WO2013140339A1 - Contrôle positif pour pcr - Google Patents

Contrôle positif pour pcr Download PDF

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Publication number
WO2013140339A1
WO2013140339A1 PCT/IB2013/052178 IB2013052178W WO2013140339A1 WO 2013140339 A1 WO2013140339 A1 WO 2013140339A1 IB 2013052178 W IB2013052178 W IB 2013052178W WO 2013140339 A1 WO2013140339 A1 WO 2013140339A1
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Prior art keywords
positive control
sequences
pcr
target
sequence
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PCT/IB2013/052178
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English (en)
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Louis WELEBOB
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Vela Operations Pte.Ltd
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Publication of WO2013140339A1 publication Critical patent/WO2013140339A1/fr

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2545/00—Reactions characterised by their quantitative nature
    • C12Q2545/10—Reactions characterised by their quantitative nature the purpose being quantitative analysis
    • C12Q2545/101—Reactions characterised by their quantitative nature the purpose being quantitative analysis with an internal standard/control

Definitions

  • the present invention relates to positive controls for monitoring the reliability and accuracy of a nucleic acid amplification reaction, e.g. PCR, in particular RT-PCR.
  • PCR nucleic acid amplification reaction
  • RT-PCR nucleic acid amplification reaction
  • methods and positive controls are particularly useful for monitoring the integrity of reagents used in such nucleic acid amplification reactions.
  • the inventive principle of manufacturing and using these positive controls is generally applicable to (RT-) PCR assays, particularly to multiplex assays.
  • PCR Polymerase chain reaction
  • PC positive controls
  • RNA targets in RT-PCR assays are usually prepared using vector plasmids carrying an insert corresponding to the target sequence, wherein the target sequence is transcribed and afterwards isolated from the reverse transcription reaction mixtures. RNA positive controls are added in free form to the reaction vessels used for the analysis of nucleic acids isolated from material of interest and are co-amplified with target nucleic acids isolated from said material.
  • RNA targets which means that RNA has been synthesized using suitable RNA polymerases, e.g. T7-polymerase.
  • RNA polymerases e.g. T7-polymerase
  • each additional step results in an equal number of additional pipetting steps. If only one pipetting step in the preparation of a reaction mixture can be avoided, in this example this would amount to 10,000 pipetting steps that are not necessary. Over several years, and in the thousands of laboratories performing routine diagnostic, economization would be extraordinary.
  • the invention provides tools and methods that overcome the obstacles in the prior art.
  • Other objects achieved by the present invention is the easy purification of positive control mRNA synthesized from positive control constructs for RT-PCR.
  • the positive controls are highly stable, e.g. under conditions of at least 12 hours, 18 hours, 24 hours, 48 hours or even longer storage time at temperatures up to 37°C.
  • the target genes represent genes found in natural organisms, i.e. their structure should permit detection at high sensitivity without the disadvantages of too short sequences serving as controls for RT-PCR that are used in the prior art.
  • the present invention provides constructs that may serve as tools, i.e. positive control, which overcome the disadvantages of the prior art constructs. These constructs have proven to be very stable, highly flexible with respect to the target genes that can be cloned into the constructs, and generally surprisingly sensitive and reliable for real-time RT-PCR reactions.
  • the present invention thus relates, inter alia, to:
  • a positive control construct for amplification reactions of nucleic acids comprising at least two nucleotide sequences serving as target nucleic sequences of interest in a nucleic acid amplification reaction, wherein at least one target nucleic sequence comprises a polyadenosine tail.
  • polynucleotide sequence according to any one of item 1 to 3, further comprising a promoter sequence.
  • the positive control construct according to any one of items 1 to 7, wherein said target sequences of interest are separated by nucleotide sequences encoding a polyadenosine- tail, or wherein a polyadenosine tail is found at the 3 ' end of at least one target sequence in the positive control.
  • each target sequences comprises two primer binding sequences and a sequence which hybridizes specifically with a probe for use in real time (RT-)PCR assays, wherein at least one primer binding sequence and a sequence specifically hybridizing with a probe in real time (RT-)PCR assays are separated by a nucleotide sequence, which is not specifically bound by said primers that specifically bind to the primer binding sequences or by said probe specifically hybridizing with said sequence specifically recognized by a probe.
  • Said additional sequences between primer binding sites and probe binding sites may be at least 5 or 10 or 15 or 20 or 25 or 30 or 35 or 40, 50, 60, 70, 80, 90 or about 100 nucleotides long. Longer sequences are also possible.
