CA2665650A1 - Primers and probes for detection of influenza type a viruses of subtype h1 - Google Patents
Primers and probes for detection of influenza type a viruses of subtype h1 Download PDFInfo
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- CA2665650A1 CA2665650A1 CA 2665650 CA2665650A CA2665650A1 CA 2665650 A1 CA2665650 A1 CA 2665650A1 CA 2665650 CA2665650 CA 2665650 CA 2665650 A CA2665650 A CA 2665650A CA 2665650 A1 CA2665650 A1 CA 2665650A1
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- 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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The present invention relates to primers and probes for detecting influenza virus type A strains of subtype H1, as well as kits including the probes and primers and methods of using the probes and primers.
Description
PRIMERS AND PROBES FOR DETECTION OF
INFLUENZA TYPE A VIRUSES OF SUBTYPE HI
FIELD OF THE INVENTION
The present invention relates to primers and probes for detecting influenza virus type A strains of subtype H1, as well as kits including the probes and primers and methods of using the probes and primers.
DESCRIPTION OF THE FIGURES
Figure 1 shows the nucleotide sequence of the hemagglutinin (HA) gene of the influenza A virus (A/California/04/2009(H1N1)); Genbank GQ117044.
DESCRIPTION OF THE INVENTION
Recent increased circulation of pathogenic influenza type A strains, such as H1 N1, has raised serious concerns about the pandemic threat of these viruses.
In this connection, the present inventors have thus identified primers and probes particularly useful for detecting influenza virus type A strains of subtype 1-11. The present invention also relates to kits including the probes and primers and methods of using the probes and primers in the detection of the HA (subtype 1) genes of influenza A viruses of human health significance.
1. Definitions The term "about" used in connection with a nucleotide position within a particular nucleotide sequence, means that the intended nucleotide position can vary of 1 to 5 positions, upstream or downstream.
A primer related to short nucleic acid molecules, such as a DNA
oligonucleotide, for example sequences of at least 15 nucleotides, which can be annealed to a complementary target nucleic acid molecule (i.e., the HA nucleic acid sequence of an influenza A virus) by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. A primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify a target nucleic acid molecule (such as a portion of a HA nucleic acid), wherein the sequence of the primer is specific for the target nucleic acid molecule, for example so that the primer will hybridize to the target nucleic acid molecule under very high stringency hybridization conditions.
A probe relates to an isolated nucleic acid capable of hybridizing to a target nucleic acid (i.e. the HA nucleic acid sequence of an influenza A virus). A detectable label or reporter molecule can be attached to a probe. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
Fluorescent agent or fluorophore relates to a chemical compound, which when excited by exposure to a particular stimulus such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light). For instance, a fluorophore may be, but not limited to, a dark quencher, such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACK HOLE QUENCHERS.TM. (Glen Research), ECLIPSE.TM. Dark Quencher (Epoch Biosciences), or IOWA BLACK.TM. (Integrated DNA Technologies).
A sample, such as a biological sample, is a sample obtained from a plant or animal subject. As used herein, biological samples include all clinical samples useful for the detection of influenza type A infection in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin;
tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid;
prostate fluid; pus; bone marrow aspirates; bronchoalveolar levage; tracheal aspirates; sputum; nasopharyngeal aspirates; oropharyngeal aspirates; and saliva.
I
INFLUENZA TYPE A VIRUSES OF SUBTYPE HI
FIELD OF THE INVENTION
The present invention relates to primers and probes for detecting influenza virus type A strains of subtype H1, as well as kits including the probes and primers and methods of using the probes and primers.
DESCRIPTION OF THE FIGURES
Figure 1 shows the nucleotide sequence of the hemagglutinin (HA) gene of the influenza A virus (A/California/04/2009(H1N1)); Genbank GQ117044.
DESCRIPTION OF THE INVENTION
Recent increased circulation of pathogenic influenza type A strains, such as H1 N1, has raised serious concerns about the pandemic threat of these viruses.
In this connection, the present inventors have thus identified primers and probes particularly useful for detecting influenza virus type A strains of subtype 1-11. The present invention also relates to kits including the probes and primers and methods of using the probes and primers in the detection of the HA (subtype 1) genes of influenza A viruses of human health significance.
1. Definitions The term "about" used in connection with a nucleotide position within a particular nucleotide sequence, means that the intended nucleotide position can vary of 1 to 5 positions, upstream or downstream.
