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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6816—Hybridisation assays characterised by the detection means
  • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
• • 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
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates

Definitions

• 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.
• 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.
• 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
• 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
• 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
• 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
• PspGI restriction endonuclease
• 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.
• 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), Cal ⁇ lO (Biosearch).
• Exemplified quenchers are: BHQl (Biosearch), BHQ2 (Biosearch), Iowa Black FQ (IDTdna), Iowa Black RQ (IDTdna), Eclipse (Epoch/Nanogen), Qsy series (Molecular Probes).
• the 5 'end region of the first and/or of the second oligonucleotide comprises spacer regions to 5'end and/or 3'end regions.
• 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.
• a reverse transcription step is foreseen to synthesize cDNA.
• thermostable reverse transcriptase 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.
• 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).
• HCV Hepatitis C Virus
• HBV Hepatitis B Virus
• HAV Hepatitis A Virus
• HIV Human Immunodeficiency Virus
• HMV Human Cytomegalovirus
• EBV Epstein Barr Virus
• VZV Varicella Zoster Virus
• HHV Human Herpes Viruses
• HPV Human Papilloma Virus
• 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
• 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.
• 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.
• the coupled detection system is a fluorophore-quencher system.
• the 5'end region of the first and of the second oligonucleotide further comprises spacer regions at its 5' and/or 3 'ends.
• 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, HIV 5 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.
• 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
• 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.
• the temperature resistant DNA polymerase is able to exercise its polymerizing activity only at a high temperature.
• the temperature-resistant DNA polymerase is obtained by biochemical or recombinant techniques from ⁇ termus filiformis.
• the kit further comprises a reverse transcriptase enzyme, preferably a thermostable reverse transcriptase enzyme.
• the kit comprises a thermostable enzyme exerting both a reverse transcriptase and a DNA polymerase activity.
• 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.
• FIG. 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.
• 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 17 Quantification of the HCMV DNA sequences using the assay of the invention in the variant with anchor.
• 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).
• Ct threshold cycle derived from 17A
• FIG. 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 10 5 -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.
• FIG. 25 Quantification of the HAV RNA sequences using the assay of the invention.
• 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/ ⁇ l) of HAV genomic RNA (R 0.991).
• Ct threshold cycle derived from 25A
• Ct 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).
• EtBr Ethidium Bromide fluorescein
• HCMV control DNA (Tebu-bio, 08-701-000) referred to as "target"
• Reaction buffer the stock 10x solution is constituted as follows: 100 mM TrisHCl, 500 mM
• SALF SpA Sterile apyrogenic water
• Test tubes pipettes and tips (Eppendorf, Finnpipette, ART) for the preparation of samples,
• Nanodrop Spectrophotometer ND- 100 Nanodrop Technologies, Wilmington, DE
• Thermal cycler equipped with a fluorescence detector, Chromo-4 (BioRad), "thermal cycler"
• the final volume of the reaction mixture must be % of total reaction volume (i.e. 15 ⁇ l of reaction mixture + 5 ⁇ l 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)
• target dilutions Prepare the target dilutions ("target dilutions") from the purchased solution ("target”).
• baseline subtraction 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").
• 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.
• Figure 15A To determine the linear quantification range and develop a calibration curve for quantification purposes, 10-fold serial dilutions of HCMV genomic DNA consisting of 2x10 6 - 2XlO 1 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 ⁇ to 2XlO 1 .
• 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 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.
• EBV control DNA (Tebu-bio, 08-702-000) referred to as "target" Primers: DA75QF and EG30QF, synthesised by IDT-DNA Technologies, Coralville, IA,
• Reaction buffer the stock 10x solution is constituted as follows: 100 niM TrisHCl, 500 mM
• Taq recombinant DNA polymerase 5U/ ⁇ l Fermentas
• Taq Pol Taq recombinant DNA polymerase 5U/ ⁇ l
• SALF SpA Sterile apyrogenic water
• Test tubes pipettes and tips (Eppendorf, Finnpipette, ART) for the preparation of samples,
• Nanodrop Spectrophotometer ND-100 Nanodrop Technologies, Wilmington, DE
• Thermal cycler equipped with a fluorescence detector, Chromo-4 (BioRad), "thermal cycler"
• the final volume of the reaction mixture must be 3 A of total reaction volume (i.e. 15 ⁇ l of reaction mixture + 5 ⁇ l 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)
• target dilutions Prepare the target dilutions ("target dilutions") from the purchased solution ("target”).
• amplification cycles 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.