  • the positive control construct according to any one of items 1 to 10 wherein each target gene sequence has a length of about 100 to about 500 nucleotides, preferably a length of about 100 to about 300 nucleotides, more preferably a length of about about 100 to about 200 nucleotides.
  • the positive control construct according to any one of items 1 to 12 for use in diagnostic assays for the detection of one or more genes selected from cancer-related genes, oncogenes, viral genes, bacterial genes, fungal genes, or genes involved in human diseases selected from autoimmune diseases, inflammatory diseases, infectious diseases, metabolic diseases, degenerative diseases, neurologic diseases, diseases of heart, bones, joints, muscles, brain, reproductive organs, blood, lymphatic tissues, glandular tissues, breast, digestive tracts, respiratory tract, or skin.
  • cancer-related genes selected from cancer-related genes, oncogenes, viral genes, bacterial genes, fungal genes, or genes involved in human diseases selected from autoimmune diseases, inflammatory diseases, infectious diseases, metabolic diseases, degenerative diseases, neurologic diseases, diseases of heart, bones, joints, muscles, brain, reproductive organs, blood, lymphatic tissues, glandular tissues, breast, digestive tracts, respiratory tract, or skin.
  • a method for the amplification of nucleic acids comprising the use of at least one positive control construct for amplification reaction according to items 1 to 13.
  • a kit comprising any of the positive control constructs of items 1 to 12.
  • Figure 1 Universal PC Design Concept for DNA (A) and RNA (B). Fragments are subsequently cloned into a plasmid.
  • Figure 5 Detection of gene C ofNorovirus using PC-I.
  • Figure 6 Detection of gene D of Influenza using PC-II.
  • the invention provides a polynucleotide sequence, i.e. a construct (plasmid) comprising at least two different nucleotide sequences serving as targets of interest in an amplification reaction.
  • a construct plasmid
  • one of the at least two target sequences is designed to serve as a control for RT-PCR.
  • the polynucleotide sequence may comprise natural or modified nucleotides, i.e. nucleotides carrying moieties usually not found in natural organisms.
  • the at least two different nucleotide sequences comprise the genetic information encoding searched target sequences that are isolated from a sample taken from an organism, e.g. clinical sample obtained from a mammal, preferably a human being, from a plant or the environment (e.g. from water, food, drink, any type of surface or liquid source found, for example, in hospitals, laboratories, public premises, changing rooms, agricultural or aquaculture premises, stables, plant fields, filters of air conditioning devices, toilets, etc.).
  • a sample taken from an organism e.g. clinical sample obtained from a mammal, preferably a human being, from a plant or the environment (e.g. from water, food, drink, any type of surface or liquid source found, for example, in hospitals, laboratories, public premises, changing rooms, agricultural or aquaculture premises, stables, plant fields, filters of air conditioning devices, toilets, etc.).
  • Target sequences may be chosen as required in an amplification reaction. They may be selected from, e.g., sequences that are suitable to detect and/or distinguish pathogenic material such as viruses, bacteria, fungi, parasites such as Plasmodium falciparum, ticks, etc., or they may encode sequences derived from the organism of which a sample has been obtained. In the latter case, the samples can be clinical samples obtained from patients etc., preferably clinical samples of mammals such as human beings.
  • Individual target sequences in the plasmids of the invention preferably have a length of about 100 to 500, about 100 to 400, about 100 to about 300, or about 100 to about 250 nucleotides.
  • the length of the target sequences in the PC constructs of the invention may be slightly longer or slightly shorter, but these sequences are always longer than only the combined primer and probe binding sequences, i.e. they contain additional nucleotide residues at least between one primer binding site and the sequence to which the probe hybridizes.
  • the probe and primer binding sites are separated (i.e.
  • sequence in the target sequences not bound by a primer and which is also not bound by the probe by at least 10 nucleotides, preferably by about 15 nucleotides, by at least 20 nucleotides, by at least 25 nucleotides, or by longer sequences.
  • the sequence separating primer and probe on one side may be shorter or longer than the sequence separating the probe and primer binding sites on the other side of the target sequence (e.g. the 3 '-side).
  • said target nucleotide sequences serve as positive controls in nucleic acid amplification reactions, such as PCR, RT-PCR, real-time PCR, etc..
  • the target nucleotide sequences are transcribed into RNA (optionally comprising modified nucleic acid residues), preferably mRNA.
  • the target fragments present on a polynucleotide constucts of the invention are separated and/or followed by a nucleotide sequence encoding a poly A-tail (when the target nucleotide sequences consist of RNA).
