CN101724713A - Method for detecting different subtype avian influenza viruses and special kit thereof - Google Patents
Method for detecting different subtype avian influenza viruses and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting different subtype avian influenza viruses and a special kit thereof. The method comprises the following steps of:, carrying out multiple PCR using a primer pair B by a primer pair A by utilizing the cDNA of a biological sample to be tested as a template, carrying out agarose gel electrophoresis on products of the multiple PCR, and respectively carrying out gel scanning and analysis by a fluorescence imaging analyzer with the wavelength of 532 nm and 635 nm; if a fluorescence strip exists under the wavelength of 635nm, judging that the biological sample to be tested contains H3 subtype avian influenza virus; and if a fluorescence strip exists under the wavelength of 532nm, judging that the biological sample to be tested contains H6 subtype avian influenza virus. The primer pair A is a primer pair consisting of nucleotides represented by a sequence 11 and a sequence 12 in a sequence list, and the 5' end of the nucleotides represented by the sequence 12 is marked by Tamra. The primer pair B is a primer pair consisting of nucleotides represented by a sequence 3 and a sequence 4 in the sequence list, and the 5' end of the nucleotides represented by the sequence 4 is marked by Cy5. In the invention, the method has the advantages of high flux, high distinguishability, strong maneuverability, wide application range, time saving, test consumable saving, safety and the like.
Description
Technical field
The present invention relates to a kind of method and dedicated kit thereof that detects different subtype avian influenza viruses.
Background technology
PCR (Polymerase Chain Reaction, polymerase chain reaction) technology is the Protocols in Molecular Biologies that grow up of the eighties place of a kind of 20th century, is used for the specific dna fragmentation of enrichment.Its utilization isolated archaeal dna polymerase (DNA Polymerase) from the hot spring bacterium, by three-step reactions such as sex change, renaturation (annealing) and extensions, dna replication dna in the analogue body.The nucleic acid of denier can be increased rapidly at short notice and duplicate, obtain a large amount of target DNA fragments.
Need after pcr amplification is finished to be tested and appraised, just can determine whether really to have obtained accurately and reliably the expection amplified production, agarose gel electrophoresis is that the laboratory is the most frequently used, and is simple and easy to do.Nucleic acid molecule is electronegative, to positive polar motion, the nucleic acid belt electric weight difference of differing molecular size is under the molecular sieve effect of gel media under electric field, make the different nucleic acid molecule of molecular size different mobilities occur, detect its big or small purpose thereby reach separating acid fragment.Intercalative dye in the nucleic acid molecule is analyzed in the gel analysis system.
The concentration of agarose gel electrophoresis usually between 0.5-2%, the electrophoresis that is used for carrying out big fragment nucleic acid of lower concentration, the electrophoresis that is used for separating small segment of high density.The nucleic acid size that can differentiate such as 1% commonly used agarose gel electrophoresis of laboratory is 0.5kb-7kb, and 2% glue can be differentiated the nucleic acid of 0.1kb-3kb.This shows that its resolving power is not high, just can't distinguish that these several the close bands of molecular size just may only show a band in electrophoresis when nucleic acid fragment size close when identical (or).
Summary of the invention
The purpose of this invention is to provide a kind of method and dedicated kit thereof that detects different subtype avian influenza viruses.
Two Colour Fluorescence multiplex PCR (the DFM-PCR that whether contains dna fragmentation first and/or dna fragmentation second in the detection provided by the invention biological specimen to be measured, Dual Fluorescence Multiplex-PCR) method, comprise the steps: that total DNA with biological specimen to be measured as template, carries out multiplex PCR with primer to first and primer pair B; The product of multiplex PCR is carried out agarose gel electrophoresis, under 532nm and 635nm, carry out the gel scanning analysis respectively with the fluorescence imaging analyser; If have the fluorescence band under the 635nm, contain dna fragmentation second in the biological specimen to be measured, if having the fluorescence band under the 532nm, contain the dna fragmentation first in the biological specimen to be measured; Primer is to 5 ' end Tamra mark of first, and 5 ' of primer pair B is held and used the Cy5 mark; Described primer is used for the amplification of DNA fragments first to first; Described primer pair B is used for amplification of DNA fragments second.
Described agarose gel electrophoresis specifically can be 0.5-2% (quality percentage composition) agarose gel electrophoresis, as 0.5-1% (quality percentage composition) agarose gel electrophoresis, 1-1.5% (quality percentage composition) agarose gel electrophoresis, 1.5-2% (quality percentage composition) agarose gel electrophoresis etc.When described agarose gel electrophoresis was 1% (quality percentage composition) agarose gel electrophoresis, described dna fragmentation first and described dna fragmentation second differed below the 7000bp, specifically can differ below the 500bp.When described agarose gel electrophoresis was 2% (quality percentage composition) agarose gel electrophoresis, described dna fragmentation first and described dna fragmentation second differed below the 3000bp, specifically can differ below the 100bp.The sensitivity of method of the present invention be higher than far away conventional DNA detection used agarose gel electrophoresis-SYBGreenI staining.
