CN101313079A - Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of H5 and N1 genes of the influenza A virus - Google Patents
Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of H5 and N1 genes of the influenza A virus Download PDFInfo
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Abstract
The invention concerns a double pair of oligonucleotides for amplifying two target sequences localized respectively in the H5 and N1 genes of the genome of the Influenza A virus, said oligonucleotides being of a length ranging between 10 and 50 nucleotides and comprising at least a fragment of 10 consecutive nucleotides derived from the following sequences: SEQ ID No. 1: TGTATGTTGTGGAATGGCA, SEQ ID No. 2: GCCGAATGATGCCATCAA, SEQ ID No. 3: CGTGGATTGTCTCCGAAA, and SEQ ID No. 4: GGAATGCTCCTGTTATCCTGA, or their complementary sequence. The invention also concerns oligonucleotides for detecting amplicons, the use of all those sequences, a method for detecting genes H5 and N1 of the Influenza A virus. The invention is particularly applicable in the field of diagnosis.
Description
The present invention relates in the method that has labeling nucleic acid under the condition of at least a solid support.
The present invention relates to oligonucleotide, it is respectively applied for amplification and detects two target sequences that are arranged in genomic H5 of influenza A virus and N1 gene.
The invention still further relates to the purposes of utilizing these oligonucleotide, the method and being used to that is used to detect the existence of the H5 of influenza A virus and N1 gene is diagnosed the test kit of the existence of the H5 of influenza A virus and N1 gene.
Diagnose in the routine techniques of influenza being generally used for, be usually directed to agglutination, suppress the diffusion in agglutination or the agar gel.These methods are used usually and make can qualitative influenza virus A.
Optional substitute of these methods is according to IOE[International Office for Epizootics in the ovum gallinaceum of embryo stage or in mdck cell] scheme in the handbook carries out virus culture.Yet, need a couple of days could obtain qualitative results, this delay is contradictory with clinical needs or emergency situation sometimes.
Be used to detect the detection of antibody or antigenic elisa technique or immunofluorescence and also greatly developed, but these " immunology " detection methods are compared common susceptibility with conventional virus culture and specificity is lower.
The needs to quick, specificity and hypersensitivity diagnostic detection have been aroused in the appearance of the bird flue virus H 5 N 1 subtype of the recently high form of causing a disease again.For this reason, above-mentioned the whole bag of tricks is replaced by " molecule " technology gradually, such as RT-PCR technology (reverse transcription relevant with polymerase chain reaction).RT-PCR is more responsive, does not need to exist in the sample " work " virus, and the feasible thus various forms with influenza virus is classified and is divided into subclass and becomes possibility.The development of this real-time technique (" real-time RT-PCR ") has also greatly promoted its application in diagnosis A C-type virus C.
For example, D.M.Whiley etc. are at nearest disclosed document (Diagnostic Microbiology andInfectious Diseases (2005), in press) described the experiment of the wide spectrum influenza virus sub-strain that is used for detecting clinical sample in, it is based on the RT-PCR reaction that relates to 5 '-nuclease.Select two kinds of oligonucleotide and probe, itself and the proteic dna homolog of M (matrix) of 23 kinds of hypotypes of coding A virus.This detection makes that thus the influenza A virus that detects in the clinical sample becomes possibility, but on the other hand, still can not accurately detect the subclass of related type.
Recently open (Emerging Infectious Diseases (2005) vol.11 (8) such as E.K.O.Ng, p.1303-1305) based on the detection of multiple RT-PCR reaction, it comprises two steps, utilize the probe of two groups of oligonucleotide and mark, thus two zones of the HA gene of selectively targeted H5N1.Make it possible to develop the detection method that also directly detects the H5 hypotype among the human sample fast sensitively like this.This detection method confirms in clinical sample, the patient of the influenza virus infection H5N1 hypotype of the comfortable Hong Kong of described sample source and Vietnam, but still can not directly diagnose the hypotype of institute's infective virus.
S.Payungporn etc. describe (Viral Immunology (2004) vol.17 (4), p.588-593and Journal of Virological Methods (2005), in press) detects the M of H5N1 hypotype simultaneously, the method of H5 and N1 sequence, it is finished based on a real-time multiple RT-PCR detection method and a step.Corresponding M, thus the TaqMan probe of the oligonucleotide of H5 and N1 sequence and various marks is selected and be used for this detection method and detect three kinds of fluorescent signals simultaneously.H5 and N1 oligonucleotide are selected from the constant region of 50 known specific sequences of covering H5N1 virus.But should notice that this RT-PCR method is non-isothermal, is easy to pollute sometimes.
