EP1969140A2

EP1969140A2 - Reagents and method for simultaneous nucleic acid amplification and detection

Info

Publication number: EP1969140A2
Application number: EP06832332A
Authority: EP
Inventors: Daniel Adlerstein; Erlet Shehi; Alessandro Di Nicola
Original assignee: Diasorin SpA
Current assignee: Diasorin SpA
Priority date: 2005-11-23
Filing date: 2006-11-22
Publication date: 2008-09-17
Also published as: WO2007060707A3; WO2007060707A2

Abstract

The present invention relates to a method for simultaneous amplification and detection of nucleic acids, in an homogeneous environment, and to reagents for the implementation thereof. The method is advantageously utilized for detecting even very low amounts of the target nucleic acid.

Description

REAGENTS AND METHOD FOR SIMULTANEOUS NUCLEIC ACID AMPLIFICATION

AND DETECTION

* * *

BACKGROUND ART The present invention relates to a method for simultaneous amplification and detection of nucleic acids, and to reagents for the implementation thereof.

The method departs to the Polymerase-Chain-Reaction to develop a simultaneous amplification and detection reaction. Other simultaneous methods are known in the art.

EP 878554 refers to a method and a signal primer for detection of nucleic acid target sequences by fluorescence quenching mechanisms, employed in an amplification reaction for detection of target sequence amplification. Signal primers do not serve as amplification primers.

EP 881302 refers to a method wherein a signal is generated further to the target extension on to a probe. The probe is not extended during the reaction. DESCRIPTION OF THE INVENTION

The. authors of the invention set up a method that allows the simultaneous amplification and detection of a target nucleic acid sequence in an homogeneous environment. The method is advantageously utilized for detecting even very low amounts of the target nucleic acid.

Therefore it is an object of the invention a method for detecting the presence or the absence of a target nucleic acid sequence in a sample, comprising the following steps: a) contacting the sample with an oligonucleotide system under hybridization conditions such as to form a reaction mixture, in which said oligonucleotide system comprises at least a first oligonucleotide and a second oligonucleotide, wherein i) at least one of said first and said second oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; iv) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence; b) adding, with appropriate substrates and cofactors at a suitable ionic and pH environment, both a temperature resistant DNA polymerase and said double strand, site specific, high temperature resistant cleaving agent, to said reaction mixture, under predetermined reaction conditions such that, if the target nucleic acid sequence is present in the sample, said first and second oligonucleotides hybridize thereto and prime the DNA polymerase reaction to obtain a first specific amplified product; c) cycling the hybridization of said first and said second oligonucleotide to said first specific amplified product such that a second specific amplified product is extended comprising, respectively, the 5 'end region of the second or of the first oligonucleotide, forming a double stranded recognition site for said cleaving agent so that said agent specifically cleaves the recognition sequence and induces the generation of a signal by means of the coupled detection system; d) detecting the generated signal. In a preferred embodiment, the method for detecting the presence or the absence of a target nucleic acid sequence in a sample comprises the following steps: a) contacting the sample with an oligonucleotide system under hybridization conditions such as to form a reaction mixture, in which said oligonucleotide system comprises three oligonucleotides, being a first and a second primer oligonucleotides as above described, and a third "anchor" oligonucleotide, in which: i) at least one of the primer oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high

^■ temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises: - a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; and - a region comprising a sequence able to hybridize to a complementary region at the 3'end of the third "anchor" oligonucleotide; iv) said third "anchor" oligonucleotide comprises: - a 5'end region able to hybridize to the target nucleic acid sequence in a region upstream of the complementary sequence to said second oligonucleotide and - a 3'end region able to hybridize to the complementary region of said second oligonucleotide, such as to act as an anchor; v) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence; b) adding, with appropriate substrates and cofactors at a suitable ionic and pH environment, both a temperature resistant DNA polymerase and said double strand, site specific, high temperature resistant cleaving agent, to said reaction mixture, under predetermined reaction conditions such that, if the target nucleic acid sequence is present in the sample, said first, second and third oligonucleotides hybridize thereto, said second and said third oligonucleotide hybridize to each other, and said first and said second oligonucleotide prime the DNA polymerase reaction to obtain a first specific amplified product; c) cycling the hybridization of said first and said second oligonucleotide to said first specific amplified product such that a second specific amplified product is extended comprising, respectively, the 5'end region of the second or of the first oligonucleotide, forming a double stranded recognition site for said cleaving agent so that said agent specifically cleaves the recognition sequence and induces the generation of a signal by means of the coupled detection system; d) detecting the generated signal.

Preferably the double strand, site specific, high temperature resistant cleaving agent is a restriction endonuclease, more preferably, it is the enzyme PspGI, alternatively it is one of the following enzymes: TIiI, Till, BstNT, Apol, BstBI, BstEII, SmII, TspRI, Tsp45I or BsoBL The person skilled in the art will readily understand that other agents may be used.

In a preferred embodiment, the coupled detection system is a fluorophore-quencher system, but the person skilled in the art will readily understand that other systems may be used. Exemplified fluorophores are: Fluorescein, FAM, MAXn (IDTdna), Tamra, Texas red, Alexa488 (Molecular Probes), Oyster-556 (Flownamics, DeNovo), Oyster-645 (Flownamics, DeNovo), Cy3 (GE-Amersham), Cy5 (GE-Amersham), CalβlO (Biosearch). Exemplified quenchers are: BHQl (Biosearch), BHQ2 (Biosearch), Iowa Black FQ (IDTdna), Iowa Black RQ (IDTdna), Eclipse (Epoch/Nanogen), Qsy series (Molecular Probes).

In a preferred embodiment, the 5 'end region of the first and/or of the second oligonucleotide comprises spacer regions to 5'end and/or 3'end regions. hi a preferred embodiment, the temperature-resistant DNA polymerase has a diminished or absent 5'->3'exonuclease activity, alternatively it is able to exercise its polymerization activity only at high temperature, hi a further preferred embodiment of the invention the temperature-resistant DNA polymerase is obtained by biochemical or recombinant techniques from Thermits flliformis. hi the event the target nucleic acid sequence is a target RNA sequence, a reverse transcription step is foreseen to synthesize cDNA. Preferably such step may be performed by a thermostable reverse transcriptase, more preferably the thermostable reverse transcriptase is co-present in an enzyme exerting also the thermostable DNA polymerase activity, thus allowing the method to be fully homogeneous also in case of target RNAs. hi a preferred embodiment, the target nucleic acid sequence is a specific sequence for an organism, preferably a pathogenic organism, preferably a virus, more preferably a virus of the following species: Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), Hepatitis A Virus (HAV), Human Immunodeficiency Virus (HIV), Human Cytomegalovirus (HCMV), Epstein Barr Virus (EBV), Varicella Zoster Virus (VZV), Human Herpes Viruses (HHV) 1, 2, 6 and 8, Human Papilloma Virus (HPV). Another object of the invention is an oligonucleotide system comprising at least a first oligonucleotide and a second oligonucleotide, wherein: i) at least one of said first and said second oligonucleotides includes a 5'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; iv) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence; hi a preferred embodiment, the oligonucleotide system comprises three oligonucleotides, a first and a second primer oligonucleotides and a third "anchor" oligonucleotide, in which: i) at least one of the primer oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises: - a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; and - a region comprising a sequence able to hybridize to a complementary region at the 3 ' end of the third "anchor" oligonucleotide; iv) said third "anchor" oligonucleotide comprises: - a 5'end region able to hybridize to a the target DNA sequence in a region upstream of the complementary sequence to said second oligonucleotide and - a 3 'end region able to hybridize to the complementary region of said second oligonucleotide, such as to act as an anchor; v) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence.