A primer related to short nucleic acid molecules, such as a DNA
oligonucleotide, for example sequences of at least 15 nucleotides, which can be annealed to a complementary target nucleic acid molecule (i.e., the HA nucleic acid sequence of an influenza A virus) by nucleic acid hybridization to form a hybrid between the primer and the target nucleic acid strand. A primer can be extended along the target nucleic acid molecule by a polymerase enzyme. Therefore, primers can be used to amplify a target nucleic acid molecule (such as a portion of a HA nucleic acid), wherein the sequence of the primer is specific for the target nucleic acid molecule, for example so that the primer will hybridize to the target nucleic acid molecule under very high stringency hybridization conditions.
A probe relates to an isolated nucleic acid capable of hybridizing to a target nucleic acid (i.e. the HA nucleic acid sequence of an influenza A virus). A detectable label or reporter molecule can be attached to a probe. Typical labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
Fluorescent agent or fluorophore relates to a chemical compound, which when excited by exposure to a particular stimulus such as a defined wavelength of light, emits light (fluoresces), for example at a different wavelength (such as a longer wavelength of light). For instance, a fluorophore may be, but not limited to, a dark quencher, such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACK HOLE QUENCHERS.TM. (Glen Research), ECLIPSE.TM. Dark Quencher (Epoch Biosciences), or IOWA BLACK.TM. (Integrated DNA Technologies).
A sample, such as a biological sample, is a sample obtained from a plant or animal subject. As used herein, biological samples include all clinical samples useful for the detection of influenza type A infection in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin;
tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid;
prostate fluid; pus; bone marrow aspirates; bronchoalveolar levage; tracheal aspirates; sputum; nasopharyngeal aspirates; oropharyngeal aspirates; and saliva.
I
Very High Stringency relates to the detection of sequences that share at least 90%
identity. The conditions used may be: hybridization: 5x SSC at 65 C. for 16 hours;
wash twice: 2x SSC at room temperature (RT) for 15 minutes each, wash twice:
0.5x SSC at 65 C. for 20 minutes each.
High Stringency relates to the detection of sequences that share at least 80%
identity. The conditions used may be: hybridization: 5x-6x SSC at 65 C.-70 C. for 16-20 hours; wash twice: 2x SSC at RT for 5-20 minutes each, wash twice: 1 x SSC
at 55 C.-70 C. for 30 minutes each.
Low Stringency relates to the detection of sequences that share at least 50%
identity. The conditions used may be: hybridization: 6x SSC at RT to 550 C.
for 16-20 hours; wash at least twice: 2x-3x SSC at RT to 55 C. for 20-30 minutes each.
2. Probes and primers of the invention According to a first aspect of the invention, there is provided probes for the detection of a hemagglutinin nucleic acid of an influenza type A virus.
Such probes comprise a nucleic acid sequence between about 15 and about 35 nucleotides in length and are capable of hybridizing under very high stringency conditions to the nucleic acid encoding the hemagglutinin of an influenza type A
virus. More particularly, the hemagglutinin nucleic acid consists of a subtype nucleic acid sequence which is at least 90% or even at least 95% identical to the H1 nucleic acid sequence of the influenza A virus (A/California/04/2009(H1N1);
GenBank Acc. No GQ117044; see Fig 1).
Advantageously, the probe hybridizes for instance to a fragment (or portion) of the H1 nucleic acid sequence. Such a portion or fragment spans from about nucleotide 349 to about nucleotide 601 (see Fig 1). A contemplated probe according to the present invention may comprise a nucleic acid sequence at least 95% identical to the nucleotide sequence TTGCTGAGCTTTGGGTATGA. More particularly, the probe consists essentially of the nucleic acid sequence TTGCTGAGCTTTGGGTATGA.
As one skilled in the art may appreciate, the probe of the present invention may be labelled (e.g. radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled). In the case where the probe is labeled with a fluorophore, such a fluorophore may be a fluorescence quencher.
According to another aspect, there is provided primers for the amplification of a HA
nucleic acid sequence or portion thereof of an influenza type A virus. Such a primer comprises a nucleic acid 15 to 40 nucleotides in length capable of hybridizing under very high stringency conditions to an influenza virus subtype H1 nucleic acid sequence at least 90% or even at least 95% identical to the nucleotide sequence shown in Fig 1, and wherein the primer is capable of directing the amplification of the influenza type A nucleic acid.
