture probes are immobilized on one or more discrete areas of the electrophoretic medium. A plurality of mutation target molecules containing different mutation sites are contained in various mutation target molecules, an immobilized capture probe containing nucleotide sequence regions corresponding to different mutation sites or complementary to the different mutation sites. Can be detected using. (Kenney, M. et al., BioTechniques, 25 (3): 516.
-521 (1998), the teachings of which are incorporated herein in its entirety). 1
It is also possible that one mutation target molecule may contain more than one mutation site. These mutation sites can be detected by multiple capture probes designed to be complementary to one particular mutation site contained within the mutant target molecule. Multiple capture probes can be immobilized in various different capture layers for detection of one specific mutation site within the electrophoretic medium. The electrophoretic mobility of the test sample nucleic acid is affected by the degree of complementarity with the immobilized probe.
The target molecule having complete complementarity to the probe is immobilized on the electrophoretic medium via the capture probe, and the target molecule having low complementarity passes through the capture layer.
(Kenney, M. et al., BioTechniques, 25 (3): 516-.
521 (1998)).

Samples can be conveniently prepared and labeled by PCR amplification with labeled nucleotide triphosphates prior to solid phase hybridization analysis.

Detection of Target Molecules by Single Substitution In another embodiment of the invention, a strand displacement reaction (US Pat. No. 4,766,06) is used.
No. 2; U.S. Pat. No. 4,766,064; Vary, Nucleic Acid.
s Res. , 15: 6883-6897 (1987); Vary et al., Clin.
See Chemical Chemistry 32: 1696-1701 (1986), the entire teachings of which are incorporated by reference), or reverse chain substitution (see US Pat. No. 60 / 103,075, the teachings of which are incorporated herein by reference). Methods are disclosed for the detection of microorganisms obtained from test samples using (), which is incorporated by reference in its entirety)).

The displacement assay uses a partially double-stranded probe. The long single-stranded nucleic acid portion of the probe, referred to herein as a "tethered" nucleic acid, is complementary to a region of nucleotide sequence contained within the target. This tether is about 5 to about 100
It is the length of the nucleotide. The other components of this probe will be referred to hereafter in this application.
A short, single-stranded, complementary to a particular subsequence of an unlabeled tether component (the same subsequence that is complementary to a nucleotide sequence region contained within the target molecule), termed a "signal" nucleic acid. , A detectably labeled nucleic acid. This signal is about 5 to about 50 nucleotides in length. Since it is complementary to the tether, the sequence of the signal nucleic acid is identical (or very similar) in sequence with respect to a portion of the target. When the single-stranded components of the two probes are hybridized, the signal and tether single strands form a hybrid in which the signal nucleic acid is completely base paired with the tether, and the tether is not ,
A single-stranded region that is useful for hybridization to the target is included.

In conventional displacement assays, the probe is incubated with a test sample containing one or more target molecules and the target hybridizes to the single-stranded portion of the tether component. Since the target is homologous to the entire length of its tether, it initiates a homologous strand exchange reaction with the signal nucleic acid and also displaces it from its tether. As the target is long and its tether forms a more stable double-stranded molecule, its strand exchange reaction proceeds rapidly towards signal nucleic acid displacement.
(For this, see Green, C. and Tibbetts, C., Nucleic.
Acids Res. , 9: 1905-1918 (1981),
The entire teachings are incorporated herein by reference). If you are a person skilled in the art,
Experimental conditions can be found in which the displaced labeled signal nucleic acid accurately reflects the amount of target in the sample. (See U.S. Pat. No. 4,766,666.
062; U.S. Pat. No. 4,766,064; Vary, Nucleic Ac.
ids Res. , 15: 6883-6897 (1987); Vary et al., Cl.
See internal Chemistry 32: 1696-1701 (1986), the entire teachings of which are incorporated by reference).

This embodiment of the invention discloses a simplified process for performing microbiological assays using the displacement method. In particular, the tether component of the displacement probe complex pair is immobilized within the electrophoretic substrate. Thus, the target molecule becomes a probe complex (consisting of hybrid signal and tethered nucleic acid) by subjecting them to electrophoresis through a substrate containing a capture probe under conditions suitable for hybridization. Contact. When the target molecule contacts the immobilized capture probe complex, the target molecule displaces the signal component of the probe complex and hybridizes to the immobilized tethered component of the complex. After that displacement, the displaced signal probe component is detected and quantified to determine the presence and number of target molecules in its original test sample.

In another embodiment, reverse substitution is described with respect to the detection of one or more target molecules in a test sample. The displacement probe complex consists of two nucleic acids. A "signal" nucleic acid and a "tether" nucleic acid. In this complex, the signal is complementary to the target nucleic acid. The tethered nucleic acid is thus identical, or substantially similar, in sequence to the particular subsequence of its target. In one preferred embodiment of the invention, the signal nucleic acid is detectably labeled. When the target nucleic acid is electrophoresed through the electrophoretic medium containing the immobilized displacement probe complex and comes into contact with the probe complex under conditions suitable for hybridization, in that case, the signal nucleic acid Hybridizes to the label and the signal nucleic acid is removed from the tethered nucleic acid by homologous strand exchange. The product of the reverse displacement reaction is a hybrid of the signal nucleic acid and its target. This hybrid can be displaced and detected from the capture probe layer and quantified as it migrates away from the immobilized displacement probe layer.

One major advantage of these displacement methods is that the target molecule does not need to be detectably labeled. The target molecule displacement reaction results in the migration of the detectably labeled probe molecule on the electrophoretic substrate. Many different methods of labeling probes have been described, but for use with direct labels such as radioactivity, fluorescent moieties, chemiluminescent moieties, direct enzyme conjugates, and secondary or tertiary labeled molecules. There are both indirect labels such as the affinity labels of. The reverse displacement assay is not label specific and any practical label can be used.

In another embodiment of the invention, target-dependent probe displacement is detected by a change in the properties of the tether nucleic acid or by a change in the properties of the signal tether complex that is modified by the displacement reaction. be able to.