  • the polynucleotides may also comprise promoter and/or enhancer sequences that are so positioned that the target nucleic acid sequences can be more efficiently transcribed.
  • the promoter and/or enhancer sequences may be found at the N-terminal and/or C-terminal end of one or more target sequence(s).
  • the positive controls of the invention to monitor the integrity of reagents used in the amplification reaction of nucleic acids whose presence shall be detected.
  • Enzymes may lose their activity under certain non-favorable storage conditions.
  • Primers and probes, e.g. those that are fluorescently labeled may also deteriorate, thereby loosing fluorescent or other detectable moieties.
  • the universal positive controls (PC) of the invention encompass several amplicons of interest arranged in a tandem (or multi-amplicon) manner.
  • a T7 promoter or other suitable promoter for RNA polymerases may be inserted in front of the RNA amplicons.
  • the plasmid construct for use in the production of RNA may contain the necessary sequences to encode a poly A tail at the end of the RNA amplicons and one terminator after the poly A tail (cf. Figure 1).
  • the purpose for having, e.g. a T7 promoter is for the RNA polymerase to recognize the site and initiate the RNA fragment synthesis.
  • RNA fragments are separate of each other, only one T7 promoter may be used for a set of tandem RNA amplicons of interest.
  • the poly A tail that consists of 30 Adenines is for the affinity purification of full length single stranded RNA, and the RNA fragments which are incompletely synthesized and consequently not tailed by poly A are discarded.
  • the most common purification method is by affinity binding to poly dT coated beads.
  • the terminator provides a signal for the RNA polymerase to end transcription. For RNA pathogen detection assays, the same design principle is applied.
  • the differences in plasmid design are the insertions of a T7 promoter at the beginning of the RNA fragment, a poly A tail at the end of the fragment and a terminator after the poly A tail.
  • In vitro transcription is carried out using the plasmids, followed by affinity purification by poly dT coated beads. The purified full-length single stranded RNA fragments are then used as the PC in RNA assays.
  • the amplicon fragment itself is designed in such a manner that the upstream and downstream 30 nucleotides or longer flanking the amplicon in the genome sequence are included to best imitate the binding of primers and probes on target sequences (cf. Figure 2).
  • the first derivative involves a combination of DNA amplicons and RNA amplicons in the same plasmid. As the sequences before the T7 promoter and after the terminator do not exert any effect on the transcription of the RNA amplicons, DNA assays and RNA assays can share the same plasmids.
  • the second derivative is the reverse complement of the inventive design using an SP6 promoter at the end of Amplicon N, a poly T tail in front of Amplicon 1 and a terminator after the poly T tail.
  • the target sequences are arranged in such a manner that target sequences of the same assay are placed in separate plasmids.
  • one primer pair can bind to just one target fragment and generate an amplicon of one size only. This ensures the complete monitoring of every primer pair.
  • the same set of plasmids can be used across different assays.
  • assay A is designed to detect DNA or R A pathogens 1, 2 and 3 by amplifying Al, A2 and A3 in their respective genes using a 4-plex q(RT-) PCR including an extraction control (EC).
  • assay B is designed to detect DNA or RNA pathogens 4, 5 and 6 by amplifying B4, B5 and B6 in their respective genes using a 4-plex qPCR including EC.
  • plasmid #1 contains target sequences Al and B4 (upstream and downstream sequences all included)
  • plasmid #2 contains target sequences A2 and B5
  • plasmid #3 contains target sequences A3, B6 and EC amplicon.
  • the EC amplicon can be arbitrarily appointed to any one of the plasmids.
  • the same plasmids can be used as positive control in both assays when no cross reactivity is seen.
  • the target sequences can be used for RT-PCR, which means that the PCR- step is preceded by RNA-synthesis and reverse transcription steps, respectively.
  • these positive controls may be used in more than one assay.
  • the positive control comprises two different control target sequences it may be used in two separate or individual reactions and/or assays.
  • the positive control comprises three different control target sequences, it may be used in three separate or individual reactions (assays), and so forth.
  • the number of target sequences in a positive control construct can be increased as needed. It may be limited only by the capacity of a given cloning vector.
  • the transcribed nucleic acid fragments are isolated and purified, preferably using a substrate carrying a nucleotide sequence comprising a poly dT nucleotide sequence. They may also be isolated and/or purified using any other method known in the art of molecular biology (Sambrook; Molecular Cloning: A Laboratory Manual, 3 Vol.; incorporated by reference). The presence of a polyadenine tail greatly increases the yield in mRNA when poly dT -tailed substrates (e.g. oligo dT beads) are used for purification.