For instance, the primer contemplated by the present invention hybridizes to a fragment (or portion) of the H1 nucleic acid sequence shown in Fig 1. Such a portion or fragment spans from about nucleotide 349 to about nucleotide 601 (see Fig 1). Such a primer is advantageously at least 95% identical to GAGCTAAGAGAGCAATTGA or GTAGATGGATGGTGAATG.
According to a further aspect, the invention also contemplates of providing a set of primers for the amplification of an influenza subtype H1 nucleic acid. Such a set or pair of primers comprises at least one primer as defined above. More particularly, the set of primers may comprises: a forward primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GAGCTAAGAGAGCAATTGA and a reverse primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GTAGATGGATGGTGAATG, wherein the set of primers is capable of hybridizing to and directing the amplification of an influenza A subtype H1 nucleic acid.
identity. The conditions used may be: hybridization: 5x SSC at 65 C. for 16 hours;
wash twice: 2x SSC at room temperature (RT) for 15 minutes each, wash twice:
0.5x SSC at 65 C. for 20 minutes each.
High Stringency relates to the detection of sequences that share at least 80%
identity. The conditions used may be: hybridization: 5x-6x SSC at 65 C.-70 C. for 16-20 hours; wash twice: 2x SSC at RT for 5-20 minutes each, wash twice: 1 x SSC
at 55 C.-70 C. for 30 minutes each.
Low Stringency relates to the detection of sequences that share at least 50%
identity. The conditions used may be: hybridization: 6x SSC at RT to 550 C.
for 16-20 hours; wash at least twice: 2x-3x SSC at RT to 55 C. for 20-30 minutes each.
2. Probes and primers of the invention According to a first aspect of the invention, there is provided probes for the detection of a hemagglutinin nucleic acid of an influenza type A virus.
Such probes comprise a nucleic acid sequence between about 15 and about 35 nucleotides in length and are capable of hybridizing under very high stringency conditions to the nucleic acid encoding the hemagglutinin of an influenza type A
virus. More particularly, the hemagglutinin nucleic acid consists of a subtype nucleic acid sequence which is at least 90% or even at least 95% identical to the H1 nucleic acid sequence of the influenza A virus (A/California/04/2009(H1N1);
GenBank Acc. No GQ117044; see Fig 1).
Advantageously, the probe hybridizes for instance to a fragment (or portion) of the H1 nucleic acid sequence. Such a portion or fragment spans from about nucleotide 349 to about nucleotide 601 (see Fig 1). A contemplated probe according to the present invention may comprise a nucleic acid sequence at least 95% identical to the nucleotide sequence TTGCTGAGCTTTGGGTATGA. More particularly, the probe consists essentially of the nucleic acid sequence TTGCTGAGCTTTGGGTATGA.
As one skilled in the art may appreciate, the probe of the present invention may be labelled (e.g. radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled). In the case where the probe is labeled with a fluorophore, such a fluorophore may be a fluorescence quencher.
According to another aspect, there is provided primers for the amplification of a HA
nucleic acid sequence or portion thereof of an influenza type A virus. Such a primer comprises a nucleic acid 15 to 40 nucleotides in length capable of hybridizing under very high stringency conditions to an influenza virus subtype H1 nucleic acid sequence at least 90% or even at least 95% identical to the nucleotide sequence shown in Fig 1, and wherein the primer is capable of directing the amplification of the influenza type A nucleic acid.
For instance, the primer contemplated by the present invention hybridizes to a fragment (or portion) of the H1 nucleic acid sequence shown in Fig 1. Such a portion or fragment spans from about nucleotide 349 to about nucleotide 601 (see Fig 1). Such a primer is advantageously at least 95% identical to GAGCTAAGAGAGCAATTGA or GTAGATGGATGGTGAATG.
According to a further aspect, the invention also contemplates of providing a set of primers for the amplification of an influenza subtype H1 nucleic acid. Such a set or pair of primers comprises at least one primer as defined above. More particularly, the set of primers may comprises: a forward primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GAGCTAAGAGAGCAATTGA and a reverse primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GTAGATGGATGGTGAATG, wherein the set of primers is capable of hybridizing to and directing the amplification of an influenza A subtype H1 nucleic acid.