Preferably the recognition sequence is recognized by a temperature-resistant restriction endonuclease, more preferably by the enzyme PspGI, alternatively by an enzyme included in the group of TIiI, Tfil, BstNI, Apol, BstBI, BstEII, SmII, TspRI, Tsp45I or BsoBI.

Preferably, the coupled detection system is a fluorophore-quencher system.

Preferably the 5'end region of the first and of the second oligonucleotide further comprises spacer regions at its 5' and/or 3 'ends. Preferably, the target nucleic acid sequence is a specific sequence for an organism, preferably a pathogenic organism, preferably a virus, more preferably a virus of the following species:

HCV, HBV, HAV, HIV₅ HCMV, EBV, VZV, HHV 1, HHV 2, HHV 6, HHV8, HPV.

A further object of the invention is a kit for the amplification and the simultaneous detection of a target nucleic acid sequence comprising one of the oligonucleotide systems of the invention and a double strand, site specific, high temperature resistant cleaving agent.

Preferably the cleaving agent is a restriction endonuclease, more preferably, it is the enzyme

PspGI, alternatively it is TIiI, Tfil, BstNI, Apol, BstBI, BstEII, SmII, TspRI, Tsp45I or

BsoBI. Preferably the kit for the amplification and the simultaneous detection of a target nucleic acid sequence further comprises a temperature resistant DNA polymerase, preferably with a reduced or absent 5 '-> 3' exonuclease activity. In a more preferred aspect, the temperature resistant DNA polymerase is able to exercise its polymerizing activity only at a high temperature. In a further preferred embodiment of the invention the temperature-resistant DNA polymerase is obtained by biochemical or recombinant techniques from Ωtermus filiformis.

In a further aspect of the invention the kit further comprises a reverse transcriptase enzyme, preferably a thermostable reverse transcriptase enzyme.

In a preferred embodiment of the invention, the kit comprises a thermostable enzyme exerting both a reverse transcriptase and a DNA polymerase activity.

The invention shall be described with reference to non-limiting specific examples, including the following figures:

Figure 1. Schematic diagram of an embodiment of the method, with signal generated by both primers. Figure 2. Schematic diagram of the oligonucleotides of the invention, according to the method of Fig. 1.

Figure 3. Schematic diagram of an embodiment of the method, with signal generated by only one primer.

Figure 4. Schematic diagram of the oligonucleotides of the invention, according to the method of Fig. 3.

Figure 5. Schematic diagram of an embodiment of the method, in the variant with anchor, with signal generated by both primers.

Figure 6. Schematic diagram of the oligonucleotides of the invention, according to the method of Fig. 5.

Figure 7. Schematic diagram of an embodiment of the method, in the variant with anchor, with signal generated by only one primer.

Figure 8. Schematic diagram of the oligonucleotides of the invention, according to the method of Fig. 7.

Figure 9. Oligonucleotides used for the detection of HCMV DNA sequences.

Figure 10. Oligonucleotides used for the detection of EBV DNA sequences.

Figure 11. Oligonucleotides used for the detection of HCMV DNA sequences, in the variant with anchor. Figure 12. Target sequence for HCMV - SEQ ID No.16.

Figure 13. Target sequence for EBV a - SEQ ID No. 17.

Figure 14. Example of programmed cycles; the eye indicates the fluorescence detection step.

Figure 15. Quantification of the HCMV DNA sequences using the assay of the invention. A.

Amplification curves of 10-fold serial dilutions of HCMV genomic DNA consisting of 2x10⁶ — 2xlO^! target copies and a no-template control. B. Calibration curves generated using the MJ

Opticon 3 computer program in which Ct (threshold cycle derived from 15A) is plotted against the initial amount (number of copies) of HCMV genomic DNA (R = 0.999). C.

Electrophoresis on Ethidium Bromide-stained agarose gel of a reaction product. Fluorescence was detected on a Typhoon 9200 (Amersham) for Ethidium Bromide (EtBr) and for the fluorophore AlexaFluor 488 (dye).

Figure 16. Quantification of the EBV DNA sequences using the assay of the invention. A.

Amplification curves of 10-fold serial dilutions of EBV genomic DNA consisting of 2x10⁴ -

2XlO¹ target copies and a no-template control. B. Calibration curves generated using the MJ

Opticon 3 computer program in which Ct (threshold cycle derived from 16A) is plotted against the initial amount (number of copies) of EBV genomic DNA (R = 0.993). C.

Electrophoresis on Ethidium Bromide-stained agarose gel of a reaction product. Fluorescence was detected on a Typhoon 9200 (Amersham) for Ethidium Bromide (EtBr).

Figure 17. Quantification of the HCMV DNA sequences using the assay of the invention in the variant with anchor. A. Amplification curves of 10-fold serial dilutions of HCMV genomic DNA consisting of 2xlO⁴ - 2XlO¹ and a no-template. B. Calibration curves generated using the MJ Opticon 3 computer program in which Ct (threshold cycle derived from 17A) is plotted against the initial amount (number of copies) of HCMV genomic DNA (R = 0.998). C. O

Electrophoresis on Ethidium Bromide-stained agarose gel of a reaction product. Fluorescence was detected on a Typhoon 9200 (Amersham) for Ethidium Bromide (EtBr). Figure 18. Example of programmed cycles, the grey area (thermal step at 72⁰C for 31 seconds) indicates the fluorescence detection step. Figure 19. Quantification of HCMV DNA sequences using the OCEANII assay and AmpliTaq Gold DNA polymerase according to example 1. Panel A: amplification plots of 10- fold serial dilutions of HCMV DNA containing 10⁵-10 starting copy number and a no- template control (ntc); Panel B: simultaneous amplification plots of the Internal Control IC DNA occurring in the same samples of panel A. Panel C: Calibration curves generated using the SDS software where Ct (threshold cycle derived from Panel A) is plotted against starting quantity (log₁₀ of copy number) of HCMV genomic DNA.

Figure 20. Quantification of HCMV DNA sequences using the OCEANII assay and Platinum Tfi DNA polymerase according to example 2. Panel A: amplification plots of 10-fold serial dilutions of HCMV DNA containing 10⁵-10 starting copy number and a no-template control (ntc); Panel B: simultaneous amplification plots of the Internal Control IC DNA occurring in the same samples of panel A. Panel C Calibration curves generated using the SDS software where Ct (threshold cycle derived from Panel A) is plotted against starting quantity (1Og_1O of copy number) of HCMV genomic DNA. Figure 21. Schematic diagram of an embodiment of the method for the detection of RNA target sequences, in the variant with anchor, with signal generated by only one primer.

Figure 22. Schematic diagram of the oligonucleotides of the invention, according to the method of Fig. 21.

Figure 23. Oligonucleotides used for the detection of HAV RNA sequences, in the variant with anchor. Figure 24. Target sequence for HAV - SEQ ID No .18.