  • poly dT -tailed substrates e.g. oligo dT beads
  • compositions for use in amplification reactions of nucleic acids e.g. in buffers for use in amplification reactions comprising, e.g. enzymes used in nucleic acid amplification reactions, building blocks of nucleic acid sequences, e.g. nucleotides or nucleosides that can be chemically modified or those found in natural environments, minerals such as magnesium or manganese, detergents, stabilizers, etc.
  • compositions are further embodiments of the invention.
  • kits which a packaged and may comprise instructions for use. Accordingly, the present invention provides such kits.
  • the present invention relates to a method of monitoring (or controlling) an amplification reaction comprising the following steps:
  • polynucleotide sequence(s) of the invention comprising target sequence(s) of interest, or homologous sequences that are at least 50%, 55%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical (due to deletions, additions, insertions, inversions, or other modification in said target sequence(s)), or fragments thereof which maintain the capacity of serving as an amplifiable positive control sequence based on the target sequence(s) of interest suspected to be present in a sample;
  • a target specific amplification reaction using at least one pair of two nucleotide sequence fragments capable of hybridizing to said target sequence under stringent conditions as primers, which a suitable for amplification of a target sequence of interest; optionally quantifying the amount of amplification product formed using a probe that specifically hybridizes with the amplification product and can be detected, e.g. because it has been labeled, for example with at least one fluorophore.
  • the above methods preferably comprise real-time PCR (qPCR), more preferably qRT-PCR, wherein said targets of interest nucleotide sequences serve as a positive control amplicons for said amplification reaction.
  • qPCR real-time PCR
  • said positive controls are for use in multiplex amplification reactions, wherein said positive control is preferably added to the amplification reaction, when it serves as positive control for multiplex amplification of DNA.
  • positive control RNA is transcribed using a positive control construct of the invention and the transcribed RNA may then be added to an amplification mixture in free form.
  • the primers and/or probes of the invention can be labeled with a fluorescent moiety.
  • fluorescent moieties for use in real-time PCR detection are known to persons skilled in the art and are available from various commercial sources, e.g. from life technologiesTM and subsidiearies (e.g. Molecular Probes) or other suppliers of ingredients for real-time RT-PCR.
  • Kits of the invention can include primer sets specific for the amplification of nucleic acids and at least one probe hybridizing specifically with the amplification products.
  • the probe is preferably capable of specifically hybridizing with specific amplification products obtained with the primer sets of the invention.
  • Articles of manufacture can include fluoropfioric moieties for labeling the primers or probes or the primers and probes are already labeled with donor and corresponding acceptor fluorescent moieties.
  • the article of manufacture can also include a package insert having instructions thereon for using the primers, probes, and fluoropfioric moieties
  • Amplification generally involves the use of a polymerase enzyme. Suitable enzymes are known in the art, e.g. Taq Polymerase, etc.
  • An amplifying step generally includes contacting the sample or nucleic acids isolated from said sample with a pair of specific primers to produce an amplification product if a target nucleic acid molecule is present in the sample.
  • a dye-binding step generally includes contacting the amplification product with a double- stranded DNA binding dye. The method further includes detecting the presence or absence of binding of the double-stranded DNA binding dye into the amplification product.
  • the presence of binding is typically indicative of the presence of target nucleic acid in the sample, and the absence of binding is typically indicative of the absence of target nucleic acid in the sample.
  • Such a method can further include the steps of determining the melting temperature between the amplification product and the double- stranded DNA binding dye. Generally, the melting temperature confirms the presence or absence of target nucleic acid.
  • Representative double-stranded DNA binding dyes include SYBRGREEN I ® , SYBRGOLD ® , and ethidium bromide.
  • Additional embodiments of the invention i) Polynucleotide sequence comprising at least two different nucleotide sequences serving as target sequences of interest in a nucleic acid amplification reaction. ii) The polynucleotide sequence according to item i), comprising at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more different inserted nucleotide sequences serving as target sequences of interest in a nucleic acid amplification reaction.
  • a reverse transcription preferably performing a reverse transcription; performing a target specific amplification reaction using at least one pair of two nucleotide sequence fragments capable of hybridizing to said target sequence under stringent conditions as primers,
  • xiv) A positive control for amplification reactions of nucleic acids, preferably for realtime PCR, wherein said control selected from any one of items i) to viii).
  • xv) The positive control according to item xiv), wherein said control is part of a
  • composition or a kit.
  • a diagnostic tool comprising the positive control according to any one of item i) to viii).
  • a diagnostic method comprising the use of a positive control as defined in any one of items i) to viii) or xiv) to xvi).
