WO1997040188A1

WO1997040188A1 - Method to detect amplified nucleic acids and kit for the use thereof

Info

Publication number: WO1997040188A1
Application number: PCT/IT1997/000094
Authority: WO
Inventors: Maurizio Gramegna; Giuseppe Colucci
Original assignee: Clonit S.P.A.
Priority date: 1996-04-24
Filing date: 1997-04-23
Publication date: 1997-10-30
Also published as: ITRM960277A0; IT1284635B1; ITRM960277A1; AU2404597A; EP0907749A1

Abstract

A method for detecting an amplified nucleic acid in a sample comprising the following steps: i) incorporating a ligand into each of nucleic strands by means of an amplification reaction using as polymerisation primers two primers, each having said ligand, in order to obtain a nucleic acid-ligand complex; ii) allow said nucleic acid-ligand complex to denaturate into single strands; iii) incubate under denaturing conditions said nucleic acid-ligand complex with a binding reagent specific for said ligand, wherein said binding reagent is adhered to a solid substrate, in order to obtain a nucleic acid-ligand-binding reagent complex which is adhered to a solid substrate; iv) reveal said nucleic acid-ligand-binding reagent complex. Also described is a kit to detect an amplified nucleic acid according to the method of the invention.

Description

METHOD TO DETECT AMPLIFIED NUCLEIC ACIDS AND KIT FOR THE USE THEREOF

The invention concerns a method to detect amplified nucleic acids, in particular DNA, which makes use of a ligand bound to both of amplification primers to work both in the capturing and in the revealing reaction. The method is utilised for any of genomic, viral, bacterial, etc., DNA sequences, which are amplified by means of techniques to those expert in the field, as the polymerase chain reaction (PCR) , provided that oligonucleotide primers are required. The method is advantageously used also for detecting amplified DNA obtained by reverse transcription of target mRNAs, viral genomic RNAs, etc.

The PCR technology (Hoffman La Roche) is now widely accepted as a powerful tool, both in the medical diagnostic and m the research field. The amplification reaction relies on the use of specific oligonucleotide primers and of a template DNA, by means of thermostable polymerases as, for example, Taq polymerase ( Thermus aqua ticus) , Tth polymerase ( Thermus thermophilus) . A good enrichment of template DNA is obtained, by repeating cycles comprising a denaturation step, wherein the two DNA strands are separated; an annealing step, wherein primers specifically hybridise to complementary sequences of target DNA; and an elongation step, wherein the thermostable polymerase polymerises a nucleotide chain on the target template DNA. The so-called nested PCR is used when a sufficient amount of amplified DNA may not be obtained according to the previous method, and comprises a first amplification by means of a first set of two primers, and a second amplification by means of a second set of two primers, which are different from those of the first set, having a sequence which is internal to the fragment sequence obtained by the first amplification.

Patent application No. WO90/06374 in the name of Amrad Co. concerns a method for capturing on a solid substrate a target amplified DNA comprising the step of incorporating a first ligand into tne DNA by means of a PCR reaction utilising a set of two primers wherein only one primer comprises the first ligand, and to incubate the amplified DNA with a solid substrate adhered binding reagent which is specific for the first ligand. The revealing step of the captured amplified DNA is performed by means of binding to a second ligand, which is different to the first capturing ligand, with a reagent able to give rise to a detectable reaction. Primers which are not elongated during the amplification act as competitors of the amplified DNA for the binding to the solid substrate reagent, thus rendering the reaction not very sensible.

The instant invention overcomes the disadvantages of the prior art and allows to detect the amplified DNA m a very simply and sensible way. The amplified DNA is detected by incorporating a ligand molecule on each of DNA strands and further capturing on a solid substrate-adhered specific reagent for said ligand. The incorporation of the ligand in the amplified DNA is achieved by means of amplifying primers which both comprise a ligand, i.e. a biotin molecule at their 5' end.

The capturing is achieved by means of reacting said ligand with a specific molecule, preimmobilized on a solid substrate. In the case that the ligand is a biotin molecule the specific molecule may advantageously be streptavidin, which has four binding sites for biotin.

The revealing step is achieved by binding the ligand with an enzyme conjugated molecule able to catalyse a detectable reaction, i.e., peroxidase, alkaline phosphatase.

Advantages of the invention comprise the use of the same ligand both for the solid phase binding system and for the detecting system; and the introduction of a denatuπng- annealing step which render the primer saturated solid phase reagents able to capture and reveal the amplification products.