Described Two Colour Fluorescence multiple PCR method can be applicable to detect whether contain H3 subtype avian influenza virus and/or H6 subtype avian influenza virus in the biological specimen to be measured.
The present invention also protects a kind of method that whether contains H3 subtype avian influenza virus and/or H6 subtype avian influenza virus in the biological specimen to be measured that detects, comprise the steps: that the cDNA with biological specimen to be measured is a template, with primer A and primer are carried out multiplex PCR to B, the product of multiplex PCR is carried out agarose gel electrophoresis, under 532nm and 635nm, carry out the gel scanning analysis respectively with the fluorescence imaging analyser; If have the fluorescence band under the 635nm, biological specimen to be measured contains the H3 subtype avian influenza virus; If have the fluorescence band under the 532nm, biological specimen to be measured contains the H6 subtype avian influenza virus; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to A, and 5 ' of Nucleotide end shown in the sequence 12 is with Tamra (TAMRA) mark; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to B, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4.Described agarose gel electrophoresis specifically can be 0.5-2% (quality percentage composition) agarose gel electrophoresis, as 0.5-1% (quality percentage composition) agarose gel electrophoresis, 1-1.5% (quality percentage composition) agarose gel electrophoresis, 1.5-2% (quality percentage composition) agarose gel electrophoresis etc., specifically can be 2% agarose gel electrophoresis.
The reaction conditions of described multiplex PCR specifically can be: 95 ℃, 5min; 94 ℃, 30s, 52 ℃, 60s, 72 ℃, 90s, 35 circulations; 72 ℃, 10min.In the reaction system of described multiplex PCR, when reacting initial, the upstream primer that each described primer is right and the concentration of downstream primer specifically all can be 0.1mmol/L.
The present invention protects a kind of test kit that whether contains H3 subtype avian influenza virus and/or H6 subtype avian influenza virus in the biological specimen to be measured that detects simultaneously, comprise primer to A and primer to B; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to A, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to B, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4.
The present invention also protects a kind of method that detects any subtype avian influenza virus in containing in the biological specimen to be measured, comprise the steps: that total DNA with biological specimen to be measured is as template, with primer I, primer are carried out multiplex PCR to VI and primer to VII to V, primer to IV, primer to III, primer to II, primer, the product of multiplex PCR is carried out agarose gel electrophoresis, under 532nm and 635nm, carry out the gel scanning analysis respectively with the fluorescence imaging analyser; If have the fluorescence band of 641bp under the 635nm, biological specimen to be measured contains the H1 subtype avian influenza virus; If have the fluorescence band of 708bp under the 635nm, biological specimen to be measured contains the H3 subtype avian influenza virus; If have the fluorescence band of 419bp under the 635nm, biological specimen to be measured contains the H5 subtype avian influenza virus; If have the fluorescence band of 367bp under the 532nm, biological specimen to be measured contains the N1 subtype avian influenza virus; If have the fluorescence band of 338bp under the 532nm, biological specimen to be measured contains the N2 subtype avian influenza virus; If have the fluorescence band of 724bp under the 532nm, biological specimen to be measured contains the H6 subtype avian influenza virus; If have the fluorescence band of 532bp under the 532nm, biological specimen to be measured contains the H9 subtype avian influenza virus; Described primer is that the primer that Nucleotide is formed shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table is right to I, 5 ' of Nucleotide end Cy5 mark shown in the sequence 2; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to II, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4; Described primer is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right to III, 5 ' of Nucleotide end Cy5 mark shown in the sequence 6; Described primer is that the primer that Nucleotide is formed shown in the sequence 8 of Nucleotide shown in the sequence 7 of sequence table and sequence table is right to IV, 5 ' of Nucleotide end Tamra mark shown in the sequence 8; Described primer is that the primer that Nucleotide is formed shown in the sequence 10 of Nucleotide shown in the sequence 9 of sequence table and sequence table is right to V, 5 ' of Nucleotide end Tamra mark shown in the sequence 10; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to VI, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 14 of Nucleotide shown in the sequence 13 of sequence table and sequence table is right to VII, 5 ' of Nucleotide end Tamra mark shown in the sequence 14.Described agarose gel electrophoresis specifically can be 0.5-2% (quality percentage composition) agarose gel electrophoresis, as 0.5-1% (quality percentage composition) agarose gel electrophoresis, 1-1.5% (quality percentage composition) agarose gel electrophoresis, 1.5-2% (quality percentage composition) agarose gel electrophoresis etc., specifically can be 2% agarose gel electrophoresis.
The reaction conditions of described multiplex PCR specifically can be: 95 ℃, 5min; 94 ℃, 30s, 52 ℃, 60s, 72 ℃, 90s, 35 circulations; 72 ℃, 10min.In the reaction system of described multiplex PCR, when reacting initial, the upstream primer that each described primer is right and the concentration of downstream primer specifically all can be 0.1mmol/L.
