WO2011131192A1 - Nouveau procédé d'amplification de séquences nucléotidiques - Google Patents

Nouveau procédé d'amplification de séquences nucléotidiques Download PDF

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WO2011131192A1
WO2011131192A1 PCT/DK2011/000026 DK2011000026W WO2011131192A1 WO 2011131192 A1 WO2011131192 A1 WO 2011131192A1 DK 2011000026 W DK2011000026 W DK 2011000026W WO 2011131192 A1 WO2011131192 A1 WO 2011131192A1
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amplification
pcr
reaction
specific
nucleotide sequence
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PCT/DK2011/000026
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Lena Erlandsson
Anders Fomsgaard
Lars-Peter Nielsen
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Statens Serum Institut
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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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

Definitions

  • the present invention discloses a nucleotide sequence amplification method comprising a random unbiased whole genome amplification (WGA) pre-amplification reaction followed by a specific amplification performed in the same vial or tube where the two reaction mixtures are separated by a wax layer so the reaction mixture on top of the wax layer is the pre-amplification mixture and the reaction mixture under the wax layer is the specific amplification.
  • WGA whole genome amplification
  • PCR real-time polymerase chain reaction
  • Herpes Simplex virus PCR which is a widely used and validated PCR assay for cerebrospinal fluids (CSF)
  • CSF cerebrospinal fluids
  • the detection level of the routine PCR is not sensitive enough to detect the causing agent. Even if every measure has been taken to design the most sensitive primers and probes from the sequence known about a specific virus, the PCR may still not be sensitive enough for clinical situations with expected low viral load or limited amount of sample. Moreover, there appears to be an inherent limitation to the PCR amplification of small amounts from complex samples, known as the "Monte Carlo effect" (Karrer et al., 1995). Complex samples, such as a clinical sample, containing a low viral copy number close to the detection level of a PCR assay will experience large variations in amplification and reduced reproducibility.
  • One current technique used to increase the sensitivity of a virus-specific PCR-assay is nested-PCR or semi- nested-PCR, where two sets of primers are used in two successive PCR-runs.
  • the second set of primers are intended to amplify a target within the amplicon produced in the first run, using the first set of primers.
  • the first PCR reaction generates relatively short DNA products that can be difficult to use as templates in the second PCR-reaction.
  • nested-PCR therefore only results in a 10- to 100-fold increase in amplification (Perrott et al., 2009) and present a serious risk of contamination, since amplicons from the first run needs to be transferred to the second run reaction tube.
  • Phi29 DNA polymerase By isothermal multiple displacement amplification (MDA) in the presence of random primers, Phi29 DNA polymerase allows for uniform amplification across genomes with less than 3-fold bias (Dean et al., 2002; Hosono et al., 2003; Lasken and Egholm, 2003). Furthermore, the Phi29 DNA polymerase has the highest processivity rate reported, -70 000 bases every time it binds (Blanco et al., 1989) and high fidelity with an error rate of only 1 in 10 6 -10 7 bases (Esteban et al., 1993).
  • the Phi29 DNA polymerase can, in a 50 ⁇ 1 reaction, generate ⁇ 40 ⁇ of amplified DNA after 16 hours incubation at 30°C (Repli-g Mini/Midi handbook, Qiagen). This amplification is not specific and unbiased as it amplifies all DNA present in the sample.
  • a pre-amplification of a sample by using WGA prior to a specific PCR could be helpful in clinical cases where the detection limit of the routine real-time PCR assay is not sensitive enough. This could be when only very limited amount of sample material is available or where the pathogen is present at very low amounts and where detection early in a disease progression would benefit the patient with a better prognostic outcome. For example this is true for cases of suspected progressive multifocal leukoencephalopathy (PML) in the central nervous system, where an early correct detection of the causing agent JC polyomavirus in the spinal fluid increases the chances for survival (Landry et al., 2008; Linda et al., 2009).
  • PML progressive multifocal leukoencephalopathy
  • a pre-amplification before a specific PCR would be helpful to reduce the amount of sample needed for an analysis.
  • CMV congenital Cytomegalovirus
  • SNHL sensory neural hearing loss
  • the dried blood samples are revisited to establish the presence of CMV at birth (Barbi et al., 2000; de Vries et al., 2009).
  • a random nonbiased pre-amplification of purified DNA would help gain more material for a more accurate analysis testing.
