WO2010058059A1 - A method for detecting specific nucleotide sequences - Google Patents

A method for detecting specific nucleotide sequences Download PDF

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Publication number
WO2010058059A1
WO2010058059A1 PCT/FI2008/050666 FI2008050666W WO2010058059A1 WO 2010058059 A1 WO2010058059 A1 WO 2010058059A1 FI 2008050666 W FI2008050666 W FI 2008050666W WO 2010058059 A1 WO2010058059 A1 WO 2010058059A1
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Prior art keywords
sample
specific
previous
magnetic beads
nucleotide sequence
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PCT/FI2008/050666
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French (fr)
Inventor
Christian Oker-Blom
Original Assignee
University Of Jyväskylä
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Priority to PCT/FI2008/050666 priority Critical patent/WO2010058059A1/en
Publication of WO2010058059A1 publication Critical patent/WO2010058059A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates to the fields of life sciences and genetics. Specifically, the invention relates to a method for detecting biological com- ponents. More specifically, the present invention relates to a novel method for detecting a specific nucleotide sequence in a sample. Furthermore, the present invention also relates to a use of the method of the invention for identifying viruses, micro-organisms or derivatives thereof from a sample and to a use of the method of the invention for identifying mutations of nucleotide sequences from a sample.
  • WO 03/054523 A2 describes a method for measuring an areal density of magnetic nanoparticles on a substrate on a microarray.
  • Publication WO2007/092909 A2 describes a method for detecting nucleic acids by utilizing specific molecular interaction sensors.
  • the present invention provides a simple and inexpensive way as well as ready to use tools to detect specific nucleotide sequences. Compared to the above-mentioned methods, the present invention provides a more specific, more sensitive and more reliable method. Advantages of the method are also the speed by which it can be conducted as compared to e.g. conventional microbial cultivation methods and the ease by which such an analysis can be carried out. Combined assay steps of the method are elementary to one skilled in the art. This invention provides an optimal method for detecting specific nucleotide sequences.
  • the invention has a significant commercial potential.
  • a method based on detection of specific nucleotide sequences is applicable to a large variety of areas including biology and medicine.
  • By using the method of the invention or variations thereof it is possible to quickly test for the presence and/or the amount of a specific sequence, e.g. DNA and/or RNA sequence, present in a sample.
  • a specific sequence e.g. DNA and/or RNA sequence
  • different sequences can be detected simultaneously.
  • the object of the invention is to provide novel methods and means for studying samples, more specifically for identifying properties of the sample.
  • the object of the invention is also to provide a diagnostic method, which is simply based on the measurement of properties of magnetic beads as well as the specificity of nucleotide sequences.
  • the present invention relates to a novel method for detecting a specific nucleotide sequence in a sample, characterized in that the method comprises the following steps: a) a helper molecule and/or nucleotide primer specific for said nucleotide sequence are immobilized on magnetic beads; b) the magnetic beads obtained in step a) are brought into contact with the sample; c) the sample obtained in step b) is run in a lateral flow assay; d) the specific nucleotide sequences present in the sample are detected by monitoring the properties of the magnetic beads.
  • the present invention also relates to a use of the method of the invention for identifying viruses, micro-organisms or derivatives thereof from a sample. Also, the present invention relates to a use of the method of the invention for identifying mutations of nucleotide sequences from a sample.
  • the present invention provides a specific, sensitive, reliable, fast, and easy method for detecting a specific nucleotide sequence in a sample.
  • Figure 1 shows the method of the invention utilizing magnetic beads displaying chemically bound DNA/RNA sequences.
  • the single stranded DNA- primers are synthesized to contain one or several chemically modified nucleotides (e.g. carboxylated magnetic beeds/amino coupled DNA primers) enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead.
  • the DNA sequence under study contained in the sample is am- plified by using PCR and primers labelled with e.g. biotin.
  • the amplified DNA, containing biotin at its 5' and/or 3' ends hybridizes with the ssDNA presented by the magnetic beads.
  • the sample is applied onto the test strip (e.g.
  • Figure 2 shows the method of the invention utilizing magnetic beads coated with a helper molecule.
  • a helper molecule e.g. recombinant strepta- vidin/avidin, is immobilized on the surface of the magnetic bead.
  • the gene un- der study is amplified by PCR using primers conjugated with e.g. biotin or another helper molecule as explained above for Figure 1 . If the DNA is amplified, i.e.
  • the amplified nucleotide sequences, carrying biotin at both ends will bind to the said magnetic beads, and the magnetic beads will further bind to either streptavid in/avid in immobilized on the strip or another pair of helper molecule used to conjugate the other pair of primer.
  • Figure 3 shows the method of the invention utilizing detection of DNA by magnetic beads without PCR-mediated amplification of the sample.
  • Single stranded DNA-phmers are synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead.
  • the DNA contained within the sample is pre-treated with appropriate restriction enzymes and allowed to hybridize with the DNA displayed on the magnetic bead. Flanking sequences in relation to the DNA sequence displayed on the beads are immobilized on the strip, allowing specific hybridization of the DNA bound to the beads to that immobilized on the strip.
  • Figure 4 shows the method of the invention utilizing magnetic beads coated with a helper molecule and hybridization to DNA immobilized on the strip.
  • Magnetic beads are furnished with a helper molecule, e.g. recombinant streptavidin/avidin.
  • the gene under study is amplified by PCR using primers conjugated with e.g. biotin.
  • the corresponding amplified DNA binds to the beads via biotin.
  • Strips are immobilized with DNA representing e.g. protruding ends of DNA relative to that hybridized to the beads.
  • step b) i.e. the step wherein the magnetic beads obtained in step a) are brought into contact with the sample, the helper molecules immobilized on magnetic beads are bound to another helper molecule conjugated to nucleotide primers, which border the specific nucleotide sequence.
  • step b) i.e. the step wherein the magnetic beads obtained in step a) are brought into contact with the sample, the primers immobilized on magnetic beads are hybridized with the specific nucleotide sequence(s) of the sample.
  • the analysis method of the invention is based on detection of nucleotide sequences. Even only one specific nucleotide sequence present in the sample is adequate for detection method of the invention.
  • the sample may comprise a great number of nucleotide sequences, either un- defined or specific sequences. Therefore, depending on the amount of nucleotide sequences in the sample, different method steps can be applied. For example, amplification methods can be carried out when a number of nucleotide sequences is low, and a use of restriction enzymes is possible when a large number of nucleotide sequences is present in a sample.
  • the specific nucleotide sequence is a RNA and/or DNA sequence.
  • specific DNA sequences can be selected from sequences from a group consisting of genomic DNA, mtDNA, cDNA, plasmids, kosmids, BACs, YACs and HACs.
  • specific RNA sequences can be selected from sequences from a group consisting of mRNA, tRNA, rRNA, ncRNA, sgRNA, shRNA, siRNA, snRNA, miRNA, snoRNA, LNA.