The present invention protects a kind of test kit that contains following which kind of subtype avian influenza virus in the biological specimen to be measured that detects simultaneously: H1 subtype avian influenza virus, H3 subtype avian influenza virus, H5 subtype avian influenza virus, N1 subtype avian influenza virus, N2 subtype avian influenza virus, H6 subtype avian influenza virus and H9 subtype avian influenza virus; Described test kit comprise primer to I, primer to II, primer to III, primer to IV, primer to V, primer to VI and primer to VII; Described primer is that the primer that Nucleotide is formed shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table is right to I, 5 ' of Nucleotide end Cy5 mark shown in the sequence 2; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to II, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4; Described primer is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right to III, 5 ' of Nucleotide end Cy5 mark shown in the sequence 6; Described primer is that the primer that Nucleotide is formed shown in the sequence 8 of Nucleotide shown in the sequence 7 of sequence table and sequence table is right to IV, 5 ' of Nucleotide end Tamra mark shown in the sequence 8; Described primer is that the primer that Nucleotide is formed shown in the sequence 10 of Nucleotide shown in the sequence 9 of sequence table and sequence table is right to V, 5 ' of Nucleotide end Tamra mark shown in the sequence 10; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to VI, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 14 of Nucleotide shown in the sequence 13 of sequence table and sequence table is right to VII, 5 ' of Nucleotide end Tamra mark shown in the sequence 14.
The DFM-PCR technology not only can be distinguished the identical band of molecular size in the agarose gel electrophoresis, and need not nucleic acid dye in the testing process, therefore also has the advantage of Environmental Safety.Easy, safe and high-throughout technological method are provided for detecting pathogen.
Method of the present invention is based on fluorochrome label technology and multiple PCR technique.Multiplex PCR (Mutiplex PCR) is exactly to add manyly to Auele Specific Primer in same pipe, with a plurality of template reactions in the PCR pipe, thereby detects a plurality of target dna molecules simultaneously in a PCR pipe.Fluorescent substance is under the specific wavelength shooting conditions, and the electron absorption energy jump is to high energy excited state.The electronics instability of excited state, ground state is returned in transition, and energy changes into corresponding wavelength, sends fluorescence.By add different fluorescent mark (being referred to as " probe primer " here) to primer, can obtain the PCR product of different fluorescence, because the PCR fragment of molecular size identical (or close) is with being with different fluorescently-labeled probe primer amplification gained, so as long as just can distinguish the PCR product that detects these molecular sizes identical (or close) by detecting different fluorescence.Thereby make this have characteristics easily and efficiently.
Nucleic acid detection method provided by the invention has the following advantages:
1) high flux property: use suitable different fluorescent probe primers, under different excitation wavelengths, the band that the molecular size of can not differentiating on the agarose gel electrophoresis is close will be launched the fluorescence of different wave length, can do further difference to the close band of clip size on the agarose according to the color of band and judge.Therefore, DMF-PCR can analyze regular-PCR result several times to come out easy to operate saving time in primary first-order equation.
2) high resolution: when utilization DFM-PCR analyzed, owing to the high sensitivity of analytical instrument at fluorescent probe, thereby fluorescent probe primer multiplex PCR can reach higher resolving power.Therefore, under the little situation of amplification amount, general gel imaging system is differentiated limited, and after with DFM-PCR, it is sensitiveer that detected result becomes, for experiment provides better analysis foundation.
3) workable: on the regular-PCR instrument, can realize segmental amplification to be measured, fluorescent probe primer synthesis condition maturation, amplification adopts agarose gel electrophoresis and fluorescent scanning instrument to analyze.
4) applied widely: as to can be applicable to fields such as quarantine, scientific research, especially high-throughput and high sensitivity are had the field of requirement.
5) save time: can carry out multiplex PCR simultaneously, reduce the consuming time of substance regular-PCR, save time.
6) save test consumable:,, save experimental expenses so can save consumptive materials such as corresponding test reagent, articles for use owing to concentrate a tube reaction.
7) safety: owing to need not nucleic acid dye in the operation detection process, feasible Environmental Safety more.
Description of drawings
Fig. 1 is SYBGreenI dyeing detected result.
Fig. 2 is a FLA-5100 fluorescence imaging analyser 532nm scintigram.
Fig. 3 is a FLA-5100 fluorescence imaging analyser 635nm scintigram.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
10 * PCR buffer (the precious biotech firm in Dalian); DNTP (the precious biotech firm in Dalian); Taq enzyme (the precious biotech firm in Dalian); DL2000DNA Marker (the precious biotech firm in Dalian); QIAamp Viral RNA MiniKit (QIAGEN company); QIANGEN One Step test kit (QIAGEN company); DNA reclaims test kit (U.S. Omega company); T-carrier (the precious biotech firm in Dalian).
The fluorescent probe primer is synthetic by Shanghai Invitrogen company, and sequence sees Table 1:
Table 17 kind of A type influenza virus positive plasmid makes up the primer
Table 2A type influenza virus 7 heavy PCR probe primer and amplified fragments sizes
Fujifilm FLA-5100 fluorescence/chemoluminescence and isotropic substance image scan analyser; PCR instrument (MJResearch); Electrophoresis apparatus (Bio-Rad).
The structure of embodiment 1, avian influenza virus positive plasmid
One, the structure of H1 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from H1 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 2) to carrying out RT-PCR then with the primer that H1f ' in the table 1 and H1r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 15 (seeing the sequence 15 of sequence table) of sequence table in the T-carrier.