It is NASBA technology (NucleicAcid Sequence-Based Amplification) that another kind can be used for detecting the molecular method that infectant exists.For example, R.A.Collins etc. describe (Journal ofVirological Methods (2002) vol.103, p.213-225and Avian Diseases (2003) vol.47 (3), p.1069-1074) detection of bird flu H5 hypotype (detecting highly pathogenic and weak pathogenic hypotype), it utilizes and ECL (electron chemistry luminescence method) detection system bonded NASBA technology.The NASBA technology relates to the isothermal duplication of the active nucleic acid of plurality of enzymes, and it allows rapid detection H5 virus.Be fit to the rna gene group by this method amplification of nucleic acid, such as the influenza virus gene group, by the reverse transcription step is directly introduced amplified reaction.Yet, although the method for describing in this piece document make may detect and divide a little less than pathogenic bacterial strains and highly pathogenic bacterial strain, still can not specificity differentiate the H5N1 type of this bacterial strain.
Utilize the transcription amplification technology to differentiate the detection method of the influenza A virus of H5N1 form, it especially is fit to amplification and detects RNA viruses, still remains thus to be developed.In addition, multiple detection method can increase and detect the detection method of H5 and two genes of N1 simultaneously, amplification technique wherein be the transcription amplification technology such as NASBA, especially favourable.
The present invention relates to two pairs of oligonucleotide thus, and it is respectively applied for amplification and is arranged in two target sequences of genomic H5 gene of influenza A virus and N1 gene, and the oligonucleotide of the H5 gene that wherein is used to increase is to being made up of following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.1:TGTATGTTGTGGAATGGCA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.2:GCCGAATGATGCCATCAA or its complementary sequence,
Wherein be used to increase the oligonucleotide of N1 gene to forming by following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.3:CGTGGATTGTCTCCGAAA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.4:GGAATGCTCCTGTTATCCTGA or its complementary sequence.
It is right to the invention still further relates to oligonucleotide, is used for increasing being positioned at the target sequence of the genomic H5 gene of influenza A virus, and described oligonucleotide is to being made up of following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.1:TGTATGTTGTGGAATGGCA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.2:GCCGAATGATGCCATCAA or its complementary sequence.
Similarly, it is right to the invention still further relates to such oligonucleotide, and being used for increasing is positioned at the target sequence of the genomic N1 gene of influenza A virus, and described oligonucleotide is to being made up of following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.3:CGTGGATTGTCTCCGAAA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.4:GGAATGCTCCTGTTATCCTGA or its complementary sequence.
In above-mentioned three kinds of situations, oligonucleotide centering, first oligonucleotide also comprises can be by the promoter sequence of DNA-RNA-dependent polysaccharase identification.
According to above-mentioned two kinds of situations, wherein, the oligonucleotide that has added promoter sequence may increase when being arranged in the target sequence of H5 gene when making, and it is made up of following sequence basically:
SEQ ID No.5:aattctaatacgactcactataggggTGTATGTTGTGGAATGGCA, or its complementary sequence.The corresponding T7 promoter sequence of lowercase in this sequence.
As indicated above identical, when this oligonucleotide makes it possible to increase when being arranged in the target sequence of N1 gene, described oligonucleotide is made up of following sequence basically:
SEQ ID No.6:aattctaatacgactcactataggggCGTGGATTGTCTCCGAAA, or its complementary sequence.
In all situations, the length of each oligonucleotide all is 12-30 Nucleotide and the fragment that comprises at least one 16 continuous nucleotide, and preferably the length of each oligonucleotide is 15-26 Nucleotide and the fragment that comprises at least one 18 Nucleotide.
The invention still further relates to a pair of oligonucleotide, lay respectively at two target sequences in genomic H5 of influenza A virus and the N1 gene as probe in detecting, the probe that wherein is used to detect the H5 gene is made up of following sequence:
SEQ ID No.7:ACACCAAGTGTCAAACTCCAAT or its complementary sequence;
The probe that is used to detect the N1 gene is made up of following sequence:
SEQ ID No.8:GCGAAATCACATGTGTGTGCAGGGA or its complementary sequence,
Each sequence comprises at least a marking tool.
The invention still further relates to oligonucleotide as probe, described probe is used for detecting the nucleotide sequence that amplification is positioned at the amplification that target sequence produced of the genomic H5 gene of influenza A virus, described amplification is by utilizing above-mentioned oligonucleotide to carrying out, the length of described detection probes is 10-50 Nucleotide and the fragment that comprises at least one 10 continuous nucleotide that is derived from following sequence: SEQ ID No.7:ACACCAAGTGTCAAACTCCAAT, or its complementary sequence, described sequence comprises at least a marking tool.