Figure 25. Quantification of the HAV RNA sequences using the assay of the invention. A- Amplifϊcation curves of 100-fold serial dilutions of HAV genomic RNA consisting of 80 ng/μl - 8x10^"5 ng/μl and a no-template control. B- Calibration curves generated using the MJ Opticon 3 computer program in which Ct (threshold cycle derived from 25A) is plotted against the initial amount (ng/μl) of HAV genomic RNA (R = 0.991). C. Electrophoresis on Ethidium Bromide-stained agarose gel of a reaction product. Fluorescence was detected on a Typhoon 9200 (Amersham) for Ethidium Bromide (EtBr) and for the fluorophore fluorescein (dye). 1. Example Method Ocean II for HCMV

Reagents

HCMV control DNA (Tebu-bio, 08-701-000) referred to as "target"

Primers: ES69QF and ES70QF, synthesized by IDT-DNA Technologies, Coralville, IA, "primers " (the sequences are indicated in Figure 9).

Reaction buffer: the stock 10x solution is constituted as follows: 100 mM TrisHCl, 500 mM

NaCl, 500 mM KCl, pH 8.0; all reagents supplied by Sigma, "buffer"

MgCl₂ 10O mM (Sigma), "MgCl₂" dNTPs 10 mM (Fermentas) mixture, "dNTPs " Formamide 100% (Sigma), "Formamide "

BSA 10Ox (New England Biolabs), "BSA "

Taq recombinant DNA polymerase 5U/μl (Fermentas), "Taq "

PspGI 10 U/μl (New England Biolabs), "PspGI"

Sterile apyrogenic water (SALF SpA), "ddw" Materials

PCR sample cooling system (Eppendorf) for the preparation of samples, "on ice"

Test tubes, pipettes and tips (Eppendorf, Finnpipette, ART) for the preparation of samples,

"plasticware" ^■ Gloves Low profile 0.2ml reaction tubes with optical caps "strips".

Nanodrop Spectrophotometer ND- 100 (Nanodrop Technologies, Wilmington, DE),

"Nanodrop "

Thermal cycler equipped with a fluorescence detector, Chromo-4 (BioRad), "thermal cycler"

Procedure a. Sample Preparation

Dilute the primers (shipped as powder) with ddw to obtain a 100 μM stock solution. Check for correct concentration using absorbance at 260 nm, in accordance with the manufacturer's instructions. Prepare a 10 μM work dilution in ddw. It is better to store stock solutions in aliquots at -20°C, while working dilutions should be stored at 4°C. Prepare the buffer and MgCl₂ in ddw. Prepare the reaction mixture as follows: 0,375 μM of primers, buffer Ix, 2 mM MgCl₂,, 5% Formamide, 0,3 mM dNTPs, 0,025 U/μl Taq, 0,5 U/μl PspGI. The final volume of the reaction mixture must be % of total reaction volume (i.e. 15 μl of reaction mixture + 5 μl of sample). Always keep the reagents on ice. Prepare the reaction mixture for at least 6 standard samples and 1 negative sample, plus the number of unknown samples to be assayed. An example of volumes to be mixed for 6 standard samples and 1 negative sample is shown in Table 1 (all volumes are in microliters). Table 1 - Composition of the sample mixture (assay for HCMV)

Vortex the reaction mixture for 5 seconds and rapidly centrifuge for 15 seconds at low speed.

From now on, always keep the reaction mixture on ice.

Distribute 15 μl of reaction mixture in the reaction tube. Keep the reaction tubes on ice. Start adding 5 μl of sample to the strips. Always follow this order: BSA Ix in the test tube of the control sample, (close the test tube), unknown samples. Close all these tubes before continuing. Keep the reaction tubes on ice. Change the gloves.

Prepare the target dilutions ("target dilutions") from the purchased solution ("target").

Initially dilute 4*10⁵ copies/μl in BSA Ix, then make serial dilutions (1:10) in Ix BSA to 4*10⁴ copies/μl, 4*10³ copies/μl, 4*10² copies/μl, 4x10¹ copies/μl, 4 copies/μl. Place the target dilutions on ice. Change the gloves.

Add 5μl of target dilutions to the reaction tubes. Add 5 μl of the target dilutions starting from the least concentrated to the most concentrated dilution. Close all tubes. Keep the strips in ice. Discard gloves. b. Reaction

The reaction follows the set-up of the method of Figs. 1 and 2. Similar results are also obtained with the method of Figs. 3 and 4.

Program the amplification cycles to obtain the following protocol: 50 cycles of denaturation at 94°C for 10 seconds, annealing-extension-digestion at 68°C for 1 minute. Include at the end of the annealing -extension-digestion step, the fluorescence detection for the appropriate fluorophore. An example of programmed cycles is described in Figure 14. Start the protocol. If the thermal cycler requires a few minutes to heat the lid wait until the lid has reached to correct working temperature and keep the reaction tubes on ice. Place the reaction tubes in the thermal cycler immediately before the start of the cycles. c. Data Analysis

Perform the subtraction of the baseline, i.e. the value of fluorescence measured in each sample before they enter the exponential reaction phase. Since in the first 3-5 cycles there is a slight increase in fluorescence, do not set the baseline as the first measured fluorescence, but as the fluorescence measured during the first 5-15 cycles (as long as no curve grows exponentially in the cycles considered) ("baseline subtraction").

From each standard sample and from each unknown sample, for each cycle, subtract the fluorescence value measured in the same cycle, in the negative control ("blank subtraction"). Set the fluorescence threshold level to at least twice the numeric value of the standard deviation measured in the interval of cycles used for the baseline subtraction. Alternatively, try to set the threshold in such a way that the value R² (linear regression coefficient between the known numeric values of the concentrations of standard samples and the number of the cycles whereat the value exceeds the fluorescence threshold value) is as close to 1 as possible. In this case, discard a threshold that is below the value of standard deviation measured in the interval of cycles used for the baseline subtraction. d. Results

The linear quantification range of the assay for the HCMV target DNA was determined. The samples were prepared according to Table 1. The results are shown in Fig. 15. To determine the linear quantification range and develop a calibration curve for quantification purposes, 10-fold serial dilutions of HCMV genomic DNA consisting of 2x10⁶ - 2XlO¹ target copies were analyzed (Figure 15A). Figure 15B shows that the linear dynamic range was of at least 5 orders of magnitude, from a number of target copies of 2xlO^δ to 2XlO¹. The correlation coefficient of the calibration curve was greater than 0.99. The specificity of the assay for the HCMV DNA was demonstrated by the continued lack of amplification in the negative control even after 50 cycles.

The analysis of the final results of the reaction on an agarose gel stained with EtBr (Figure 15C) shows that a single specific reaction product is obtained starting from any one of the assayed dilutions. The same gel scanned on a Typhoon 9200 laser scanner (Amersham) at

532/526 run shows that the products of the PCR contain the fluorescent dye (AlexaFluor

488). In the lower part of the gel, the primers that did not react reflect the efficiency of the amplification reactions, being progressively more consumed as the number of starting copies increases. No dimers of the primers are observed, even after 50 cycles.

The sequence of the target fragment is shown in Fig. 12.

2. Example Method Ocean II for EBV

Reagents

EBV control DNA (Tebu-bio, 08-702-000) referred to as "target" Primers: DA75QF and EG30QF, synthesised by IDT-DNA Technologies, Coralville, IA,

"primers" (the sequences are indicated in Fig. 10).