  • Such methods are not limited to detection of virus DNA but could be other target DNA and/or RNA-
  • RNA-genomes Similar situations where the amount of material is limiting and contamination needs to be avoided could be when working with ancient samples, forensic samples or single-cells from culture or cell-sorting. It would also be possible to combine the described method with a suitable reverse transcription (RT)-step, thereby enabling pre-amplification plus specific real-time PCR of mRNA to look at expression patterns or of viral RNA-genomes. To be able to amplify the signal in this way would help in the monitoring of low viral load seen in situations as in certain HTV-infections for example so called "elite controllers" or patients in antiretroviral therapy. Furthermore, the pre-amplification could also be combined with a multiplex real-time PCR reaction detecting multiple pathogens, instead of a singleplex reaction as used here.
  • RT reverse transcription
  • the described method discloses a technique that, in one tube, combines a random pre-amplification (nonbiased isothermal WGA) reaction with a specific real-time PCR, performed one after the other.
  • the described method would work on any DNA of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as on cDNA of sizes >2kb (Berthet et al., 2008) (Data not shown) (Repli-g Mini/Midi handbook, Qiagen).
  • the present invention discloses a nucleotide amplification method comprising of random unbiased whole genome amplification (WGA) pre-amplification followed by a specific amplification performed in the same vial or tube.
  • WGA whole genome amplification
  • the two reaction mixtures are separated by a wax layer so the reaction mixture on top of the wax layer is the pre-amplification mixture and the reaction mixture under the wax layer is the specific amplification.
  • the WGA pre-amplification reaction is a multiple displacement amplification (MDA) or a rolling circle amplification and the specific amplification is a polymerase chain reaction (PCR).
  • MDA multiple displacement amplification
  • PCR polymerase chain reaction
  • the PCR mixture comprises primers and probes for detecting a specific DNA or RNA of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as cDNA.
  • the invention discloses a kit for performing said PCR method comprising a vial or tube containing the two reaction mixtures separated by a wax layer.
  • PCR Polymerase chain reaction
  • the PCR application employs a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermits aquaticus.
  • This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.
  • DNA oligonucleotides also called DNA primers
  • the vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting.
  • each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA.
  • the selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
  • PCR techniques are known e.g. Allele-specific PCR, Polymerase Cycling Assembly (PCA), Asymmetric PCR, Helicase-dependent amplification, Hot-start PCR, Intersequence-specific PCR (ISSR), Ligation-mediated PCR, Methylation-specific PCR (MSP), Multiplex Ligation-dependent Probe Amplification (MLPA), Multiplex-PCR, Nested PCR, Overlap-extension PCR, Quantitative PCR (Q-PCR) also known as Real-time PCR and not to be mixed up with Reverse Transcription PCR (RT-PCR). All these PCR methods will be well known to the skilled worker.
  • PCA Polymerase Cycling Assembly
  • Asymmetric PCR Helicase-dependent amplification
  • Hot-start PCR Hot-start PCR
  • ISSR Intersequence-specific PCR
  • MSP Methylation-specific PCR
  • MLP Methylation-specific PCR
  • MLPA Multiplex Ligation-dependent Probe Amplification
  • Q-PCR Quantitative PCR
  • Specific PCR product refers to a method that generates a single specific PCR product with the sequence and size predicted from the sequences of the primers and the region of nucleic acid to which the primer or probe were designed to anneal.
  • the specific PCR product can be detected in various ways known to the skilled worker or analyzed by sequencing.
  • WGA Whole Genome Amplification refers to an in vitro method that is used to amplify a genomic DNA sample, and generate large amounts of amplified DNA for further molecular genetic analyses. Different methods have been developed to amplify the whole genome, including primer extension pre-amplification (PEP), degenerate oligonucleotide primed PCR (DOP-PCR) and multiple displacement amplification (MDA).
  • PEP primer extension pre-amplification
  • DOP-PCR degenerate oligonucleotide primed PCR
  • MDA multiple displacement amplification
  • MDA Multiple displacement amplification is a non-PCR based DNA amplification method. It is an isothermal method that preferentially utilizes bacteriophage phi29 DNA polymerase (Dean et al., 2002) and random hexamers for amplification (Dean et al., 2001). The reaction starts by the annealing of random hexamer primers all over a single-stranded DNA template followed by polymerization by Phi29 DNA polymerase at each of these annealing sites. When the polymerase reaches another annealing site downstream, instead of halting, it displaces the newly produced DNA strand and continues its own strand elongation. In this way, every polymerase generates long strands of newly synthesized DNA, and further primer annealing and strand displacement on these newly synthesized templates results in a hyper-branched DNA network. This results in very high yields and high quality products.