When needs detect the genomic N1 gene of influenza A virus, the present invention relates to oligonucleotide as probe, described probe is used for detecting the nucleotide sequence that amplification is positioned at the amplification that target sequence produced that detects the genomic N1 gene of influenza A virus, described amplification is by utilizing above-mentioned oligonucleotide to carrying out, the length of described detection probes is 10-50 Nucleotide and the fragment that comprises at least one 10 continuous nucleotide that is derived from following sequence: SEQ ID No.8:GCGAAATCACATGTGTGTGCAGGGA, or its complementary sequence, described sequence comprises at least a marking tool.
In the preferred embodiment of the present invention, detection probes by " molecular signal " form, it is also referred to as molecular probe.Described molecular probe is the detection probes of single stranded oligonucleotide form, and it has loop-stem structure known in the art.This ring contains and target sequence (being generally amplicon) complementary probe sequence, and described stem becomes the arm sequence hybridizations to form by two, and wherein each sequence is positioned at an end of probe.Fluorophore is covalently attached in two arms one end, and quencher (fluorescent absorption agent) is connected in the end of other one arm.When being in unbound state in solution, molecular probe do not fluoresce.But when having complementary amplicon, they and these targets are hybridized, thereby the experience conformational change allows them to send fluorescence.When lacking target, stem structure makes fluorophore and quencher the most approaching, thereby causes fluorescence to transfer to quencher from fluorophore.Described quencher is non-fluorescent chromophore, and the energy dissipation that its future, autofluorescence was rolled into a ball is a heat.When probe runs into the molecule target, formation probe-target hybridization, described hybridization is longer and more stable than hybridization of two arms formation of described stem.The rigidity of probe-target hybridization and length exist when preventing stem structure hybridization.Therefore, molecular probe is through spontaneous conformation reconstruct, make stem structure hybridization subsolution from, fluorophore and quencher separately, thereby recover fluorescence.
More specifically, it is made up of described detection probes molecular probe, preferably is made up of following sequence:
SEQ ID 9:[6-FAM]-cgatcgaCACCAAGTGTCAAACTCCAAtcgatcg-[DabSyl], be used to detect the H5 gene,
SEQ ID 10:[6-FAM]-cgatcgGCGAAATCACATGTGTGTGCAGGGAcgatcg-[DabSyl], be used to detect the N1 gene.
Multiple detection detects H5 and N1 gene simultaneously and when carrying out in single container, preferably use two kinds of not isolabelings when needs carry out.In possible fluorescent mark, include but not limited to:
Fluorescein (FAM)
Tetrachloro-6-Fluoresceincarboxylic acid (TET)
Tetramethylrhodamin (TMR)
5-carboxyl rhodamine 6G (RHD)
Carboxyl rhodamine (ROX) and
Cyanin 5 (CY5).
Each sequence among the above-mentioned SEQ ID Nos.9 and 10 can have one of described mark, distinguishes detection signal thereby two kinds of marks of use are different permissions.
The invention still further relates in the reaction of amplification of nucleic acid and to use above-mentioned one or two pair of oligonucleotide or used as the suspicious genome that has the influenza A virus in the biological sample of probe in detecting.
The invention still further relates to the method for the nucleic acid that is used for detecting the influenza A virus that may be present in sample, wherein described sample is utilized the reaction of aforesaid oligonucleotide to amplification of nucleic acid, described being reflected under the condition that has the required amplification agent of described amplification carried out, and the existence of testing goal amplicon.
Described detection method is based on the RT-PCR amplified reaction.
Optional, described detection method is based on the transcription amplification technology.Preferably, this technology is the NASBA technology.
The invention still further relates to amplification and may be present in the method for the H5 and N1 two genes of the influenza A virus in the sample, may further comprise the steps:
-in amplification buffer, be incubated sample under the condition of following material existing:
Two kinds of amplimers, the length of each primer is 10-50 Nucleotide, a kind of promoter sequence that also comprises, and polarity another kind of and in conjunction with the primer of promoter sequence is opposite, thereby respectively with the upstream and downstream hybridization in the purpose zone of the H5 gene that is arranged in influenza A virus
Two kinds of amplimers, the length of each primer is 10-50 Nucleotide, a kind of promoter sequence that also comprises, and polarity another kind of and in conjunction with the primer of promoter sequence is opposite, thereby respectively with the upstream and downstream hybridization in the purpose zone of the N1 gene that is arranged in influenza A virus
-following reagent is added sample:
Have the active enzyme of RNA-dependent dna-polymerases,
Have the active enzyme of DNA-dependent dna-polymerases,
Have the active enzyme of RNase H,
Enzyme with DNA-RNA-dependent polymerase activity, and
-under suitable condition, keep for some time that consequent reaction mixture is enough to feasible amplification generation.