It is therefore a specific object of the invention a method for detecting an amplified nucleic acid in a sample comprising the following steps: i) incorporating a ligand into each of nucleic strands by means of an amplification reaction using as polymerisation primers two primers, each having said ligand, in order to obtain a nucleic acid-ligand complex; ii) allow the nucleic acid-ligand complex to denaturate into single strands; iii) incubate under denaturing conditions the nucleic acid- ligand complex with a binding reagent specific for said ligand, wherein the binding reagent is adhered to a solid substrate, in order to obtain a nucleic acid- ligand-binding reagent complex which is adhered to a solid substrate; iv) reveal the nucleic acid-ligand-binding reagent complex. Preferably the nucleic acid is DNA, more preferably obtained by reverse transcription of a RNA into cDNA and amplification of said cDNA.

In a preferred embodiment of the invention the ligand is biotin and the binding reagent is streptavidin. Preferably the biotin is bound to each of 5' end terminus of the two primers.

Another object of the invention is a kit to detect an amplified nucleic acid according to the described method comprising separate vials containing: - one or more amplification mix;

- a denaturing solution;

- an hybridisation solution;

- a conjugating solution; and further comprising a solid substrate adhered binding reagent. According to an application of the instant invention, a target viral RNA, i.e. the hepatitis C virus

(HCV) RNA, is reverse transcribed by means of an antisense primer which is specific for the viral genome 5' end UTR (described, for example in EP 388232), and of the AMV reverse transcriptase enzyme (AMV-RT) .

A first amplification step is performed with a sense primer of the same 5' end UTR sequence, on the reverse transcribed cDNA template. Afterwards a nested amplification is performed by means of two primers having sequences comprised in the preamplified sequence fragment of the first PCR. The nested amplification primers are 5' end labelled with biotin. The amplification product is a double strand DNA fragment having at each of 5' ends a biotin molecule available for further binding.

The nested amplification product is then reacted with a solid substrate preadhered binding reagent, i.e. streptavidin, and then captured.

A second binding reagent is then introduced, i.e. peroxidase conjugated streptavidin (SA-HRP) , which binds to the biotin molecule, being still available for reacting with a chromogen and producing a detectable colorimetric reaction.

Streptavidin comprises four biotin binding sites and is able to bind many biomolecules, as antigens, antibodies, carbohydrates, cells, DNAs, enzymes, haptens, lectins, peptides, proteins, receptors, which act as bridges for the colorimetric reaction.

Biotin is able to bind, by means of an avidinic binding, to all of available, free solid substrate adhered streptavidin sites. Therefore each streptavidin molecule is able to bind up to four biotin molecules.

By introducing a denaturation step further to the nested amplification, before to incubate the amplified product with the solid substrate, the system sensitivity is further increased. The denaturation step may be performed by means of a denaturing solution, or simply by boiling the sample for appr. 10 min.

The denaturing step allows to get single strand amplified DNAs and unutilised primers, as well. Therefore all of molecules in the denatured mix, either primers or amplified fragments, have at their 5' end a biotin molecule which is able to bind to streptavidin available sites. As a consequence the structures adhered on the solid substrate will comprise both unutilised primers and amplified fragments.

By adding the hybridisation solution the molecules will bind again to complementary strands. In this manner structures adhered on the solid substrate will be constituted partly by streptavidin bound primers hybridised to complementary amplified fragments, and partly by streptavidin bound amplified fragments hybridised to complementary primers.

The invention may utilise other binding molecules as well, as alkaline phosphatase, peptides, lectins, carbohydrates, DNA, binding proteins, etc.

The system may be used also with "multiplex" PCR, with different fluoresceines 5' end labelled primers, and detected on dishes, or with automated systems (^λVgene scanner" and sequencing) . The invention foresees also the possibility of a direct quantitation of the amplified product, by diluting either the starting target nucleic acid or the amplified nucleic acid and performing different parallel reactions.

The invention will now be described for illustrative but not limitative purposes, according to preferred embodiments, referring to the following figures: figure 1 is a representation of the complex obtained according to the invention; figure 2 is a representation of the complex obtained further to the denaturation step and to the binding step of the amplified mix to the solid phase; figure 3a is a representation of a first complex obtained further to the renaturation and to the binding step to the detecting molecule; figure 3b is a representation of a second complex obtained further to the renaturation and to the binding step to the detecting molecule; figure 3c is a representation of a third complex obtained further to the renaturation and to the binding step to the detecting molecule. Amplification with biotinilated primers and detection on microtiter dishes - kit

Reverse transcription mix: 50 mM Tris-HCl pH 8.2, 70 mM KCl, 10 mM MgCl₂, 4 mM DTT, 20 U RNAsin, 0.4 % NP-40, 100 μM dNTPs, 25 pmoles antisense primer (final volume: 25μl) .