  • Rolling circle amplification fRCA refers to prolonged extension of an oligonucleotide primer annealed to a circular template DNA producing a continuous sequence of tandem copies of the circle (a concatemer).
  • Phi29 polymerase is a DNA polymerase from the phage Phi29 with unique properties. By isothermal MDA in the presence of random primers, Phi29 DNA polymerase allows for uniform amplification across genomes with less than 3-fold bias (Dean et al., 2002; Hosono et al., 2003; Lasken and Egholm, 2003).
  • Phi29 DNA polymerase has the highest processivity rate reported, -70 000 bases every time it binds (Blanco et al., 1989) and high fidelity with an error rate of only 1 in 10 6 -10 7 bases (Esteban et al., 1993).
  • Repli-g A commercially available kit from Qiagen for WGA by MDA through the used of Phi29 DNA polymerase and random hexamer primers. According to the Repli-g Midi kit-protocol (Qiagen), the reaction should be run for 16 hours to reach maximum yield of amplified DNA ( ⁇ 40 ⁇ g in 50 ⁇ 1 when starting with lOng, Qiagen). As for any Phi29 DNA polymerase-reaction, smaller fragments are inefficiently amplified by the Phi29 DNA polymerase (Berthet et al., 2008) (Repli-g Mini/Midi handbook, Qiagen).
  • Tube for PCR reactions refers to any container suitable for holding PCR reagents and test sample during PCR amplifications.
  • the container will have a tightly fitting lid which blocks vapour escape and contamination.
  • Wax include greases and refers to an organic substance, solid at temperatures below about 40°C, which melts at somewhat higher temperatures to form a liquid with a lower density than water. Waxes tend to adhere to solids thereby sealing and underlying water layer when placed together in a tube.
  • Typical waxes are a mixture of high molecular weight hydrocarbons (greases) and as pure compounds eicosane C20H42, octacosane C28H180O8, cetyl palmitate C32H64o2 and pentaerythritol tetrabehenate C93H180O8.
  • Waxes can be prepared by mixing pure or mixed waxes with one another or with greases or oils in any ratios which preserve the relative hardness and stickiness characteristic of a wax.
  • the present invention discloses a method that, in one tube, combines random nonbiased pre-amplification with a specific PCR, preferably a real-time PCR.
  • the pre-amplification (MDA by Phi29 DNA polymerase) is run before the specific PCR in the same tube.
  • the two reactions are separated from each other by a wax- layer. Combining the two reactions in one tube removes the need for transfer of pre-amplified material to the PCR-reaction and the risk of contamination between samples and from the environment. Any transfer of material from one tube to another poses a serious risk of contamination in a routine laboratory.
  • the pre- amplification is run to completion to avoid any surplus of oligonucleotides that can interfere with the following specific PCR reaction.
  • the Hot Start Taq polymerase present in the PCR reaction mix is inactive at 30°C.
  • 15-minutes incubation at 95°C inactivated the Phi29 DNA polymerase, activated the Hot Start Taq polymerase and melted the wax so that the two reactions could be mixed.
  • the virus-specific real-time PCR-reaction was run for 45 cycles. All steps are performed after each other in a thermal PCR cycler.
  • the described method can increase the signal-intensity of a specific real-time PCR at least 100xl0 6 -fold and that the pre-amplification can make detection possible of samples normally under the detection level of a specific real-time PCR (Figure 3B).
  • the described method would work on any nucleic acid of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as on cDNA of sizes >2kb (Berthet et al., 2008) (Repli-g Mini/Midi handbook, Qiagen).
  • NA nucleic acid
  • various nucleic acid (NA)-purification methods or protocols can be applied, known to the skilled worker.
  • NA can be purified from pelleted sample or from supernatants depending on where the NA of interest is situated in the sample. If analysing tissue samples, they need to be homogenized prior to NA purification.
  • Liquid clinical samples such as cerebrospinal fluid (CSF), urine and serum, are normally first centrifuged and the resulting supernatant used for NA purification.
  • Other clinical samples, such as various swabs are usually first transferred into a suitable liquid prior to centrifugation and the resulting supernatant used for NA purification.