5 3. Method of use The probes and primers of the invention may be used in many ways in the detection of infection caused by an influenza type A strain of subtype H1.
For instance and according to another aspect of the invention, the probes and primers contemplated by the present invention are advantageously useful for the detection of the H1 nucleic acid sequence of an influenza type A virus in a sample obtained from a subject that has or is suspected of having an influenza type A
infection. Thus, the primers and probes contemplated by the present invention can be used to diagnose if a subject has an influenza type A infection and/or discriminate between the viral type and/or subtype the subject is infected with.
Therefore, the probes and primers of the invention may be used in methods for detecting and amplifying H1 nucleic acid sequences derived from influenza type A
strains. The steps of detecting and amplifying can be done separately or simultaneously.
In the case where the H1 nucleic acids of an influenza type A virus present in a sample are amplified prior to using a hybridization probe for detection, one in the art will understand that it can be advantageous to amplify a portion of the H1 nucleic acid, then detect the presence of the amplified H1 nucleic acid. The primers contemplated by the present invention can be used to amplify a region that is at least about 50 to at least about 250, or more base pairs in length to produce amplified H1 specific nucleic acids. Any nucleic acid amplification method can be used to detect the presence of influenza in a sample. Non-limiting examples of such methods which can be used to amplify the H1 nucleic acid sequences are, polymerase chain reaction (PCR), real-time PCR, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR) or ligase chain reaction. Techniques for nucleic acid amplification are well-known to those of skill in the art.
For instance and according to another aspect of the invention, the probes and primers contemplated by the present invention are advantageously useful for the detection of the H1 nucleic acid sequence of an influenza type A virus in a sample obtained from a subject that has or is suspected of having an influenza type A
infection. Thus, the primers and probes contemplated by the present invention can be used to diagnose if a subject has an influenza type A infection and/or discriminate between the viral type and/or subtype the subject is infected with.
Therefore, the probes and primers of the invention may be used in methods for detecting and amplifying H1 nucleic acid sequences derived from influenza type A
strains. The steps of detecting and amplifying can be done separately or simultaneously.
In the case where the H1 nucleic acids of an influenza type A virus present in a sample are amplified prior to using a hybridization probe for detection, one in the art will understand that it can be advantageous to amplify a portion of the H1 nucleic acid, then detect the presence of the amplified H1 nucleic acid. The primers contemplated by the present invention can be used to amplify a region that is at least about 50 to at least about 250, or more base pairs in length to produce amplified H1 specific nucleic acids. Any nucleic acid amplification method can be used to detect the presence of influenza in a sample. Non-limiting examples of such methods which can be used to amplify the H1 nucleic acid sequences are, polymerase chain reaction (PCR), real-time PCR, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR) or ligase chain reaction. Techniques for nucleic acid amplification are well-known to those of skill in the art.
In the case where the steps of detecting and amplifying are done simultaneously, one in the art will understand that it will allow to rapidly confirm the detection of an influenza type A virus, to exclude the presence of an influenza strain representing the "common flu" and to display the presence of the influenza type A strain responsible for the infection. An example of such a method is described in the Example section.
4. Kit of the invention The present invention further provides kits for use within the detection methods contemplated herein. Such kits typically comprise two or more components necessary for performing a detection assay. Components may be compounds, reagents, containers and/or equipment.
For instance and according to another aspect of the invention, there is provided a kit for detecting an influenza subtype H1 nucleic acid in a sample. Such a kit comprises a probe and/or a primer or pair of primers as defined above and instructions for hybridizing the probe to the influenza subtype H1 nucleic acid within the sample.
Additional components that may be present within such kits include other oligonucleotides (probes or primers) and/or a detection reagent or container to facilitate the detection of H1 nucleic acids derived from influenza type A
strains.