Subsequently, different forms of detection often use labeled probe nucleic acids, others using labeled tether nucleic acids and still others using labeled tethers and labeled probes. And others may use unlabeled probes and unlabeled tethers.

Detection of probe displacement can be accomplished by, but not limited to, a value related to any chemical or physical property of the substance, or other means including detecting a change in the value. is not. Such values or changes in values include, but are not limited to, the following: That is, physical measurement methods (mass or density measurement method, mass spectrometry method, plasmon (extrachromosomal genetic determinant) resonance method), optical detection (luminescence, absorption, fluorescence, phosphorescence, luminescence, chemiluminescence, polarization) , Refractive index change, etc.), electrical conductivity, absorption or release of other electromagnetic energy, induced changes in radioactivity and solution properties (viscosity, turbidity, optical rotation), etc.

Detection Schemes Detection of specific binding reactions, eg, detection of immobilized microbial target molecules bound to capture probes, can be accomplished in a number of different ways.

The test molecule can be detectably labeled prior to the binding reaction. Suitable labels for direct target labeling may be by focused absorption (eg, in bright shades), radioactivity, fluorescence, phosphorescence, chemiluminescence, or catalysis.
Direct target labeling of nucleic acid samples using modified polynucleotides can be accomplished by those skilled in the art by a number of well-known enzymatic methods (Sambro).
Ok et al., "Molecular Cloning: Lab Manual (Molecular Clon
ing: A Laboratory Manual) ", 2nd edition, Cold S
pring Harbor Press, 1989, Review, Cold Spring Harbor, NY).

Alternatively, the target molecule uses a ligand capable of being recognized by a second specific binding entity, which is either itself labeled or capable of producing a detectable signal. And indirectly labeled.

One example of such an indirect system is labeling with biotinylated polynucleotides. In this system, the sample is enzymatically labeled using standard nucleic acid labeling techniques and biotinylated polynucleotides. The resulting biotin-modified nucleic acid can be detected by biotin-specific binding of streptavidin or avidin protein molecules. Streptavidin or avidin molecules should be attached to fluorescent labels such as fluorescein or reporter enzymes, such as alkaline phosphatase or horseradish peroxidase, which can be used to produce chemiluminescent or colorimetric signals by appropriate substances. (For review, see Keller and Manak
, "DNA Probes", Second Edition, published by Macmillan Publishing,
1993, London; Pershing et al., Ed., "Diagnostic Diagnostic Molecular Biology-Principles and Applications" (Diagnostic Molecular Microbiology:
Principles and Applications) ", Americ
an Society for Microbiology, 1993, Washington, D .; C. checking).

Another useful detection system is the digoxigenin system, which uses an anti-digoxigenin antibody conjugated to alkaline phosphatase, which recognizes digoxigenin-dUTP incorporated into nucleic acids. (Ausubel, FM, Ed., Current Protocols in Molecular Biology (Current Protocols
in Molecular Biology) ", Volume 1, §§ 3.18.1-
3.19.6 (1995); the entire teachings of which are incorporated herein by reference in their entirety).

Detectably labeled hybridization probes can also be used as indirect target labels. For example, the target nucleic acid is hereafter “sandwich”
Hybridization with a detectably labeled probe, called a probe, allows for indirect labeling prior to electrophoresis. The sandwich probe is designed to hybridize to a region of the microbial target that does not overlap with the region recognized by the capture probe. This sandwich probe is designed to remain associated with the target during electrophoresis and cannot bind directly to the capture probe.

Sandwich probes can also be used to label target molecules after electrophoretic capture. In this labeling method, the unlabeled target is subjected to electrophoresis and first hybridizes to the capture probe. After that,
The sandwich probe is electrophoresed through the capture layer. In short, the captured target then acts as a new "capture" probe for the sandwich probe. This capture target sandwich probe complex can then be detected by the sandwich probe label.

Blotting can also be adapted for detection of target-bound capture probes. For example, the detection surface is juxtaposed to a separation medium having bound sample components, which sample components then migrate to the detection surface, optionally assisted by chemical means such as solvent or reagent modification. Here, the transferred sample components are
It is detected by known means such as optical detection of intercalating staining, or by detection of radioactivity obtained from the hybridizing radioactive species.

Various optical techniques can be used to detect the presence of sample components bound to the capture probe. For example, if the capture probes are arranged in a linear array, the position and intensity of each signal will mechanically or optically result in a single detector along the array of detectable signals. It can be measured by scanning. Alternatively, a linear array of detectable signals may be provided by juxtaposing the array detector with the array of detectable signals or optically imaging all or part of the signal ray on the array detector. Therefore, it can be detected by the linear array detector.

A number of detection schemes can be used when the capture probes with a detectable signal are arranged as a two-dimensional array. A single detector can be used to measure the signal at each time point by mechanical or optical scanning, or by any combination. Alternatively, a linear optical detection array can be used to detect a set of signals by parallel or optical imaging, and multiple sets of such signals can be mechanically or optically signaled. It can be detected by scanning the array or detector. Alternatively, the two-dimensional array of capture probes can be wholly or by a two-dimensional optical area detector by juxtaposing the optical signal array obtained from the immobilized capture probe or by optical image of the optical signal array. It can be optically detected in part.

When the capture probes are arranged as a three-dimensional array, detection of individual signals can be arranged by the techniques described above by first physically taking subsections of one or more arrays. . Alternatively, optical techniques such as confocal microscopy techniques can be used whereby one or many detectable signals are imaged and interference from the other is detected to a minimum and other The signal is subsequently detected after optical adjustment.

Features and other details of the invention will now be described in more detail and pointed out in the examples. Specific embodiments of the invention are shown by way of illustration and not limitation as to the invention. The main features of this invention can be used in various embodiments without departing from the scope of the invention.