  • a number of commercially available kits can be used for the NA purification step. It is recommended that in all cases where Phi29 DNA polymerase-amplification is used down-stream of the NA purification step, carrierRNA should not be included in the purification process since it seems to interfere with the downstream amplification (Repli-g Midi handbook, Qiagen).
  • the extracted viral NA should be stored at -20°C or immediately used.
  • the pre-amplification used here is preferably a random isothermal MDA-reaction performed by the Pni29 DNA polymerase together with random hexamer primers, but other isothermal amplification-reactions, e.g. primase-based WGA (pWGA; Rapisome, BioHelix Corp.) could also work in this set-up.
  • WGA primase-based WGA
  • Various commercial kits utilizing the Phi29 DNA polymerase is available for purchase, we decided to use Repli-g from Qiagen but others would work equally well.
  • the concentration of the Phi29 DNA polymerase and the other ingredients such as primers, nucleotides and buffer is only known to the manufacturer.
  • the pre-amplification reaction is set up in a total volume of ⁇ and the reaction contains, in addition to the polymerase and the random primers, also nucleotides and a suitable buffer that supports the polymerase.
  • the reaction contains, in addition to the polymerase and the random primers, also nucleotides and a suitable buffer that supports the polymerase.
  • the sample DNA needs to be single-stranded, which in the Repli-g kit is achieved by the use of chemical denaturation described in the Repli-g protocol. Other kits use heat- denaturation at 95°C, which is also a suitable method.
  • the reaction is isothermal and performed at 30°C.
  • the pre-amplification is not specific; any suitable DNA present in the reaction will be amplified
  • the real-time PCR reaction performed after the random pre-amplification can be a multi-plex or single-plex specific PCR. This means that it generates one or several specific PCR products with the sequence and size predicted from the sequences of the primers/probe and the region of DNA to which they were designed to anneal to.
  • the pre-amplification and the real-time PCR are combined in the same tube, but separated physically by a wax-layer.
  • the tube is placed in a thermo cycler and the pre-amplification performed first, followed by a heating step and then the real-time PCR reaction for a set number of cycles, typically 40-45.
  • the thermo cycler detects the fluorescence generated during each of the PCR cycles, which is then analysed by the machines software to generate curves and Ct-values to detect the DNA present in the sample.
  • the pre-amplification is not specific and amplifies any DNA present in the sample, while the real-time PCR reaction is specific.
  • DNA of any origin can be analysed in a real-time PCR reaction, as long as there is enough known sequence to make it possible to design specific primers and probe for a region within the DNA-fragment.
  • the origin of the DNA can be both non-cellular (viral) and cellular (bacterial, archae, eukaryotic).
  • the described method could be helpful in clinical cases where the detection limit of the routine real-time PCR assay is not sensitive enough. This could be when the virus is present at very low amounts and where detection early in a disease progression would benefit the patient with a better prognostic outcome. This is true for cases of suspected progressive multifocal leukoencephalopathy (PML) in the central nervous system, where an early correct detection of the causing agent JC polyomavirus in the spinal fluid increases the chances for survival (Landry et al., 2008; Linda et al., 2009).
  • PML progressive multifocal leukoencephalopathy
  • a pre-amplification before a specific PCR would be helpful to reduce the amount of sample needed for an analysis.
  • CMV congenital Cytomegalovirus
  • SNHL sensori neural hearing loss
  • Torque Teno virus (1 TV) appears to be ubiquitous in humans, elicits seemingly innocuous infections and does not exhibit seasonal fluctuations or epidemic spikes. TTV is transmitted primarily via the fecal-oral route and has been tested as an appropriate indicator of viral pathogens in drinking water (Griffin et al., 2008). TTV is a small circular DNA virus and is easily amplified by the use of MDA using the Phi29 DNA polymerase, thereby being a suitable candidate for the use of a combined pre-amplification and specific realtime PCR method to analyse drinking water quality around the world. Due to the increased sensitivity, the described method is predicted to have significant value and impact when screening blood and blood components for viruses such as hepatitis B, hepatitis C and human
  • HIV immunodeficiency virus
  • the Phi29 DNA polymerase does not efficiently amplify DNA fragments of less than 2kb in size (Berthet et al., 2008) (Repli-g Mini/Midi handbook, Qiagen), but works well on DNA of any origin that is 2kb or larger.
