EP1601787A1 - Bandelette d'essai a reactif sec et detection d'acide nucleique - Google Patents

Bandelette d'essai a reactif sec et detection d'acide nucleique

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Publication number
EP1601787A1
EP1601787A1 EP03769703A EP03769703A EP1601787A1 EP 1601787 A1 EP1601787 A1 EP 1601787A1 EP 03769703 A EP03769703 A EP 03769703A EP 03769703 A EP03769703 A EP 03769703A EP 1601787 A1 EP1601787 A1 EP 1601787A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
oligonucleotide
test strip
detection
dry reagent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03769703A
Other languages
German (de)
English (en)
Inventor
Theodoros Christopoulos
Penelope Ioannou Amarantidou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Automated Analysers and Diagnostic Reagents Medicon Hellas SA
Original Assignee
Automated Analysers and Diagnostic Reagents Medicon Hellas SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Automated Analysers and Diagnostic Reagents Medicon Hellas SA filed Critical Automated Analysers and Diagnostic Reagents Medicon Hellas SA
Publication of EP1601787A1 publication Critical patent/EP1601787A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5023—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures with a sample being transported to, and subsequently stored in an absorbent for analysis

Definitions

  • 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.
  • DNA and RNA specific nucleic acid sequences
  • hybridization methods are 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.
  • 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.
  • 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)
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • This method can give information on the quantity of nucleic acid in the sample, via densitometric scanning of the colored lines on the strip.
  • 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.
  • Detecting nucleic acids by use of the proposed method does not require use of specific equipment and dangerous materials.
  • This method is reliable and permits evaluation of results by minimally trained personnel.
  • 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.
  • Picture 1 is a real picture of a dry reagent strip, presenting a positive and a negative result.
  • 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.
  • T 30 - SH oligo 100 pmol/ ⁇ L 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.
  • the stabilization is achieved by gradual addition of NaCI, up to a final concentration of 90mM. 80 ⁇ l NaCI 900mM are first added and new additions are performed every
  • the mixture is placed into 50ml centrifuge tubes, per 5ml aliquots and centrifuged at
  • 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
  • Absorbent membrane Schleicher and Schuell 2992 (20x5mm) or equivalent. This membrane is used without pretreatment.
  • Test membrane Pall and Gelman Predator laminated (25x5mm) (pore size 0.45 ⁇ m) 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
  • Oligonucleotide conjugated gold nanoparticles are loaded on the conjugate pad (membrane 33 Glass), at a density of 7.2 fmol (4.5xl0 9 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 ⁇ g per 4mm by Biojet Dispenser BJQ 3000 or equivalent.
  • 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.
  • a mixture consisting of 4uL TdT buffer 5x, 7 uL dATP lOmM, 7uL poly (A) 30 oligo 100 pmol/ ⁇ L 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/ ⁇ L is loaded at a density of 1.2 pmol per 4mm by Biojet Dispenser BJQ 3000 or equivalent.
  • 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.
  • a mixture consisting of 4uL TdT buffer 5x, 7 uL dATP lOmM, 7uL oligonucleotide-probe-1 100 pmol/ ⁇ L and 1.5uL TdT 20U/uL is prepared and incubated at 37°C for 1 hour.
  • oligonucleotide-probe-1 1 ⁇ l NaCI 900mM and 1 pmol oligonucleotide-probe-1, specific for the target DNA, are added to 10 ⁇ l 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 lO ⁇ L 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.
  • 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 mixture is heated at 95°C for 2min and incubated at 37°C for 5min. A 5 ⁇ l aliquot of the mixture is placed on the sample application zone.
  • the strip is placed into an eppendorf tube containing 200 ⁇ l 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.
  • 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).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
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  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à une bandelette d'essai à réactif sec dans laquelle sont intégrées des nanoparticules d'or conjuguées à des oligonucléotides, et à un procédé de préparation de cette bandelette d'essai destinée à la détection et/ou la détermination d'un acide nucléique. Les nanoparticules d'or conjuguées à des oligonucléotides sont stabilisées sous forme sèche et elles conservent toutes leurs propriétés après réhydratation (mobilité, capacité d'hybridation et état colloïdal). La présence d'un acide nucléique dans l'échantillon est détectée par la formation de deux bandes rouges sur les zones d'essai et de référence, formation qui résulte de l'agrégation de nanoparticules d'or au niveau de la zone d'essai et de référence du fait de l'interaction avec la protéine de liaison, la streptavidine, et les oligonucléotides complémentaires respectivement. L'invention constitue un système de détection d'acide nucléique rapide, exceptionnellement sensible, facile à utiliser et ne nécessitant aucun équipement spécial. Ce système est conçu pour permettre une détection efficace de toute sorte d'acide nucléique, la spécificité étant déterminée uniquement par la séquence oligonucléotide-sonde-1.
EP03769703A 2002-11-07 2003-11-07 Bandelette d'essai a reactif sec et detection d'acide nucleique Withdrawn EP1601787A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GR20020100485A GR1004393B (el) 2002-11-07 2002-11-07 Μεθοδος ανιχνευσης ειδικων αλληλουχιων νουκλεικων οξεων σε ταινια ξηρων αντιδραστηριων
GR2002100485 2002-11-07
PCT/GR2003/000052 WO2004042084A1 (fr) 2002-11-07 2003-11-07 Bandelette d'essai a reactif sec et detection d'acide nucleique