EXAMPLES Example 1 Gel-Based Sandwich Assay for Bacterial Contamination in Platelet Concentrate 250 μL of platelet concentrate suspected of microbial contamination was 250 mL of buffer containing Escherichia coli (E. coli) cells (pH 7). 0.0 mM 200 mM sodium phosphate buffer, 2 mM EDTA, 2% sodium dodecyl sulfate (SDS
), 0.1% heparin). The E. coli cells
Liquid LB broth 2 with bacterial colonies obtained from LB agar plate culture medium.
It was prepared by ingesting mL and was added with shaking and grown overnight at 37 ° C. (Maniatis et al., “Molecular Cloning: Lab Manual (Molec).
urular Cloning: A Laboratory Manual), 2nd Edition, Cold Spring Harbor Press, 1989,
Cold Spring Harbor City, New York). Next morning, overnight culture medium was fresh L
It was diluted 100-fold with B medium and grown to an optical absorbance of 2.4 at 600 nm. The culture medium is a microcentrifuge (10,000
xg) and pelleted in ice-cold TE buffer (10 mM Tris-HCl, p.
H8.0, 1 mM EDTA) to a density of 4 × 10 ⁹ cells / mL.

An aliquot of platelet concentrate was infused with washed E. coli cells as follows. That is, 1) Platelet concentrate 1.75 mL 2) pH 6.8 1M sodium phosphate buffer 0.35 mL 3) 10 mM aurintricarboxylic acid 0.36 mL (Rnase inhibitor, S
(igma Chemical Co., St. Louis, MO) 4) 10% wt / v SDS 0.35 mL 5) 0.5M Na ₂ EDTA 0.035 mL 6) 10% low molecular weight heparin (Sigma Chemical Co.) 0.03
5 mL

An aliquot (450 μL) of the buffer / platelet mixture was applied into a screw-cap microcentrifuge tube and a 50 μL aliquot of a serial dilution of washed E. coli cells was added. The test tubes were mixed briefly and heated in an aluminum heating block at 130-140 ° C for 10 minutes. The resulting lysate was centrifuged in a microcentrifuge (10,000 xg) for 10 minutes at room temperature.

An aliquot of the lysate is used in the hybridization reaction as follows. 1) Lysed and infused platelet concentrate 24 μL 2) 10 × conjugated hybridization buffer 4 μL (1M NaCl, p
H8.3 337 Mm Tris-borate, 10 mM MgCl ₂ , 1 mM Zn
Cl ₂ , pH 6.8, 30 mM sodium phosphate buffer) 3) 10 μM adapter polynucleotide sequence (5′-GCT GCT T)
CC TTC CGG ACC TGA GTG AAT ACG TTC C
CG GGC CT-3 ′ [SEQ ID NO: 1]), 2 μl 4) 300 nM alkaline phosphatase (AP) conjugated sandwich probe 6 μL (5′-AP-NH ₂ -GGCA CAC GCG TCA TCT)
GCC TTC-3 ′ [SEQ ID NO: 2]), 6 μl 5) 10 mM aurintricarboxylic acid 1 μL 6) Water 1 μL Hybridization was carried out at 53 ° C. for 10 minutes. Incidentally, as used in this application, the term polynucleotide is interchangeable with the phrase "polynucleotide sequence".

In the adapter probe, 21 nucleotides at the 5′-most terminal side are 4.5 in E. coli.
41-m which is complementary to bases 65 to 41 of S RNA, and the 3'-most 20 nucleotides are complementary to the gel-immobilized capture probe polynucleotide
er DNA polynucleotide. The bacterial target polynucleotide used in this example is a nucleotide sequence obtained from E. coli 4.5S RNA and is as follows: That is, GGGGGCTCTG TTGGTTCTCC CGCAACGCTA CT
CTGTTTAC CAGGTCAGGT CCGGAAGGAA GCAGC
CAAGG CAGATGACGC GTGTGCCGGG ATGTGAGC
TGG CAGGGGCCCCCC ACCC (Genbank accession number #A
E000151 U00096; bases 10899-11013; SEQ ID NO: 3)
.

Hybridized samples were 90 mM pH 8.3 without EDTA.
It was loaded on a vertical 5% polyacrylamide gel (29: 1, monomer: bis wt / wt) containing Tris borate buffer. The gel was poured into 3 sections. The center layer of the gel contains immobilized capture probe polynucleotide (or simply capture probe, 5'-acrylamide-AGG CCC GGG AAC.
GTA TTC AC-3 '[SEQ ID NO: 4]). The acrylamide modified probe was included in the acrylamide gel mixture upon polymerization with a polyacrylamide substrate. The acrylamide groups are commercially available and available as acrylamide phosphoramidite (Acrydite ™, Mosaic Techn).
(Biology, Boston, Mass.) was added to the capture probe. The concentration of the capture probe in the capture layer was 4 μM. The upper and lower sections of the gel contained no capture probe. The gel is about 0.75
The thickness was 10 mm, the length was 10 cm, and the width was 10 cm. Electrophoresis 90 at 200V
Min at a gel temperature of 34 ° C. (Penguin device, Owl S
Scientific, Woburn, Mass.).

After electrophoresis, the gel was removed from the glass electrophoresis plate and the AP buffer (2.4 M diethylamine-HCl buffer, pH 11, 1 mM MgCl ₂ ,
It was washed in 0.1 mM ZnCl ₂ ) for 10 minutes. The gel is then Atto
phos chemiluminescent AP substrate (Attophos, Lumigen, Boehr
(Inner Mannheim Biochemicals, Indianapolis, Ind.) was soaked in 15 mL for 10 minutes. Fluorescent AP product was imaged using a two-dimensional fluorescence scanner (Fluorimager 595, 595).
Molecular Dynamics, Sunnyvale, CA
e).

The fluorescence image of the gel is shown in FIG. The location of the trapping layer extending horizontally across the gel is indicated by the bracketed 7's. The sample was loaded in the following manner. Lanes 1:10 ⁷ pre-hybridized lysate containing E. coli cells, lane 2: no loaded sample, lanes 3:10 ⁶ pre-hybridized lysate containing E. coli cells, lane 4: lane 4: Pre-hybridized lysate containing 10 ⁵ E. coli cells, lane 5: 10 pre-hybridized lysate containing ⁴ E. coli cells, lane 6: pre-hybridized lysate without addition of E. coli cells .