Reaction buffer: the stock 10x solution is constituted as follows: 100 niM TrisHCl, 500 mM

NaCl, 500 mM KCl, pH 8.0; all reactants supplied by Sigma, "buffer"

MgCl₂ 100 mM (Sigma), "MgCl₂" dNTPs 10 mM (Fermentas) mixture, "dNTPs"

BSA 10Ox (New England Biolabs), "BSA"

Taq recombinant DNA polymerase 5U/μl (Fermentas), "Taq Pol"

PspGI 10 U/μl (New England Biolabs), "PspGI"

Sterile apyrogenic water (SALF SpA), "ddw" Materials

PCR sample cooling system (Eppendorf) for the preparation of samples, "on ice"

"plasticware"

Gloves Low profile 0.2ml reaction tubes with optical caps, "strips".

Nanodrop Spectrophotometer ND-100 (Nanodrop Technologies, Wilmington, DE),

"Nanodrop"

Procedure A. Sample Preparation

Dilute the primers (shipped as powder) with ddw to obtain a 100 μM stock solution. Check for correct concentration using absorbance at 260 nm, in accordance with the manufacturer's instructions. Prepare a 10 μM work dilution in ddw. It is better to store stock solutions in aliquots at -20°C, while working dilutions should be stored at 4°C. Prepare the buffer and MgCl₂ in ddw. Prepare the reaction mixture as follows: 0.375 μM of primers, buffer Ix, 2 mM MgCl₂, 0.3 dNTPs, 0.025 U/μl Taq, 0.5 U/μl PspGI. The final volume of the reaction mixture must be ³A of total reaction volume (i.e. 15 μl of reaction mixture + 5 μl of sample). Always keep the reagents on ice. Prepare the reaction mixture for at least 4 standard samples and 1 negative sample, plus the number of unknown samples to be assayed. An example of volumes to be mixed for 4 standard samples and 1 negative sample is shown in Table 2 (all volumes are in microliters). Table 2 - Composition of the sample mixture (assay for EBV)

From now on, always keep the reaction mixture on ice.

Distribute 15 μl of reaction mixture in the strip. Keep the strips on ice.

Start adding 5 μl of sample to the reaction tubes. Always follow this order: BSA Ix in the test tube of the negative control, (close the test tube), unknown samples. Close all these tubes before continuing. Keep the strips on ice. Change the gloves.

Initially dilute l*10⁴ copies/μl in BSA Ix, then make serial dilutions (1:10) in Ix BSA to

4*10³ copies/μl, 4*10² copies/μl, 4XlO¹ copies/μl, 4 copies/μl. Place the target dilutions on ice. Change the gloves.

Add 5μl of target dilutions to the reaction tubes. Add 5 μl of the target dilutions starting from the least concentrated to the most concentrated dilution. Close all tubes. Keep the strips on ice. Discard gloves. b. Reaction ^

The reaction follows the set-up of the method of Figs. 1 and 2. Similar results are obtained with the method of Figs. 3 and 4.

Program the amplification cycles to obtain the following protocol: 50 cycles of: denaturation at 94°C for 10 seconds, annealing-extension-digestion at 65 °C for 1 minute. Include at the end of the annealing-extension-digestion step, the fluorescence detection for the appropriate fluorophore.

Start the protocol. If the thermal cycler requires a few minutes to heat the lid wait until the lid has reached to correct working temperature and keep the reaction tubes on ice. Place the reaction tubes in the thermal cycler immediately before the beginning of the cycles. c. Data Analysis

The linear quantification range of the assay for the EBV target DNA was determined. The samples were prepared according to Table 2. The results are shown in Fig. 16. To determine the linear quantification range and develop a calibration curve for quantification purposes, 10-fold serial dilutions of EBV genomic DNA consisting of 2x10⁴ - 2XlO¹ target copies were analyzed (Figure 16A). Figure 12B shows that the linear dynamic range was of at least 4 orders of magnitude, from a target copy number of 2x10⁴ to 2XlO¹. The correlation coefficient of the calibration curve was greater than 0.99. The specificity of the assay for the EBV DNA was demonstrated by the continued lack of amplification in the negative control even after 50 cycles.

The analysis of the final results of the reaction on an agarose gel stained with EtBr (Figure

16C) shows that a single specific reaction product is obtained starting from any one of the assayed dilutions. In the lower part of the gel, the primers that did not react reflect the efficiency of the amplification reactions, being progressively more consumed as the number of starting copies increases. No dimers of the primers are observed, even after 50 cycles.

The sequence of the target fragment is shown in Fig. 13.

3. Example Method Ocean II for HCMV - Variant with Anchor

Reagents HCMV control DNA (Tebu-bio, 08-701-000) referred to as "target"

Primers: ES75, synthesised by Sigma-Proligo, Hamburg, Germany and ES70QF, IDT-DNA

Technologies, Coralville, IA, "primers" (the sequences are indicated in Fig. 11).

Anchor: ES73d, synthesised by Sigma-Proligo, Hamburg, Germany.

Reaction buffer: the stock 10x solution is constituted as follows: 100 mM TrisHCl, 500 mM NaCl, 500 mM KCl, pH 8.0; all reactants supplied by Sigma, "buffer"

MgCl₂ 100 mM (Sigma), "MgCl₂" dNTPs 10 mM (Fermentas) mixture, "dNTPs"

BSA 10Ox (New England Biolabs), "BSA"

Taq recombinant DNA polymerase 5U/μl (Fermentas), "Taq Pol" PspGI 10 LVμl (New England Biolabs), "PspGF

Sterile apyrogenic water (SALF SpA), "ddw"

Materials

PCR sample cooling system (Eppendorf) for the preparation of samples, "on ice"

Test tubes, pipettes and tips (Eppendorf, Finnpipette, ART) for the preparation of samples, "plasticware"

Gloves

Low profile 0.2ml reaction tubes with optical caps, "strips".

Nanodrop Spectrophotometer ND- 100 (Nanodrop Technologies, Wilmington, DE),

"Nanodrop" Thermal cycler equipped with a fluorescence detector, Chromo-4 (BioRad), "thermal cycler"

Procedure a. Sample Preparation

Dilute the primers (shipped as powder) with ddw to obtain a 100 μM stock solution. Check for correct concentration using absorbance at 260 nm, in accordance with the manufacturer's instructions. Prepare a 10 μM work dilution in ddw. It is better to store stock solutions in aliquots at -20°C, while working dilutions should be stored at 4°C. Prepare the buffer and MgCl₂ in ddw. Prepare the reaction mixture as follows: 0.375 μM of primers, buffer Ix, 2 mM MgCl₂, 0.3 dNTPs, 0.025 U/μl Taq, 0.5 U/μl PspGI. The final volume of the reaction mixture must be ³Λ of total reaction volume (i.e. 15 μl of reaction mixture + 5 μl of sample). Always keep the reagents on ice. Prepare the reaction mixture for at least 4 standard samples and 1 negative sample, plus the number of unknown samples to be assayed. An example of volumes to be mixed for 4 standard samples and 1 negative sample is shown in Table 3 (all volumes are in microliters). Table 3 - Composition of the sample mixture (assay for HCMV - variant with anchor)

From now on, always keep the reaction mixture on ice.