  • nucleotide sequences can also be selected from a group comprising nucleic acid analog sequences consisting of GNA, PNA, TNA and morpholino.
  • Specific nucleotide sequences may also include, but are not limited to, fragments of sequences, oligonucleotides and polynucleotides.
  • a specific nucleotide sequence to be detected may have preferably at least 5, more preferably at least 25, more preferably at least 100 and most preferably at least 200 nucleotides.
  • nucleotide sequence refers to any nucleotide se- quence having chemical modifications.
  • Any nucleic acid sequence can be unique and a sequence can be specific for any organism, virus or derivative thereof.
  • organism refers to any living thing such as an animal, plant, fungus, or micro-organism. It is well-known that nucleotide sequences vary for example between organisms, species, breeds, subclasses and individuals. Any of the unique i.e. specific sequences can be used in the method of the invention. To find out these specific sequences, any conventional biotechnical or bioinfor- matic methods can be utilized.
  • the method of the invention can be used for detecting any specific nucleotides in samples comprising biological material.
  • the target nucleotides can originate from a sample under investigation or the target nucleotides can originate from other organisms or samples.
  • micro-organisms typically represent clear application areas within the fields of both biology and medicine.
  • the method can be used for detecting any micro-organisms, or combinations thereof.
  • micro-organism refers to any small organism or virus comprising nucleotide sequences.
  • a micro-organism can be selected from the group consisting of viruses, bacteria, protozoa, fungi and mold.
  • primer refers to a polynucleotide or oligonucleotide, preferably comprising 2-1000 nucleotides of DNA or RNA, more preferably 10-50 nucleotides and most preferably 15-35 nucleotides.
  • the primer can be in a single stranded or double stranded form. In a preferred embodiment of the invention, the primers are single stranded.
  • Covalent bonding includes but is not limited to ⁇ -bonding, ⁇ - bonding, metal-metal bonding, agostic interactions, and three-center two-electron bonds.
  • Non-covalent bonds include but are not limited to the following: hydrogen bonds, ionic interactions, ionic bonds, Van der Waals interactions, hydrophobic bonds, lipophilic interactions and hydrophilic interactions.
  • primers for PCR can for example be conjugated with helper molecules (see figure 1 ).
  • primers for PCR are designed for the nucleotide sequence bordering the specific nucleotide sequence.
  • helper molecule refers to a molecule linking at least two other molecules together. At least one helper molecule can be utilized in order to link other molecules. Helper molecules can also be used as pairs (e.g. both biotin and avidin may act as helper molecules in linking two other molecules together).
  • helper molecule can be selected from a group consisting of any protein such as streptavidin and avidin, antibody or receptor selectively binding to molecules, nucleic acid sequences, any biological compound such as carbohydrate or vitamin such as biotin, amino groups, carboxyl groups and synthetic polymers such as polyethers (e.g. polyethylene glycol (PEG)).
  • helper molecules can also be produced by any synthetic or recombinant techniques.
  • the helper molecule is selected from the group consisting of streptavidin, avidin and biotin. Furthermore, in a preferred embodiment of the invention the helper molecule pairs are streptavidin-biotin and/or avidin-biotin.
  • a helper molecule can be chemically or biologically immobilized on magnetic beads. Chemical or biological immobilization includes but is not limited to the above-mentioned covalent or non-covalent bonding. Helper molecules can also be immobilized on a device or platform such as a test strip used for lateral flow.
  • the use of 5' and/or 3' amino coupled PCR products and carboxylated beeds can be combined with e.g. bioti- nylated primers and strips sensitized with streptavidin or avidin.
  • magnetic bead refers to a nanoparti- cle or microparticle, which comprises magnetic material.
  • the magnetic features of the beads may be present all the time or appear in the presence of a magnetic field. Any magnetic beads, which do not interfere with the nucleotide hy- bridization or any other method steps, can be utilized in the method of the invention.
  • the magnetic beads comprise one or more materials selected from the group consisting of paramagnetic, superparamagnetic, ferromagnetic, anti- ferromagnetic and ferrimagnetic material and artificial magnetic material generally termed magnetic metamaterial, combinations thereof and hybrid materials.
  • Magnetic beads suitable for use in the method of the present invention include but are not limited to Co, Co alloys, Fe, ferrites, Cobalt nitride, Cobalt oxide, Co-Pd, Co-Pt, Co-Fe, Iron, Iron alloys, Fe-Au, Fe-Cr, Fe-N, Fe3O4, Fe-Pd, Fe-Pt, Fe-Zr-Nb-B, Mn-N, Nd-Fe-B, Nd-Fe-B-Nb-Cu, Ni, and Ni alloys.
  • magnetic beads can also comprise different layers or can be coated to achieve any additional properties. Different tags may be added to the beads for example for additional detection possibilities.
  • the properties of the magnetic beads, which can be monitored, can be any detectable properties such as magnetic properties, dyes, labels, colors or radioactive properties.
  • the magnetic beads can be of any size. However, typically the size (a mean diameter) of magnetic beads varies from 2 nm to 5 ⁇ m, preferably from 10 nm to 1 ⁇ m, more preferably from about 5 nm to about 700 nm, more preferably from about 5 nm to about 500 nm, and most preferably from about 50 nm to about 400 nm.
  • a shape of magnetic beads may vary and include but is not limited to spherical shape, disks, rods, coils, and fibers.
  • lateral flow tests can be used to detect the presence or absence of a target analyte in a sample.
  • many analytes including the nucleic acid products can be detected.
  • On the same platform such as a strip, one or several analytes can be tested simultaneously.
  • lateral flow refers to liquid flow in a material. At least part of the components of the sample is transported with the flow laterally through the material. In a lateral flow assay, one or more analytes are separated from a sample or from other analytes.
  • lateral flow binding assay is also used for these lateral flow assays, which are based on the binding of an analyte and any suitable binding particle, such as a helper molecule or molecules as described in page 6.
  • the binding may be achieved for example by placing the suitable binding particle on a plat form on which the sample is to be added.
  • Detection of an analyte can be direct visualization, e.g. visualization of a colour on a strip, or indirect visualization, e.g. monitoring the magnetism of a sample on a strip.
  • a lateral flow assay can be used for determining the presence, ab- sence, amount or concentration of a particular analyte, i.e. nucleotide sequence, in a fluid sample. Lateral flow tests can be used for the qualitative, semiquantitative or quantitative detection of analytes.
  • Lateral flow binding assay platforms or devices may comprise a fluid sample receiving section, an analyte detection section and/or an absorption section. Lateral flow assays are preferably on a flat surface, such as a strip, capillaries, channels, microfluidic channels or matrixes.
  • Strips are usually made of synthetic materials such as nylon or polymers, ceramic materials, glass fiber or of natural materials such as cellulose or their derivatives such as nitrocellulose.
  • the use of magnetic particles in a lateral flow method is usually accompanied by an electronic reader to assess the test result. Test results can usually be read after 0.5-20 minutes, preferably after 1-15 minutes, most preferably after 1-10 minutes of the application of a sample on a strip.