The results show AP activity in the capture layer of lanes 1, 3 and 4. Trace AP activity is observed in the high control image in lane 5. Lane 6 shows a bottom out of AP activity above background. In conclusion, the sandwich assay described in this application is sensitive enough to detect 10 ⁴ -10 ⁵ bacterial cells.

Example 2 Strand Displacement Detection of Bacterial RNA by Gel Hybridization Enterobacter cloacae
, Pseudomonas aeruginos
a), Serratia marcescens
, Bacillus cereus, Escherichia coli (Esch)
erichia coli), Stahirococcus epidermidis (Stap)
All bacterial RNAs from Hylococcus epidermidis, Staphylococcus aureus were purified by extraction from cells cultured using heated phenol. 5mL nutrition broth (Difco, Fisher Scientific
Company, Pittsburgh, PA) culture medium was grown overnight at 37 ° C starting with single isolated colonies obtained from nutrient agar plates. Then, after this overnight culture, 100 mL of nutrient broth was inoculated into the overnight culture medium. This new culture medium was allowed to grow for 4 hours, after which the bacteria were centrifuged (60
Harvested by using 00x g). Bacterial cells were resuspended in 5 mL sodium acetate buffer (pH 5.2, 20 mM sodium acetate, 1 mM
EDTA). It was preheated to the suspended cells (70 ° C, 1: 1).
, Volume: volume, pre-equilibrated with water) 500 μL of 10% SDS and 5 mL of phenol-chloroform were added, followed by vigorous shaking. The mixture was then incubated at 70 ° C. for 10 minutes with intermittent shaking. The mixture containing bacterial cells was then centrifuged at 8000 xg for 10 minutes to separate the hydrophilic and hydrophobic phases. The aqueous phase was transferred to a new tube and re-extracted using phenol / chloroform as described above. RNA was then recovered from the aqueous phase by two successive ethanol precipitations using a 10% volume solution of 3M sodium acetate (pH 5.2, 3M sodium acetate) and 2.5 volumes of ethanol. The recovered RNA is then approximately 1-2.
mL TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
It was thawed and stored at -20 ° C. RNA concentration was measured by absorbance at 260 nm, assuming 33 μg / mL per OD ₂₆₀ unit. RNA concentrations ranged from 1-3 μg / μL in the final solution.

The electrophoretic gel was poured into three sections. An immobilizable tether capture probe polynucleotide (5'-acrylamide-GG is included in the middle layer of the gel).
T AAG GTT CTT CGC GTT GCA TCG AAT TA
A ACC ACA TGC TCC ACC GCT TGT G-3 ′ [SEQ ID NO: 5]) as a fluorescent signal polynucleotide (5′-Cy3-CAC AA
G CGG TGG AGC ATG TGG TTT-3 ′ [SEQ ID NO: 6]
, Where Cy3 indicates the position of the fluorescent phosphoramidite) and which is immobilized and comprises a partially double-stranded displacement probe complex. The acrylamide modified displacement complex was included in the acrylamide gel mixture during polymerization and immobilized in the layer by copolymerization with a polyacrylamide substrate. Acrylamide groups are commercially available and available as acrylamide phosphoramidite (Acrydite ™, Mosaic Technology).
, Inc., Boston, Mass.) to the tethered polynucleotides. The concentration of displacement probe complex in the capture layer was 2 μM. The upper and lower sections of the gel contained no capture probe. The gel was approximately 0.75 mm thick x 10 cm long x 10 cm wide.

A sample of bacterial RNA or bacterial target polynucleotide was hydrolyzed by treatment with 100 mM NaOH for 30 minutes at 50 ° C. to thereby cleave the RNA into small pieces, and thereby the molecules of 16S rRNA. The secondary structure was reduced. The E. coli 16S rRNA nucleotide sequence used in this example is as follows. That is, 5'-CAC AAG CGG TGG AG
CATG TGG TTT AAT TCG ATG CAA CGC GA
A GAA CCT TAC C-3 '(Genbank accession number: # M243
86; rRNA nucleotide sequence position 933-984, [SEQ ID NO: 7]). The 5'-end of this 16S rRNA obtained from E. coli shared partial sequence homology with the fluorescent signal polynucleotide described above. E. coli 16S
The rRNA nucleotide sequence had a higher degree of complementarity to the immobilized capture probe than the fluorescent signal due to the increased base pairs due to the capture probe polynucleotide. Based on this difference, which is basically a difference in affinity when the target polynucleotide contacts the capture probe / signal polynucleotide complex, the target polynucleotide replaces the signal polynucleotide and a new complex is formed. It will be formed between the target polynucleotide and the capture probe polynucleotide.

After neutralization, the sample was run on neutral 1x TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA) 6% polyacrylamide gel (29: 1 monomer: bisacrylamide, wt: wt). I ran it. Approximately 30-4 of all RNA
0 μg was run in each lane of the gel. Negative and positive control samples consisting of the human 18S rRNA sequence and the E. coli 16S rRNA sequence were respectively T7.
Produced by in vitro transcription of the appropriate vector using RNA polymerase. These in vitro transcripts are also hydrolyzed and neutralized by NaOH,
It was also electrophoresed in parallel with a bacterial RNA standard sample. Electrophoresis is 35
Performed at 100 V using a gel temperature of ° C (Penguin device, Owl
(Scientific, Woburn, Mass.). After electrophoresis,
The gel was removed from the glass plate and stained with Cy3 stained fluorescence (Amersham).
Pharma Biotech, Inc., Piscataway, NJ, Gel Scanner (Fluorimager 595, Molecular)
Dynamics, Inc., Sunny Valley, Calif.).

FIG. 6 illustrates a fluorescence image of the gel. Approximately 30-40 μg of RNA was added to each lane, except for the lane containing the in vitro transcription control. Approximately 8 pmol of human in vitro transcript; and 4 pmol of E. coli in vitro transcript were used as controls (right lane). As can be seen, the E. coli in vitro transcript and all other RNAs were able to displace the fluorescent signal polynucleotide depicted in the signal layer from its displacement complex. In contrast, the human 18S rRNA in vitro transcript was unable to displace the signal due to the lack of a nucleotide sequence complementary to its replacement complex. Therefore, the displacement assay illustrated is capable of detecting bacterial RNA with a wide range of phylogenetic diversity without interference from human rRNA.