Listed four kinds of enzymes hereinbefore, had two kinds even three kinds of above-mentioned active a kind of enzymes but may utilize fully; In this case, three kinds of uses even two kinds of enzymes are possible and are encompassed in the scope of the present invention.In addition, necessary when establishing other factors that increase required, such as Nucleotide.Described factor is well known by persons skilled in the art.
At last, the invention provides and be used for detecting the H5 of the influenza A virus that may be present in sample and the test kit of N1 gene, it comprises:
-two couple as indicated above amplification oligonucleotide or primer, two zones of its be used to increase purpose H5 and N1,
-two kinds of oligonucleotide, but it is a form aforesaid mark or mark, and have basically and the H5 of amplification or at least a portion complementary nucleotide sequence of N1 nucleotide sequence,
-carry out the required reagent of amplified reaction.
Term " complementary basically " but mean hybridization between at least a portion of the oligonucleotide (being also referred to as detection probes) of mark or mark and nucleotide sequence that is increased or amplicon, the specificity of this hybridization and selectivity are enough high, allow the testing goal amplicon.
In addition, carrying out the required reagent of amplified reaction is the reagent that is used for the NASBA amplification.
Term " detectable label " mean at least a mark, it can directly produce detectable signal.For example, the existence of vitamin H is considered to direct mark, because it is detectable, although may subsequently it be combined with the streptavidin of mark.The non-limiting of these marks is listed as follows:
Generation can be by colorimetry/fluorescence for example or luminous detection the enzyme of signal, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or glucose-6-phosphate dehydrogenase;
Chromophoric group such as fluorescent chemicals/luminophor or dye composition,
Can by electron microscope method or by their electrical properties such as conductivity, amperometry, the electron dense group that voltage analysis or impedance detect,
But detection moiety, for example size is enough to induce the molecule of the detected change of its physics and/or chemical property; This detection can be by optical means such as diffraction, surface plasma body resonant vibration, and surface modification (surface variation), contact angle change (contact angle variation), or physical method is such as the atomic power spectroscope, or tunnel effect carries out,
The radioactivity molecule such as
32P,
35S or
125I.
Preferably, described mark is not the radioactivity mark, thereby avoids the safety issue relevant with these marks.
In the specific embodiments of the present invention, described mark is that electrochemistry is detectable, and concrete described mark is the derivative of iron complex body, such as ferrocene.
Term " nucleic acid " means a series of at least two kinds of deoxyribonucleotides or ribonucleotide, the optional Nucleotide that comprises at least a modification, the for example at least a Nucleotide that comprises the base of modification, such as inosine, methyl-5-Deoxyribose cytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, or any other allows the modified base of hybridization.These polynucleotide also can be modified in the level of internucleotide linkage, thiophosphatephosphorothioate for example, H-phosphonate or alkyl-phosphonate, or in the modification of main chain level, for example, α-oligonucleotide (FR 2 607 507) or PNA (M.Egholm et al., J.Am.Chem.Soc., 114,1895-1897,1992) or 2 '-O-alkyl ribose and LNAs (BW.Sun et al., Biochemistry, 4160-4169,43,2004).Nucleic acid can be natural or synthetic, oligonucleotide, and polynucleotide, nucleic acid fragment, ribosome-RNA(rRNA), messenger RNA(mRNA), transfer RNA, or the nucleic acid that obtains by following enzymatic amplification technology:
PCR (polymerase chain reaction) is described in patent US-A-4, and 683,195, US-A-4,683,202 and US-A-4,800,159, and derivative RT-PCR (reverse transcription PCR), specifically in the single step reaction pattern, as described in patent EP-B-0.569.272,
LCR (ligase chain reaction), open and for example patent application EP-A-0.201.184,
RCR (reparation chain reaction) is described in patent application WO-A-90/01069,
3SR (independently keeping dubbing system) sees patent application WO-A-90/06995,
NASBA (based on the amplification of nucleotide sequence) sees patent application WO-A-91/02818,
TMA (amplification of transcriptive intermediate), patent US-A-5,399,491 and
RCA (rolling circle amplification) (US-6,576,448).
The term amplicon refers to the nucleic acid by the generation of enzymatic amplification technology.
Each of these modifications use capable of being combined.
Before above disclosed amplification and the detection step is purification step.Term " purification step " specifically mean the separation between the cellular component that discharges in the cleavage step before the nucleic acid of described microorganism and the nucleic acid purification.These cleavage step are known; For example, can utilize the cleavage method of describing in the patent application:
-WO-A-00/60049, by ultrasonic degradation,
-WO-A-00/05338, cracking of blended magnetic and mechanical lysis,
-WO-A-99/53304, electric cracking and
-WO-A-99/15621, mechanical lysis.