  • specific sequences in the sample cause the magnetic beads to bind to singles- tranded flanking sequences of the specific sequence immobilized on the assay strip.
  • the magnetism of the sample is monitored. Therefore, the detection method itself can be based on magnetic beads. In one preferred embodiment of the method of the invention the monitoring is a quantitative or qualitative measurement. For example, the higher the magnetism the more amplified nucleotide sequences a sample contains. The test can naturally also be designed as a +/- test.
  • Real-time detection is also possible in the lateral flow method.
  • the magnetism can be measured during the lateral flow.
  • a sensitive magnetometric reader is used for monitoring the magnetism of the sample.
  • monitoring the magnetism of the sample is done by utilizing inductance.
  • a change in inductance correlating to the content of the magnetically labeled specific sequence is monitored.
  • the document WO2007122293 presents a device for the qualitative or quantitative measurement of a magnetically labelled analyte.
  • the device includes a coil arrangement, formed of at least one measuring coil and a reference coil arranged in connection with it, for measuring the analyte from a sample absorbed in a test base. From the signal of the coil arrangement a change in inductance correlating to the content of the magnetically labelled analyte is arranged to be detected.
  • the change in inductance is arranged to be detected from a change ( ⁇ A, ⁇ ) in amplitude and/or phase appearing in the output signal of the coil arrangement, which is arranged to be measured at the frequency of the input signal.
  • the device includes a coil arrangement compensating error signals, for example, for compensating error signals caused by the environment and/or magnetic particles bound unspecifically to the test base.
  • Document WO2007122293 also relates to a corresponding method for the qualitative or quantitative measurement of an analyte, in which a test base is used to measure the analyte, and in which method a sample is absorbed into the test base, and the test base is analysed using a coil arrangement, from the signal of which a change in inductance correlating to the content of the magnetically labelled analyte is detected.
  • the change in inductance is detected from a change ( ⁇ A, ⁇ ) in amplitude and/or phase appearing in the output signal of the coil arrangement, which is measured at the frequency of the input signal and, in addition, error signals caused by the environment and/or magnetic particles bound unspecifically to the test base are compensated by a coil arrangement.
  • the coil arrangement includes at least one measuring coil and a reference coil arranged in connection with it, and in order to increase the inductive reactance of the measuring coil to be greater than the resistance, the measurement is performed using a measuring frequency of 10 6 — 10 8 Hz.
  • the compensation can be performed using a differential coil arrangement and /or the sample can be absorbed to a test base, which is integrated interactively with at least part of the coil arrangement.
  • sample refers to any sample compris- ing living material, viruses or derivatives thereof.
  • the sample can be taken from any living organism such as eukaryotes, prokaryotes, animals, human beings, and plants such as crops or fruits.
  • a sample comprising biological material can be a tissue sample from a body, preferably fluid such as blood, or for example urine, saliva, serum, plasma, whole blood, feces, spu- turn, exudate.
  • the sample is a biological sample.
  • the sample may also contain, in addition to biological material, any non-biological material e.g. water, sand, plastic, or metal.
  • the sample may be any environmental sample such as a sample of water, soils, dust, vegetation, or food, or environmental swabs such as from food processing plants.
  • the sample of the invention may be untreated or treated before using in the method of the invention.
  • DNA/RNA may have been extracted from the sample for the method of the invention.
  • said DNA/RNA sample may have been pre-treated with e.g. restriction enzymes in order to cut the DNA into appropriate pieces or the specific sequence under study may have been amplified by the polymerase chain reaction (PCR) using appropriate primers.
  • PCR polymerase chain reaction
  • the sam- pie has been pre-treated by amplifying the specific sequence of the sample by PCR.
  • the specific nucleotide sequence has been amplified by the nucleotide primers bordering the specific nucleotide sequence.
  • the sample has been pre-treated with a restriction enzyme or enzymes.
  • the nucleotides of the sample can be extracted before the amplification or restriction enzyme treatment. However, the extraction step is optional.
  • the method of the invention enables rapid and effective screening, detection or analysis of a multitude of nucleotide sequences.
  • the method is usable in diagnostics as well as in any research or analysis.
  • the method of the invention enables the discovery or identification of for example viruses, organisms, species, breeds, subclasses and individuals.
  • the single stranded DNA-phmers are synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 1 ).
  • the DNA sequence under study contained in the sample is amplified by using PCR and primers labelled with e.g. biotin.
  • the amplified DNA, containing biotin at its 5' and/or 3' ends hybridizes with the ssDNA presented by the magnetic beads.
  • the sample is applied onto the test strip (e.g. Magnasense) where recombinant streptavidin/avidin has been immobilized. Beads to which DNA has hybridized will bind to the immobilized streptavidin/avidin and monitoring of the magnetism e.g. by employing the technology provided by Magnasense is conducted.
  • Magnetic beads coated with a helper molecule A helper molecule, e.g. recombinant streptavidin/avidin, is immobilized on the surface of the magnetic bead (Fig. 2).
  • the gene under study is amplified by PCR using primers conjugated with e.g. biotin or another helper molecule. If the DNA is amplified, i.e. if the DNA sequence under study was present in the sample, the magnetic beads will bind to either streptavidin/avidin immobilized on the strip or an other pair of helper molecule used to conjugate the other pair of primer.
  • Single stranded DNA-phmers are synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 3).
  • the DNA contained within the sample is pre-treated with appropriate restriction en- zymes and allowed to hybridize with the DNA displayed on the magnetic bead. Flanking sequences in relation to the DNA sequence displayed on the beads are immobilized on the strip, allowing specific hybridization of the DNA bound to the beads to that immobilized on the strip.
  • Magnetic beads are furnished with a helper molecule, e.g. recombinant streptavidin/avidin (Fig. 4).
  • the gene under study is amplified by PCR using primers conjugated with e.g. biotin.
  • the corresponding amplified DNA binds to the beads via biotin.
  • Strips are immobilized with DNA representing e.g. pro- truding ends of DNA relative to that hybridized to the beads.
  • the single stranded DNA-primers were synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 1 ).
  • the DNA sequence under study contained in the sample was amplified by using PCR and primers labelled with biotin.
  • the amplified DNA, containing biotin at its 5' and/or 3' ends hybridized with the ssDNA presented by the magnetic beads.
  • the sample was applied onto the test strip (e.g. Magnasense) where recombinant streptavidin had been immobilized. Beads to which DNA had hybridized bound to the immobilized streptavidin and monitoring of the magnet- ism by employing the technology provided by Magnasense was conducted. The higher was the magnetism the more the sample contained amplified DNA.
  • the test strip e.g. Magnasense
  • Example 2 Magnetic beads coated with a helper molecule
  • helper molecule recombinant streptavidin
  • Fig. 2 A helper molecule, recombinant streptavidin, was immobilized on the surface of the magnetic bead (Fig. 2).