Two, the structure of H3 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from H3 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 2) to carrying out RT-PCR then with the primer that H3f ' in the table 1 and H3r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 16 (seeing the sequence 16 of sequence table) of sequence table in the T-carrier.
Three, the structure of H5 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from H5 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 2) to carrying out RT-PCR then with the primer that H5f ' in the table 1 and H5r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 17 (seeing the sequence 17 of sequence table) of sequence table in the T-carrier.
Four, the structure of H6 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from H6 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 2) to carrying out RT-PCR then with the primer that H6f ' in the table 1 and H6r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 18 (seeing the sequence 18 of sequence table) of sequence table in the T-carrier.
Five, the structure of H9 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from H9 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 2) to carrying out RT-PCR then with the primer that H9f ' in the table 1 and H9r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 19 (seeing the sequence 19 of sequence table) of sequence table in the T-carrier.
Six, the structure of N1 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from N1 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 2) to carrying out RT-PCR then with the primer that N1f ' in the table 1 and N1r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 20 (seeing the sequence 20 of sequence table) of sequence table in the T-carrier.
Seven, the structure of N2 positive plasmid
With the method that QIAamp Viral RNA MiniKit provides to specifications, from N2 subtype avian influenza virus standard strain, extract the avian influenza virus geneome RNA.Use QIANGEN One Step test kit operation instructions to react (reaction conditions sees Table 3) to carrying out RT-PCR then with the primer that N2f ' in the table 1 and N2r ' form.Reclaim test kit with DNA and reclaim the PCR product, be connected in the T-carrier.The carrier that reclaims after the RT-PCR product connects is checked order, and sequencing result shows, inserted the DNA shown in the sequence 21 (seeing the sequence 21 of sequence table) of sequence table in the T-carrier.
RT-PCR reaction conditions in table 3 step 1 to seven
Embodiment 2, application Two Colour Fluorescence multiplex PCR detect the avian influenza virus plasmid of different subtype
One, multiplex PCR
The positive-virus plasmid of seven kinds of hypotypes is mixed as template; In the mixture, the concentration of H1 positive plasmid is 1 * 10
6The concentration of copy, H3 positive plasmid is 1.2 * 10
5The concentration of copy, H5 positive plasmid is 8 * 10
6The concentration of copy, H6 positive plasmid is 8 * 10
5The concentration of copy, H9 positive plasmid is 1 * 10
7The concentration of copy, N1 positive plasmid is 1.2 * 10
6The concentration of copy, N2 positive plasmid is 6 * 10
6Copy subtype virus plasmid.
Adopt 7 couples of primers (H1f, H1r of table 2; H3f, H3r; H5f, H5r; H6f, H6r; H9f, H9r; N1f, N1r; N2f, N2r) carry out multiplex PCR.The multi-PRC reaction system sees Table 4, and loop parameter sees Table 5.
Adopt 7 couples of primers (H1f, H1r of table 2 respectively; H3f, H3r; H5f, H5r; H6F, H6R; H9f, H9r; N1f, N1r; N2f, N2r) carry out substance PCR.Substance PCR reaction system sees Table 6, and loop parameter sees Table 7.
Table 4 multi-PRC reaction system
| ??10×PCR?Buffer | ??5uL |
| ??dNTP?Mix(2.5mM?each) | ??4uL |
| ??H1f(10mM) | ??0.5uL |
| ??H1r(10mM) | ??0.5uL |
| ??H3f(10mM) | ??0.5uL |
| ??H3r(10mM) | ??0.5uL |
| ??H5f(10mM) | ??0.5uL |
| ??H5r(10mM) | ??0.5uL |
| ??H6f(10mM) | ??0.5uL |
| ??10×PCR?Buffer | ??5uL |
| ??H6r(10mM) | ??0.5uL |
| ??H9f(10mM) | ??0.5uL |
| ??H9r(10mM) | ??0.5uL |
| ??N1f(10mM) | ??0.5uL |
| ??N1r(10mM) | ??0.5uL |
| ??N2f(10mM) | ??0.5uL |
| ??N2r(10mM) | ??0.5uL |
| Dna profiling | ??3.5uL |
| The Taq enzyme | ??0.5uL |
| ??ddH 2O | ??30uL |
| Amount to | ??50uL |
Table 5 multiplex PCR loop parameter
Table 6 substance PCR reaction system
| ??10×PCR?Buffer | ??5uL |
| ??dNTP?Mix(2.5mM?each) | ??4uL |
| Upstream primer (f) | ??0.5uL |
| Downstream primer (r) | ??0.5uL |
| Dna profiling | ??0.5uL |
| ??10×PCR?Buffer | ??5uL |
| The Taq enzyme | ??0.5uL |
| ??ddH 2O | ??39uL |
| Amount to | ??50uL |
Table 7 substance PCR loop parameter
Get 5 μ L multiple PCR products and carry out 2% agarose electrophoresis (5V/cm) (free nucleic acid dyestuff), carry out scanning analysis with FLA-5100 fluorescence imaging analyser after electrophoresis is finished.7 every pipes of substance PCR product are got 5 μ L, carry out 2% agarose electrophoresis (5V/cm) (free nucleic acid dyestuff), carry out scanning analysis with FLA-5100 fluorescence imaging analyser after electrophoresis is finished.Get 5 μ L multiple PCR products and carry out 2% agarose electrophoresis (5V/cm) (nucleic acid dye is arranged), carry out SYBGreenI dyeing after electrophoresis is finished and detect.7 every pipes of substance PCR product are got 5 μ L, carry out 2% agarose electrophoresis (5V/cm) (nucleic acid dye is arranged), carry out SYBGreenI dyeing after electrophoresis is finished and detect.