Distribute 15 μl of reaction mixture in the strip, keep the strips on ice.

Add 5μl of target dilutions to the reaction tubes. Add 5 μl of the target dilutions starting from the least concentrated to the most concentrated dilution. Close all tubes. Keep the strips on ice. Discard the gloves. b. Reaction

The reaction follows the set-up of the method of Figs. 7 and 8. Similar results are also obtained with the method of Figs. 5 and 6. Program the amplification cycles to obtain the following protocol: 45 cycles of: denaturation at 94°C for 10 seconds, annealing-extension- digestion at 65°C for 1 minute. Include at the end of the annealing-extension-digestion step, the fluorescence detection for the appropriate fluorophore.

The linear quantification range of the assay for the HCMV target DNA was determined. The samples were prepared according to Table 3. The results are shown in Fig. 17. To determine the linear quantification range and develop a calibration curve for quantification purposes, 10-fold serial dilutions of HCMV genomic DNA consisting of 2xlO⁴ - 2x10* target copies were analyzed (Figure 17A). Figure 17B shows that the linear dynamic interval was of at least 4 orders of magnitude, from a number of target copies of 2x10⁴ to 2XlO¹. The correlation coefficient of the calibration curve was greater than 0.99.

The specificity of the assay for the HCMV DNA was demonstrated by the continued lack of amplification in the negative control even after £Q 45 cycles.

The analysis of the final results of the reaction on an agarose gel stained with EtBr (Figure 17C) shows that a single specific reaction product is obtained starting from any one of the assayed dilutions. In the terminal part of the gel, the primers that did not react reflect the efficiency of the amplification reactions, being progressively more consumed as the number of starting copies increases. No dimers of the primers are observed, even after 45 cycles.

The sequence of the fragment is shown in Fig. 12.

Further examples refer to different assay protocols with the same DNA templates and oligonucleotide sets but different DNA polymerases and optimised reaction conditions.

Instrument platform and procedures used are common. Protocols refer to duplex amplifications, where, along the reaction for the detection of the target DNA, an analogous second specific system is present for the detection of the Internal Control DNA, provided that the fluorophore/quencher pair is different to allow signal detection at a different wavelength without interfering with the detection of the target.

Example 4 Amplification of HCMV DNA using OCEANTI assay with anchor optimized with AB Gold polymerase

The following procedure was optimized for the use with AmpliTaq Gold DNA Polymerase

(Applied Biosystems).

Reagents

Plasmid DNA containing HCMV DNA control (region UL122-exon4 coding region for Immediate Early Antigen 1), referred to as "target dilutions ".

Plasmid DNA containing a portion of the human Beta-Globin gene referred to as "Internal

Control-IC".

Oligonucleotides: (for sequences see below)

ES73d (Anchor) ES75QF (Forward primer labelled with FAM)

ES 89 (Reverse Primer)

AD84d (Anchor for the Internal Control)

AD86QR (Forward primer labelled with MAXn for the Internal Control) AD70 (Reverse Primer for the Internal Control)

All oligonucleotides were synthesized by IDT DNA technologies, Coralville, IA, "primers "

(sequences attached).

Reaction buffer: GeneAmp 10x PCR Gold buffer, (Applied Biosystems), "buffer" 25mM MgCl₂ solution (Applied Biosystems), "MgCl₂ "

12.5mM dNTPs mix w.dUTP (Applied Biosystems), "dNTPs"

AmpliTaq GoIdONA Polymerase recombinant 5U/μL, (Applied Biosystems), "Taq Pol"

PspGI lOU/μL (New England Biolabs), "PspGI"

DMSO 100% solution, purchased from Sigma Aldrich, "DMSO" Sterile apyrogen water, "ddw"

Tris-EDTA Dilution buffer/Negative Control: "TE"

ROX reference dye, Invitrogen "ROX"

Amperase Uracyl-N-Glycosidase, (Applied Biosystems) "UNG"

Instrument and software ABI Prism 7500 thermalcycler, "real-time or thermal cycler" and appropriate plasticware

(tubes, strips, 96/wells plates, optical caps) (Applied Biosystems).

SDS - sequence detection software (Applied Biosystems).

Procedure

Sample preparation Reaction

Data analysis,

1) Sample preparation:

Dilute primers (shipped as powder) to a lOOμM stock solution with TE. Check for correct concentration using absorbance at 260 nm, according to manufacturer's instructions. It is better to store stock solutions in aliquots at -20⁰C, while working dilutions should be stored at

4°C.

Prepare reaction mix as follows: 0.150μM primers, Ix buffer, 2.5mM MgCl₂, 5% DMSO,

ImM dNTPs, 0.025U/μL Taq, 0.5U/μL PspGI, O.OlU/μL UNG, 0.06mM ROX, 0.25pg/μL

Internal control IC DNA₅. Final volume of the reaction mix must be Vz of the total reaction volume (i.e. 20μL reaction mix + 20μL sample). Prepare mix at least for 5 standard samples and 1 negative sample, plus the number of unknown samples to test. An example of volumes to mix for 5 standards and 1 negative sample is shown in Table 4 (all volumes in microlitres).

Table 4 — Sample mix composition.

Vortex reaction mix for 5 seconds and quickly spin down for 15 seconds at low speed.

Dispense 20μL of reaction mix in the tubes. Start adding 20μL of samples to the tubes. Close all tubes before continuing. Change gloves. Add 20μL of target dilutions to the tubes. Add the target dilutions starting from the less concentrated one to the most concentrated one. Close all the tubes. Discard gloves.

2) Reaction.

The reaction follows the method scheme of Figure 7 and 8.

Program the amplification cycles in order to obtain the following protocol: 2 minutes at 50°C, 10 minutes at 95°C, 60 cycles described as: denaturation at 95°C for 10 seconds, annealing at

65°C for 20 seconds, extension-digestion at 72⁰C for 32 seconds; a final incubation at 12°C for 2 minutes. Set reaction volume at 40μL; Mode: 9600 emulation; set the fluorescence collection step within the extension-digestion step for both fluorophores (see oligonucleotides sequence below). An example of programmed cycles is described in Figure 18. 3) Data analysis with SDS software.

Set manually threshold at 0.02 fluorescence a.u. (arbitrary units) for the FAM channel and to

0.01 a.u. for the MAXn channel. Enable manual baseline subtraction from cycle 3 to cycle 15.

4) Sequences of oligonucleotides used.

ES75QF - SEQ ID No.7 (modifications: Iowa black FQ on base 1, internal fluorescein-dT on base 11)

5 '-/SEBFQ/TTTCCAGGTT/iFluorT/GAGGCGAGTGTAACGGGCCATCGCCGA-S '

ES73d - SEQ ID No.8

(modifications: 3' terminal dideoxy- cytidine) 5'-CTGCTCTCCTAGTGTGGATGACCATCACTCGCCT/3ddC/-3'

ES89 - SEQ ID No.9

(no modifications)

5 '-TCCAGGTTTAGGTGACACCAGAGAATCAGAGGAGC-S '

AD86QR - SEQ ID No.10 (modifications: Iowa black FQ on base 1 , internal fluorescein-dT on base 11)

5'-/5IbFQ/TTTCCAGGTT/iMAXN/

GTGTGTGTGGCTAGAACCGAGGTAGAGTTTTCATCCATT-S'

AD84d - SEQ ID No.11

(modifications: 3' terminal dideoxy- cytidine) 5'-CCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTATCCACACACAC

AA/3ddC/-3'

AD70 - SEQ ID No.12

(no modifications)

5'-TTTCCAGGTTCACAGCTTGGTAAGCATATTGAAGATCG-S' 5) Results

The linear quantification range of the assay for HCMV target DNA was assessed. Samples were prepared according to Table 4. Results are shown in Figure 19. To determine the linear range and to develop a calibration curve for purposes of quantification, ten-fold serial dilutions of HCMV genomic DNA consisting of 10⁵-10 target copy number were analyzed . Specificity of the assay for HCMV target was demonstrated by the consistent lack of amplification in the no-template control even after 60 cycles. (Fig. 19 Panel A).