Example 3: Mutation Detection Using Solid Phase Analysis A polynucleotide containing a 5'-terminal acrydite group was used in this example (Research Genetics, Huntsville, AL).
e; Eurogentec, Belgium, Seraing; Operon Te
chnologies, Alameda, Calif.). Unmodified and 5'-fluorolein labeled polynucleotides were isolated from Ransom Hill B
Obtained from iosciences, Inc. (Ramona, Calif.).
The lyophilized polynucleotide was a TE buffer (10 mM Tris-pH 8.3).
HCl, 1 mM EDTA) and stored frozen at -20 ° C. Concentrations were determined from A ₂₆₀ nm readings (assuming 33 μg / mL oligonucleotide per optical density (OD) unit). All concentrations are relative to the oligonucleotide chain.

[0100]   The sequences of the polynucleotides used in this example are described below. "Fl"
And "Ac" represent fluorescein and acrydite modifications, respectively. Fluo
Hybridization using rescein-labeled target molecule (viz, capture)
The capture probe used in the first experiment designed to demonstrate the efficiency of
c-GTA CCA TAA CAG CAA GCC TCA-3 '[SEQ ID NO:
No. 8], and the target polynucleotides used were as follows.
That is, (i) left complementary lane, 5'-F1-TGA GGC TTG CT. G   TTA TGG TAC-3 '[SEQ ID NO: 9]; (ii) right complementary lane
5'-Fl-TGA GGC TTG CTT  TTA TGG TAC-3
'[SEQ ID NO: 10]; (iii) non-complementary 5'-Fl-ATT ACG T
TG ATA TTG CTG ATT A-3 '[SEQ ID NO: 11].
(See Figure 7). The two complementary polynucleotides are 12 bases from the 5'end.
Different at a single position (underlined). Non-stringent gel conditions used (ie 23 ° C)
Nucleotide sequence difference between the two complementary polynucleotides at gel temperature)
No differences are identified (see Figure 7).

In the next experiment, a series of target molecules containing different plural and singular base mismatches were run using the same capture probe as used in previous experiments, and the target molecules were Fluorescein-labeled complement (compl.): 5'-Fl-TG
A GGC TTG CTG TTA TGG TAC-3 ′ [SEQ ID NO: 12
] (See FIG. 8). Various mutant target molecules are labeled in Figure 8 above by the type of mispairing with the capture probe and the distance from the 5'end of the target molecule. The mismatch is identified by a base in the target molecule followed by a base in the target molecule separated by a colon. For example, c: t,
5 target molecules used in lanes, 5'-Fl-TGA G T C TTG CT
G TTA TGG TAC-3 ′ [SEQ ID NO: 13], and c: t, 5
A: a, 7; a: c, target used in lane 14 is 5'-Fl-TG
A G T C A TG CTG T C A TGG TAC-3 '[ SEQ ID NO: 14
]. The underlined position is different from the perfectly complementary target molecule. The non-complementary target molecule in this experiment is the same as that used in the previous experiment.

In the next experiment, solid phase hybridization was used to detect mutations in the PCR product, in particular the human immunodeficiency virus (HIV) reverse transcriptase codon 41 mutation: 5'-Ac-GT ACA GAG C. TG GAA AAG G-
It was used to detect mutations in HIV reverse transcriptase using the capture probe used to identify 3 '[SEQ ID NO: 15] (the mutation position is underlined). The capture probe used to identify the codon 215-219 mutation was 5
'-Ac-GG CT C TA C ACA CCA GAC C AA-3' [ SEQ ID NO: 16] and which was (a variant position are underlined) (see FIG. 9).

Next, the ability to detect polymorphisms was investigated. The capture probe is as follows. That is, (i) A ′, 5′-Ac-GTT TTC AGC TCC
ACC TAC CAC AAG T-3 ′ [SEQ ID NO: 17], (ii) B
', 5'-Ac-GTT TTA GCT CCA ACT ACC ACA
AGT T-3 ′ [SEQ ID NO: 18], (iii) C ′, 5′-Ac-GTT
TTG CAC CTC AAA GCT GTT CCG T-3 ′ [SEQ ID NO: 19], and (iv) D ′, 5′-Ac-GTT TTG TTC AT.
G CCG CCC ATG CAG G-3 ′ [SEQ ID NO: 20], and
The target molecule was as follows. That is, (i) A, 5'-Fl-AC
TTG TGG TAG TTG GAG CTG-3 ′ [SEQ ID NO: 21]
, (Ii) B, 5'-F1-AA CTT GTG GTA GTT GGA
GCT-3 ′ [SEQ ID NO: 22], (iii) C, 5′-Fl-AC GGA
ACA GCT TTG AGG TGC-3 ′ [SEQ ID NO: 23], (iv)
D, 5'-F1-CC TGC ATG GGC GGC ATG AAC-3
'[SEQ ID NO: 24]. Target molecules A and B share and share a common nucleotide sequence of 19 bases (see Figure 10).

An acrylamide gel (5% -20% total acrylamide, 29: 1 monomer: bis-acrylamide) was loaded with 0.5x TBE buffer (45 mM Tris-borate, pH 8.3, 1 mM EDTA). 7% of 10% aqueous ammonium persulfate
And was prepared and polymerized by adding 2 μL of N, N, N ′, N′-tetramethylenediamine (TEMED) per mL of gel solution. Standard mini-gel system (Mini-PROTEAN® II, 8x10c
m-plate (Bio-Rad) and Penguin ™, 10 × 10 cm
Plates (Owl Scientific, Woburn, Mass.) Were used with spacers ranging in thickness between 0.8 and 1.5 mm.

Typically, the gel was cast in three sections. First, the lower unmodified gel was poured in and allowed to polymerize again. The capture layer containing the acrydite modified capture probe was then polymerized on the unmodified gel. The volume of the trapping layer was approximately 0.5 cm to ensure uniform polymerization of the layer. Acrydite capture probe (10 μM final concentration) is combined with non-polymerized gel solution prior to catalyst addition. After polymerization of the capture layer, the gel surface was thoroughly washed with 0.5x TBE and the rest of the gel cassette was filled with unmodified gel.

