WO2004042084A1

WO2004042084A1 - Dry reagent strip and nucleic acid detection

Info

Publication number: WO2004042084A1
Application number: PCT/GR2003/000052
Authority: WO
Inventors: Theodoros Christopoulos; Penelope Ioannou Amarantidou
Original assignee: AUTOMATED ANALYSERS AND DIAGNOSTIC REAGENTS MEDICON HELLAS SA
Current assignee: AUTOMATED ANALYSERS AND DIAGNOSTIC REAGENTS MEDICON HELLAS SA
Priority date: 2002-11-07
Filing date: 2003-11-07
Publication date: 2004-05-21
Anticipated expiration: 2005-05-07
Also published as: EP1601787A1; GR1004393B; AU2003278398A1

Abstract

This invention provides a dry reagent test strip having oligonucleotide conjugated gold nanoparticles as an integral part and a method for the preparation of said test strip for the detection and/or determination of a nucleic acid. Oligonucletide conjugated gold nanoparticles are stabilized in dry form and retain all their properties after rehydration (mobility, hybridization capacity and colloidal state). The presence of a nucleic acid in the sample is detected by the formation of two red bands on the test and control zones, as a result of aggregation of gold nanoparticles at the test and control zone by the interaction with binding protein streptavidin and the complementary oligonucleotides, respectively. This invention represents an exceptionally sensitive and fast nucleic acid detection system, easy to use, with no need for special equipment. It has been designed so that to be effective in the detection of any kind of a nucleic acid, the specificity being determined solely by the sequence of the nucleotide- probe-1.

Description

DRY REAGENT STRIP AND NUCLEIC ACID DETECTION

This invention relates to a dry reagent test strip having oligonucleotide conjugated gold nanoparticles as an integral part and a method for the preparation of said test strip for the detection and/or determination of a nucleic acid. The analysis of specific nucleic acid sequences (DNA and RNA), by hybridization methods is common practice in many disciplines like molecular diagnosis, food quality control, agriculture, criminology, environmental control etc. (Christopoulos,T.K. (1999) Nucleic acid analysis. Anal. Chem., 71, 425R-438R). DNA and RNA amplification techniques, like polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), that provide exponential amplification of trace quantities of specific sequences of DNA/RNA from complex mixtures, are usually the basic step of contemporary methods for the analysis of nucleic acids. The analysis of DNA/RNA amplification products is performed by several laboratory techniques like electrophoresis, capillary electrophoresis, hybridization in microtiter plates etc. These techniques are time consuming, they require special equipment and often employ hazardous dyes. Specially skilled and trained personnel must perform these techniques and evaluate the results. In addition, electrophoretic methods, as opposed to hybridization techniques, are not specific to the nucleic acid sequence, as they only provide information on the size of analyzed DNA.

Vertical or lateral flow dry reagent strips represent a simple and rapid method for the analysis of DNA/RNA which do not require special equipment and skilled operators Analysis of nucleic acids by using dry reagent strips is based a) on hybridization between complementary strands of nucleic acid which determines the specificity of the test and b) interactions of antigen-antibody type or those of haptens with specific proteins such as antibodies or binding proteins such as streptavidin or avidin which determines the carrier of the analytical signal (Klepp J, Biochemica, 2, 2000). In dry reagent strips the above mentioned interactions take place in flow (after rehydration of the dry reagents by developing solution and their development along the strip due to capillary forces). On the other hand, interactions that take place in flow allow the separation between bound and unbound reagents and thus dry reagent strips are characterized as chromatographic test strips. In general, a dry reagent lateral or vertical flow strip which is used either in immunoassays or in nucleic acid assays consists of i) an absorbent pad which comes in contact with a sample solution or developing solution, ii) a conjugate pad where the detection moiety is reversible immobilized, iii) a membrane where the analytical signal is observed and iv) an absorbent pad for absorbing the excess of liquid (Short Guide for developing immunochromatographic Test Strips, Millipore Corp. Bedford MA, 1996; Jones KD, Lateral flow strip test technology The Latex Course, 2000; Chandler J et al, IVD Technology, March 2001)

The principle of DNA detection with dry reagent strips is similar to the principle of PCR ELISA. Nucleic acid labeled through PCR with haptens such as biotin or digoxigenin is hybridized with an oligonucleotide probe labeled with digoxigenin or biotin, respectively. As detection moieties colored particles as latex microspheres or gold nanoparticles conjugated with antibodies are used (Klepp J, Biochemica, 2, 2000).