Fig. 19 Panel C shows that the dynamic linear range was at least 5 orders of magnitude, from

10⁵ to 10 target copy number per reaction. The correlation coefficient of the calibration curve

(R²) was greater than 0.99. Fig. 19 Panel B shows the concomitant amplification of the Internal Control IC DNA (duplex reaction) in the same samples shown in Panel A. As expected, the cycle threshold is almost identical among all the samples, although different end point values are reached, according to the level of amplification achieved in Panel A. These data confirms that a duplex amplification reaction occurs in each sample.

Example 5 Amplification of HCMV DNA using OCEANII variant with anchor assay optimized with Invitrogen Platinum Tfi DNA polymerase.

The following procedure was optimized for the use with Platinum Tfi DNA Polymerase

(mvitrogen).

Reagents

Control-IC".

Oligonucleotides:

ES73d (Anchor) ES75QF (Forward primer labelled with FAM)

ES89 (Reverse Primer)

AD84d (Anchor for the Internal Control)

AD86QR (Forward primer labelled with MAXn for the Internal Control)

AD70 (Reverse Primer for the Internal Control) All oligonucleotides were synthesized by IDT DNA technologies, Coralville, IA, "primers "

(sequences attached).

Reaction buffer: 5x Platinum Tfi reaction buffer, (Invitrogen), "buffer"

5OmM MgCl₂ (hivitrogen), "MgCl₂"

12.5mM dNTPs mix w.dUTP (Applied Biosystems), "dNTPs " Platinum Tfi DNA Polymerase 5U/μL, (Invitrogen), "Tfi Pol"

PspGI lOU/μL (New England Biolabs), "PspGI"

Sterile apyrogen water, "ddw"

Tris-EDTA Dilution buffer/Negative Control: "TE"

ROX reference dye, Invitrogen "ROX" Amperase Uracyl-N-Glycosidase, (Applied Biosystems) "UNG"

Instrument and software

ABI Prism 7500 thermalcycler, "real-time or thermal cycler" and appropriate plasticware

(tubes, strips, 96/wells plates, optical caps) (Applied Biosystems). SDS - sequence detection software (Applied Biosystems).

Procedure

Sample preparation

Reaction

Data analysis

1) Sample preparation:

Dilute primers (shipped as powder) to a lOOμM stock solution with TE. Check for correct concentration using absorbance at 260 nm, according to manufacturer's instructions. It is better to store stock solutions in aliquots at -2O⁰C, while working dilutions should be stored at

4°C.

ImM dNTPs, 0.025U/μL Tfi, 0.5U/μL PspGI, O.OlU/μL UNG, 0.06mM ROX, 0.25pg/μL

Internal control IC DNA₅. Final volume of the reaction mix must be ¹A of the total reaction volume (i.e. 20μL reaction mix + 20μL sample). Prepare mix at least for 5 standard samples and 1 negative sample, plus the number of unknown samples to test. An example of volumes to mix for 5 standards and 1 negative sample is shown in Table 5 (all volumes in microlitres).

Table 5 - Sample mix composition.

Vortex reaction mix for 5 seconds and quickly spin down for 15 seconds at low speed. Dispense 20μL of reaction mix in the tubes. Start adding 20μL of samples to the tubes. Close all tubes before continuing. Change gloves.

Add 20μL of target dilutions to the tubes. Add the target dilutions starting from the less concentrated one to the most concentrated one. Close all the tubes. Discard gloves. 2) Reaction.

The reaction follows the method scheme of Figure 7 and 8.

Program the amplification cycles in order to obtain the following protocol: 2 minutes at 50°C, 2 minutes at 95°C, 60 cycles described as: denaturation at 95°C for 15 seconds, annealing at 60°C for 30 seconds, extension-digestion at 68°C for 32 seconds; a final incubation at 12⁰C for 2 minutes. Set reaction volume at 40μL; Mode: 9600 emulation; set the fluorescence collection step within the extension-digestion step for both fluorophores (see oligonucleotides sequence above).

3) Data analysis with SDS software.

Set manually threshold at 0.02 fluorescence a.u. (arbitrary units) for the FAM channel and to 0.01 a.u. for the MAXn channel. Enable manual baseline subtraction from cycle 3 to cycle 15.

4) Sequences of oligonucleotides used.

The oligonucleotides used were the same as in Example 4.

5) Results

The linear quantification range of the assay for HCMV target DNA was assessed. Samples were prepared according to Table 5. Results are shown in Figure 20. To determine the linear range and to develop a calibration curve for purposes of quantification, ten-fold serial dilutions of HCMV genomic DNA consisting of 10⁵-10 target copy number were analyzed

(Fig. 20 Panel A).

Fig. 20 Panel C shows that the dynamic linear range was at least 5 orders of magnitude, from 10⁵ to 10 target copy number per reaction. The correlation coefficient of the calibration curve

(R²) was greater than 0.99. Specificity of the assay for HCMV target was demonstrated by the consistent lack of amplification in the no-template control even after 60 cycles.

Fig. 20 Panel B shows the concomitant amplification of the Internal Control IC DNA (duplex reaction) in the same samples shown in Panel A. As expected, the cycle threshold is almost identical among all the samples, although different end point values are reached, according to the level of amplification achieved in Panel A. These data confirms that a duplex amplification reaction occurs in each sample. 2

Example 6. Method for the detection of HAV RNA sequences- Variant with Anchor

Reagents

HAV control RNA extracted from surnatant of infected monkey kidney (VERO) cells, with

QIAamp Viral RNA from Qiagen (Hilden, Germany), referred to as "target" Primers: AG2, synthesized by Sigma-Proligo, Hamburg, Germany and AGlQF, HDT-DNA

Technologies, Coralville, IA, "primers" (the sequences are indicated in Fig. 23).

Anchor: AGId, synthesized by Sigma-Proligo, Hamburg, Germany.

Superscript™ III One-Step RT-PCR System with Platinum® Taq DNA polymerase Kit from Invitrogen (Invitrogen, Carlsbad, CA, USA), Invitrogen Kit Reaction buffer: 2x Reaction Mix provided in the Invitrogen Kit, constituted as follows:

0.4mM of each dNTP, 3.2 mM MgSO₄ , referred to as "Reaction mix"

5mM Magnesium Sulfate provided in the Invitrogen Kit, "MgSO₄"

BSA 10Ox (New England Biolabs), "BSA"

Superscript III RT/ Platinum Taq Mix provided in the Invirrogen Kit, "RT/ Taq pol" PspGI 10 U/μl (New England Biolabs), "PspGF

Sterile apyrogenic water (SALF SpA), "ddw"

Materials

PCR sample cooling system (Eppendorf) for the preparation of samples, "on ice"

Gloves

Low profile 0.2ml reaction tubes with optical caps, "strips".