Three, substance PCR and multiplex PCR amplification are relatively
SYBGreenI dyeing detected result is seen Fig. 1.FLA-5100 fluorescence imaging analyser 532nm scintigram is seen Fig. 2.FLA-5100 fluorescence imaging analyser 635nm scintigram is seen Fig. 3.
Show 6 electrophoretic bands in the SYBGreenI colored graph of multiple PCR products.By with the comparative analysis of substance PCR band, can judge because H3, H6 hypotype PCR product band molecular weight are close (being respectively 708bp and 724bp), in the multiple PCR products, H3 hypotype and H6 hypotype PCR product band overlap, thereby can't be distinguished by regular-PCR and agarose gel electrophoresis.
Under 532nm, multiple PCR products detects 4 bands (Fig. 2).With the contrast of substance PCR product, detected as can be known four bands are respectively H6, H9, N1 and four kinds of hypotypes of N2 of 5 ' Tamra mark.
Under 635nm, multiple PCR products detects three bands (Fig. 3).With the contrast of substance PCR product, detected as can be known three bands are respectively H1, H3 and the H5 hypotype of CY5 mark.
Sequence table
＜110〉China Inst. of Quarantine Inspection Sciences
＜120〉a kind of method and dedicated kit thereof that detects different subtype avian influenza viruses
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gtcctatgca?aacaacaaag?agaaagaagt?ccttgtacta?tggggtgttc?atcacccgcc??240
taacataggg?gaccaaaggg?ccctctatca?tacagaaaat?gcttatgtct?ctgtagtgtc??300
ttcacattat?agcagaagat?tcaccccaga?aatagccaaa?agacccaagg?tgagagatca??360
ggaaggaaga?atcaactact?actggactct?gctggaaccc?ggggatacaa?taatatttga??420
ggcaaatgga?aatctaatag?cgccaaggta?tgctttcgca?ctgagtagag?gctttggatc??480
aggaatcatc?acctcaaatg?caccaatgga?tgaatgtgat?gcgaagtgtc?aaacacctca??540
gggagctata?aacagcagtc?ttcctttcca?gaatgtacac?ccagtcacaa?taggagagtg??600
tc?????????????????????????????????????????????????????????????????602
<210>16
<211>669
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>16
tgttaccctt?atgatgtgcc?agattatgcc?tcccttaggt?cactagttgc?ctcatcaggc????60
accctggagt?ttatcaatga?agactttaat?tggactggag?tcgctcagaa?tggggacagc????120
tatgcttgca?aaaggggatc?tgttaaaagt?ttctttagta?gattgaattg?gttgcacaaa????180
tcagaataca?aatatccagc?gctgaacgtg?actatgccaa?acaatgacaa?atttgacaag????240
ttgtacattt?ggggggttca?ccacccgagc?acggacagag?aacaaaccag?cctatatatt????300
cgagcatcag?ggaaagtcac?agtctctacc?aaaagaagcc?aacaaactgt?aatcccgaat????360
atcgggtcca?gaccctgggt?aaggggtctg?tccagtagaa?taagcatcta?ttggacaata????420
gtaaaaccgg?gagacatact?tttgattaat?agcaccggga?atctaattgc?tcctcggggt????480
tacttcaaaa?tacggactgg?gaaaagctcg?ataatgaggt?cagatgcacc?cattggcacc????540
tgcagttctg?aatgcatcac?tccaaatgga?agcatcccca?atgacaaacc?ttttcaaaat????600
gtaaacagga?tcacatatgg?ggcatgtccc?agatatgtta?agcaaaacac?tctgaaattg????660
gcaacaggg????????????????????????????????????????????????????????????669
<210>17
<211>380
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>17
agtgaattgg?aatatggtaa?ctgcaacacc?aagtgtcaaa?ctccaatggg?ggcgataaac????60
tctagtatgc?cattccacaa?catacaccct?ctcaccatcg?gggaatgccc?caaatatgtg????120
aaatcaaaca?gattagtcct?tgcgactggg?ctcagaaata?gccctcaaag?agagagaaga????180
agaaaaaaga?gaggactatt?tggagctata?gcaggtttta?tagagggagg?atggcaggga????240
atggtagatg?gttggtatgg?gtaccaccat?agcaatgagc?aggggagtgg?atacgctgca????300
gacaaagaat?ccactcaaaa?ggcaatagat?ggagtcacca?ataaggtcaa?ctcgatcatt????360
gacaaaatga?acactcagtt????????????????????????????????????????????????380
<210>18
<211>685
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>18
aaggcactta?ttggatcagg?ggaaagggta?gagaggtttg?agatgtttcc?caagagtaca?????60
tgggcaggag?tggacaccag?cagtggagta?acaagctctt?gttcctttgg?tagtggttca????120
tctttctaca?ggaatctcct?atggataata?aaacacaggt?catcaggata?tccagtaatt????180
aagggaactt?tcagaaacac?tggagacaag?tcagttctct?atttttgggg?tgtgcaccat????240
cctactgaca?caactgagca?aaacattctg?tatggttctg?gtaatcgata?cgttagaatg????300
ggaactgaaa?gcatgaattt?tgccaggagc?ccagaaattg?cggcaaggcc?tgctgtgaat????360
ggtcagagag?gcagaattga?ttatttctgg?tccgttttaa?aaccagggga?aaccttgaat????420