  • the gene under study was amplified by PCR using primers conjugated with biotin.
  • the magnetic beads bound to either streptavidin immobilized on the strip or another pair of helper molecule used to conjugate the other pair of primer.
  • Single stranded DNA-primers were synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 3).
  • the DNA contained within the sample was pre-treated with appropriate restriction enzymes and allowed to hybridize with the DNA displayed on the magnetic bead. Flanking sequences in relation to the DNA sequence displayed on the beads were immobilized on the strip, allowing specific hybridization of the DNA bound to the beads to that immobilized on the strip.
  • Example 4 Magnetic beads coated with a helper molecule and hybridizing to DNA immobilized on the strip
  • Magnetic beads were furnished with a helper molecule, e.g. recombinant streptavidin (Fig. 2).
  • the gene under study is amplified by PCR using primers conjugated with e.g. biotin.
  • the corresponding amplified DNA binds to the beads via biotin.
  • Strips are immobilized with DNA representing e.g. protruding ends of DNA relative to that hybridized to the beads.
  • Both membranes is coated with a plastic film
  • 0.5XSSC-buffer (1 .5 M NaCI, 0.15 M tri-sodium citrate pH 7), 300 ⁇ l per wash.
  • the beads are captured by using the magnetic stand whereafter the washing solution is carefully removed.
  • the beads are resus- pended in 100 ⁇ l of 10X SSC or 0.5XSSC-buffer.
  • the biotinylated PCR product (50 ⁇ l) and the washed streptavidin- coated beads (10 ⁇ l) are incubated for 2 hrs or overnight at + 4°C with gentle rotation of the tube.
  • the binding reaction should be complete (biotin/strep- tavidin complexes).
  • the beads are collected by placing the tube into the magnetic stand until the beads are attached to the inner wall of the tube (approximately 60 seconds). The washed beads are then resuspended in 100 ⁇ l of 0.2% Tween 20/TBS pH 7.5.
  • the detection zone (the sence oligo) is set up on the nitrocellulose membrane (7x7 cm) by using a plastic ruler.
  • the solution containing the sence oligo (100 pmol/ ⁇ l) is stamped on the nitrocellulose membrane 3 cm from the upper side, giving a zone width of approximately 1-2 mm.
  • the amino coupled PCR products bind to carboxylated beads.
  • the Lateral Flow strip is composed of roughly 7-9 components. These are:
  • conjugate pad contains detection beads (conjugate) adsorbed with antibodies or antigens specific to the analyte being detected 3. Detection Conjugate

Abstract

The present invention relates to the fields of life sciences and genetics. Specifically, the invention relates to a method for detecting biological components. More specifically, the present invention relates to a novel method for detecting a specific nucleotide sequence in a sample. Furthermore, the present invention also relates to a use of the method of the invention for identifying viruses, micro-organisms or derivatives thereof from a sample and a use of the method of the invention for identifying mutations of nucleotide sequences from a sample.

Description

A method for detecting specific nucleotide sequences
Field of the invention
The present invention relates to the fields of life sciences and genetics. Specifically, the invention relates to a method for detecting biological com- ponents. More specifically, the present invention relates to a novel method for detecting a specific nucleotide sequence in a sample. Furthermore, the present invention also relates to a use of the method of the invention for identifying viruses, micro-organisms or derivatives thereof from a sample and to a use of the method of the invention for identifying mutations of nucleotide sequences from a sample.
Background of the invention
Several methods can be used for detecting nucleotides from a variety of samples. Various methods provide possibilities for both qualitative and quantitative determination of specific sequences. In previous detection methods, magnetic nanoparticles have been combined with nucleic acids, followed by magnetic detection. Examples of these publications include WO 2006/131892 A2, WO2008/020381 A2, US2005/0100930 A1 and WO01 /14591 A1. However, lateral flow assays have not been used in these publications. Furthermore, WO 03/054523 A2 describes a method for measuring an areal density of magnetic nanoparticles on a substrate on a microarray. Publication WO2007/092909 A2, on the other hand, describes a method for detecting nucleic acids by utilizing specific molecular interaction sensors.
Compared to the above-mentioned methods, the present invention provides a simple and inexpensive way as well as ready to use tools to detect specific nucleotide sequences. Compared to the above-mentioned methods, the present invention provides a more specific, more sensitive and more reliable method. Advantages of the method are also the speed by which it can be conducted as compared to e.g. conventional microbial cultivation methods and the ease by which such an analysis can be carried out. Combined assay steps of the method are elementary to one skilled in the art. This invention provides an optimal method for detecting specific nucleotide sequences.
The invention has a significant commercial potential. A method based on detection of specific nucleotide sequences is applicable to a large variety of areas including biology and medicine. By using the method of the invention or variations thereof it is possible to quickly test for the presence and/or the amount of a specific sequence, e.g. DNA and/or RNA sequence, present in a sample. In addition, different sequences can be detected simultaneously.
Therefore, the improvements of the present invention enhance the analysis of the samples.
Brief description of the invention
The object of the invention is to provide novel methods and means for studying samples, more specifically for identifying properties of the sample.
The object of the invention is also to provide a diagnostic method, which is simply based on the measurement of properties of magnetic beads as well as the specificity of nucleotide sequences.
The present invention relates to a novel method for detecting a specific nucleotide sequence in a sample, characterized in that the method comprises the following steps: a) a helper molecule and/or nucleotide primer specific for said nucleotide sequence are immobilized on magnetic beads; b) the magnetic beads obtained in step a) are brought into contact with the sample; c) the sample obtained in step b) is run in a lateral flow assay; d) the specific nucleotide sequences present in the sample are detected by monitoring the properties of the magnetic beads.
Furthermore, the present invention also relates to a use of the method of the invention for identifying viruses, micro-organisms or derivatives thereof from a sample. Also, the present invention relates to a use of the method of the invention for identifying mutations of nucleotide sequences from a sample.
The present invention provides a specific, sensitive, reliable, fast, and easy method for detecting a specific nucleotide sequence in a sample.
Brief description of the figures
Figure 1 shows the method of the invention utilizing magnetic beads displaying chemically bound DNA/RNA sequences. The single stranded DNA- primers are synthesized to contain one or several chemically modified nucleotides (e.g. carboxylated magnetic beeds/amino coupled DNA primers) enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead. The DNA sequence under study contained in the sample is am- plified by using PCR and primers labelled with e.g. biotin. The amplified DNA, containing biotin at its 5' and/or 3' ends hybridizes with the ssDNA presented by the magnetic beads. The sample is applied onto the test strip (e.g. Magna- sense) where recombinant streptavid in/avid in has been immobilized. Beads to which DNA has hybridized will bind to the immobilized streptavid in/avid in and monitoring of the properties of magnetic beads e.g. by employing the technology provided by Magnasense is conducted.