• baseline subtraction 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").
• 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.
• Figure 16A To determine the linear quantification range and develop a calibration curve for quantification purposes, 10-fold serial dilutions of EBV genomic DNA consisting of 2x10 4 - 2XlO 1 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 2x10 4 to 2XlO 1 .
• 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.
• 16C shows that a single specific reaction product is obtained starting from any one of the assayed dilutions.
• 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.
• 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"
• SALF SpA Sterile apyrogenic water
• Nanodrop Spectrophotometer ND- 100 Nanodrop Technologies, Wilmington, DE
• the final volume of the reaction mixture must be 3 ⁇ of total reaction volume (i.e. 15 ⁇ l of reaction mixture + 5 ⁇ l 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)
• target dilutions Prepare the target dilutions ("target dilutions") from the purchased solution ("target”).
• 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.
• baseline subtraction 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").
• 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.
• Figure 17A To determine the linear quantification range and develop a calibration curve for quantification purposes, 10-fold serial dilutions of HCMV genomic DNA consisting of 2xlO 4 - 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 2x10 4 to 2XlO 1 .
• the correlation coefficient of the calibration curve was greater than 0.99.
• 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.
• Plasmid DNA containing HCMV DNA control (region UL122-exon4 coding region for Immediate Early Antigen 1), referred to as "target dilutions ".
• AD86QR Forward primer labelled with MAXn for the Internal Control
• AD70 Reverse Primer for the Internal Control
• Reaction buffer GeneAmp 10x PCR Gold buffer, (Applied Biosystems), "buffer” 25mM MgCl 2 solution (Applied Biosystems), "MgCl 2 "
• DMSO 100% solution purchased from Sigma Aldrich, "DMSO” Sterile apyrogen water, "ddw”
• ROX reference dye Invitrogen "ROX"
• Dilute primers (shipped as powder) to a lOO ⁇ M 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 -20 0 C, while working dilutions should be stored at
• reaction mix as follows: 0.150 ⁇ M primers, Ix buffer, 2.5mM MgCl 2 , 5% DMSO,
• ImM dNTPs 0.025U/ ⁇ L Taq, 0.5U/ ⁇ L PspGI, O.OlU/ ⁇ L UNG, 0.06mM ROX, 0.25pg/ ⁇ L
• Final volume of the reaction mix must be Vz of the total reaction volume (i.e. 20 ⁇ L reaction mix + 20 ⁇ L sample).
• Vz of the total reaction volume (i.e. 20 ⁇ L reaction mix + 20 ⁇ L sample).
• An example of volumes to mix for 5 standards and 1 negative sample is shown in Table 4 (all volumes in microlitres).
• Dispense 20 ⁇ L of reaction mix in the tubes start adding 20 ⁇ L of samples to the tubes. Close all tubes before continuing. Change gloves. Add 20 ⁇ L 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.
• AD86QR - SEQ ID No.10 (modifications: Iowa black FQ on base 1 , internal fluorescein-dT on base 11)
• 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 10 5 -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
• 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.
• Plasmid DNA containing HCMV DNA control (region UL122-exon4 coding region for Immediate Early Antigen 1), referred to as "target dilutions ".
• AD86QR Forward primer labelled with MAXn for the Internal Control
• Reaction buffer 5x Platinum Tfi reaction buffer, (Invitrogen), "buffer"
• ROX reference dye Invitrogen "ROX” Amperase Uracyl-N-Glycosidase, (Applied Biosystems) "UNG”
• Dilute primers (shipped as powder) to a lOO ⁇ M 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 -2O 0 C, while working dilutions should be stored at
• reaction mix as follows: 0.150 ⁇ M primers, Ix buffer, 2.5mM MgCl 2 , 5% DMSO,
• ImM dNTPs 0.025U/ ⁇ L Tfi, 0.5U/ ⁇ L PspGI, O.OlU/ ⁇ L UNG, 0.06mM ROX, 0.25pg/ ⁇ L
• Final volume of the reaction mix must be 1 A of the total reaction volume (i.e. 20 ⁇ L reaction mix + 20 ⁇ L sample).
• An example of volumes to mix for 5 standards and 1 negative sample is shown in Table 5 (all volumes in microlitres).
• Vortex reaction mix for 5 seconds and quickly spin down for 15 seconds at low speed. Dispense 20 ⁇ L of reaction mix in the tubes. Start adding 20 ⁇ L of samples to the tubes. Close all tubes before continuing. Change gloves.
• the oligonucleotides used were the same as in Example 4.
• Fig. 20 Panel C shows that the dynamic linear range was at least 5 orders of magnitude, from 10 5 to 10 target copy number per reaction.
• Panel B shows the concomitant amplification of the Internal Control IC DNA (duplex reaction) in the same samples shown in Panel A.
• 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.
• Target QIAamp Viral RNA from Qiagen (Hilden, Germany), referred to as "target” Primers: AG2, synthesized by Sigma-Proligo, Hamburg, Germany and AGlQF, HDT-DNA
• Anchor AGId, synthesized by Sigma-Proligo, Hamburg, Germany.
• reaction mix 0.4mM of each dNTP, 3.2 mM MgSO 4 , referred to as "Reaction mix"
• SALF SpA Sterile apyrogenic water
• Nanodrop Spectrophotometer ND- 100 Nanodrop Technologies, Wilmington, DE
• the final volume of the reaction mixture must be % of total reaction volume (i.e. 15 ⁇ l of reaction mixture + 5 ⁇ l 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).
• the starting target solution is 80ng/ ⁇ l.
• 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.
• thermostable DNA polymerase jTth DNA polymerase from Applied Biosystems, Foster City, CA, USA
• polymerization reactions i.e. reverse transcription of RNA to cDNA and the subsequent amplification using the PCR process

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