gtagaatcta?atgggaattt?agtagcccct?tggtatgcat?acagatttgt?cagcaaagat????480
agtaaaggag?ccgtgttcag?gtctaattta?ccaatcgaga?actgtgatgc?cacatgccag????540
actattgaag?gagtgataag?aaccaacaaa?acatttcaga?atgtgagccc?tctgtggata????600
ggagaatgcc?caaaatatgt?aaaaagtgag?agtctaaggc?ttgcaacagg?actaaggaat????660
gttccacaaa?ttgaaactag?aggac????685
<210>19
<211>493
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>19
aagagaatgg?tcctatatcg?tcgaaagacc?atcggctgtt?aatggattgt?gttaccccgg?????60
aaatgtagaa?aacctagaag?aactaaggtc?actttttagt?tctgctagtt?cttaccaaag????120
aatccagatc?tttccagaca?caatctggaa?tgtgtcttat?agtggaacaa?gcaaagcatg????180
ttcagattca?ttctacagga?gcatgagatg?gttgactcaa?aagaacaacg?cttaccctgt????240
tcaagacgcc?caatacacaa?ataatagagg?aaagaacatt?ctgttcatgt?ggggtataaa????300
tcacccaccc?actgatactg?cgcagacaaa?tctgtacaca?aggactgaca?caacaacaag????360
tgtggcaaca?gaagatataa?ataggacctt?caaacccttg?atagggccaa?ggcctcttgt????420
caatggtctg?caaggaagga?ttgattatta?ttgggcggta?ttgaaaccag?gtcagacatt????480
gcgagtaaga?tcc???????????????????????????????????????????????????????493
<210>20
<211>328
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>20
tcccacttgg?aatgcagaac?tttcttttta?actcagggag?ccttgctgaa?tgacaagcac?????60
tccaatggga?ctgttaaaga?cagaagccct?cacagaacat?taatgagttg?tccggtgggt????120
gaggctcctt?ccccatataa?ctcaaggttt?gagtctgttg?cttggtcagc?aagtgcttgc????180
catgatggca?ccagttggtt?gacaattgga?atttctggcc?cagacaatgg?ggctgtggct????240
gtattgaagt?acaatggcat?aataacagac?actatcaaga?gttggaggaa?caacatactg????300
agaactcaag?agtctgaatg?tgcatgtg???????????????????????????????????????328
<210>21
<211>299
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>21
atagcatggt?ccagctcaag?ttgtcacgat?ggaaaagcat?ggctgcatgt?ttgtgtaacg?????60
ggggatgatg?aaaatgcaac?tgctagcttc?atttacaatg?ggaggcttgt?agatagtatt????120
ggttcatggt?ccaaaaaaat?cctcaggacc?caggagtcgg?aatgcgtttg?tatcaatgga????180
acttgtacag?tagtaatgac?tgatgggagt?gcttcaggaa?aagctgatac?taaaatacta????240
ttcattgagg?aggggaaaat?cgttcatact?agctcattgt?caggaagtgc?tcagcatgt?????299
Claims (10)
1. one kind is detected the Two Colour Fluorescence multiple PCR method that whether contains dna fragmentation first and/or dna fragmentation second in the biological specimen to be measured, comprises the steps: that total DNA with biological specimen to be measured as template, carries out multiplex PCR with primer to first and primer pair B; The product of multiplex PCR is carried out agarose gel electrophoresis, under 532nm and 635nm, carry out the gel scanning analysis respectively with the fluorescence imaging analyser; If have the fluorescence band under the 635nm, contain dna fragmentation second in the biological specimen to be measured, if having the fluorescence band under the 532nm, contain the dna fragmentation first in the biological specimen to be measured; Primer is to 5 ' end Tamra mark of first, and 5 ' of primer pair B is held and used the Cy5 mark; Described primer is used for the amplification of DNA fragments first to first; Described primer pair B is used for amplification of DNA fragments second.
2. one kind is detected the method that whether contains H3 subtype avian influenza virus and/or H6 subtype avian influenza virus in the biological specimen to be measured, comprise the steps: that the cDNA with biological specimen to be measured is a template, with primer A and primer are carried out multiplex PCR to B, the product of multiplex PCR is carried out agarose gel electrophoresis, under 532nm and 635nm, carry out the gel scanning analysis respectively with the fluorescence imaging analyser; If have the fluorescence band under the 635nm, biological specimen to be measured contains the H3 subtype avian influenza virus; If have the fluorescence band under the 532nm, biological specimen to be measured contains the H6 subtype avian influenza virus; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to A, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to B, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4.