The non-denaturing acrylamide gel was 0.5 × TBE at an applied voltage of 100-150V.
It was run in buffer (gel length 8-10 cm). Gel should be 1
Pre-migrated for 5-20 minutes. Fluorescein-labeled target molecule was mixed with sucrose-containing buffer and loaded without prior heating. Labeled PCR product, usually 5 μL from a 50 μL reaction, was raised to 75% formamide (volume / volume) and heated at 90 ° C. for 2 minutes before loading.

A plasmid containing the mutation (pKRT67 / 70/215/219, pKRT
41/215/219) and a wild type (pKRTwt) subclone of the HIV reverse transcriptase gene (HIV-RT) was used. The plasmid sample is Hin
Limited by dIII and 10 ⁶ copies of the restricted material were used as the initial PCR target. Amplification at 94 ° C for 10 seconds, 50 ° C for 10 seconds, and 72 ° C
Was performed for 30 cycles consisting of 1 minute. The reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl ₂ , 0.
． 5% Tween® 20,50 μM dCTP, dTTP and d
ATP, 32 μM dGTP, 16 μM fluorescein-dGTP (NEN L
if Science Products, Boston, Mass.)
, 0.005 U / μL AmpliTaq® DNA Polymerase
(Perkins Elmer, Norwalk, CT) and 1μ
M amplification primers are included. The 321-base pair (bp) target molecule containing the codons 215 to 219 region of HIV-RT was labeled with the primer 5′-ATG AGA C.
AC CAG GGA TTA GA-3 '[SEQ ID NO: 25], and 5'-
TAG GCT GTA CTG TCC ATT TAT-3 ′ [SEQ ID NO:
26] was used for amplification. 249-b containing the codon 41 region of HIV-RT
The p target molecule is the primer 5′-GGA TGG CCC AAA AGT T
A-3 ′ [SEQ ID NO: 27], and 5′-CCT GCG GGA TGT
Amplified using GGT ATT C-3 '[SEQ ID NO: 28].

FIG. 7 illustrates the efficiency of hybridization using a fluorescein labeled target molecule. The results show that the complementary sample (C lane) was completely immobilized on the capture layer containing the capture probe, while the non-complementary target molecule (N lane) was not retained. .

FIG. 8 illustrates a series of target molecules containing different multiple and single base mismatches using migration of capture probes in acrydite gels at various temperatures. The high stringency achieved by the acridite gel assay is due to the smearing of the capture layer to the capture layer because mutant targets near the ends have weakened binding under conditions where the perfectly complementary target forms a tight band. smear) (and pass through). FIG. 8A shows typical gel migration at 40 ° C., while
FIG. 8B provides a graphical representation of the entire dataset. As can be seen in Figure 8B, the mismatches were divided into several classes. In most cases, a gel temperature of 40 ° C. was destabilized sufficiently to prevent trapping. Some still partially 4
It was retained at 0 ° C, but was effectively removed from the capture layer at 45 ° C. Only one mismatch was not completely removed from the acquisition layer at 45 ° C. However, the variants are localized near the ends (20 of 21 base target molecules), and mismatches near these ends are usually not discernible without kinetic lysis analysis. difficult.

FIG. 9 illustrates and illustrates the use of acridite gels in typing mutations in PCR products. In particular, two sets of mutations in HIV reverse transcriptase were detected. The codon 41 mutation is a single nucleotide change, while codons 215-21
In 9, the mutation involves the substitution of 4 nucleotides. Fluorescein labeled PC
The R product is prepared from a plasmid target molecule with the HIV-RT fragment, denatured with formamide, and then loaded on a gel containing capture probes specific for HIV-RT mutations. In both cases, the mutant probe-complementary strand was efficiently captured, whereas the wild-type product did not. Thus, in both cases, the mutated gene is clearly distinguishable from the wild-type gene.

FIG. 10 illustrates polymorphic detection using multiple capture layers. Target molecules "A" and "B" share a common 19-base sequence and both are efficiently captured by the first "A" capture layer. Targets "A" and "B" with "B" capture layer, as best seen in lanes when "A" and "B" targets were loaded simultaneously (Figure 10, leftmost and rightmost lanes). Complementarity can be observed from the overflow of the target caused by the slight overloading of the "A" capture layer. Targets "C" and "D" show perfect specificity for layers "C" and "D", respectively.

While the present invention has been shown and described in detail with reference to certain embodiments thereof,
Those skilled in the art will appreciate that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Will be done.

[Brief description of drawings]

FIG. 1 is a schematic illustration showing the various steps involved in detecting a bacterial target molecule.

FIG. 2 is a schematic illustration illustrating the preparation of potential bacterial target molecules obtained from a platelet-containing source.

[Figure 3]   FIG. 3 is a schematic diagram illustrating a sandwich assay.

[Figure 4]   FIG. 4 is a schematic illustration illustrating a strand displacement assay.

FIG. 5 is a photograph of a gel showing the results of a sandwich assay for target molecules.

[Figure 6]   FIG. 6 is a photograph of a gel showing the results of a single displacement assay on a target molecule.

FIG. 7: Results of displacement assay using capture probe immobilized in discrete layers in the medium, two molecules complementary to the capture probe and one non-complementary target molecule. Is a photograph of a gel showing.

8A and B illustrate analysis of variants using solid phase technology. FIG. 8A is a gel obtained from gel analysis detecting mutations in target molecules. FIG. 8B is a profile for the 11 different target molecules used in this experiment.

FIG. 9 is a photograph of a gel showing the results of an experiment for detecting a variant in a PCR product, particularly in the HIV reverse transcriptase gene.