First amplification mix: 67 mM Tris-HCl pH 8.8, 16.6 mM (NH„)₂S0₄, 200 μM dNTPs, 1.5 mM MgCl₂, 10 mM β- mercaptoethanol, 25 pmoles sense primer (final volume: 75 μl). Nested amplification mix: 67 mM Tris-HCl pH 8.8,

16.6 mM (NH₄)₂SCM, 200- μM dNTPs, 1.5 mM MgCl₂, 10 mM ^β- mercaptoethanol, 25 pmoles biotinilated nested primers (final volume: 97 μl) .

Denaturing solution: 80 mM EDTA, 0.4 N NaCl. Hybridisation solution: 80 mM sodium phosphate

(dibasic), 10 mM sodium phosphate (monobasic), 2.5 M sodium thiocianate, 0.125 % Tween-20, pH 5.

Washing solution: 7 mM sodium phosphate (monobasic) , 3 mM sodium phosphate (dibasic), 150 mM NaCl, 1 mM EDTA, 0.125 % Tween-20, pH 7.

Conjugating solution: 10 mM Tris-HCl pH 7.6, peroxidase conjugated streptavidin.

Colorimetric reagent: 1 g/1 3, 3'-5, 5'-tetra- methylbenzidine (BM Blue POD Substrate) (Boehringer Mannheim GmbH) .

Stop solution: sulphuric acid 5%. Preparation of the solid substrate: ELISA microtiter dishes (maxi Sorp, Pierce) are used by pipetting 200 μl streptavidin 2 μg/ml in each well and by overnight incubating at 4°C. Microtiter dishes are available from Pierce.

RNA extraction may be performed by means of the standard phenol-chloroform method (Sambrook, Fritsch, Maniatis, Molecular Cloning A Laboratory Manual, CSH) or of one of commercially available kits.

3 μl of sera or extracted RNA were added to 22 μl of the reverse transcription mix, prepared as above, to a final volume of 25 μl, and a denaturation step was performed at 92°C 3 sec. 8 U AMV-RT were added and the mix was incubated at 42°C for 60 min.

Therefore to 75 μl of the first amplification mix, 2 U Taq polymerase (Hoffman La Roche) were added and the amplification was performed with the following cycles:

Further to the first amplification 97 μl of the nested amplification mix were put in another vial and 3 μl of the first amplified product were added together with 2 U Taq polymerase; the same cycles were again performed but with a total number of 24 cycles instead of 34.

At the end of the amplification:

- 20 μl of the amplified mix were taken and chemically denatured by adding an equal volume (20 μl) of denaturing solution, and incubating at room temperature for 10 min;

- the prepared amplified denatured mix was added;

- 100 μl of hybridisation solution were added and incubated at 37°C for 30 min;

- wells were washed 6 times with a washing solution (by leaving the solution 30 sec each time) ; - further to a 1:5000 in 10 mM Tris-HCl pH 7.6 dilution, peroxidase-conjugated streptavidin was added, according to the manufacturer, and incubated at 37°C for 30 min;

- washings were repeated as above; - 100 μl POD (colorimetric substrate) were added and colour was developed for 10-20 min at room temperature;

- the colorimetric reaction was stopped by adding 100 μl of stop solution;

- the microtiter was read at 450 nm referring to a wavelength of 492 nm.

Claims

1. Method for detecting an amplified nucleic acid in a sample comprising the following steps: i) incorporating a ligand into each of nucleic strands by means of an amplification reaction using as polymerisation primers two primers, each having said ligand, in order to obtain a nucleic acid-ligand complex; ii) allow said nucleic acid-ligand complex to denaturate into single strands; iii) incubate under denaturing conditions said nucleic acid- ligand complex with a binding reagent specific for said ligand, wherein said binding reagent is adhered to a solid substrate, in order to obtain a nucleic acid- ligand-binding reagent complex which is adhered to a solid substrate; iv) reveal said nucleic acid-ligand-binding reagent complex.

2. Method for detecting an amplified nucleic acid according to claim 1 wherein said nucleic acid is DNA.

3. Method for detecting an amplified nucleic acid according to claim 2 wherein said amplified DNA is obtained by reverse transcription of a RNA into cDNA and amplification of said cDNA.

4. Method for detecting an amplified nucleic acid according to claim 3 wherein said RNA is the hepatitis C virus (HCV) RNA.

5. Method for detecting an amplified nucleic acid according to claim 4 wherein said HCV RNA is the 5' end UTR RNA.

6. Method for detecting an amplified nucleic acid according to any of previous claims wherein said ligand is biotin and said binding reagent is streptavidin.

7. Method for detecting an amplified nucleic acid according to claim 6 wherein said biotin is bound to each of 5' end terminus of said two primers.

8. Kit to detect an amplified nucleic acid according to the method of any of previous claims comprising separate vials containing:

- one or more amplification mix;

- a denaturing solution;

- an hybridisation solution;

- a conjugating solution; and further comprising a solid substrate adhered binding reagent.