Gold nanoparticles possess certain characteristics that render them applicable to dry reagent strip tests. They have a controlled size (20 - 50 nm) and are fairly stable in liquid and dry form. Solutions of colloidal gold have a characteristic plasmon absorbance band at 520 nm (red color). Conjugation products of colloidal gold to proteins have been used in immunochromatographic strip systems (Weller,M.G. (2000), Fresenius 1 Anal. Chem., 366, 635-645, Kasahara,Y., Ashihara,Y. (1997) Clin. Chim. Ada, 267, 87-102). Disadvantages in using antibodies conjugated with microspheres as detection moieties include the high cost of monoclonal antibodies used in conjugation reactions, removing of antibodies from the surface while drying the conjugate or during the test procedure as well as problems with non-specific binding of microsphere conjugates on the membrane (Chandler J et al, IVD Technology, March 2001). In the present invention, gold colloidal nanoparticles (40nm) conjugated with oligonucleotides are used as detection moieties in dry reagent strips for the detection or determination of nucleic acids.

Conjugation products of colloidal gold to oligonucleotides have been mainly used in the formation of nanostractures (Mirkin,C.A., Letsinger,R.L, Mucic,R.C. and Storhoff,J . (1996), Nature, 382, 607-609, Alivisatos,A.P., Johnsson,K.P., Peng,X., Wilson,T.E., Loweth,C ., Bruchez,M.P. and Schultz,P.G. (1996), Nature, 382, 609- 612) and for the analysis of nucleic acids in solution (Elghanian,R., Storhoff,J ., Mucic,R.C, Letsinger,R.L and Mirkin,C.A. (1997) Science, 211, 1078-1080). In the present invention we exploit the special characteristics of gold particles (stability and specificity) and the well established chemistry of gold particle conjugation to oligonucleotides and stabilization of conjugates (Elghanian,R., Storhoff,JJ Mucic,R.C, Letsinger,R.L. and Mirkin,C.A. (1997) Science, 277, 1078- 1080), for the development of a system for the detection of nucleic acids that combines the excellent specificity of DNA hybridization reactions with the concurrent labeling of the reaction product with a colored label (gold nanoparticles). The stability of conjugated gold nanoparticles in dry form and the preservation of their functionality after rehydration allow for their use as a part of dry reagent strip for the rapid analysis of nucleic acids. This invention provides a method for the analysis of nucleic acids, by use of oligonucleotide conjugated gold nanoparticles, which form part of a dry reagent strip. The first part of the invention is related to a method for the stabilization of oligonucleotide conjugated gold nanoparticles (prepared by methods known in the literature) in dry form such that after rehydration these particles retain their ability to hybridize with complementary strands in flow as well as their stability in high salt concentration solutions where hybridization of nucleic acids takes place and includes: (a) conditions under which conjugation reaction of gold nanoparticles with SH- modified oligonucleotides take place b) stabilization of the conjugation product against high salt concentration c) composition for oligonucleotide gold conjugate diluent associated with the good quality of conjugate in dry form and its ability to be fully released from the conjugated pad d) composition of the developing solution which reflects the interactions between the components of the test and eliminates any non-specific binding on the membrane. The second part of the invention relates to the use of oligonucleotide conjugated gold nanoparticles for the analysis of nucleic acids with dry reagent strip (dipstick) which comprises: a) hybridization of DNA to an oligonucleotide-probe 1. DNA is labeled with a ligand, like biotin during the amplification step. One part of the oligonucleotide- probe-1 is complementary to a part of the DNA. The second part of the oligonucleotide-probe-1 consists of a sequence complementary to an oligonucleotide- probe-2, conjugated to gold nanoparticles. The reaction is performed under specific conditions that promote the hybridization of DNA to oligonucleotide-probe-1. b) application of the hybridization product on the sample application zone on the dry reagent strip and soaking the low end of the strip in a development buffer. As buffer develops by capillary action across the strip, DNA hybridized to oligonucleotide-probe-1 is mixed with gold nanoparticles conjugated to oligonucleotide-probe-2 that are immobilized in dry form on the conjugate zone on the strip. Mixing is done under conditions that promote both the reconstitution of gold nanoparticles and their release from the membrane and the hybridization of the complex DNA-oligonucleotide-probe-1 to gold nanoparticles conjugated to oligonucleotide-probe-2. c) in flow interaction of the biotinylated DNA-gold nanoparticles complex with a binding protein streptavidin immobilized on the test line on the strip and capturing on the test zone forming a distinct red color due to accumulation of gold nanoparticles,. A red band on the test zone indicates the presence of nucleic acid in the sample, while absence of the red band indicates the absence of nucleic acid in the sample. d) in flow interaction of the free (unbound) oligonucleotide-probe-2 conjugated gold nanoparticles with an oligonucleotide-probe-3, having a sequence complementary to the sequence of oligonucleotide-probe-2 and is immobilized on the control line on the strip forming a distinct red color due to accumulation of gold nanoparticles. A red band on the control zone indicates correct development of the test and should appear regardless of the presence of nucleic acid in the sample. This invention presents the following advantages: i) The detection of nucleic acid is very fast, as only 10 minutes are required for a positive or negative result, as compared to some hours with other techniques like agarose gel electrophoresis and others ii) The sensitivity is 8 times better than that of agarose gel electrophoresis with ethidium bromide dying of DNA. Hi) Contrary to electrophoretic techniques, this method allows for the verification of a given nucleotide sequence, as it is based on the hybridization of target DNA to oligonucleotide-probe-1, that is specifically designed for the target DNA. Non-specific amplification products are not detected, since hybridization occurs only between the specific product and oligonucleotide-probe-1. iv) This method can give information on the quantity of nucleic acid in the sample, via densitometric scanning of the colored lines on the strip. v) The strip has been designed so that it can be used for the detection of any type of nucleic acid. Specificity is only defined by the oligonucleotide-probe-1. vi) Detecting nucleic acids by use of the proposed method does not require use of specific equipment and dangerous materials. vii) This method is reliable and permits evaluation of results by minimally trained personnel. viii) Gold nanoparticles conjugated with oligonucleotides are very stable reagents, with well-established chemistry for production and stabilization and can be used in dry form as part of a dry reagent strip. After reconstitution they regain all their liquid form properties. ix) Running the test in flow permits the effective separation of bound from unbound gold particle-oligonucleotide complexes. The method for detection of specific nucleic acid sequences on a dry reagent strip using oligonucleotide gonjugated gold nanoparticles is detailed below, in relation to the attached drawings, where: a) drawing 1 shows the strip and its parts and b) drawings 2, 3 and 4 present the reactions taking place.