Nanodrop Spectrophotometer ND- 100 (Nanodrop Technologies, Wilmington, DE),

Procedure a. Sample Preparation

Dilute the primers (shipped as powder) with ddw to obtain a 100 μM stock solution. Check for correct concentration using absorbance at 260 run, in accordance with the manufacturer's instructions. Prepare a 10 μM work dilution in ddw. It is better to store stock solutions in aliquots at -20⁰C, while working dilutions should be stored at 4°C. Prepare the reaction mixture as follows: 0.2 μM of primers, 0.02 μM of anchor, 2x Reaction Mix (0.4mM of each dNTP, 3.2 mM MgSO₄), 0.2mM Magnesium Sulfate, lμl Superscript III RT/ Platinum Taq Mix , 0.5 U/μl PspGI.

The final volume of the reaction mixture must be % of total reaction volume (i.e. 15 μl of reaction mixture + 5 μl of sample). Always maintain the reagents on ice. Prepare the reaction mixture for at least 4 standard samples and 1 negative sample, plus the number of unknown samples to be assayed. An example of volumes to be mixed for 4 standard samples and 1 negative sample is shown in Table 6 (all volumes are in microliters).

Table 6 - Composition of the sample mixture (assay for HAV - variant with anchor)

Vortex the reaction mixture for 5 seconds and rapidly centrifuge for 15 seconds at low speed. From now on, always keep the reaction mixture on ice.

Distribute 15 μl of reaction mixture in the strip. Maintain the strips on ice.

Start adding 5 μl of sample to the reaction tubes. Always follow this order: BSA Ix in the test tube of the negative control, (close the test tube), unknown samples. Close all these tubes before continuing. Maintain the strips in ice. Change the gloves. Prepare the target dilutions ("target dilutions") from the starting target solution.

The starting target solution is 80ng/μl. Make serial dilutions (1:100) in H₂O to δxlO^ng/μl,

8xlO^'3ng/μl, 8xlO^"5ng/μl . Place the target dilutions on ice.

Change the gloves.

Add 5μl of indicated target dilutions to the reaction tubes. Add 5 μl of the target dilutions starting from the least concentrated to the most concentrated dilution. Close all tubes.

Maintain the strips on ice. Discard gloves. b. Reaction

The reaction follows the set-up of the method of Figs. 21 and 22. Similar results are also obtained with the method when signal was both generated by both primers. The thermal- cycling program consist of two parts: the first part represent retro-transcription protocol (production of cDNA) at 60°C for 30 min, denaturation at 94°C for 2 min and the second part represent the protocol of 40 amplification cycles: denaturation at 95°C for 15 seconds, annealing-extension-digestion at 65°C for 30 seconds and extension to 68°C for 1 min. Final extension step at 68°C for 5 minutes. Include at the end of the annealing-extension-digestion step, the fluorescence detection for the appropriate fluorophore.

Start the protocol. If the thermal cycler requires a few minutes to heat the lid wait until the lid has reached to correct working temperature and keep the reaction tubes on ice. Place the reaction tubes in the thermal cycler immediately before the beginning of the cycles. c. Data Analysis Perform the subtraction of the baseline, i.e. the value of fluorescence measured in each sample before they enter the exponential reaction phase. Since in the first 3-5 cycles there is a slight increase in fluorescence, do not set the baseline as the first measured fluorescence, but as the fluorescence measured during the first 5-15 cycles (as long as no curve grows exponentially in the cycles considered) ("baseline subtraction"). From each standard sample and from each unknown sample, for each cycle, subtract the fluorescence value measured in the same cycle, in the negative control ("blank subtraction"). Set the fluorescence threshold level to at least twice the numeric value of the standard deviation measured in the interval of cycles used for the baseline subtraction. Alternatively, try to set the threshold in such a way that the value R² (linear regression coefficient between the known numeric values of the concentrations of standard samples and the number of the cycles whereat the value exceeds the fluorescence threshold value) is as close to 1 as possible. In this case, discard a threshold that is below the value of standard deviation measured in the interval of cycles used for the baseline subtraction. d. Results The linear quantification range of the assay for the HAV target RNA was determined. The samples were prepared according to Table 6. The results are shown in Fig. 25. To determine the linear quantification range and develop a calibration curve for quantification purposes, 100-fold serial dilutions of HAV genomic RNA consisting of 80 ng/μl - 8xlO^"5 ng/μl were analyzed (Figure 25A). Figure 25B shows that the linear dynamic interval was of at least 6 orders of magnitude, from 80 ng/μl - 8xlO^"5 ng/μl target RNA. The correlation coefficient of the calibration curve was greater than 0.99.

The specificity of the assay for the HAV RNA was demonstrated by the continued lack of amplification in the negative control even after 40 cycles. The analysis of the final results of the reaction on an Ethidium Bromide-stained agarose gel (Figure 25 C) shows that a single specific reaction product is obtained starting from any one of the assayed dilutions. The same gel scanned on a Typhoon 9200 laser scanner (Amersham) at 532/526 nm shows that the products of the PCR contain the fluorescent dye (fluorescein). In the lower part of the gel, the primers that did not react reflect the efficiency of the amplification reactions, being progressively more consumed as the starting target RNA increases. No dimers of the primers are observed. The sequence of the target fragment is shown in Fig. 24.

Similar results were obtained using a single thermostable DNA polymerase (jTth DNA polymerase from Applied Biosystems, Foster City, CA, USA) for both polymerization reactions (i.e. reverse transcription of RNA to cDNA and the subsequent amplification using the PCR process) instead of a mix of two different enzymes.

Claims

1. A method for detecting the presence or the absence of a target nucleic acid sequence in a sample, comprising the following steps: a) contacting the sample with an oligonucleotide system under hybridization conditions such as to form a reaction mixture, in which said oligonucleotide system comprises at least a first oligonucleotide and a second oligonucleotide, wherein: i) at least one of said first and said second oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; iv) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence; b) adding, with appropriate substrates and cofactors at a suitable ionic and pH environment, both a temperature resistant DNA polymerase and said double strand, site specific, high temperature resistant cleaving agent, to said reaction mixture, under predetermined reaction conditions such that, if the target nucleic acid sequence is present in the sample, said first and second oligonucleotide hybridize thereto and prime the DNA polymerase reaction to obtain a first specific amplified product; c) cycling the hybridization of said first and said second oligonucleotide to said first specific amplified product such that a second specific amplified product is extended comprising, respectively, the 5 'end region of the second or of the first oligonucleotide, forming a double stranded recognition site for said cleaving agent so that said agent specifically cleaves the recognition sequence and induces the generation of a signal by means of the coupled detection system; d) detecting the generated signal.