3. method as claimed in claim 2 is characterized in that: the reaction conditions of described multiplex PCR is: 95 ℃, 5min; 94 ℃, 30s, 52 ℃, 60s, 72 ℃, 90s, 35 circulations; 72 ℃, 10min.
4. as claim 2 or 3 described methods, it is characterized in that: in the reaction system of described multiplex PCR, when reacting initial, the upstream primer that each primer is right and the concentration of downstream primer are 0.1mmol/L.
5. one kind is detected the test kit that whether contains H3 subtype avian influenza virus and/or H6 subtype avian influenza virus in the biological specimen to be measured, comprise primer to A and primer to B; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to A, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to B, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4.
6. one kind is detected the method that contains following any subtype avian influenza virus in the biological specimen to be measured: H1 subtype avian influenza virus, H3 subtype avian influenza virus, H5 subtype avian influenza virus, N1 subtype avian influenza virus, N2 subtype avian influenza virus, H6 subtype avian influenza virus and H9 subtype avian influenza virus comprise the steps:
With the cDNA of biological specimen to be measured as template, with primer I, primer are carried out multiplex PCR to VI and primer to VII to V, primer to IV, primer to III, primer to II, primer, the product of multiplex PCR is carried out agarose gel electrophoresis, under 532nm and 635nm, carry out the gel scanning analysis respectively with the fluorescence imaging analyser; If have the fluorescence band of 641bp under the 635nm, biological specimen to be measured contains the H1 subtype avian influenza virus; If have the fluorescence band of 708bp under the 635nm, biological specimen to be measured contains the H3 subtype avian influenza virus; If have the fluorescence band of 419bp under the 635nm, biological specimen to be measured contains the H5 subtype avian influenza virus; If have the fluorescence band of 367bp under the 532nm, biological specimen to be measured contains the N1 subtype avian influenza virus; If have the fluorescence band of 338bp under the 532nm, biological specimen to be measured contains the N2 subtype avian influenza virus; If have the fluorescence band of 724bp under the 532nm, biological specimen to be measured contains the H6 subtype avian influenza virus; If have the fluorescence band of 532bp under the 532nm, biological specimen to be measured contains the H9 subtype avian influenza virus; Described primer is that the primer that Nucleotide is formed shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table is right to I, 5 ' of Nucleotide end Cy5 mark shown in the sequence 2; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to II, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4; Described primer is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right to III, 5 ' of Nucleotide end Cy5 mark shown in the sequence 6; Described primer is that the primer that Nucleotide is formed shown in the sequence 8 of Nucleotide shown in the sequence 7 of sequence table and sequence table is right to IV, 5 ' of Nucleotide end Tamra mark shown in the sequence 8; Described primer is that the primer that Nucleotide is formed shown in the sequence 10 of Nucleotide shown in the sequence 9 of sequence table and sequence table is right to V, 5 ' of Nucleotide end Tamra mark shown in the sequence 10; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to VI, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 14 of Nucleotide shown in the sequence 13 of sequence table and sequence table is right to VII, 5 ' of Nucleotide end Tamra mark shown in the sequence 14.
7. method as claimed in claim 6 is characterized in that: the reaction conditions of described multiplex PCR is: 95 ℃, 5min; 94 ℃, 30s, 52 ℃, 60s, 72 ℃, 90s, 35 circulations; 72 ℃, 10min.
8. as claim 6 or 7 described methods, it is characterized in that: in the reaction system of described multiplex PCR, when reacting initial, the upstream primer that each primer is right and the concentration of downstream primer are 0.1mmol/L.
9. one kind is detected the test kit that contains following which kind of subtype avian influenza virus in the biological specimen to be measured: H1 subtype avian influenza virus, H3 subtype avian influenza virus, H5 subtype avian influenza virus, N1 subtype avian influenza virus, N2 subtype avian influenza virus, H6 subtype avian influenza virus and H9 subtype avian influenza virus;
Described test kit comprise primer to I, primer to II, primer to III, primer to IV, primer to V, primer to VI and primer to VII; Described primer is that the primer that Nucleotide is formed shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table is right to I, 5 ' of Nucleotide end Cy5 mark shown in the sequence 2; Described primer is that the primer that Nucleotide is formed shown in the sequence 4 of Nucleotide shown in the sequence 3 of sequence table and sequence table is right to II, 5 ' of Nucleotide end Cy5 mark shown in the sequence 4; Described primer is that the primer that Nucleotide is formed shown in the sequence 6 of Nucleotide shown in the sequence 5 of sequence table and sequence table is right to III, 5 ' of Nucleotide end Cy5 mark shown in the sequence 6; Described primer is that the primer that Nucleotide is formed shown in the sequence 8 of Nucleotide shown in the sequence 7 of sequence table and sequence table is right to IV, 5 ' of Nucleotide end Tamra mark shown in the sequence 8; Described primer is that the primer that Nucleotide is formed shown in the sequence 10 of Nucleotide shown in the sequence 9 of sequence table and sequence table is right to V, 5 ' of Nucleotide end Tamra mark shown in the sequence 10; Described primer is that the primer that Nucleotide is formed shown in the sequence 12 of Nucleotide shown in the sequence 11 of sequence table and sequence table is right to VI, 5 ' of Nucleotide end Tamra mark shown in the sequence 12; Described primer is that the primer that Nucleotide is formed shown in the sequence 14 of Nucleotide shown in the sequence 13 of sequence table and sequence table is right to VII, 5 ' of Nucleotide end Tamra mark shown in the sequence 14.