  • the target nucleic acid is a nucleic acid derived from a pathogen, an oncogene, a healthy control sample or from an extraction control.
  • Primers and probes can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights, Inc., Cascade, Colorado).
  • Important features when designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size amplification product to facilitate detection, similar melting temperatures for the members of a pair of primers, and the length of each primer (i.e., the primers need to be long enough to anneal with sequence-specificity and to initiate synthesis but not so long that fidelity is reduced during oligonucleotide synthesis).
  • oligonucleotide primers are 15 to 30 nucleotides in length. Designing oligonucleotides to be used as hybridization probes can be performed in a manner similar to the design of primers, although the members of a pair of probes preferably anneal to an amplification product. As with oligonucleotide primers, oligonucleotide probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15 to 30 nucleotides in length.
  • multiplex assay refers to multiple assays that are carried out simultaneously, in which detection and analysis steps are generally performed in parallel.
  • a multiplex assay may also be an assay that is suitable to simultaneously amplify and identify different target nucleic acids of interest.
  • a multiplex assay would be for example, a molecular assay that simultaneously screens for nucleic acids that are indicative of a gastrointestinal pathogen, and/or a respiratory and/or an oncogene.
  • probe or “detection probe” refers to an oligonucleotide that forms a hybrid structure with a target sequence contained in a molecule (i.e., a "target molecule") in a sample undergoing analysis, due to complementarity of at least one sequence in the probe with the target sequence.
  • target molecule a target molecule
  • the nucleotides of any particular probe may be
  • deoxyribonucleotides ribonucleotides, and/or synthetic nucleotide analogs.
  • primer refers to an oligonucleotide that is capable of acting as a point of initiation for the 5' to 3' synthesis of a primer extension product that is complementary to a nucleic acid strand.
  • the primer extension product is synthesized in the presence of appropriate nucleotides and an agent for polymerization such as a DNA polymerase in an appropriate buffer and at a suitable temperature.
  • the primers may comprise only natural nucleotide residues or at least one or more nucleotide analogs, e.g. chemically modified analogs, PNAs, etc.
  • target amplification refers to enzyme -mediated procedures that are capable of producing billions of copies of nucleic acid target.
  • enzyme-mediated target amplification procedures known in the art include PCR. Whenever PCR is mentioned, this term is also meant to refer to RT-PCR when the sequences to be amplified are present as RNA molecules, e.g. in certain diagnostic applications targeting RNA viruses.
  • RNA complementary DNA
  • cDNA complementary DNA
  • RNA PCR reverse transcriptase PCR
  • a sample of DNA is mixed in a solution with a molar excess of at least two oligonucleotide primers of that are prepared to be complementary to the 3' end of each strand of the DNA duplex; a molar excess of nucleotide bases (i.e., dNTPs); and a heat stable DNA polymerase, (preferably Taq polymerase), which catalyzes the formation of DNA from the oligonucleotide primers and dNTPs.
  • dNTPs nucleotide bases
  • a heat stable DNA polymerase preferably Taq polymerase
  • At least one is a forward primer that will bind in the 5' to 3' direction to the 3' end of one strand of the denatured DNA analyte and another is a reverse primer that will bind in the 3' to 5' direction to the 5' end of the other strand of the denatured DNA analyte.
  • the solution is heated to 94-96°C to denature the double- stranded DNA to single-stranded DNA.
  • the primers bind to separated strands and the DNA polymerase catalyzes a new strand of analyte by joining the dNTPs to the primers.
  • each extension product serves as a template for a complementary extension product synthesized from the other primer.
  • sequence being amplified doubles after each cycle, a theoretical amplification of a huge number of copies may be attained after repeating the process for a few hours; accordingly, extremely small quantities of DNA may be amplified using PCR in a relatively short period of time.
  • RNA complementary DNA
  • cDNA complementary DNA
  • reverse transcriptases are known to those of ordinary skill in the art as enzymes found in retroviruses that can synthesize complementary single strands of DNA from an mRNA sequence as a template.
  • a PCR used to amplify RNA products is referred to as reverse transcriptase PCR or "RT-PCR.”
  • real-time PCR and “real-time RT-PCR,” refer to the detection of PCR products via a fluorescent signal generated by the coupling of a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates.
  • Examples of commonly used probes are TAQMAN ® probes, Molecular Beacon probes, SCORPION ® probes, and SYBR ® Green probes. Briefly, TAQMAN ® probes, Molecular Beacons, and SCORPION ® probes each have a fluorescent reporter dye (also called a "fluor”) attached to the 5' end of the probes and a quencher moiety coupled to the 3' end of the probes.