Those skilled in the art can utilize other known cleavage methods, such as heat-shocked or osmotic shock or utilize chaotropic agent such as guanidinesalt to handle (patent US-A-5,234,809).
But this step is condensed nucleic acid usually.For example, can utilize solid support,, be attached to the nucleic acid of these magnetic-particles thus by the washing step purifying such as magnetic-particle (referring to patent US-A-4,672,040 and US-A-5,750,338).This nucleic acid purification step of described nucleic acid that increases subsequently if desired is especially favourable.The embodiment of these especially favourable magnetic-particles is described in patent application WO-A-97/45202 and WO-A-99/35500.
Term " solid support " comprises that all can adhere to the material of nucleic acid.Synthetic materials or natural materials, it is optional through chemically modified, can be used as solid support, polysaccharide especially, such as based on cellulosic material, for example paper/derivatived cellulose such as cellulose acetate and nitrocellulose or dextran; Polymkeric substance, multipolymer, the especially monomer of styrene-based type, natural cellulose is such as cotton, and synthon are such as nylon; Mineral substance is such as silica gel, quartz, glass, pottery; Rubber; Magnetic-particle; Metal derivative, gel etc.Solid support can be the form of microtiter plate, the form of film, particle form or flat substantially glass or the form of silicon plate, or derivatives thereof.
Can in a test tube, carry out whole process (getting access to the amplicon that can be used for hybridizing from sample), artificial or automatic machine operation.
Appended example is represented specific embodiment and is not understood to and limits the scope of the invention.
Can carry out many group contrasts in these embodiments.It at first is the negative control that utilizes water; In this case, do not detect the signal of H5 or N1.Secondly, utilize the specificity contrast of influenza H3N2 RNA; Wherein do not detect the signal of H5 or N1.
Embodiment 1: the test of assessment H5N1 primer on from the H5N1 RNA of Aisa people's clinical sample
Right sequence of oligonucleotide (N1_P1 and N2_P2 are used for amplification and detect the N1 sequence, and H5_P1 and H5_P2 are used for amplification and detect the H5 sequence) and the detection probes (molecular probe N1 and molecular probe H5) that exists with the molecular probe form are as follows:
| N1_P2 | 5’-GGAATGCTCCTGTTATCCTGA-3’ |
| N1_P1 | 5’-AATTCTAATACGACTCACTATAGGGGCGTGGATTGTCTCCGAAA-3’ |
| N1 probe | 5’-CGATCGGCGAAATCACATGTGTGTGCAGGGACGATCG-3’ |
| H5_P2 | 5’-GCCGAATGATGCCATCAA-3’ |
| H5_P1 | 5’-AATTCTAATACGACTCACTATAGGGGTGTATGTTGTGGAATGGCA-3’ |
| H5 probe | 5’-CGATCGACACCAAGTGTCAAACTCCAATCGATCG-3’ |
The corresponding T7 promoter sequence of sequence shown in the boldface letter by t7 rna polymerase identification, carries out the NASBA technology in the concurrent present P1 oligonucleotide.
Described sample utilizes the miniMAG system handles, described in operation scheme.Used kit is
-NucliSens lysate, bioM é rieux B.V. (Boxtel, Holland), lot number 200295 and
-NucliSens magnetic extraction agent, bioM é rieux B.V., lot number #200297.
Detect the operation scheme of test
According to the basic test kit of NucliSens EasyQ (bioM é rieux B.V., Boxtel, Holland, Batch No.#285006) explanation, prepare two kinds of reaction mixtures, a kind of amplification and detection H5 sequence (" mixture " H5) of being used for, another kind is used for amplification and detects N1 sequence (" mixture N1 ").11 μ l water in brief, the KCl of 13 μ l 1.2M, the oligonucleotide (H5_P1 and H5_P2 or N1_P1 and N1_P2, stoste is 10 μ M) and the suitable detection probes (molecular probe H5 or molecular probe N1, stoste is 2 μ M) of 0.8 μ l that are respectively 4 μ l add 64 μ l diluents.The every kind of mixture that is respectively 10 μ l adds 5 μ lRNA targets.Simultaneously, the preparation enzyme solution, the reaction mixture with in the 5 μ l enzyme solution adding test tube makes that final volume is 20 μ l.Thereby sample allows down by isothermal NASBA technology (Kievits, T et al.J.Virol.Methods (1991) vol.35 (3), p.273-286) amplification and testing goal sequence with being placed on the reaction conditions that the basic test kit of NucliSens EasyQ recommends.