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EP1601787A1 true EP1601787A1 (fr) 2005-12-07

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EP (1) EP1601787A1 (fr)
AU (1) AU2003278398A1 (fr)
GR (1) GR1004393B (fr)
WO (1) WO2004042084A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009034842A1 (fr) * 2007-09-11 2009-03-19 Kaneka Corporation Procédé de détection d'acide nucléique, et coffret de détection d'acide nucléique
EP2406374A4 (fr) 2008-06-02 2014-03-19 Brookhaven Science Ass Llc Assemblage et désassemblage contrôlables de systèmes de nanoparticules par des protéines et des agents d'adn
CN101762574B (zh) * 2008-12-23 2013-07-31 中国科学院上海微系统与信息技术研究所 一种增强纳米金稳定性的方法及应用其的生物检测的方法
CN113138269B (zh) * 2021-04-20 2024-03-26 江南大学 一种检测卡那霉素的适配体胶体金侧向层析试纸
CN113406329B (zh) * 2021-06-15 2024-05-28 江南大学 一种检测小分子物质的通用型适配体胶体金侧向层析试纸
ES2932995A1 (es) * 2021-07-22 2023-01-30 Ocupharm Diagnostics Sl Sistema de detección de parásitos oculares

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CA2223705A1 (fr) * 1998-02-25 1999-08-25 William Jia Methode de dosage en une etape pour determiner le produit final de produits d'amplification nucleotidique, comme l'amplification en chaine par polymerase (acp)
GB0016833D0 (en) * 2000-07-07 2000-08-30 Lee Helen Improved dipstick assays (2)

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GR1003966B (el) 2001-10-16 2002-08-06 Εμβαπτιζομενος χαρτης αποξηραμενων αντιδραστηριων και μεθοδος για την ανιχνευση η και προσδιορισμο ειδικων αλληλουχιων νουκλεικων οξεων

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ALIVISATORS A.P. ET AL.: "Organization of nanocrystal molecules using DNA", NATURE, vol. 382, 1996, pages 609 - 611, XP002222113
LETSINGER R.L. ET AL.: "Use of hydrophobic substituents in controlling self-assembly of oligonucleotides", J AM CHEM SOC, vol. 115, 1993, pages 7535 - 7536, XP003017496
MEDICON HELLAS S.A.: "Development of dry reagents for detection of DNA and applications in molecular diagnostics", TECHNICAL BULLETIN, 1999, pages 1 - 26+10 PG-S DRAWINGS, XP003017495
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Also Published As

Publication number Publication date
AU2003278398A1 (en) 2004-06-07
WO2004042084A1 (fr) 2004-05-21
GR1004393B (el) 2003-11-28

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