10. whether the described method of claim 1 contains the application in H3 subtype avian influenza virus and/or the H6 subtype avian influenza virus in detecting biological specimen to be measured.
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| CN200910243330A CN101724713A (en) | 2009-12-21 | 2009-12-21 | Method for detecting different subtype avian influenza viruses and special kit thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910243330A CN101724713A (en) | 2009-12-21 | 2009-12-21 | Method for detecting different subtype avian influenza viruses and special kit thereof |
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|---|---|
| CN101724713A true CN101724713A (en) | 2010-06-09 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875979A (en) * | 2010-05-21 | 2010-11-03 | 扬州大学 | Multi-PCR (Polymerase Chain Reaction) detection method of H5 subtype avian influenza virus variant strain |
| CN102260749A (en) * | 2011-04-25 | 2011-11-30 | 吉林农业大学 | H5, H7 and H9 subtype avian influenza virus detection kit |
| CN102304591A (en) * | 2011-10-08 | 2012-01-04 | 中国农业大学 | PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof |
| CN102337352A (en) * | 2010-07-16 | 2012-02-01 | 中山大学达安基因股份有限公司 | Kit for detecting multiple influenza viruses by polymerase chain reaction (PCR) microarray |
| CN104313181A (en) * | 2014-10-09 | 2015-01-28 | 广州维伯鑫生物科技有限公司 | Bicolor fluorescent quantitative PCR (polymerase chain reaction) kit and detection method for jointly detecting H4 and H6 subtypes of avian influenza |
| CN106086234A (en) * | 2016-06-17 | 2016-11-09 | 广西壮族自治区兽医研究所 | Triple RT PCR kit of H9 hypotype AIV, H6 hypotype AIV and AIV and application thereof |
| CN107326103A (en) * | 2017-08-28 | 2017-11-07 | 聊城大学 | A kind of triple RT PCR specificity amplification primers groups and the RT PCR detection methods of triple identifications |
| WO2023077490A1 (en) * | 2021-11-06 | 2023-05-11 | 江汉大学 | Combination of mnp markers of influenza a, b and c viruses, primer pair combination, kit, and uses of combination, primer pair combination and kit |
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- 2009-12-21 CN CN200910243330A patent/CN101724713A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875979A (en) * | 2010-05-21 | 2010-11-03 | 扬州大学 | Multi-PCR (Polymerase Chain Reaction) detection method of H5 subtype avian influenza virus variant strain |
| CN101875979B (en) * | 2010-05-21 | 2012-09-12 | 扬州大学 | Multi-PCR (Polymerase Chain Reaction) detection method of H5 subtype avian influenza virus variant strain |
| CN102337352A (en) * | 2010-07-16 | 2012-02-01 | 中山大学达安基因股份有限公司 | Kit for detecting multiple influenza viruses by polymerase chain reaction (PCR) microarray |
| CN102337352B (en) * | 2010-07-16 | 2013-08-14 | 中山大学达安基因股份有限公司 | Kit for detecting multiple influenza viruses by polymerase chain reaction (PCR) microarray |
| CN102260749A (en) * | 2011-04-25 | 2011-11-30 | 吉林农业大学 | H5, H7 and H9 subtype avian influenza virus detection kit |
| CN102260749B (en) * | 2011-04-25 | 2012-12-19 | 吉林农业大学 | H5, H7 and H9 subtype avian influenza virus detection kit |
| CN102304591A (en) * | 2011-10-08 | 2012-01-04 | 中国农业大学 | PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof |
| CN102304591B (en) * | 2011-10-08 | 2013-06-05 | 中国农业大学 | PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof |
| CN104313181A (en) * | 2014-10-09 | 2015-01-28 | 广州维伯鑫生物科技有限公司 | Bicolor fluorescent quantitative PCR (polymerase chain reaction) kit and detection method for jointly detecting H4 and H6 subtypes of avian influenza |
| CN106086234A (en) * | 2016-06-17 | 2016-11-09 | 广西壮族自治区兽医研究所 | Triple RT PCR kit of H9 hypotype AIV, H6 hypotype AIV and AIV and application thereof |
| CN107326103A (en) * | 2017-08-28 | 2017-11-07 | 聊城大学 | A kind of triple RT PCR specificity amplification primers groups and the RT PCR detection methods of triple identifications |
| WO2023077490A1 (en) * | 2021-11-06 | 2023-05-11 | 江汉大学 | Combination of mnp markers of influenza a, b and c viruses, primer pair combination, kit, and uses of combination, primer pair combination and kit |
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Application publication date: 20100609 |