In order to confirm this detection test, used RNA target is as follows:
-H5N1: influenza virus A hypotype H5N1 Vietnam 1194/2004
-influenza virus A: hypotype H3N2,
-H5 contrasts RNA: hypotype H5N3,
-influenza virus B.
Suitably (positive and negative) contrast comprises in test.
In NucliSens EasyQ system, after amplification and the detection, obtain following result:
| Virus | NASBA H5 result | NASBA N1 result |
| Negative control | Negative | Negative |
| Influenza virus A H3N2 | Negative | Negative |
| Negative | Negative | Negative |
| Flu A H5N1 Vietnam 1194/2004 | Positive | Positive |
| Negative control | Negative | Negative |
| Flu A H5N1 Vietnam 1194/2004 | Positive | Positive |
| Flu A H5N3 Duck contrasts virus | Positive | Negative |
These results show that H5N1 detection probes and oligonucleotide are specific and have detected their target H5 and N1 separately really.
Embodiment 2: the detection probes of assessment H5N1 and the test of primer in multiple detection
In multiple detection, used molecular probe N1 CY5 mark, and molecular probe H5 FAM mark.
In order to confirm described sequence functional in multiple detection, used target is synthetic transcript, and it makes up from reorganization H5N1RNA.It is RNA, only comprises H5 and N1 zone.It is as assessing with reference to RNA at the amplimer of H5 and N1 and the efficacy levels of detection probes (because it can obtain in a large number for synthetic transcript, thereby needn't use very expensive reorganization H5N1 RNA).
According to the explanation of the basic test kit of NucliSens EasyQ (bioM é rieux B.V., Boxtel, Holland, Batch No.#285006), preparation detects the single reaction mixture of H5 gene and N1 gene simultaneously.In brief, following material is joined in the 64 μ l thinners: 11 μ l water, the KCl of 13 μ l 1.2M, being respectively the solution of oligonucleotide of 8 μ l and the solution of molecular probe (contains 5 μ M oligonucleotide H5_P1 and H5_P2 and is used for H5,20 μ M oligonucleotide N1_P1 and N1_P2, be used for N1,2 μ M molecular probe H5-FAM and 2 μ M molecular probe N1-CY5).10 μ l mixtures add 5 μ l RNA targets (the H5N1 transcript, 1000 the copy/NASBA).
Concentration difference between H5 primer and the N1 primer approximately and sensitivity differences be inversely proportional to.The concentration of H5 primer is hanged down 4 times than the concentration of N1 primer thus.
Simultaneously, the preparation enzyme solution, the reaction mixture with in the 5 μ l enzyme solution adding test tube makes that final volume is 20 μ l.Thereby sample allows down by isothermal NASBA technology (Kievits with the reaction conditions that is placed on the basic test kit recommendation of the NucliSens EasyQ, T et al.J.Viro1.Methods (1991) vol.35 (3), p.273-286) amplification and testing goal sequence.
The results are shown in Figure 1.These results have shown that in vitro to detect H5 and N1 effect for synthetic transcript simultaneously very good single.
Sequence table
＜110〉Biomerieux SA
＜120〉be used to diagnose the oligonucleotide of the existence of the H5 of influenza A virus and N1 gene, its purposes, detection method and test kit
<130>H5N1 PI
<140>FR06/00843
<141>2006-01-30
<150>FR05/11974
<151>2005-11-25
<160>10
<170>PatentIn version 3.3
<210>1
<211>19
<212>DNA
<213>Influenza A virus
<400>1
tgtatgttgt ggaatggca 19
<210>2
<211>18
<212>DNA
<213>Inf1uenza A virus
<400>2
gccgaatgat gccatcaa 18
<210>3
<211>18
<212>DNA
<213>Influenza A virus
<400>3
cgtggattgt ctccgaaa 18
<210>4
<211>21
<212>DNA
<213>Influenza A virus
<400>4
ggaatgctcc tgttatcctg a 21
<210>5
<211>45
<212>DNA
<213>Influenza A virus
<400>5
aattctaata cgactcacta taggggtgta tgttgtggaa tggca 45
<210>6
<211>44
<212>DNA
<213>Influenza A virus
<400>6
aattctaata cgactcacta taggggcgtg gattgtctcc gaaa 44
<210>7
<211>22
<212>DNA
<213>Influenza A virus
<400>7
acaccaagtg tcaaactcca at 22
<210>8
<211>25
<212>DNA
<213>Influenza A virus
<400>8
gcgaaatcac atgtgtgtgc aggga 25
<210>9
<211>34
<212>DNA
<213>Influenza A virus
<400>9
cgatcgacac caagtgtcaa actccaatcg atcg 34
<210>10
<211>37
<212>DNA
<213>Influenza A virus
<400>10
cgatcggcga aatcacatgt gtgtgcaggg acgatcg 37
Claims (21)
1. two pairs of oligonucleotide, it is respectively applied for two target sequences that amplification is arranged in genomic H5 gene of influenza A virus and N1 gene, and the oligonucleotide of the H5 gene that wherein is used to increase is to being made up of following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.1:TGTATGTTGTGGAATGGCA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.2:GCCGAATGATGCCATCAA or its complementary sequence,
Wherein be used to increase the oligonucleotide of N1 gene to forming by following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.3:CGTGGATTGTCTCCGAAA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.4:GGAATGCTCCTGTTATCCTGA or its complementary sequence.