Picture 1 is a real picture of a dry reagent strip, presenting a positive and a negative result. As depicted in drawing 1 the strip consists of a backing membrane (1), a wick membrane (2), a conjugate membrane (3), the sample application zone (4), the main membrane (5), which includes the test zone (6) and control zone (7), a wick membrane (8) and a handle (9). The series of reactions is described in drawings 2, 3 and 4 as follows: Drawing 2. Biotinylated amplification product (10) is mixed with a complementary oligonucleotide-probe 1 (11), the mixture is heated to 95°C for 2 min (denaturation of DNA) and then incubated at 37°C for 5 min, resulting to hybridization of DNA to oligonucleotide-probe 1 (12). Oligonucleotide-probe-1, in addition to the complementary sequence, bears a poly-adenine (~70-120 bases) tail at 3' end (13). Drawing 3. Hybridization mixture is applied on the sample application zone, on the strip (4). Gold nanoparticles (14) bear on their surface poly-thymine (100-150 bases) oligonucleotide-probe-2 (15). The strip end (2) is then soaked in development buffer, which develops by capillary action across the strip, reconstitutes and carries the gold nanoparticles along the strip. As particles move across the sample application zone (4), they bind to the hybridization product via adenine-thymine hybridization (16). Drawing 4. The hybridization product bears a biotin group at the 5 'end (17). As the mixture moves across the test zone (6), where streptavidin (18) is immobilized, hybridized and free DNA will be immobilized by a streptavidin-biotin interaction (19). Immobilization of the product results to a red band formation on the test zone. The mixture then passes across the control zone (7) where oligonucleotide-probe-3, a poly-adenine oligonucleotide, is bound (20). Free gold nanoparticles are then immobilized via adenine-thymine hybridization (21), forming a red band on the control zone.

The detection of DNA is confirmed by the presence of two red zones on the strip. In the absence of nucleic acid a red zone is formed only on the control line (7). In cases where a red band is visible only on the test line, the test must be repeated.

Picture 1. A positive and a negative result are pictured.