  • the sensitivity and specificity of any given specific PCR vary depending on the robustness of the primers and the genome variability or mutations in the primer-binding region of the DNA fragment being analysed, as well as the efficiency of the reaction.
  • a detection limit with maintained reproducibility at 10 copies per real-time PCR reaction have been reported for a JC polyomavirus-specific real-time PCR(Ryschkewitsch et al., 2004).
  • FIG. 1 A. Schematic picture showing how the AmpliWax works as a physical barrier to separate the random pre-amplification reaction (Repli-g) and the specific real-time PCR reaction. B. AmpliWax does not interfere with the JC-specific real-time PCR reaction.
  • Figure 3 A. Real-time PCR only on single sample of 100-fold and duplicate samples of 6400-fold diluted JC viral DNA. B. Sixteen hours of Repli-g amplification + real-time PCR on single sample of 100-fold and duplicate samples of 6400-fold diluted JC viral DNA.
  • FIG. 4 A. Sixteen hours of Repli-g amplification + real-time PCR on HPV16-positive clinical sample. B Sixteen hours of Repli-g amplification followed by a HSV1 -specific real-time PCR on a HSV1 -positive clinical sample. EXAMPLES
  • Example 1 Detection of the circular 5kb-large double-stranded DNA genome of the JC polvomavirus Virus sample
  • JC polyomavirus-positive cerebrospinal fluid received for routine diagnostic analysis at the Department of Virology, Statens Serum Institut, Copenhagen, Denmark (accredited and quality-controlled Danish National reference diagnostic routine laboratory (ISO 17025); www.ssi.dk).
  • the JC polyomavirus is a circular double-stranded DNA virus of 5kb, which may cause fatal progressive multifocal leukoencephalopathy (PML) in immunodeficient or immunosuppressed patients (Padgett et al., 1971).
  • NA nucleic acid
  • Real-time PCR reaction mix specific for JC virus was prepared, either using described primers and probe (MacKenzie et al., 2003; Ryschkewitsch et al., 2004) or primers and probe from an in-house real-time PCR assay.
  • JCT-1 (5 ' -AGA GTG TTG GGA TCC TGT GTT TT-3 '; SEQ ID NO 1
  • JCT-2 (5 '-GAG AAG TGG GAT GAA GAC CTG TTT-3 ' ; SEQ ID NO 2)
  • JCT-1.1 5 - FAM-TCA TCA CTG GCA AAC ATT TCT TCA TGG C-TAMRA-3 SEQ ID NO 3) where FAM is the fluorescence reporter dye fluorescein and TAMRA is the fluorescence quencher dye tetramethylrhociarnine.
  • PCR primers and internal probe were: JC-F (5'-TGA ACC AAA AGC TAC ATA GGT AAG TAA TG-3'; SEQ ID NO 4), JC-R (5'-AAT CCT GTG GCA GCA G-3'; SEQ ID NO 5) and JC-P (5'-FAM-TTC ATG GGT GCC GCA CTT GCA-BHQl-3'; SEQ ID NO 6) where BHQl is the fluorescent dye "black hole quencher-1".
  • PCR reaction mix containing 15 ⁇ 1 of 2x QuantiTect Multiplex PCR NoROX Master mix (contains HotStarTaq DNA polymerase, Qiagen), 500nM of each primer and ⁇ probe was put in a 0,2ml PCR-micro tube.
  • One pellet of AmpliWax PCR Gem 50 was added on top of the PCR reaction mix, the tube put in a cycler and incubated at 60°C for 5 min to melt the wax followed by cooling at 37°C to solidify the wax on top of the PCR reaction mix.
  • the random Phi29-amplification reaction was prepared by making a ⁇ -Repli-g Midi reaction (only 1/5 of the normal reaction) according to manufacturer's protocol (Repli-g Midi kit, Qiagen). Shortly, ⁇ of purified viral DNA was mixed with ⁇ of denaturation-solution and incubated at room temperature for 3 minutes. Two microliters of stop solution was added and the sample mixed. Thereafter, 5.8 ⁇ 1 of Repli-g Midi reaction buffer and 0.2 ⁇ 1 of Phi29 DNA polymerase was added and the sample vortexed. After a short centrifugation, the Repli-g reaction was added on top of the solidified AmpliWax, the tube closed and put in a thermal cycler (Mx3005P, Stratagene).