Figure 2 shows the method of the invention utilizing magnetic beads coated with a helper molecule. A helper molecule, e.g. recombinant strepta- vidin/avidin, is immobilized on the surface of the magnetic bead. The gene un- der study is amplified by PCR using primers conjugated with e.g. biotin or another helper molecule as explained above for Figure 1 . If the DNA is amplified, i.e. if the DNA sequence under study was present in the sample, the amplified nucleotide sequences, carrying biotin at both ends, will bind to the said magnetic beads, and the magnetic beads will further bind to either streptavid in/avid in immobilized on the strip or another pair of helper molecule used to conjugate the other pair of primer.
Figure 3 shows the method of the invention utilizing detection of DNA by magnetic beads without PCR-mediated amplification of the sample. Single stranded DNA-phmers are synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead. The DNA contained within the sample is pre-treated with appropriate restriction enzymes and allowed to hybridize with the DNA displayed on the magnetic bead. Flanking sequences in relation to the DNA sequence displayed on the beads are immobilized on the strip, allowing specific hybridization of the DNA bound to the beads to that immobilized on the strip.
Figure 4 shows the method of the invention utilizing magnetic beads coated with a helper molecule and hybridization to DNA immobilized on the strip. Magnetic beads are furnished with a helper molecule, e.g. recombinant streptavidin/avidin. The gene under study is amplified by PCR using primers conjugated with e.g. biotin. The corresponding amplified DNA binds to the beads via biotin. Strips are immobilized with DNA representing e.g. protruding ends of DNA relative to that hybridized to the beads.
Detailed description of the invention In one preferred embodiment of the method, more than one specific nucleotide sequence is detected simultaneously. In one preferred embodiment of the method of the invention, in step b), i.e. the step wherein the magnetic beads obtained in step a) are brought into contact with the sample, the helper molecules immobilized on magnetic beads are bound to another helper molecule conjugated to nucleotide primers, which border the specific nucleotide sequence.
In one preferred embodiment of the method of the invention, in step b), i.e. the step wherein the magnetic beads obtained in step a) are brought into contact with the sample, the primers immobilized on magnetic beads are hybridized with the specific nucleotide sequence(s) of the sample.
Specific nucleotide sequences
The analysis method of the invention is based on detection of nucleotide sequences. Even only one specific nucleotide sequence present in the sample is adequate for detection method of the invention. On the other hand, the sample may comprise a great number of nucleotide sequences, either un- defined or specific sequences. Therefore, depending on the amount of nucleotide sequences in the sample, different method steps can be applied. For example, amplification methods can be carried out when a number of nucleotide sequences is low, and a use of restriction enzymes is possible when a large number of nucleotide sequences is present in a sample. In one preferred embodiment of the method of the invention the specific nucleotide sequence is a RNA and/or DNA sequence. For example, specific DNA sequences can be selected from sequences from a group consisting of genomic DNA, mtDNA, cDNA, plasmids, kosmids, BACs, YACs and HACs. For example, specific RNA sequences can be selected from sequences from a group consisting of mRNA, tRNA, rRNA, ncRNA, sgRNA, shRNA, siRNA, snRNA, miRNA, snoRNA, LNA. Furthermore, nucleotide sequences can also be selected from a group comprising nucleic acid analog sequences consisting of GNA, PNA, TNA and morpholino.
Specific nucleotide sequences may also include, but are not limited to, fragments of sequences, oligonucleotides and polynucleotides. A specific nucleotide sequence to be detected may have preferably at least 5, more preferably at least 25, more preferably at least 100 and most preferably at least 200 nucleotides.
Furthermore, the nucleotide sequence refers to any nucleotide se- quence having chemical modifications. Any nucleic acid sequence can be unique and a sequence can be specific for any organism, virus or derivative thereof. As used herein the expression "organism" refers to any living thing such as an animal, plant, fungus, or micro-organism. It is well-known that nucleotide sequences vary for example between organisms, species, breeds, subclasses and individuals. Any of the unique i.e. specific sequences can be used in the method of the invention. To find out these specific sequences, any conventional biotechnical or bioinfor- matic methods can be utilized.
The method of the invention can be used for detecting any specific nucleotides in samples comprising biological material. The target nucleotides can originate from a sample under investigation or the target nucleotides can originate from other organisms or samples.
Typically, sequences of micro-organisms represent clear application areas within the fields of both biology and medicine. In a preferred embodiment of the invention, the method can be used for detecting any micro-organisms, or combinations thereof. As used herein the expression "micro-organism" refers to any small organism or virus comprising nucleotide sequences. For example a micro-organism can be selected from the group consisting of viruses, bacteria, protozoa, fungi and mold.
Primers
As used herein, the expression "primer" or "nucleotide primer" refers to a polynucleotide or oligonucleotide, preferably comprising 2-1000 nucleotides of DNA or RNA, more preferably 10-50 nucleotides and most preferably 15-35 nucleotides. The primer can be in a single stranded or double stranded form. In a preferred embodiment of the invention, the primers are single stranded.
Primers can be attached to the magnetic beads by covalent or non- covalent bonds. Covalent bonding includes but is not limited to σ-bonding, π- bonding, metal-metal bonding, agostic interactions, and three-center two-electron bonds. Non-covalent bonds include but are not limited to the following: hydrogen bonds, ionic interactions, ionic bonds, Van der Waals interactions, hydrophobic bonds, lipophilic interactions and hydrophilic interactions.
It is possible to attach primers to any molecules by any conventional chemical or biological conjugation such as using antibody-antigen, receptor- molecule or biotin-avidin interaction. Primers for PCR can for example be conjugated with helper molecules (see figure 1 ). In a preferred embodiment of the invention, primers for PCR are designed for the nucleotide sequence bordering the specific nucleotide sequence.
Helper molecules
A used herein the expression "helper molecule" refers to a molecule linking at least two other molecules together. At least one helper molecule can be utilized in order to link other molecules. Helper molecules can also be used as pairs (e.g. both biotin and avidin may act as helper molecules in linking two other molecules together).
For example, a helper molecule can be selected from a group consisting of any protein such as streptavidin and avidin, antibody or receptor selectively binding to molecules, nucleic acid sequences, any biological compound such as carbohydrate or vitamin such as biotin, amino groups, carboxyl groups and synthetic polymers such as polyethers (e.g. polyethylene glycol (PEG)). In addition to using natural helper molecules, helper molecules can also be produced by any synthetic or recombinant techniques.
In one preferred embodiment of the method of the invention the helper molecule is selected from the group consisting of streptavidin, avidin and biotin. Furthermore, in a preferred embodiment of the invention the helper molecule pairs are streptavidin-biotin and/or avidin-biotin.
A helper molecule can be chemically or biologically immobilized on magnetic beads. Chemical or biological immobilization includes but is not limited to the above-mentioned covalent or non-covalent bonding. Helper molecules can also be immobilized on a device or platform such as a test strip used for lateral flow.
In one embodiment of the invention, the use of 5' and/or 3' amino coupled PCR products and carboxylated beeds can be combined with e.g. bioti- nylated primers and strips sensitized with streptavidin or avidin.