  • a fluorescent reporter dye also called a "fluor”
  • the proximity of the fluor and the quencher molecules prevents the detection of fluorescent signal from the probe; during PCR, when the polymerase replicates a template on which a probe is bound, the 5 '-nuclease activity of the polymerase cleaves the probe thus, increasing fluorescence with each replication cycle.
  • SYBR Green ® probes binds double-stranded DNA and upon excitation emit light; thus as PCR product accumulates, fluorescence increases. In the context of the present invention, the use of TAQMAN ® probes is preferred.
  • complementary and substantially complementary refer to base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double- stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single- stranded nucleic acid to be sequenced or amplified.
  • Complementary nucleotides are, generally, A and T (or A and U), and G and C.
  • sequence lengths listed are illustrative and not limiting and that sequences covering the same map positions, but having slightly fewer or greater numbers of bases are deemed to be equivalents of the sequences and fall within the scope of the invention, provided they will hybridize to the same positions on the target as the listed sequences. Because it is understood that nucleic acids do not require complete
  • the probe and primer sequences disclosed herein may be modified to some extent without loss of utility as specific primers and probes.
  • sequences having homology of about 80%, 85%, 90%, 95%, 96%, 97%, 98% or more fall within the scope of the present invention provided they are still capable of hybridizing with target nucleic.
  • hybridization of complementary and partially complementary nucleic acid sequences may be obtained by adjustment of the hybridization conditions to increase or decrease stringency, i.e., by adjustment of hybridization temperature or salt content of the buffer.
  • hybridizing conditions is intended to mean those conditions of time, temperature, and pH, and the necessary amounts and concentrations of reactants and reagents, sufficient to allow at least a portion of complementary sequences to anneal with each other.
  • time, temperature, and pH conditions required to accomplish hybridization depend on the size of the oligonucleotide probe or primer to be hybridized, the degree of complementarity between the oligonucleotide probe or primer and the target, and the presence of other materials in the hybridization reaction admixture.
  • the actual conditions necessary for each hybridization step are well known in the art or can be determined without undue experimentation.
  • label refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) signal, and that can be attached to a nucleic acid or protein via a covalent bond or noncovalent interaction (e.g., through ionic or hydrogen bonding, or via immobilization, adsorption, or the like). Labels generally provide signals detectable by fluorescence, chemiluminescence, radioactivity, colorimetry, mass
  • labels include fluorophores, chromophores, radioactive atoms, electron- dense reagents, enzymes, and ligands having specific binding partners.
  • sample as used in its broadest sense to refer to any biological sample from any human or veterinary subject, from a plant or from the environment that may be tested for the presence or absence of target nucleic acids.
  • the samples from human or veterinary subjects may include, without limitation, tissues obtained from any organ, such as for example, lung tissue; and fluids obtained from any organ such as for example, blood, plasma, wound swabs, serum, lymphatic fluid, synovial fluid, cerebrospinal fluid, amniotic fluid, amniotic cord blood, tears, saliva, and nasopharyngeal washes.
  • patient as used herein is meant to include both human and veterinary patients.
  • the amplification primers and detection probes of the present invention are set forth in the sequence listing.
  • the target nucleic acid is selected from RNA and DNA.
  • the nucleic acid is RNA
  • it is amplified using real time RT-PCR.
  • the nucleic acid is DNA
  • it is amplified using real time PCR. It is possible to use the positive controls of the present invention and corresponding methods in parallel for monitoring of nucleic acid amplifications of DNA and RNA, since one of the at least two target nucleic acid sequences in the inventive construct is designed as positive control for RT-PCR as highlighted by the presence of a poly A-tail carried by at least one target gene found on the constructs of the invention.
  • the assay is a component of a devices that is suitable in fully automated laboratories comprising an arrangement, e.g. for extracting nucleic acids from a sample (e.g. using the epMotion System of Eppendorf International, and preferably the proprietary SentosaTM platform of VelaDx), optionally capable of reverse transcribing isolated nucleic acids, performing amplification reactions using the assay components described herein and quantitatively and qualitatively detecting nucleic acid targets, e.g. using real-time PCR.
  • an arrangement e.g. for extracting nucleic acids from a sample (e.g. using the epMotion System of Eppendorf International, and preferably the proprietary SentosaTM platform of VelaDx), optionally capable of reverse transcribing isolated nucleic acids, performing amplification reactions using the assay components described herein and quantitatively and qualitatively detecting nucleic acid targets, e.g. using real-time PCR.