  • the following program was run: 30°C for 16 hours to run the Repli-g reaction; 95°C for 15 minutes to inactivate the Phi29 DNA polymerase, to activate the Taq polymerase and to melt the wax so that the Repli-g product was mixed with the PCR reaction mix; followed by a 45 cycle-real- time PCR with 95°C for 15 seconds and 60°C for 1 minutes.
  • the assay was performed in an Mx3005P-cycler (Stratagene).
  • Example 2 Detection of the circular 8kb-large double-stranded DNA genome of the HPV-16 and the linear 152kb-large double-stranded DNA genome of the HSV1
  • HPV-16-positive cervix smears and HSV-1 -positive swabs received for routine diagnostic analysis at the Department of Virology, Statens Serum Institut, Copenhagen, Denmark (accredited and quality-controlled Danish National reference diagnostic routine laboratory (ISO 17025); www.ssi.dk).
  • the Human papilloma virus-16 (HPV-16) is a circular double-stranded DNA virus of 8kb, which may cause cervical cancer in women.
  • Human herpes simplex-1 virus is a linear double-stranded DNA virus of 152kb.
  • NA nucleic acid
  • Real-time PCR reaction mix specific for HPV-16 virus was prepared, using primers and probe from an in- house real-time PCR assay.
  • In-house primers and internal probe were: HPV16-E6-12F (5'- CGA CCC AGA AAG TTA CCA CAG TT-3'; SEQ ID NO 7), HPV16-E6-12R (5'- TGT TGC TTG CAG TAC ACA CAT TCT A-3'; SEQ ID NO 8) and HPV16-E6-12P (5'-FAM- CAC AGA GCT GCA AAC AAC TAT ACA TGA TAT AAT-BHQ I-3'; SEQ ID NO 9).
  • HSVl -LPs (5'-TGT GGT GTT TTT GGC ATC AT-3'; SEQ ID NO 10)
  • HSVl-LPas (5'- CCG ACA AGA ACC AAA AGG AA -3'; SEQ ID NO 1 1)
  • HSVl-LPprobe (5'-FAM- CAT GCG TGC CGT TGT TCC CA-BHQ1-3'; SEQ ID NO 12).
  • PCR reaction mix containing 15 ⁇ 1 of 2x QuantiTect Multiplex PCR NoROX Master mix (contains HotStarTaq DNA polymerase, Qiagen), 500nM of each primer and lOOnM probe was put in a 0,2ml PCR-micro tube.
  • One pellet of AmpliWax PCR Gem 50 was added on top of the PCR reaction mix, the tube put in a cycler and incubated at 60°C for 5 min to melt the wax followed by cooling at 37°C to solidify the wax on top of the PCR reaction mix.
  • the random Phi29-amplification reaction was prepared by making a ⁇ -Repli-g Midi reaction (only 1/5 of the normal reaction) according to manufacturer's protocol (Repli-g Midi kit, Qiagen). Shortly, ⁇ ⁇ of purified viral DNA was mixed with ⁇ of denaturation-solution and incubated at room temperature for 3 minutes. Two microliters of stop solution was added and the sample mixed. Thereafter, 5.8 ⁇ 1 of Repli-g Midi reaction buffer and 0.2 ⁇ 1 of Phi29 DNA polymerase was added and the sample vortexed.
  • the Repli-g reaction was added on top of the solidified AmpliWax, the tube closed and put in a thermal cycler (Mx3005P, Stratagene).
  • the following program was run: 30°C for 16 hours to run the Repli- g reaction; 95°C for 15 minutes to inactivate the Phi29 DNA polymerase, to activate the Taq polymerase and to melt the wax so that the Repli-g product was mixed with the PCR reaction mix; followed by a 45 cycle- real-time PCR with 95°C for 15 seconds and 60°C for I minutes.
  • the assay was performed in an Mx3005P- cycler (Stratagene).
  • Cytomegalovirus DNA detection in Guthrie cards a powerful tool for diagnosing congenital infection. J Clin Virol 17, 159-65.
  • Torque teno virus an improved indicator for viral pathogens in drinking waters. Virol J 5, 112.