Magnetic beads
As used herein the expression "magnetic bead" refers to a nanoparti- cle or microparticle, which comprises magnetic material. The magnetic features of the beads may be present all the time or appear in the presence of a magnetic field. Any magnetic beads, which do not interfere with the nucleotide hy- bridization or any other method steps, can be utilized in the method of the invention.
The magnetic beads comprise one or more materials selected from the group consisting of paramagnetic, superparamagnetic, ferromagnetic, anti- ferromagnetic and ferrimagnetic material and artificial magnetic material generally termed magnetic metamaterial, combinations thereof and hybrid materials. Magnetic beads suitable for use in the method of the present invention include but are not limited to Co, Co alloys, Fe, ferrites, Cobalt nitride, Cobalt oxide, Co-Pd, Co-Pt, Co-Fe, Iron, Iron alloys, Fe-Au, Fe-Cr, Fe-N, Fe3O4, Fe-Pd, Fe-Pt, Fe-Zr-Nb-B, Mn-N, Nd-Fe-B, Nd-Fe-B-Nb-Cu, Ni, and Ni alloys.
Furthermore, magnetic beads can also comprise different layers or can be coated to achieve any additional properties. Different tags may be added to the beads for example for additional detection possibilities. The properties of the magnetic beads, which can be monitored, can be any detectable properties such as magnetic properties, dyes, labels, colors or radioactive properties.
The magnetic beads can be of any size. However, typically the size (a mean diameter) of magnetic beads varies from 2 nm to 5 μm, preferably from 10 nm to 1 μm, more preferably from about 5 nm to about 700 nm, more preferably from about 5 nm to about 500 nm, and most preferably from about 50 nm to about 400 nm. A shape of magnetic beads may vary and include but is not limited to spherical shape, disks, rods, coils, and fibers.
Lateral flow
A sample and therefore also specific nucleotide sequences present in the sample are exposed to lateral flow. Lateral flow tests can be used to detect the presence or absence of a target analyte in a sample. In addition, many analytes including the nucleic acid products can be detected. On the same platform such as a strip, one or several analytes can be tested simultaneously. As used herein, "lateral flow" refers to liquid flow in a material. At least part of the components of the sample is transported with the flow laterally through the material. In a lateral flow assay, one or more analytes are separated from a sample or from other analytes.
The term lateral flow binding assay is also used for these lateral flow assays, which are based on the binding of an analyte and any suitable binding particle, such as a helper molecule or molecules as described in page 6. The binding may be achieved for example by placing the suitable binding particle on a plat form on which the sample is to be added. Detection of an analyte can be direct visualization, e.g. visualization of a colour on a strip, or indirect visualization, e.g. monitoring the magnetism of a sample on a strip.
A lateral flow assay can be used for determining the presence, ab- sence, amount or concentration of a particular analyte, i.e. nucleotide sequence, in a fluid sample. Lateral flow tests can be used for the qualitative, semiquantitative or quantitative detection of analytes.
Lateral flow binding assay platforms or devices may comprise a fluid sample receiving section, an analyte detection section and/or an absorption section. Lateral flow assays are preferably on a flat surface, such as a strip, capillaries, channels, microfluidic channels or matrixes.
Strips are usually made of synthetic materials such as nylon or polymers, ceramic materials, glass fiber or of natural materials such as cellulose or their derivatives such as nitrocellulose. The use of magnetic particles in a lateral flow method is usually accompanied by an electronic reader to assess the test result. Test results can usually be read after 0.5-20 minutes, preferably after 1-15 minutes, most preferably after 1-10 minutes of the application of a sample on a strip.
In one preferred embodiment of the method, during lateral flow, specific sequences in the sample cause the magnetic beads to bind to helper molecules immobilized on the assay strip.
In one preferred embodiment of the method, during lateral flow, specific sequences in the sample cause the magnetic beads to bind to singles- tranded flanking sequences of the specific sequence immobilized on the assay strip.
Devices, detection and measurements
In one preferred embodiment of the method, the magnetism of the sample is monitored. Therefore, the detection method itself can be based on magnetic beads. In one preferred embodiment of the method of the invention the monitoring is a quantitative or qualitative measurement. For example, the higher the magnetism the more amplified nucleotide sequences a sample contains. The test can naturally also be designed as a +/- test.
Real-time detection is also possible in the lateral flow method. For example the magnetism can be measured during the lateral flow. In one preferred embodiment of the method a sensitive magnetometric reader is used for monitoring the magnetism of the sample.
In one preferred embodiment of the method monitoring the magnetism of the sample is done by utilizing inductance. In one preferred embodiment of the method of the invention, a change in inductance correlating to the content of the magnetically labeled specific sequence is monitored.
Various devices and methods can be utilized in the method of the invention. However, most preferable are the device and the method described in the patent appl ication WO2007/122293 A1.
Magnasense has introduced a sensitive magnetometric reader for lateral flow assays. The document WO2007122293 presents a device for the qualitative or quantitative measurement of a magnetically labelled analyte. The device includes a coil arrangement, formed of at least one measuring coil and a reference coil arranged in connection with it, for measuring the analyte from a sample absorbed in a test base. From the signal of the coil arrangement a change in inductance correlating to the content of the magnetically labelled analyte is arranged to be detected. The change in inductance is arranged to be detected from a change (ΔA, ΔΦ) in amplitude and/or phase appearing in the output signal of the coil arrangement, which is arranged to be measured at the frequency of the input signal. In addition, the device includes a coil arrangement compensating error signals, for example, for compensating error signals caused by the environment and/or magnetic particles bound unspecifically to the test base. Document WO2007122293 also relates to a corresponding method for the qualitative or quantitative measurement of an analyte, in which a test base is used to measure the analyte, and in which method a sample is absorbed into the test base, and the test base is analysed using a coil arrangement, from the signal of which a change in inductance correlating to the content of the magnetically labelled analyte is detected.
The change in inductance is detected from a change (ΔA, ΔΦ) in amplitude and/or phase appearing in the output signal of the coil arrangement, which is measured at the frequency of the input signal and, in addition, error signals caused by the environment and/or magnetic particles bound unspecifically to the test base are compensated by a coil arrangement. In WO2007122293, the coil arrangement includes at least one measuring coil and a reference coil arranged in connection with it, and in order to increase the inductive reactance of the measuring coil to be greater than the resistance, the measurement is performed using a measuring frequency of 106— 108 Hz. Furthermore, the compensation can be performed using a differential coil arrangement and /or the sample can be absorbed to a test base, which is integrated interactively with at least part of the coil arrangement.
Sample
As used herein the expression "sample" refers to any sample compris- ing living material, viruses or derivatives thereof. For example, the sample can be taken from any living organism such as eukaryotes, prokaryotes, animals, human beings, and plants such as crops or fruits. A sample comprising biological material can be a tissue sample from a body, preferably fluid such as blood, or for example urine, saliva, serum, plasma, whole blood, feces, spu- turn, exudate. In a preferred embodiment of the invention, the sample is a biological sample.