FIG. 10 is a gel photograph showing the results of one experiment using multiple capture layer gels.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 21/27 G01N 21/41 Z 4B063 21/41 21/59 Z 4B065 21/59 21/64 F 21 / 64 G 27/06 Z 27/06 33/483 F 27/447 33/53 M 33/483 33/566 33/53 33/58 A 33/566 C12N 15/00 ZNAA 33/58 G01N 27/26 325B 315F 315G 325E 315K (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, U , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA , CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU , SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF F terms (reference) 2G043 AA01 BA16 CA03 DA02 EA01 EA02 GA07 GB21 2G045 BB01 BB20 CA02 CA24 CA25 CA26 CB01 CB03 CB04 CB09 CB12 CB21 CB26 DA12 DA13 DA14 DA36 FB02 FB04 FB05 FB07 GC04 GC06 GC07 GC09 GC11 GC16 GC30 JA04 2G059 AA05 BB12 CC16 DD03 DD04 EE01 EE02 EE04 FF12 HH02 KK04 2G060 AA07 AD06 AE40 AF08 HC06 KA09 4B024 AA11 CA02 CA04 CA09 GA11 HA14 4B063 QA01 QA18 QQ05 QQ06 QQ07 QQ10 QR32 QR35 QR55 QR56 QS35 QX02 QX07 4B065 AA01X AA15X AA29X AA41X AA46X AA48X AA49X AA53X BD02 BD50 CA46

Claims (45)