Method procedure

Preparation and stabilization of oligonucleotide conjugated gold nanoparticles Colloidal gold nanoparticles of at least 40nm (British-Biocell) are used for better sensitivity of the strip assay i) Gold nanoparticles- oligonucleotide probe- 2 conjugation

10ml gold particles are mixed with 900 pmol oligonucleotide (T₃₀-SH), which has been enzymatically elongated with the addition of ~70-120 thymine groups (T) by terminal transferase (TdT) (Oligonucleotide to gold particles 600/1 moles). 8 μl pyridine (absolute) is added, the mixture is stirred and kept at 4°C for 24 h. ii) Tailing of(T)₃₀-SH oligonucleotide

For the tailing reaction a mixture consisting of 4uL TdT buffer 5x, 7 uL dTTP lOmM,

7uL T₃₀- SH oligo 100 pmol/μL and 1.5uL TdT 20U/uL is prepared and incubated at 37°C for 1 hour. The reaction mixture is purified with Sephadex-25 microcolumn to remove DTT. The product of two tailing reactions is then added to 10ml gold particle solution.

Hi) Stabilization of conjugated gold particles

The stabilization is achieved by gradual addition of NaCI, up to a final concentration of 90mM. 80μl NaCI 900mM are first added and new additions are performed every

2h. After the fourth addition (NaCI concentration 30mM) steps of 160μl NaCI 900mM can be used. After every addition the particles solution is kept at 4°C. When the NaCI concentration reaches 90mM, the solution is kept at 4°C for at least 24h, but not more than 3 days. By using the stabilization pretreatment the following are achieved a) an increased number of oligonucleotides bound to the gold surface and b) stability of oligonucleotide conjugated gold nanoparticles in high salt concentration solutions where hybridization of nucleic acids takes place. iv) Gold particles centrifugation.

The mixture is placed into 50ml centrifuge tubes, per 5ml aliquots and centrifuged at

2800g for 45min. The supernatant is carefully removed. The procedure is repeated for one more time, with centrifugation for 27 minutes. The supernatant is removed carefully and the pellet is redispersed with 300μL of a solution containing sucrose

30%, Tween20 0,25%, SDS 0,25%, NaCI 45mM. Careful aspiration of the supernate is critical in order to achieve the removal of the non reacted oligonucleotide. The presence of free oligonucleotide may cause decrease in test sensitivity. When greater amount of oligonucleotide is used in conjugation reaction an additional step of washing the gold pellet could be performed.

Dry reagent strip

The strip is a dry reagent, lateral flow device. Its construction includes gluing several membranes together, according to drawing 1, on a self-adhesive backing membrane

(1). The membranes used are listed below: Absorbent membrane: Schleicher and Schuell 2992 (20x5mm) or equivalent. This membrane is used without pretreatment.

Conjugate membrane'. Schleicher and Schuell Glass 33 (20x5mm) or equivalent. This membrane is used without pretreatment.

Test membrane: Pall and Gelman Predator laminated (25x5mm) (pore size 0.45μm) or equivalent. This membrane is used without pretreatment.

Wicking membrane. Schleicher and Schuell GB003 (20x5mm) or equivalent. This membrane is used without pretreatment.

A special device is used for the attachment of the above membranes onto the backing (Biodot Clamshell Laminator LM 5000, or equivalent), with a specially made nest. Strips are then cut to the required width by a special cutter (Biodot Guillotine

Cutting Module CM 4000, or equivalent).

Loading of the reagents on the strip a) Loading of gold conjugate

Oligonucleotide conjugated gold nanoparticles are loaded on the conjugate pad (membrane 33 Glass), at a density of 7.2 fmol (4.5xl0⁹ gold particles) per 4mm using a special spraying device (Biodot AirJet Dispenser AJQ 3000, or equivalent).

The amount of gold nanoparticles loaded on the strip is of importance for the good performance of the test. Overloading of conjugate may create several problems such as increasing the possibility false-positive signals, as well as increasing the likelihood of backflow of gold after the test period has elapsed. A good-quality gold conjugate should not need to be used in excess. After application of gold nanoparticles, the conjugate pad is allowed to dry at ambient temperature, b) Loading of streptavidin