  • the present invention relates to a composition
  • a composition comprising any of the above mentioned positive controls.
  • the composition comprises also ingredients, e.g. enzymes, buffers and deoxynucleotides necessary for reverse transcription and/or PCR, preferably for qualitative and/or quantitative RT-PCR.
  • the composition may be stored in the refrigerator in a liquid state or deep-frozen in a suitable medium, or it may be lyophilized and reconstituted before use and which may further comprises detectable probes and/or an internal control.
  • the present invention further provides a kit comprising the assay of the invention and optionally instructions for use.
  • PC-I was designed to comprise R A gene A of Influenza, R A gene B from a Respiratory Synthicial Virus (RSV) strain and RNA gene C of Norovirus (NV).
  • RSV Respiratory Synthicial Virus
  • NV Norovirus
  • the three polynucleotide sequences referred to above were cloned into pUC57 plasmid, further comprising a T7 polymerase promoter and a poly A-tail located at the 3 '-end of NV gene C.
  • the sequence of genes in the plasmid is as follows:
  • a second real-time PCR assay was performed with specific primer-probe combinations for RSV (gene B) using PC-I as template.
  • a PCR reaction was set-up using Sentosa SA RSV RT-PCR Test kit (Vela Diagnostic). Rotor-gene Q (Qiagen) was used for RT-PCR and the reaction volume set up to 25 ⁇ .
  • the PCR conditions are shown in Table 1 above.
  • Target gene B of RSV was detected in Cycling A. Green channel.
  • the result of the detection of RNA gene B in the RSV assay using PC-I is shown in Figure 4.
  • a third real-time RT-PCR assay was run with the primer-probe combinations for Norovirus assay (gene C), using PC-I as template.
  • a PCR reaction was set-up using Sentosa SA
  • Norovirus RT-PCR Test kit (Vela Diagnostic). Rotor-gene Q (Qiagen) was used for RT-PCR and the reaction volume setup to 25 ⁇ ⁇ . The PCR reaction was run at the same conditions set forth in Table 1 above. Target RNA gene C for Norovirus was detected in Cycling A.Green channel. The result of the detection of NV gene C is shown in Figure 5.
  • PC-I is suitable for specifically detecting target RNA gene A in Influenza assay, RNA gene B in RSV assay and gene C in Norovirus assay without affecting the sensitivity of the detection in all three assay.
  • PC-II comprises RNA gene D of Influenza, RNA gene B from a different strain of RSV than in PC-I, and RNA gene E of Norovirus.
  • the three genes referred to above were cloned into pUC57 plasmid as backbone further comprising a T7 polymerase promoter located 5 ' to gene D and a poly A-tail at the 3 '-end.
  • the sequence of genes in the plasmid is as follows:
  • a first real-time PCR was run with primer-probe combinations specific for Influenza gene D using PC-II as template.
  • a PCR reaction was set-up using the Sentosa SA Influenza A, B, 2009 HlNl RT-PCR Test kit (Vela Diagnostic). Rotor-gene Q (Qiagen) was used for RT-PCR and the reaction volume set up to 25 ⁇ ⁇ .
  • the PCR reaction was run at the conditions set forth in Table 1.
  • Target RNA gene D for Influenza was detected in Cycling A. Green channel. The result is shown in Figure 6.
  • a second real-time PCR assay was run with specific primer-probe combinations for RSV (gene B from different strain than used in PC-I) using PC-II as template.
  • a PCR reaction was set up using the Sentosa SA RSV RT-PCR Test kit (Vela Diagnostic). Rotor-gene Q (Qiagen) was used for RT-PCR and the reaction volume setup to 25 ⁇ ⁇ .
  • the PCR conditions are set forth in the Table 1.
  • Target DNA gene B for RSV was detected in Cycling A. Green channel and the result of is shown in Figure 7.
  • a third real-time PCR assay was run with primer-probe combinations specific for Norovirus gene E using PC-II as template.
  • a PCR reaction was set-up using Sentosa SA Norovirus RT- PCR Test kit (Vela Diagnostic). Rotor-gene Q (Qiagen) was used for RT-PCR and the reaction volume set up to 25 ⁇ ⁇ .
  • the PCR conditions are the same as in Table 1 above.
  • Target gene E for Norovirus was detected in Cycling A.Orange channel. The result is shown in Figure 8.
  • the PCR performance of PC-II in Influenza, RSV and Norovirus assays is summarized in the following Table 3.
  • PC-II is well-suited for the specific detection of target RNA gene D of Influenza, RNA gene B of RSV and gene E of Norovirus in respective assays without affecting the sensitivity.