EXAMPLE: Real-time PCR of Type A influenza virus Human and H1N1sw viruses Kits QlAamp Viral RNA (QIAGEN mini Kit 50) Ref: QIAGEN 52 904 SuperScriptTM III Platinum One-Step Quantitative RT-PCR System Ref:
Invitrogen 11732-020 Non acetyled BSA 10% Ref: Invitrogen P2046 Primers and probes Name Sequences Mers PCR Pdt Ref GAPDH-6Fw GAAGGTGAAGGTCGGAGT 18 3 GAPDH-231Rv GAAGATGGTGATGGGATTTC 20 226 bp 3 GAPDH-202Probe(-) CAAGCTTCCCGTTCTCAGCC 20 3 GRAM/7Fw CTTCTAACCGAGGTCGAAACGTA 23 2 GRAM/161Rv GGTGACAGGATTGGTCTTGTCTTTA 25 202 bp 2 GRAM probe/52/+ TCAGGCCCCCTCAAAGCCGAG [5']HEX [3']BHQ-1 21 2 Hlh-678Fw CACCCCAGAAATAGCCAAAA 20 1 H1h-840Rv TCCTGATCCAAAGCCTCTAC 20 163 bp 1 Hlh-715probe CAGGAAGGAAGAATCAACTA [5']Fam [3']BHQ-1 20 1 H3h-177Fw GAGCTGGTTCAGAGTTCCTC 20 1 H3h-388Rv GTGACCTAAGGGAGGCATAATC 22 211 by 1 H3h-306Probe TTTTGTTGAACGCAGCAAAG [5']Fam [3']BHQ-1 20 1 N2h-1150 Fw GTCCAMACCTAAYTCCAA 18 1 N2h-1344 Rv GCCACAAAACACAACAATAC 20 194 bp 1 N2h-1290 probe CTTCCCCTTATCAACTCCACA [5' ]HEX [3' ]BHQ-1 21 1 Nth-1134 Fw TGGATGGACAGATACCGACA 20 1 Nlh-1275 Rv CTCAACCCAGAAGCAAGGTC 20 142 bp 1 Nlh-1206 probe CAGCGGAAGTTTCGTTCAACAT [5']Fam [3']BHQ-1 22 1 GRswH1-349Fw GAGCTAAGAGAGCAATTGA 19 GRswH1-601Rv GTAGATGGATGGTGAATG 18 253bp 1 GRswH1-538Probe(-) TTGCTGAGCTTTGGGTATGA [5']Fam [3']BHQ-1 20 lo Positive control for M and GAPDH real-time RT-PCR
Positive control for M real-time RT-PCR is an in vitro transcribed RNA derived from strain A/Paris 650/06(HIN1). The transcript contains the Open Reading Frame of the M gene (from the ATG to nt 982 ) as negative strand. Each microtube contains 1011 copies of target sequences diluted in yeast tRNA, and lyophilised.
4. Kit of the invention The present invention further provides kits for use within the detection methods contemplated herein. Such kits typically comprise two or more components necessary for performing a detection assay. Components may be compounds, reagents, containers and/or equipment.
For instance and according to another aspect of the invention, there is provided a kit for detecting an influenza subtype H1 nucleic acid in a sample. Such a kit comprises a probe and/or a primer or pair of primers as defined above and instructions for hybridizing the probe to the influenza subtype H1 nucleic acid within the sample.
Additional components that may be present within such kits include other oligonucleotides (probes or primers) and/or a detection reagent or container to facilitate the detection of H1 nucleic acids derived from influenza type A
strains.
EXAMPLE: Real-time PCR of Type A influenza virus Human and H1N1sw viruses Kits QlAamp Viral RNA (QIAGEN mini Kit 50) Ref: QIAGEN 52 904 SuperScriptTM III Platinum One-Step Quantitative RT-PCR System Ref:
Invitrogen 11732-020 Non acetyled BSA 10% Ref: Invitrogen P2046 Primers and probes Name Sequences Mers PCR Pdt Ref GAPDH-6Fw GAAGGTGAAGGTCGGAGT 18 3 GAPDH-231Rv GAAGATGGTGATGGGATTTC 20 226 bp 3 GAPDH-202Probe(-) CAAGCTTCCCGTTCTCAGCC 20 3 GRAM/7Fw CTTCTAACCGAGGTCGAAACGTA 23 2 GRAM/161Rv GGTGACAGGATTGGTCTTGTCTTTA 25 202 bp 2 GRAM probe/52/+ TCAGGCCCCCTCAAAGCCGAG [5']HEX [3']BHQ-1 21 2 Hlh-678Fw CACCCCAGAAATAGCCAAAA 20 1 H1h-840Rv TCCTGATCCAAAGCCTCTAC 20 163 bp 1 Hlh-715probe CAGGAAGGAAGAATCAACTA [5']Fam [3']BHQ-1 20 1 H3h-177Fw GAGCTGGTTCAGAGTTCCTC 20 1 H3h-388Rv GTGACCTAAGGGAGGCATAATC 22 211 by 1 H3h-306Probe TTTTGTTGAACGCAGCAAAG [5']Fam [3']BHQ-1 20 1 N2h-1150 Fw GTCCAMACCTAAYTCCAA 18 1 N2h-1344 Rv GCCACAAAACACAACAATAC 20 194 bp 1 N2h-1290 probe CTTCCCCTTATCAACTCCACA [5' ]HEX [3' ]BHQ-1 21 1 Nth-1134 Fw TGGATGGACAGATACCGACA 20 1 Nlh-1275 Rv CTCAACCCAGAAGCAAGGTC 20 142 bp 1 Nlh-1206 probe CAGCGGAAGTTTCGTTCAACAT [5']Fam [3']BHQ-1 22 1 GRswH1-349Fw GAGCTAAGAGAGCAATTGA 19 GRswH1-601Rv GTAGATGGATGGTGAATG 18 253bp 1 GRswH1-538Probe(-) TTGCTGAGCTTTGGGTATGA [5']Fam [3']BHQ-1 20 lo Positive control for M and GAPDH real-time RT-PCR
Positive control for M real-time RT-PCR is an in vitro transcribed RNA derived from strain A/Paris 650/06(HIN1). The transcript contains the Open Reading Frame of the M gene (from the ATG to nt 982 ) as negative strand. Each microtube contains 1011 copies of target sequences diluted in yeast tRNA, and lyophilised.
Positive control for GAPDH real-time RT-PCR is an in vitro transcribed RNA.The transcript contains the Open Reading Frame of the M gene (from the 6 (ATG = 1) to nt 231 ) as negative strand. Each microtube contains 1011 copies of target sequences diluted in yeast tRNA, and lyophilised.
Reconstitution of transcribed RNA
Add 100 pl of distilled water to obtain a solution at a concentration of 109 copies/pl.
Store at -80 C.
Dilute in H2O to prepare a master bank at 2x106 copies/pl. Store at -80 C.
From this prepare a working bank of reagent at 2x104 copies/pI in order to avoid freeze/thaw cycles. Working tubes may be stored at -20 C for less than one week.
NUCLEIC ACID EXTRACTION
RNA is extracted from specimens using the QlAamp Viral RNA kit (QIAGEN Mini Kit 50 ref 52904). RNA extracted from 200 pI of original sample, is eluted in 60 pl of elution buffer.
MIX PREPARATION FOR ALL SEPARATE PRIMER/PROBE COMBINATIONS
Primers and probes described below were validated under the following conditions.
RT-PCR Mix kit:
= Invitrogen SuperscriptTM III Platinum One-Step qRT-PCR system (ref: 11732-088) Real-time PCR equipments:
= LightCycler 1.5 or 2.0 (Capillaries) = LightCycler 480 Adjustments may be required for the use of other kits or other real-time PCR
instruments.
Reconstitution of transcribed RNA
Add 100 pl of distilled water to obtain a solution at a concentration of 109 copies/pl.
Store at -80 C.
Dilute in H2O to prepare a master bank at 2x106 copies/pl. Store at -80 C.
From this prepare a working bank of reagent at 2x104 copies/pI in order to avoid freeze/thaw cycles. Working tubes may be stored at -20 C for less than one week.
NUCLEIC ACID EXTRACTION
RNA is extracted from specimens using the QlAamp Viral RNA kit (QIAGEN Mini Kit 50 ref 52904). RNA extracted from 200 pI of original sample, is eluted in 60 pl of elution buffer.
MIX PREPARATION FOR ALL SEPARATE PRIMER/PROBE COMBINATIONS
Primers and probes described below were validated under the following conditions.
RT-PCR Mix kit:
= Invitrogen SuperscriptTM III Platinum One-Step qRT-PCR system (ref: 11732-088) Real-time PCR equipments:
= LightCycler 1.5 or 2.0 (Capillaries) = LightCycler 480 Adjustments may be required for the use of other kits or other real-time PCR
instruments.