[Claims] 1. The following steps: (a) introducing a microbiological target molecule into an electrophoretic medium containing one or more capture probes immobilized in at least one region; (b) Subjecting the electrophoretic medium to an electric field to effect electrophoretic migration of the microbiological target molecule in at least one region of the electrophoretic medium containing immobilized capture probes; and (c) immobilizing within the medium. At least one of an electrophoretic medium containing an immobilized capture probe, the method comprising the step of: detecting the presence of a microbiological target molecule or a microbiological target molecule / capture probe complex.
Detection of the microbiological target molecule or the microbiological target molecule / capture probe complex within one of the regions is indicative of the presence of a microorganism in the test sample. A method for detecting the presence or absence of microorganisms in a test sample, comprising the step of detecting the presence or absence. 2. The method of claim 1, wherein prior to step (a), the test sample is treated to release one or more target molecules from the microorganism and / or host. 3. The method according to claim 1, wherein the microorganism is selected from the group consisting of bacteria, viruses, fungi and parasites. 4. The bacterium is Serratia marcescens, Staphylococcus epidermidis, Stahirococcus aureus, Pseudomonas erginosa, Escherichia coli, Bacillus cereus, Enterobacter cloake, Streptococcus pyogenes, Staphylococcus waneri, Streptococcus.・ (Α
-(Hemolyric) ((-hemolyric), Streptococcus pneumoniae, Streptococcus mitis, Salmonella, Serratia liquefaciens (liquifa)
ciens), Klebsiella, Propionibacterium acnes, Yersinia enterocolitica, Pseudomonas fluorescens, Pseudomonas putida and combinations thereof. 5. The method of claim 1, wherein the microbiological target molecule is selected from the group consisting of nucleic acids, proteins, peptides and combinations thereof. 6. The method of claim 5, wherein the nucleic acid is selected from the group consisting of DNA and RNA. 7. The test sample is selected from the group consisting of blood, plasma, platelets, red blood cells, urine, feces, sweat, tracheal exudate, aqueous humor, vitreous humor, skin, hair, cells, tissue and organ culture systems. The method according to claim 1, wherein 8. The method of claim 1, wherein the capture probe is selected from the group consisting of nucleic acids, modified nucleic acids, nucleic acid analogs and combinations thereof. 9. The method of claim 1, wherein the electrophoretic medium is a solution containing at least one polymer. 10. The polymer is polyacrylamide, poly (N, N-dimethylacrylamide) polymer, poly (hydroxyethyl cellulose) polymer,
10. The method of claim 9 selected from the group consisting of poly (ethylene oxide) polymers, poly (vinyl alcohol) polymers and combinations thereof. 11. The method of claim 1, wherein the medium is a gel formed from at least one polymer. 12. The method according to claim 11, wherein the electrophoretic medium is formed by using at least one polymer selected from the group consisting of polyacrylamide polymers, agarose polymers, starch polymers and combinations thereof. 13. The following steps: (a) contacting one or more microbiological target molecules from a test sample with at least one adapter polynucleotide under conditions suitable for hybridization, thereby causing an adapter. / Forming a hybrid / target hybridization complex, forming an adapter / target hybridization complex; (b) one or more of the hybridization complex of (a) immobilized in at least one region thereof. Introducing into an electrophoretic medium containing a capture probe polynucleotide; (c) subjecting the electrophoretic medium to an electric field to provide the adapter / on at least one region of the electrophoretic medium containing the immobilized capture probe polynucleotide. Effecting electrophoretic migration of the target complex; and (d) the medium. Detecting the presence of an adapter / target / capture probe complex immobilized in the body, the step of detecting the presence or absence of one or more microbiological target molecules in a test sample using an adapter molecule. A method of detecting the presence or absence of microorganisms in a test sample, including. 14. The method of claim 13, wherein the microorganism is selected from the group consisting of bacteria, viruses, fungi and parasites. 15. The method according to claim 13, wherein the microbiological target molecule is selected from the group consisting of nucleic acids, proteins and peptides. 16. The method of claim 15, wherein the nucleic acid is selected from the group consisting of DNA and RNA. 17. The test sample is selected from the group consisting of blood, plasma, platelets, red blood cells, urine, feces, sweat, tracheal exudate, aqueous humor, vitreous humor, skin, hair, cells, tissue and organ culture systems. 14. The method of claim 13, wherein 18. The method of claim 13, wherein the adapter polynucleotide is SEQ ID NO: 1. 19. A sandwich comprising in step (a) an adapter / target hybridization complex containing a nucleotide sequence complementary to said target molecule which does not overlap with the binding sequence region between said adapter polynucleotide and target molecule. 2. Mixing with a probe under conditions suitable for hybridization.
3. The method described in 3. 20. The method of claim 19, wherein the sandwich probe polynucleotide is bound to alkaline phosphatase. 21. The method of claim 20, wherein the sandwich probe polynucleotide is SEQ ID NO: 2. 22. The method of claim 13, wherein the microbiological target polynucleotide is SEQ ID NO: 3. 23. The immobilized capture probe polynucleotide is SEQ ID NO:
14. The method according to claim 13, which is 4. 24. (a) Microbiology used to hybridize with a capture probe polynucleotide at least one capture probe polynucleotide containing a nucleotide sequence region complementary to a region of a microbiological target molecule. At least one signal polynucleotide containing a nucleotide sequence partially homologous to the target nucleotide sequence region is contacted under conditions suitable for hybridization of the capture probe polynucleotide and the signal polynucleotide, thereby capturing. Forming at least one capture probe / signal polynucleotide complex which forms a probe / signal polynucleotide complex;
(B) introducing the microbiological target molecule into an electrophoretic medium containing one or more kinds of capture probes immobilized in at least one region; (c) subjecting the electrophoretic medium to an electric field to immobilize it. Effecting electrophoretic migration of the microbiological target molecule within at least one region of the electrophoretic medium containing an immobilized capture probe polynucleotide; and (d) at least one release in a signal layer within the medium. One or more microbiology in a test sample using a signal polynucleotide substitution reaction, comprising detecting the presence of a signal polynucleotide, thereby indicating the presence of one or more microbiological target molecules in the test sample. A method for detecting the presence or absence of a microorganism, comprising the step of detecting the presence or absence of a target molecule. 25. The method of claim 24, wherein the capture probe polynucleotide is SEQ ID NO: 5. 26. The signal polynucleotide is SEQ ID NO: 6.
4. The method described in 4. 27. The method of claim 24, wherein the microbiological target molecule is SEQ ID NO: 7. 28. The following steps: (a) introducing one or more putative mutant target molecules into an electrophoretic medium containing one or more capture probes immobilized in at least one region; (B) subjecting the electrophoretic medium to an electric field to effect electrophoretic migration of one or more putative mutant target molecules in at least one region of the electrophoretic medium containing immobilized capture probes; and (c). Detecting the presence of a putative mutant target molecule / capture probe complex immobilized in the medium, comprising: at least one nucleotide in one or more of the putative mutant target molecule or molecules present in the test sample. A method for identifying a sequence mutation site. 29. The method of claim 28, wherein the capture probe is immobilized within one or more discrete areas of the electrophoretic medium. 30. The method of claim 28, wherein the capture probe is selected from the group consisting of nucleic acids, modified nucleic acids, nucleic acid analogs and combinations thereof. 31. The method of claim 28, wherein the capture probe is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 15 and SEQ ID NOs: 16-20. 32. The method of claim 28, wherein the putative mutant target molecule is from the group consisting of bacterial, viral, fungal and parasite molecules. 33. The method of claim 32, wherein the putative mutant target molecule is a nucleic acid. 34. The method of claim 33, wherein the putative mutant target molecule is selected from the group consisting of DNA and RNA. 35. The method of claim 35, wherein the putative mutant target molecule is selected from the group consisting of SEQ ID NOs: 9-14 and SEQ ID NOs: 21-24. 36. The method according to claim 35, wherein the putative mutant target molecule is synthesized using a primer selected from the group consisting of SEQ ID NOs: 25-28. 37. The test sample according to claim 28, wherein the test sample is selected from the group consisting of blood, urine, feces, sweat, tracheal exudate, aqueous humor, vitreous humor, skin, hair, cells, tissues and organ culture systems. Method. 38. (a) A step of introducing a microbial target molecule into an electrophoretic medium containing a capture probe complex immobilized therein, wherein the capture probe complex is a tether nucleic acid. A labeled signal nucleic acid molecule hybridized with the molecule; (b) subjecting the electrophoretic medium to an electric field to electrophorese the microbial target molecule in at least one region of the electrophoretic medium containing an immobilized capture probe. (C) contacting the target molecule with the capture probe complex of step (a) under conditions suitable for hybridization to form a new hybrid complex, wherein the signal nucleic acid The molecule replaces the target molecule, thereby creating a new hive containing the target molecule hybridized with the tethered nucleic acid molecule. Forming a head complex; and detecting (d) is the signal nucleic acid, thereby comprising the step of indicating the presence of the target molecule in a test sample, using a displacement assay, the detection method of the presence or absence of microbial target molecule in a test sample. 39. The method of claim 38, wherein the capture probe complex is immobilized in a discontinuous layer of electrophoretic medium. 40. The label is radioactive, fluorescent, chemiluminescent, phosphorescent, luminescent,
The selected from the group consisting of an affinity label, an enzyme conjugate and a combination thereof.
8. The method according to 8. 41. The detection means is selected from the group consisting of mass measurement, density measurement, mass spectrometric analysis, plasmon resonance, emission, polarization, refractive index, electrical conductivity, viscosity, turbidity and optical rotation. The method described. 42. (a) A step of introducing a microbial target molecule into an electrophoretic medium containing a capture probe complex immobilized therein, wherein the capture probe complex is hybridized with a tethered nucleic acid molecule. (B) subjecting the electrophoretic medium to an electric field to effect electrophoretic transfer of the microbial target molecule to at least one region of the electrophoretic medium containing immobilized capture probes. Step; (c) forming a target-signal hybrid complex by contacting the target molecule with the capture probe complex of step (a) under conditions suitable for hybridization, wherein the signal nucleic acid molecule Is replaced with the target molecule,
And a hybridisation, thereby forming a target-signal hybrid complex; and (d) detecting the target-signal hybrid complex, thereby indicating the presence of the target molecule in the test sample, a reverse displacement assay. A method for detecting the presence or absence of a microbial target molecule in a test sample using. 43. The method of claim 42, wherein the capture probe complex is immobilized in a discontinuous layer of electrophoretic medium. 44. The label is radioactive, fluorescent, chemiluminescent, phosphorescent, luminescent,
The selected from the group consisting of an affinity label, an enzyme conjugate and a combination thereof.
2. The method described in 2. 45. The means for detecting is selected from the group consisting of mass measurement, density measurement, mass spectrometric analysis, plasmon resonance, emission, polarization, refractive index, electrical conductivity, viscosity, turbidity and optical rotation. The method described.