  • 6 PC plasmids are required for above- mentioned six different RNA target sequences in three assays.
  • inventive design only 3 PC plasmids are required.
  • PC-III Positive Control - III
  • pUC57 plasmid as backbone comprising DNA gene H in an MTB ⁇ Mycobacterium tuberculosis complex) assay and RNA gene I in Influenza assay under control of a T7 promoter and carrying a 3 '-poly A-tail. Both DNA and RNA assays work equally well using PC-III. Compared with conventional designs, the number of the plasmids for the two assays was reduced by 50%. The design of the plasmid is illustrated as below:
  • a second real-time PCR assay was run with specific primer-probe combinations to detect Influenza Gene I using PC-III as template.
  • a PCR reaction was set up using the Sentosa SA Influenza A, B, 2009 H1N1 RT-PCR Test kit (Vela Diagnostic). Rotor-gene Q (Qiagen) was used for RT-PCR and the reaction volume set up to 25 ⁇ ⁇ .
  • the PCR reaction was run at conditions referred to in the table 1 above.
  • Target RNA gene I was detected in Cycling A Orange channel as shown in Figure 10.
  • PC-III can be used to specifically detect gene H in an MTB DNA assay, and target gene I in an Influenza RNA assay without affecting the sensitivity of the detection in both assay.
  • the number of PC plasmids required for above-mentioned assays is reduced by 50% using the inventive design.
  • the design of both, RNA and DNA targets in one PC plasmid is functional and the performance of PC in both assays works equally well.

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Abstract

La présente invention concerne des outils servant comme contrôles positifs dans des réactions d'amplification d'acides nucléiques, en particulier dans des PCR en temps réel, et leurs méthodes d'utilisation ainsi que des compositions et des kits comprenant les outils de contrôle positif.
PCT/IB2013/052178 2012-03-19 2013-03-19 Contrôle positif pour pcr WO2013140339A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002096925A1 (fr) * 2001-05-25 2002-12-05 Maine Medical Center Research Institute Compositions et procedes relatifs a une construction d'adn de regulation
EP1624076A1 (fr) * 2004-08-06 2006-02-08 Assistance Publique - Hopitaux De Paris Procédé de RT-PCR pour la détection quantitative d'HDV
US20060177818A1 (en) * 2005-02-09 2006-08-10 Bernd Hoffmann Method of detection of classical swine fever
EP1795614A1 (fr) * 2005-12-08 2007-06-13 Simo Nikkari Procédé de diagnostic et produits pouvant être utilisés dans celui-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002096925A1 (fr) * 2001-05-25 2002-12-05 Maine Medical Center Research Institute Compositions et procedes relatifs a une construction d'adn de regulation
EP1624076A1 (fr) * 2004-08-06 2006-02-08 Assistance Publique - Hopitaux De Paris Procédé de RT-PCR pour la détection quantitative d'HDV
US20060177818A1 (en) * 2005-02-09 2006-08-10 Bernd Hoffmann Method of detection of classical swine fever
EP1795614A1 (fr) * 2005-12-08 2007-06-13 Simo Nikkari Procédé de diagnostic et produits pouvant être utilisés dans celui-ci

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHARREL REMY N ET AL: "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism", BMC MICROBIOLOGY, BIOMED CENTRAL LTD, GB, vol. 4, no. 1, 17 May 2004 (2004-05-17), pages 21, XP021002570, ISSN: 1471-2180, DOI: 10.1186/1471-2180-4-21 *
DAS AMARESH ET AL: "Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, vol. 44, no. 9, 1 September 2006 (2006-09-01), pages 3065 - 3073, XP002501623, ISSN: 0095-1137, DOI: 10.1128/JCM.00639-06 *
HOFMANN MARTIN A: "Construction of an infectious chimeric classical swine fever virus containing the 5'UTR of bovine viral diarrhea virus, and its application as a universal internal positive control in real-time RT-PCR", JOURNAL OF VIROLOGICAL METHODS, ELSEVIER BV, NL, vol. 114, no. 1, 1 December 2003 (2003-12-01), pages 77 - 90, XP002557233, ISSN: 0166-0934, [retrieved on 20031020], DOI: 10.1016/J.JVIROMET.2003.09.004 *
M. KODANI ET AL: "Engineered Combined-Positive-Control Template for Real-Time Reverse Transcription-PCR in Multiple-Pathogen-Detection Assays", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 50, no. 3, 14 December 2011 (2011-12-14), pages 1057 - 1060, XP055067594, ISSN: 0095-1137, DOI: 10.1128/JCM.05987-11 *

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