LiahtCycler 1.5 or 2.0 (Capillaries) Mix: Vol (PI) [final]
H2O PPI : 1.06 Reaction mix 2X: 10 3 mM Mg MgSO4 (50mM) : 0.24 0.6 mM Mg Forward Primer (10pM): 1 0.5 pM
Reverse Primer (10pM): 1 0.5 pM
Probe (10pM): 0.4 0.2 pM
BSA non acetylated (10mg/ml) 0.5 0.25 mg/ml Superscriptlll RT/Platinum Taq Mix : 0.8 Final Volume: 15 pl LiahtCycler 480 (96) Mix: Vol (pl) [final]
H2O PPI : 1.56 Reaction mix 2X: 10 3 mM Mg MgSO4 (50mM) : 0.24 0.6 mM Mg Forward Primer (10pM): 1 0.5 pM
Reverse Primer (10pM): 1 0.5 pM
Probe (10pM): 0.4 0.2 pM
Superscriptlll RT/Platinum Taq Mix: 0.8 Final Volume: 15 PI
15 pi of reaction mix + 5 pl of RNA samples I
Each real-time RT-PCR assay includes in addition of unknown samples:
= Two negative samples bracketing unknown samples during RNA extraction (negative extraction controls) = Five quantification positive controls (in duplicate) including 104, 103 and 10 copies of in vitro synthesized H5 or M RNA transcripts.
= One negative amplification control.
Presence or absence of inhibitors in extracted RNA samples is checked by spiking experiments. To this end, 103 copies of H5 or M RNA transcripts are diluted in RNA
samples previously found negative by RT-PCR. Ct of spiked samples must be equal ( no more than 3 Ct) to the Ct obtained for 103 copies of H5 or M RNA
transcripts tested without RNA samples, in the same operating conditions.
AMPLIFICATION CYCLES (LIGHTCYCLER SYSTEM) Reverse transcription 45 C 15 min x1 Denaturation 95 C 3 min x1 Amplification 95 C 10 sec 55 C 10 sec 72 C 20 sec x50 Cooling 40 C 30 sec x1 SENSITIVITY
Sensitivity, in terms of 95% hit rate is about 100 copies of RNA transcript per reaction (this amount of target sequences is always detected), the probability to detect lower amounts of virus decreases, but samples containing 10 copies could be detected.
H2O PPI : 1.06 Reaction mix 2X: 10 3 mM Mg MgSO4 (50mM) : 0.24 0.6 mM Mg Forward Primer (10pM): 1 0.5 pM
Reverse Primer (10pM): 1 0.5 pM
Probe (10pM): 0.4 0.2 pM
BSA non acetylated (10mg/ml) 0.5 0.25 mg/ml Superscriptlll RT/Platinum Taq Mix : 0.8 Final Volume: 15 pl LiahtCycler 480 (96) Mix: Vol (pl) [final]
H2O PPI : 1.56 Reaction mix 2X: 10 3 mM Mg MgSO4 (50mM) : 0.24 0.6 mM Mg Forward Primer (10pM): 1 0.5 pM
Reverse Primer (10pM): 1 0.5 pM
Probe (10pM): 0.4 0.2 pM
Superscriptlll RT/Platinum Taq Mix: 0.8 Final Volume: 15 PI
15 pi of reaction mix + 5 pl of RNA samples I
Each real-time RT-PCR assay includes in addition of unknown samples:
= Two negative samples bracketing unknown samples during RNA extraction (negative extraction controls) = Five quantification positive controls (in duplicate) including 104, 103 and 10 copies of in vitro synthesized H5 or M RNA transcripts.
= One negative amplification control.
Presence or absence of inhibitors in extracted RNA samples is checked by spiking experiments. To this end, 103 copies of H5 or M RNA transcripts are diluted in RNA
samples previously found negative by RT-PCR. Ct of spiked samples must be equal ( no more than 3 Ct) to the Ct obtained for 103 copies of H5 or M RNA
transcripts tested without RNA samples, in the same operating conditions.
AMPLIFICATION CYCLES (LIGHTCYCLER SYSTEM) Reverse transcription 45 C 15 min x1 Denaturation 95 C 3 min x1 Amplification 95 C 10 sec 55 C 10 sec 72 C 20 sec x50 Cooling 40 C 30 sec x1 SENSITIVITY
Sensitivity, in terms of 95% hit rate is about 100 copies of RNA transcript per reaction (this amount of target sequences is always detected), the probability to detect lower amounts of virus decreases, but samples containing 10 copies could be detected.