2. A method for detecting the presence or the absence of a target nucleic acid sequence in a sample comprises the following steps: a) contacting the sample with an oligonucleotide system under hybridization conditions such as to form a reaction mixture, in which said oligonucleotide system comprises three oligonucleotides, the first and the second primer oligonucleotides being as claimed in claim 1 and a third "anchor" oligonucleotide, in which: i) at least one of the primer oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises: - a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; and - a region comprising a sequence able to hybridize to a complementary region at the 3 ' end of the third "anchor" oligonucleotide; iv) said third "anchor" oligonucleotide comprises: - a 5'end region able to hybridize to the target nucleic acid sequence in a region upstream of the complementary sequence to said second oligonucleotide and - a 3 'end region able to hybridize to the complementary region of said second oligonucleotide, such as to act as an anchor; v) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence; b) adding, with appropriate substrates and cofactors at a suitable ionic and pH environment, both a temperature resistant DNA polymerase and said double strand, site specific, high temperature resistant cleaving agent, to said reaction mixture, under predetermined reaction conditions such that, if the target nucleic acid sequence is present in the sample, said first, second and third oligonucleotides hybridize thereto, said second and said third oligonucleotide hybridize to each other, and said first and said second oligonucleotide prime the DNA polymerase reaction to obtain a first specific amplified product; c) cycling the hybridization of said first and said second oligonucleotide to said first specific amplified product such that a second specific amplified product is extended comprising, respectively, the 5 'end region of the second or of the first oligonucleotide, forming a double stranded recognition site for said cleaving agent so that said agent specifically cleaves the recognition sequence and induces the generation of a signal by means of the coupled detection system; d) detecting the generated signal.

3. The method according to claim 1 or 2 wherein the double strand, site specific, high temperature resistant cleaving agent is a restriction endonuclease.

4. The method according to claim 3 wherein the restriction endonuclease belongs to the group of PspGI, TIiI, Tfil, BstNI, Apol, BstBI, BstEII, SmII, TspRI, Tsp45I or BsoBI.

5. The method according to claim 1 or 2 wherein the coupled detection system is a fluorophore-quencher system.

6. The method according to any of previous claims wherein the 5'end region of the first and/or of the second oligonucleotide comprises spacer regions to 5'end and/or 3 'end regions.

7. The method according to claim 1 or 2 wherein the temperature-resistant DNA polymerase has a diminished or absent 5'->3'exonuclease activity.

8. The method according to claim 1 or 2 wherein the temperature-resistant DNA polymerase is able to exercise its polymerization activity only at high temperature.

9. The method according to claim 1 or 2 wherein the temperature-resistant DNA polymerase is obtained by biochemical or recombinant techniques from Thermus filiformis.

10. The method according to claim 1 or 2 wherein the target nucleic acid sequence is a target RNA sequence and said target RNA sequence is reverse transcribed to cDNA before peforming the steps of claim 1 or 2.

11. The method according to claim 1 wherein the reverse transcription step is performed by a thermostable reverse transcriptase.

12. The method according to claim 11 wherein the thermostable reverse transcriptase is co- present in an enzyme exerting also the thermostable DNA polymerase activity allowing the method to be fully homogeneous.

13. The method according to any of previous claims wherein the target DNA sequence is a specific sequence for an organism.

14. The method according to claim 10 wherein the organism is a pathogenic organism.

15. The method according to claim 11 wherein the pathogenic organism is a virus.

16. The method according to claim 12 wherein the virus belongs to the following species: Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), Hepatitis A Virus (HAV), Human Immunodeficiency Virus (HIV), Human Cytomegalovirus (HCMV), Epstein Barr Virus (EBV), Varicella Zoster Virus (VZV), Human Herpes Viruses (HHV) 1, 2, 6 and 8, Human Papilloma Virus (HPV).

17. An oligonucleotide system comprising at least a first oligonucleotide and a second oligonucleotide, wherein: i) at least one of said first and said second oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; iv) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence.

18. An oligonucleotide system according to claim 17 comprising three oligonucleotides, a first and a second primer oligonucleotides and a third "anchor" oligonucleotide, in which: i) at least one of the primer oligonucleotides includes a 5 'end region comprising a recognition sequence being able to be cleaved by a double strand, site specific, high temperature resistant cleaving agent, said 5 'end region having covalently linked at its extremities a coupled detection system so that when the recognition sequence is cleaved by said double strand, site specific, high temperature resistant cleaving agent, a signal is generated; ii) said first oligonucleotide comprises a 3 'end region able to hybridize to a complementary region of one extremity of one strand of the target nucleic acid sequence; iii) said second oligonucleotide comprises: - a 3 'end region able to hybridize to a complementary region of the opposite extremity of the other strand of the target nucleic acid sequence; and - a region comprising a sequence able to hybridize to a complementary region at the 3 'end of the third "anchor" oligonucleotide; iv) said third "anchor" oligonucleotide comprises: - a 5'end region able to hybridize to a the target nucleic acid sequence in a region upstream of the complementary sequence to said second oligonucleotide and - a 3 'end region able to hybridize to the complementary region of said second oligonucleotide, such as to act as an anchor; v) said first and said second oligonucleotide are designed such as to have substantially the same melting temperature when hybridized to the respective complementary regions of the target nucleic acid sequence.

19. The oligonucleotide system according to claim 17 or 18 wherein the recognition sequence is recognized by a temperature-resistant restriction endonuclease.

20. The oligonucleotide system according to claim 19 wherein the temperature-resistant restriction endonuclease belongs to the group of: PspGI, TIiI, TfLI. BstNI, Apol, BstBI, BstEII, SmII, TspRI, Tsp45I or BsoBI.

21. The oligonucleotide system according to claim 17 or 18 wherein the coupled detection system is a fluorophore-quencher system.

22. The oligonucleotide system according to claim 17 or 18 wherein the 5 'end region of the first and of the second oligonucleotide further comprises spacer regions at its 5' and/or 3 'ends.

23. The oligonucleotide system according to claim 17 to 22 wherein the nucleic acid target sequence is a specific sequence for an organism.

24. The oligonucleotide system according to claim 23 wherein the organism is a pathogenic organism.

25. The oligonucleotide system according to claim 24 wherein the pathogenic organism is a virus.

26. The oligonucleotide system according to claim 25 wherein the virus belongs to one of the following species: HCV, HBV, HAV, HIV, HCMV, EBV, HHV 1, HHV 2, HHV 6, HHV8, HPV.

27. A kit for the amplification and the simultaneous detection of a target nucleic acid sequence comprising one of the oligonucleotide systems of the invention and a double strand, site specific, high temperature resistant cleaving agent.

28. The kit according to claim 27 wherein the cleaving agent is a restriction endonuclease.

29. The kit according to claim 28 wherein the restriction endonuclease belongs to the group of PspGI, TIiI, Tfil, BstNI, Apol, BstBI, BstEII, SmII, TspRI, Tsp45I or BsoBI.

30.^' The kit according to claim 27 to 29 further comprising a temperature resistant DNA polymerase.

31. The kit according to claim 30 wherein the temperature resistant DNA polymerase has a reduced or absent 5'->3' exonuclease activity.

32. The kit according to claim 31 wherein the temperature resistant DNA polymerase is able to exercise its polymerizing activity only at a high temperature.

33. The kit according to claim 30 wherein the temperature resistant DNA polymerase is obtained by biochemical or recombinant techniques from Thermus fitiformis.

34. The kit according to claim 27 to 30 further comprising a reverse transcriptase enzyme.

35. The kit according to claim 34 wherein the reverse transcriptase enzyme is thermostable.

36. The kit according to claim 35 comprising a thermostable enzyme exerting both a reverse transcriptase and a DNA polymerase activity.