However, the sample may also contain, in addition to biological material, any non-biological material e.g. water, sand, plastic, or metal. For example, the sample may be any environmental sample such as a sample of water, soils, dust, vegetation, or food, or environmental swabs such as from food processing plants.
The sample of the invention may be untreated or treated before using in the method of the invention. For example, DNA/RNA may have been extracted from the sample for the method of the invention. Furthermore, for example, said DNA/RNA sample may have been pre-treated with e.g. restriction enzymes in order to cut the DNA into appropriate pieces or the specific sequence under study may have been amplified by the polymerase chain reaction (PCR) using appropriate primers.
In one preferred embodiment of the method of the invention the sam- pie has been pre-treated by amplifying the specific sequence of the sample by PCR.
In one preferred embodiment of the method of the invention, the specific nucleotide sequence has been amplified by the nucleotide primers bordering the specific nucleotide sequence. In one preferred embodiment of the method of the invention the sample has been pre-treated with a restriction enzyme or enzymes. According to one preferred embodiment of the method, the nucleotides of the sample can be extracted before the amplification or restriction enzyme treatment. However, the extraction step is optional.
Utility The method of the invention enables rapid and effective screening, detection or analysis of a multitude of nucleotide sequences. The method is usable in diagnostics as well as in any research or analysis.
Furthermore, the method of the invention enables the discovery or identification of for example viruses, organisms, species, breeds, subclasses and individuals.
Rapid analysis of the biological or medical samples becomes available through novel method of the invention, and furthermore opens the way for more comprehensive, profound, and accurate analysis of the sample.
Examples of embodiments of the invention There are several ways to conduct assays based on the technology of this invention. Four examples describing the basic principles of the invention are presented below but are not intended to be limiting in any way.
Magnetic beads displaying chemically bound DNA/RNA sequences
The single stranded DNA-phmers are synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 1 ). The DNA sequence under study contained in the sample is amplified by using PCR and primers labelled with e.g. biotin. The amplified DNA, containing biotin at its 5' and/or 3' ends hybridizes with the ssDNA presented by the magnetic beads. The sample is applied onto the test strip (e.g. Magnasense) where recombinant streptavidin/avidin has been immobilized. Beads to which DNA has hybridized will bind to the immobilized streptavidin/avidin and monitoring of the magnetism e.g. by employing the technology provided by Magnasense is conducted.
Magnetic beads coated with a helper molecule A helper molecule, e.g. recombinant streptavidin/avidin, is immobilized on the surface of the magnetic bead (Fig. 2). The gene under study is amplified by PCR using primers conjugated with e.g. biotin or another helper molecule. If the DNA is amplified, i.e. if the DNA sequence under study was present in the sample, the magnetic beads will bind to either streptavidin/avidin immobilized on the strip or an other pair of helper molecule used to conjugate the other pair of primer.
Detection of DNA by magnetic beads without PCR-mediated amplification of the sample
Single stranded DNA-phmers are synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 3). The DNA contained within the sample is pre-treated with appropriate restriction en- zymes and allowed to hybridize with the DNA displayed on the magnetic bead. Flanking sequences in relation to the DNA sequence displayed on the beads are immobilized on the strip, allowing specific hybridization of the DNA bound to the beads to that immobilized on the strip.
Magnetic beads coated with a helper molecule and hybridizing to DNA immobilized on the strip
Magnetic beads are furnished with a helper molecule, e.g. recombinant streptavidin/avidin (Fig. 4). The gene under study is amplified by PCR using primers conjugated with e.g. biotin. The corresponding amplified DNA binds to the beads via biotin. Strips are immobilized with DNA representing e.g. pro- truding ends of DNA relative to that hybridized to the beads.
The present invention is illustrated by the following examples, which are not intended to be limiting in any way.
Example 1. Magnetic beads displaying chemically bound DNA/RNA se- quences
The single stranded DNA-primers were synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 1 ). The DNA sequence under study contained in the sample was amplified by using PCR and primers labelled with biotin. The amplified DNA, containing biotin at its 5' and/or 3' ends hybridized with the ssDNA presented by the magnetic beads.
The sample was applied onto the test strip (e.g. Magnasense) where recombinant streptavidin had been immobilized. Beads to which DNA had hybridized bound to the immobilized streptavidin and monitoring of the magnet- ism by employing the technology provided by Magnasense was conducted. The higher was the magnetism the more the sample contained amplified DNA.
Example 2. Magnetic beads coated with a helper molecule
A helper molecule, recombinant streptavidin, was immobilized on the surface of the magnetic bead (Fig. 2). The gene under study was amplified by PCR using primers conjugated with biotin. The magnetic beads bound to either streptavidin immobilized on the strip or another pair of helper molecule used to conjugate the other pair of primer.
Example 3. Detection of DNA by magnetic beads without PCR-mediated amplification of the sample
Single stranded DNA-primers were synthesized to contain one or several chemically modified nucleotides enabling binding to and thereby display and exposure of the corresponding DNA on the magnetic bead (Fig. 3). The DNA contained within the sample was pre-treated with appropriate restriction enzymes and allowed to hybridize with the DNA displayed on the magnetic bead. Flanking sequences in relation to the DNA sequence displayed on the beads were immobilized on the strip, allowing specific hybridization of the DNA bound to the beads to that immobilized on the strip.
Example 4. Magnetic beads coated with a helper molecule and hybridizing to DNA immobilized on the strip
Magnetic beads were furnished with a helper molecule, e.g. recombinant streptavidin (Fig. 2). The gene under study is amplified by PCR using primers conjugated with e.g. biotin. The corresponding amplified DNA binds to the beads via biotin. Strips are immobilized with DNA representing e.g. protruding ends of DNA relative to that hybridized to the beads.
Example 5. General information
In the above-mentioned examples, following products and proceedings were utilized: a) Products
Product Cat. No. Supplier
Streptavidin magnetic particles BE-M08/0.3 Merck/Estapor
Polystyrene magnetic particles Streptavidin from Streptomyces
HanoRapid NC membrane 51515 Hanomy
HiFlow Plus HFB12004 Millipore
Both membranes is coated with a plastic film
Dynazyme Il DNA Polymerase F-501 L Finnzymes
Dynazyme 10xBuffer F-511 Finnzymes
DNTP mix F-560L Finnzymes
Oliαos Biomers
Escherichia coli: 16S rDNA sequence
Forward 5\ 3' or both biotin conjugated
5N (biotin) CAG CCA CAC TGG AAC TGA GA 20bp
Reverse 3N GTG CAA TAT TCC CCA CTG CT 20 bp Sence GCC TCC CGT AGG AGT CTG GAC CGT G 25 bp
b) PCR:
+95°C 5 min
+95°C 30 sec
+52°C 1 min (depending on the Tm 0C of the oligo)
+700C 1 min → total 35 cycles
+72°C 5 min
+ 4°C
Amplification was verified by gel electrophoresis. c) Washing of magnetic beads:
An amount of 100 μl of streptavidin coated magnetic beads are captured by placing the tube into a magnetic stand until the beads have been collected/attached at the side wall of the tube (approximately 60 seconds). The magnetic beads are washed three times with 10XSSC-buffer or
0.5XSSC-buffer (1 .5 M NaCI, 0.15 M tri-sodium citrate pH 7), 300 μl per wash.