Streptavidin is diluted in a 5% sucrose solution at concentration 2mg/mL and is loaded on membrane at a density of 1.6 μg per 4mm by Biojet Dispenser BJQ 3000 or equivalent. The presence of sucrose in solution eliminates the diffusion of streptavidin on membrane thus increasing the sensitivity of the test. Loading ofpoly(A) oligonucleotide. First, poly (A)₃₀ oligonucleotide is tailed with dATP. For tailing reaction, a mixture consisting of 4uL TdT buffer 5x, 7 uL dATP lOmM, 7uL poly (A)₃₀ oligo 100 pmol/μL and 1.5uL TdT 20U/uL is prepared and incubated at 37°C for 1 hour. The tailed product diluted in water at concentration 2 pmoL/μL is loaded at a density of 1.2 pmol per 4mm by Biojet Dispenser BJQ 3000 or equivalent. After reagents application, the membrane is placed in the oven at 70°C for 30 minutes. After drying the assembly of parts is performed using a special device (Biodot Clamshell Laminator LM, or equivalent). Membranes are cut to the appropriate width by a Biodot Guillotine Cutting Module CM 4000, or equivalent. Strips are then kept in aluminum foils, in the presence of dessicant (Dritablets^®). Reagents Oligonucleotide-probe-1.

The Oligonucleotide-probe-1 is designed according to the target sequence. A tail consisting of adenine nucleotides (~70-120 bases) is then added enzymatically to the 3' end. For the tailing reaction a mixture consisting of 4uL TdT buffer 5x, 7 uL dATP lOmM, 7uL oligonucleotide-probe-1 100 pmol/μL and 1.5uL TdT 20U/uL is prepared and incubated at 37°C for 1 hour. The use of dry reagent strip

1 μl NaCI 900mM and 1 pmol oligonucleotide-probe-1, specific for the target DNA, are added to 10 μl PCR product. NaCI is added in order to increase the salt concentration necessary for hybridization reaction. The amount of oligonucleotide- probe-1 in hybridization mixture is crucial and should not exceed lpmol per lOμL of PCR mixture. Larger amount of probe may result in lower band intensity. This is because, at high levels, the amount of poly-(dA) oligonucleotide exceeds the binding capacity of the poly(dT)-conjugated nanoparticles. As a consequence, poly(dA) oligonucleotide that is hybridized to the target sequence competes with the free (unhybridised) poly(dA) oligo for binding to limited poly(dT). The higher the amount of poly(dA) oligo used, the larger the fraction of nanoparticles that binds to the free probe. The mixture is heated at 95°C for 2min and incubated at 37°C for 5min. A 5 μl aliquot of the mixture is placed on the sample application zone. The strip is placed into an eppendorf tube containing 200μl development buffer, so that approximately 10mm are soaked. The results can be read in 10 minutes. Positive and negative results appear as in picture 1. If the strip is left in the development buffer, results can be read at any time after 10 minutes. If the strip is removed from the solution, results must be read immediately after 10 minutes.

Applications The following application is given as an example and is not restrictive: Detection of RT-PCR product of PSA mRNA (messenger RNA of the Prostate Specific Antigen). Biotinylated RT-PCR product (mRNA PSA 233bp) is hybridized initially with a sequence specific oligonucleotide-probe-1 and then is hybridized in flow with gold nanoparticles conjugated with oligonucleotide-probe-2. The detection is achieved by the formation of a red color on the test zone due to the biotin-streptavidin interaction. The test is specific, as far as the initial hybridization is only possible if the oligonucleotide-probe-1 is of complementary sequence with the target DNA sequence. The detection limit of this procedure is 0,3 ng (2 x 10^"15 mol).

Claims

1. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid, having (1) a contact end for contacting the developing solution of the test, (2) a conjugate pad area where gold nanoparticles having oligonucleotides attached thereto are reversibly immobilized, (3) a sample application zone, (4) an analytical membrane which comprises (5) a test zone where the binding protein streptavidin is immobilized and (6) a control zone where oligonucleotides having sequence complementary to the sequence of oligonucleotides attached to the gold nanoparticles are immobilized, and (7) an absorbent pad which serves for withholding of the developing solution, and the said strip is characterized in that the oligonucleotide conjugated gold nanoparticles are subjected to pretreatment with NaCI prior to their loading on the strip in order to achieve their stabilization in dry form, the preservation of their ability for hybridization after rehydration and their stability in solutions of high salt concentration.

2. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claim 1 further characterized in that, after conjugation with oligonucleotides, gold nanoparticles are subjected to pretreatment with the addition of at least 90mM NaCI.

3. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 and 2 wherein the pretreatment of oligonucleotide conjugated gold nanoparticles is performed by increasing stepwise the NaCI concentration as follows. Increases of 7.5 mM/step for steps 1-4 followed by increases of 15 mM/step up to a final NaCI concentration of at least 90mM.

4. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 3 which is further characterized in that after each addition of NaCI an incubation period of the mixture of about 2h at 4 °C is required.

5. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 4 wherein the reconstitution/application solution of oligonucleotide conjugated gold nanoparticles contains 30% sucrose, 0.25% non ionic detergent Tween20, 0.25% anionic detergent SDS and at least 45mM NaCI. In this solution oligonucleotide conjugated gold nanoparticles are quite stable in dry form, are easily rehydrated and detached from the conjugate pad.

6. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 5 wherein oligonucleotide conjugated gold nanoparticles on dry reagent strip are stable for at least 12 months.

7. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 6 further characterized in that a limited amount of gold conjugate is applied onto conjugate pad which is about 7.2 fmol or 4.5X10⁹ gold nanoparicles and the size of the gold nanoparticles is about 40nm. Overloading of conjugate results to false-positive signals, as well as increases the likelihood backflow of the gold nanoparticles after the test period has elapsed. A good-quality gold conjugate should not need to be used in excess. The gold nanoparticles size relates to the detectability of the test which is better with gold paricles about 40nm and higher.

8. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 7 wherein the amount of streptavidin immobilized on the test zone of the analytical membrane is at least 1.6 μg and the streptavidin diluent is a 5% sucrose solution in water. The presence of sucrose eliminates the diffusion of streptavidin on the membrane thus increasing the test sensitivity.

9. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 8 further characterized in that the amount of oligonucleotide immobilized on the control zone is at least 1.2 pmol and the diluent of the oligonucleotide is water.

10. Dry reagent test strip and method for the preparation of said test strip for the detection and/or determination of a nucleic acid of claims 1 to 9 wherein the oligonucleotide probe solution used for the conjugation with gold nanoparticles does not contain substances with SH-groups, like DTT (dithiothreitol). These substances may displace oligonucleotide probe from gold surface. Purification could be performed by using Sephadex-25 microcolumns.

11. Method for the rapid detection and/or determination of a nucleic acid with the dry reagent test strip of claims 1-10 wherein biotinylated PCR or RT-PCR product is hybridized initially with an oligonucleotide-probe-1 having a sequence one part of which is complementary to the sequence of a nucleic acid and the second part is a poly(dA) (poly-adeninosine) oligonucleotide (~70-100b) and then is hybridized in flow with gold particles conjugated with oligonucleotide-probe-2 which consists of ~ 100-150 thymine bases. The detection is achieved by the formation of a red color on the test zone, by interaction of the biotin label to streptavidin immobilized on the strip. The successful performance of the test is controlled by the formation of a red color on the control zone due to the interaction of poly(dT) oligonucleotide-probe-2 attached to the gold surface with the poly(dT) oligonucleotide-probe-3 immobilized on the control zone. The method is further characterized in that the amount of oligonucleotide-probe-1 used for hybridization with a nucleic acid should not exceed lpmol per lOμl of PCR product. Larger amount of probe may result in lower band intensity. This is because, at high concentration levels, the amount of poly-(dA) oligonucleotide exceeds the binding capacity of the poly(dT)-conjugated nanoparticles. As a consequence, poly(dA) oligonucleotide that is hybridized to the target sequence competes with the free (unhybridised) poly(dA) oligo for binding to limited poly(dT). The higher the amount of poly(dA) oligo used, the larger the fraction of nanoparticles that bind to the free probe. It is within the scope of the present invention that the hybridization assay can be performed in a sandwich-type configuration where the target DNA is unlabeled and hybridized with two specific probes one of which is biotinylated while the other carries a poly(dA) tail.

12. Method for the rapid detection and/or determination of a nucleic acid of claim 11 with the dry reagent test strip of claims 1-10 further characterized in that the developing solution of the test contains a phosphate buffer, pH 7.4, 150mM NaCI, up to 4% glycerol and 1% anionic surfactant SDS.

13. Method for the rapid detection and/or determination of a nucleic acid with the dry reagent test strip of claims 1-12 further characterized in that the quantity of a nucleic acid is determined by measuring the density of the test zone of the developed strip by scanning densitometry.

14. Use of the method for the rapid detection and/or determination of a nucleic acid with the dry reagent test strip of claims 1-13 for the determination of infectious agents (viral or bacterial DNA), genetically modified organisms (GMOs), DNA mutations, micrometastasis etc.