References:
1. National Influenza Center (Northern-France), Institut Pasteur, Paris.
2. Wong et al., 2005, J. Clin. Pathol. 58;276-280.
3. National Influenza Center (Southern-France), CHU, Lyon
1. National Influenza Center (Northern-France), Institut Pasteur, Paris.
2. Wong et al., 2005, J. Clin. Pathol. 58;276-280.
3. National Influenza Center (Southern-France), CHU, Lyon
Claims (15)
1. A probe for the detection of an influenza nucleic acid comprising a nucleic acid sequence between about 15 and about 35 nucleotides in length capable of hybridizing under very high stringency conditions to an influenza virus subtype H1 nucleic acid sequence at least 90% identical to the nucleotide sequence shown in figure 1.
2. The probe according to claim 1, wherein the probe hybridizes to all or part thereon of the portion spanning from about nucleotide 349 to about nucleotide 601 of the nucleotide sequence shown in figure 1.
3. The probe according to claim 1 or 2, wherein the probe comprises a nucleic acid sequence at least 95% identical to the nucleotide sequence TTGCTGAGCTTTGGGTATGA.
4. The probe according to any one of claims 1 to 3, wherein the probe consists essentially of the nucleic acid sequence TTGCTGAGCTTTGGGTATGA.
5. The probe according to any one of claims 1 to 3, wherein the probe is labeled.
6. The probe according to claim 4, wherein the probe is radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled.
7. The probe according to claim 5, wherein the probe is labeled with a fluorophore.
8. The probe according to claim 6, wherein the probe is labeled with a fluorescence quencher.
9. A primer for the amplification of an influenza nucleic acid sequence comprising a nucleic acid 15 to 40 nucleotides in length capable of hybridizing under very high stringency conditions to an influenza virus subtype H1 nucleic acid sequence at least 90% identical to the nucleotide sequence shown in figure 1, and wherein the primer is capable of directing the amplification of the influenza nucleic acid.
10. The primer according to claim 9, wherein the primer hybridizes to all or part thereon of the portion spanning from about nucleotide 349 to about nucleotide 601 of the nucleotide sequence shown in figure 1.
11. The primer according to claim 9 or 10, wherein the primer is at least 95%
identical to GAGCTAAGAGAGCAATTGA or GTAGATGGATGGTGAATG.
identical to GAGCTAAGAGAGCAATTGA or GTAGATGGATGGTGAATG.
12. A set of primers for the amplification of an influenza subtype H1 nucleic acid comprising at least one primer according to any one of claims 9 to 11.
13. The set of primers according to claim 12, wherein the set of primers comprises: a forward primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GAGCTAAGAGAGCAATTGA
and a reverse primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GTAGATGGATGGTGAATG, wherein the set of primers is capable of hybridizing to and directing the amplification of the influenza subtype H1 nucleic acid.
and a reverse primer 15 to 40 nucleotides in length comprising a nucleic acid sequence at least 95% identical to GTAGATGGATGGTGAATG, wherein the set of primers is capable of hybridizing to and directing the amplification of the influenza subtype H1 nucleic acid.
14. A kit for detecting an influenza subtype H1 nucleic acid in a sample, comprising a probe according to any one of claims 1 to 9 and/or a primer according to any one of claims 9 to 11, and instructions for hybridizing the probe to the influenza subtype H1 nucleic acid within the sample.
15. Use of a probe according to any one of claims 1 to 9 or a primer according to any one of claims 9 to 11, for the detection of an influenza subtype H1 nucleic acid.
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|---|---|---|---|
| CA 2665650 CA2665650A1 (en) | 2009-05-08 | 2009-05-08 | Primers and probes for detection of influenza type a viruses of subtype h1 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2665650 CA2665650A1 (en) | 2009-05-08 | 2009-05-08 | Primers and probes for detection of influenza type a viruses of subtype h1 |
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| Publication Number | Publication Date |
|---|---|
| CA2665650A1 true CA2665650A1 (en) | 2010-11-08 |
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| Application Number | Title | Priority Date | Filing Date |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107529560A (en) * | 2017-09-14 | 2018-01-02 | 奥斯汀生命科学技术公司 | Avian influenza virus H7N9 hypotype fluorescence RT PCR primers group, probe groups, kit and method |
-
2009
- 2009-05-08 CA CA 2665650 patent/CA2665650A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107529560A (en) * | 2017-09-14 | 2018-01-02 | 奥斯汀生命科学技术公司 | Avian influenza virus H7N9 hypotype fluorescence RT PCR primers group, probe groups, kit and method |
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