2. oligonucleotide is right, is used for increasing being positioned at the target sequence of the genomic H5 gene of influenza A virus, and described oligonucleotide is to being made up of following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.1:TGTATGTTGTGGAATGGCA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.2:GCCGAATGATGCCATCAA or its complementary sequence.
3. oligonucleotide is right, is used for increasing being positioned at the target sequence of the genomic N1 gene of influenza A virus, and described oligonucleotide is to being made up of following oligonucleotide:
-the first oligonucleotide, its length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, described 10 continuous nucleotides be derived from SEQ ID No.3:CGTGGATTGTCTCCGAAA or its complementary sequence and
-the second oligonucleotide, length are 10-50 Nucleotide, comprise the fragment of at least one 10 continuous nucleotide, and described 10 continuous nucleotides are derived from SEQ ID No.4:GGAATGCTCCTGTTATCCTGA or its complementary sequence.
4. the oligonucleotide of one of claim 1-3 is right, and it is characterized in that described first oligonucleotide also comprises can be by the promoter sequence of DNA-RNA-dependent polysaccharase identification.
5. the oligonucleotide of claim 4 is right, it is characterized in that described can be the T7 promoter sequence by the promoter sequence of DNA-RNA-dependent polysaccharase identification.
6. claim 4 or one of 5 oligonucleotide are right, wherein, described first oligonucleotide can increase when being arranged in the target sequence of H5 gene when making, described first oligonucleotide is characterised in that it is made up of following sequence basically: SEQ ID No.5AATTCTAATACGACTCACTATAGGGGTGTATGTTGTGGAATGGCA, or its complementary sequence.
7. the oligonucleotide of one of claim 4-6 is right, wherein, described first oligonucleotide can increase when being arranged in the target sequence of N1 gene when making, described first oligonucleotide is characterised in that it is made up of following sequence basically: SEQ ID No.6AATTCTAATACGACTCACTATAGGGGCGTGGATTGTCTCCGAAA, or its complementary sequence.
8. the oligonucleotide of one of claim 1-7 is right, the length that it is characterized in that each oligonucleotide is 12-30 oligonucleotide, and comprise the fragment of at least one 16 continuous nucleotide, preferably each oligonucleotide length is 15-26 Nucleotide and the fragment that comprises at least one 18 Nucleotide.
9. oligonucleotide is right, and it is used for detecting two target sequences that lay respectively at genomic H5 of influenza A virus and N1 gene as probe, and the probe that wherein is used to detect the H5 gene is made up of following sequence:
SEQ ID No.7:ACACCAAGTGTCAAACTCCAAT or its complementary sequence;
The probe that is used to detect the N1 gene is made up of following sequence:
SEQ ID No.8:GCGAAATCACATGTGTGTGCAGGGA or its complementary sequence,
Each sequence comprises at least a marking tool.
10. oligonucleotide, it is as probe, be used for detecting the nucleotide sequence that is positioned at the amplification that target sequence produced that detects the genomic H5 gene of influenza A virus by amplification, the oligonucleotide of described amplification by utilizing one of claim 2 and 4-8 is to carrying out, and the length of described detection probes is 10-50 Nucleotide and comprises the fragment that at least one is derived from 10 continuous nucleotides of following sequence:
SEQ ID No.7:ACACCAAGTGTCAAACTCCAAT, or its complementary sequence, described sequence comprises at least a marking tool.
11. oligonucleotide, it is as probe, be used for detecting the nucleotide sequence that is positioned at the amplification that target sequence produced of the genomic N1 gene of influenza A virus by amplification, the oligonucleotide of described amplification by utilizing one of claim 3-8 is to carrying out, and the length of described detection probes is 10-50 Nucleotide and comprises the fragment that at least one is derived from 10 continuous nucleotides of following sequence:
SEQ ID No.8:GCGAAATCACATGTGTGTGCAGGGA, or its complementary sequence, described sequence comprises at least a marking tool.