Abstract

Le procédé courant choisi pour l'identification de virus dans de nombreux laboratoires de diagnostic est la réaction en chaîne de la polymérase (PCR) en temps réel spécifique, où des paires d'amorces spécifiques de la séquence sont utilisées pour chaque virus ou pour un groupe de virus. Ceci est un procédé rapide, sensible et spécifique qui est facile à mettre en œuvre, mais peut présenter une sensibilité limitée qui peut conduire à des résultats de faux négatifs. Selon l'invention, une pré-amplification aléatoire de l'échantillon avant une PCR spécifique pourrait être utile dans des cas cliniques où la limite de détection de l'essai de PCR en temps réel n'est pas suffisamment sensible. Ceci pourrait avoir lieu quand seulement une quantité très limitée de l'échantillon est disponible ou lorsque le pathogène est présent en quantités très faibles. La présente invention concerne un procédé d'amplification de nucléotides comprenant une pré-amplification aléatoire non biaisée du type amplification du génome entier (WGA), suivie par une amplification spécifique réalisée dans le même flacon ou tube. Les deux mélanges réactionnels sont séparés par une couche de cire afin que le mélange réactionnel au-dessus de la couche de cire soit le mélange de pré-amplification et le mélange réactionnel au-dessous de la couche de cire soit l'amplification spécifique. La mise en œuvre d'une pré-amplification aléatoire avant la PCR en temps réel augmenterait considérablement la sensibilité de la PCR en temps réel spécifique lors de tests d'échantillons pour lesquels on s'attend à un faible nombre de copies ou d'échantillons précieux ou non récupérables. La mise en œuvre des deux réactions l'une après l'autre dans le même tube réduit a un minimum le risque de contamination de la PCR puisqu'aucun transfert d'échantillon pré-amplifié n'est nécessaire. Le procédé décrit fonctionnerait sur tout ADN de n'importe quelle origine, provenant à la fois de sources non cellulaires (virus) et de sources cellulaires (bactéries, archées, eucaryotes) ainsi que sur de l'ADNc.
PCT/DK2011/000026 2010-04-22 2011-04-14 Nouveau procédé d'amplification de séquences nucléotidiques WO2011131192A1 (fr)

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WO2013192100A1 (fr) 2012-06-18 2013-12-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Procédés et compositions pour la détection du virus jc
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CN111548918A (zh) * 2020-05-14 2020-08-18 上海交通大学 一种能实现多体系兼容的全封闭一体式反应管
CN114196522A (zh) * 2022-02-18 2022-03-18 深圳市尚维高科有限公司 一种一步法核酸检测方法及其所用的密封性核酸检测装置

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WO2013143133A1 (fr) * 2012-03-30 2013-10-03 深圳华大基因科技服务有限公司 Procédé d'amplification d'un génome entier et application associée
CN104114703A (zh) * 2012-03-30 2014-10-22 深圳华大基因科技服务有限公司 全基因组扩增方法及其应用
WO2013192100A1 (fr) 2012-06-18 2013-12-27 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Procédés et compositions pour la détection du virus jc
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WO2014018093A1 (fr) * 2012-07-26 2014-01-30 Illumina, Inc. Compositions et procédés pour l'amplification d'acides nucléiques
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EP3744855A1 (fr) * 2014-07-07 2020-12-02 Institut Pasteur Transcriptome de papillomavirus à large spectre génétique et génotypique utile en tant que biomarqueur des stades du cancer associés au papillomavirus
US10808284B2 (en) 2014-07-07 2020-10-20 Institut Pasteur Broad range gene and genotype papillomavirus transcriptome as a biomarker of papillomavirus-associated cancer stages
WO2016005905A3 (fr) * 2014-07-07 2016-04-28 Institut Pasteur Transcriptome de papillomavirus à large spectre génétique et génotypique utile en tant que biomarqueur des stades du cancer associés au papillomavirus
US11530452B2 (en) 2014-07-07 2022-12-20 Institut Pasteur Broad range gene and genotype papillomavirus transcriptome as a biomarker of papillomavirus-associated cancer stages
EP3436605A4 (fr) * 2016-03-31 2019-08-21 Perkinelmer Health Sciences (Australia) Pty Ltd Amplification de séquences cibles
WO2020156102A1 (fr) * 2019-01-31 2020-08-06 中国科学院西北生态环境资源研究院 Kit de réactifs pour méthode ic-rt-lamp pour détecter le virus de la jaunisse nécrotique de la laitue et son procédé de détection
CN111548918A (zh) * 2020-05-14 2020-08-18 上海交通大学 一种能实现多体系兼容的全封闭一体式反应管
CN114196522A (zh) * 2022-02-18 2022-03-18 深圳市尚维高科有限公司 一种一步法核酸检测方法及其所用的密封性核酸检测装置

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