After each wash, the beads are captured by using the magnetic stand whereafter the washing solution is carefully removed. The beads are resus- pended in 100 μl of 10X SSC or 0.5XSSC-buffer. The biotinylated PCR product (50 μl) and the washed streptavidin- coated beads (10 μl) are incubated for 2 hrs or overnight at + 4°C with gentle rotation of the tube.
After incubation, the binding reaction should be complete (biotin/strep- tavidin complexes). The beads are collected by placing the tube into the magnetic stand until the beads are attached to the inner wall of the tube (approximately 60 seconds). The washed beads are then resuspended in 100 μl of 0.2% Tween 20/TBS pH 7.5.
d) Preparation on the immunostrips: The detection zone (the sence oligo) is set up on the nitrocellulose membrane (7x7 cm) by using a plastic ruler. The solution containing the sence oligo (100 pmol/μl) is stamped on the nitrocellulose membrane 3 cm from the upper side, giving a zone width of approximately 1-2 mm.
The membrane is then air dried for approximately 20 min whereafter it is soaked for 1 hour in a blocking solution (3% BSA/0, 2% Tween 20/TBS pH 7.5) followed by washing with 0.2% Tween 20/TBS pH 7.5. The membrane is then cut into strips of about 7 cm in length and 4 mm in width. e) Carboxylated paramagnetic beads:
The amino coupled PCR products bind to carboxylated beads.
Cat. No. M 1 -020/50 polystyrene beads Merck/Estapor
Forward 5'amino
5 " CAG CCA CAC TGG AAC TGA GA 20bp
Reverse 3 " GTG CAA TAT TCC CCA CTG CT 20 bp Sence GCC TCC CGT AGG AGT CTG GAC CGT G 25 bp
f) Covalent conjugation of magnetic beads:
Wash 125 μl of carboxylated magnetic beads twice in 0.5 ml of buffer
A (10 mM NaH2PO4 pH 6.0) by placing the tube in the magnetic stand until the beads have collected at the side of the tube (approximately 60 seconds). Dissolve 30 mg of carbodiimide (1 -ethyl-3-(3-Dimethylaminopropyl) carbodiimide) and 18 mg of NHS (N-hydroxysuccinimide) in 3 ml of buffer A.
Add 1 .25 ml of this solution to the pellet of magnetic beads.
Place the tube under agitation for 15 minutes at room temperature.
Wash the beads with 0.5 ml of buffer B (2 mM HCI). Resuspend the pellet of beads with 0.75 ml of buffer C (20 mM
NaH2PO4 /Na2HPO4 pH 7.5)
Add immediately the amino conjugated PCR product 100-200 μl
(approximately) and mix. Incubate the tube under agitation for 2 hours at room temperature. Wash the coated beads three times with buffer C and resuspend in an appropriate buffer e.g. 20 mM NaH2PO4 /Na2HPO4 pH 7.5, 100 mM Glycin,
1 % BSA.
g) The basic components of the lateral flow/ICS test:
In one of its more common forms the Lateral Flow strip is composed of roughly 7-9 components. These are:
1. Sample Pad
2. Conjugate Pad- The conjugate pad contains detection beads (conjugate) adsorbed with antibodies or antigens specific to the analyte being detected 3. Detection Conjugate
4. Solid-phase Membrane
5. Test and control reagent lines
6. Absorbent Pad 7. Plastic-adhesive backing card
The following components are "optional" and are not necessary or included in many lateral flow platforms.
1. Laminate Tape
2. A Strip housing/Cassette
(RDT info, http://www.rapid-diagnostics.org/tech-lateral.htm, 1.11.2008)

Claims

Claims
1. A method for detecting a specific nucleotide sequence in a sample, characterized in that the method comprises the following steps: a) a helper molecule and/or nucleotide primer specific for said nucleotide sequence are immobilized on magnetic beads; b) the magnetic beads obtained in step a) are brought into contact with the sample; c) the sample obtained in step b) is run in a lateral flow assay; d) the specific nucleotide sequences present in the sample are detected by monitoring the properties of the magnetic beads.
2. The method according to claim ^ characterized by detecting more than one specific nucleotide sequence simultaneously.
3. The method according to claim 1 or 2, characterized in that in step b) the helper molecules immobilized on magnetic beads are bound to another helper molecule conjugated to nucleotide primers, which border the specific nucleotide sequence.
4. The method according to claim 1 or 2, characterized in that in step b) the primers immobilized on magnetic beads are hybridized with the specific nucleotide sequence(s) of the sample.
5. The method according to any one of the previous claims, characterized in that the sample has been pre-treated by amplifying the specific sequence of the sample by PCR.
6. The method according to claim 5, characterized in that the specific nucleotide sequence has been amplified by the nucleotide primers bordering the specific nucleotide sequence.
7. The method according to any one of the previous claims, characterized in that the sample has been pre-treated with a restriction enzyme or enzymes.
8. The method according to any one of the previous claims, characterized in that during lateral flow, specific sequences in the sample cause the magnetic beads to bind to helper molecules immobilized on the assay strip.
9. The method according to any one of the previous claims, char- acterized in that during lateral flow, specific sequences in the sample cause the magnetic beads to bind to singlestranded flanking sequences of the specific sequence immobilized on the assay strip.
10. The method according to any one of the previous claims, characterized in that the specific nucleotide sequence is a RNA and/or DNA sequence.
11. The method according to any one of the previous claims, characterized in that the helper molecule is selected from the group consisting of streptavidin, avidin and biotin.
12. The method according to any one of the previous claims, char- acterized in that monitoring is a quantitative or qualitative measurement.
13. The method according to any one of the previous claims, characterized in that the magnetism of the sample is monitored.
14. The method according to any one of the previous claims, characterized in that a sensitive magnetometric reader is used for monitoring the magnetism of the sample.
15. The method according to any one of the previous claims, characterized in that monitoring the magnetism of the sample is done by utilizing inductance.
16. The method according to any one of the previous claims, char- acterized in that, a change in inductance correlating to the content of the magnetically labeled specific sequence is monitored.
17. Use of the method according to any one of claims 1-16 for identifying viruses, micro-organisms or derivatives thereof from a sample.
18. Use of the method according to any one of claims 1-16 for iden- tifying mutations of nucleotide sequences from a sample.
PCT/FI2008/050666 2008-11-18 2008-11-18 A method for detecting specific nucleotide sequences WO2010058059A1 (en)

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