12. the detection probes of claim 9-11 definition is characterized in that it is made up of molecular probe.
13. the detection probes of one of claim 9-12 definition, it is characterized in that it is made up of molecular probe, preferably form: SEQ ID 9:[6-FAM by following sequence]-cgatcgaCACCAAGTGTCAAACTCCAAtcgatcg-[DabSyl], be used to detect the H5 gene, SEQ ID 10:[6-FAM]-cgatcgGCGAAATCACATGTGTGTGCAGGGAcgatcg-[DabSyl], be used to detect the N1 gene.
14. the purposes of one or two pair of oligonucleotide of one of claim 1-8, it is used for the reaction of amplification of nucleic acid or is used for detecting the suspicious genome that is present in the influenza A virus of biological sample as probe.
15. detect the method for the nucleic acid that may be present in the influenza A virus in the sample, wherein described sample is utilized the reaction of the oligonucleotide of one of claim 1-8 to amplification of nucleic acid, described being reflected under the condition that has the required amplification agent of described amplification carried out, and the existence of testing goal amplicon.
16. the method for claim 15 is characterized in that used amplified reaction is RT-PCR.
17. the method for claim 15 is characterized in that used amplified reaction is the transcription amplification technology.
18. the method for claim 17 is characterized in that used amplified reaction is the NASBA technology.
19. amplification may be present in the method for the H5 and N1 two genes of the influenza A virus in the sample, may further comprise the steps:
-in amplification buffer, be incubated sample under the condition of following material existing:
Two kinds of amplimers, the length of each primer is 10-50 Nucleotide, and a kind of promoter sequence that also comprises is another kind of opposite with the polarity of the primer that is combined with promoter sequence, thereby respectively with the upstream and downstream hybridization in the purpose zone of the H5 gene that is arranged in influenza A virus
Two kinds of amplimers, the length of each primer is 10-50 Nucleotide, and a kind of promoter sequence that also comprises is another kind of opposite with the polarity of the primer that is combined with promoter sequence, thereby respectively with the upstream and downstream hybridization in the purpose zone of the N1 gene that is arranged in influenza A virus
-following reagent is added sample:
Have the active enzyme of RNA-dependent dna-polymerases,
Have the active enzyme of DNA-dependent dna-polymerases,
Have the active enzyme of RNase H,
Enzyme with DNA-RNA-dependent polymerase activity, and
-under suitable condition, keep for some time that consequent reaction mixture is enough to feasible amplification generation.
May be present in the H5 of the influenza A virus in the sample and the test kit of N1 gene 20. detect, comprise
The described two pairs of oligonucleotide of one of-claim 1-8,
The described two kinds of oligonucleotide of one of-claim 9-13, it is maybe can being labeled of mark, and has basically at least a portion complementary nucleotide sequence with the nucleotide sequence of amplification,
-carry out the required reagent of amplified reaction.
21. the test kit of claim 20, wherein carrying out the required reagent of amplified reaction is NASBA amplification reagent.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0511974A FR2902429A1 (en) | 2005-11-25 | 2005-11-25 | Double pair of oligonucleotides to amplify two target sequences localized respectively in H5 gene and N1 genes of the genome influenza A virus, i.e. each the pair of oligonucleotides contains first and second oligonucleotides |
| FR0511974 | 2005-11-25 | ||
| FR0600843 | 2006-01-30 | ||
| FR0600843A FR2902430B1 (en) | 2005-11-25 | 2006-01-30 | OLIGONUCLEOTIDES, USE, DETECTION METHOD AND KIT FOR DIAGNOSING THE PRESENCE OF INFLUENZA VIRUS H5 AND N1 GENES |
| PCT/FR2006/051218 WO2007060366A1 (en) | 2005-11-25 | 2006-11-23 | Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of h5 and n1 genes of the influenza a virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101313079A true CN101313079A (en) | 2008-11-26 |
| CN101313079B CN101313079B (en) | 2014-02-26 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200680043972.2A Active CN101313079B (en) | 2005-11-25 | 2006-11-23 | Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of H5 and N1 genes of the influenza A virus |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101313079B (en) |
| FR (1) | FR2902429A1 (en) |
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2005
- 2005-11-25 FR FR0511974A patent/FR2902429A1/en active Pending
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- 2006-11-23 CN CN200680043972.2A patent/CN101313079B/en active Active
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| Publication number | Publication date |
|---|---|
| FR2902429A1 (en) | 2007-12-21 |
| CN101313079B (en) | 2014-02-26 |
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