CN1635163A - A method for detecting H5N1 subtype avian influenza virus and its special kit - Google Patents
A method for detecting H5N1 subtype avian influenza virus and its special kit Download PDFInfo
- Publication number
- CN1635163A CN1635163A CN 200410088716 CN200410088716A CN1635163A CN 1635163 A CN1635163 A CN 1635163A CN 200410088716 CN200410088716 CN 200410088716 CN 200410088716 A CN200410088716 A CN 200410088716A CN 1635163 A CN1635163 A CN 1635163A
- Authority
- CN
- China
- Prior art keywords
- influenza virus
- avian influenza
- sequence
- test sample
- subtype avian
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 45
- 241001473385 H5N1 subtype Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 18
- 239000002773 nucleotide Substances 0.000 claims abstract description 18
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 8
- 102000005348 Neuraminidase Human genes 0.000 claims abstract description 6
- 108010006232 Neuraminidase Proteins 0.000 claims abstract description 6
- 101710154606 Hemagglutinin Proteins 0.000 claims abstract description 3
- 101710093908 Outer capsid protein VP4 Proteins 0.000 claims abstract description 3
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 claims abstract description 3
- 101710176177 Protein A56 Proteins 0.000 claims abstract description 3
- 239000000185 hemagglutinin Substances 0.000 claims abstract description 3
- 238000012360 testing method Methods 0.000 claims description 37
- 238000006243 chemical reaction Methods 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000003757 reverse transcription PCR Methods 0.000 claims description 17
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 9
- 239000002532 enzyme inhibitor Substances 0.000 claims description 9
- 108091092562 ribozyme Proteins 0.000 claims description 9
- 239000013641 positive control Substances 0.000 claims description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 4
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 4
- 230000004087 circulation Effects 0.000 claims description 3
- 230000001351 cycling effect Effects 0.000 claims description 2
- 208000002979 Influenza in Birds Diseases 0.000 abstract description 3
- 206010064097 avian influenza Diseases 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 241000712431 Influenza A virus Species 0.000 abstract 1
- 238000003771 laboratory diagnosis Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 17
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 238000002156 mixing Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 102000006382 Ribonucleases Human genes 0.000 description 9
- 108010083644 Ribonucleases Proteins 0.000 description 9
- 239000006166 lysate Substances 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 8
- 235000011187 glycerol Nutrition 0.000 description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- SOIFLUNRINLCBN-UHFFFAOYSA-N ammonium thiocyanate Chemical compound [NH4+].[S-]C#N SOIFLUNRINLCBN-UHFFFAOYSA-N 0.000 description 6
- 230000003321 amplification Effects 0.000 description 6
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 6
- 229960005542 ethidium bromide Drugs 0.000 description 6
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 6
- 238000003199 nucleic acid amplification method Methods 0.000 description 6
- 239000007974 sodium acetate buffer Substances 0.000 description 6
- 238000013019 agitation Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000012160 loading buffer Substances 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 229920000742 Cotton Polymers 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 108010029566 avian influenza A virus hemagglutinin Proteins 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 210000003555 cloaca Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention disclosed a detection method for H5N1 subtype avian influenza virus and special purpose reagent case thereof. The reagent case comprises avian influenza A virus general primer NPF and NPL, specificity primer H5F and H5L of H5 subtype avian influenza virus hemagglutinin and specificity primer N1F and N1L of N1 subtype avian influenza virus neuraminidase; said NPF processes nucleotide sequence of sequence1 in the sequence table, said NPL processes nucleotide sequence of sequence2 in the sequence table, said H5F processes nucleotide sequence of sequence3 in the sequence table, said H5L processes nucleotide sequence of sequence4 in the sequence table, said N1F processes nucleotide sequence of sequence5 in the sequence table, said N1F processes nucleotide sequence of sequence6 in the sequence table. The invention can be used not only for laboratory diagnosis and scientific research, but also for large-scale epidemiology survey.
Description
Technical field
The present invention relates to the method and the dedicated kit thereof of a kind of H5N1 of detection subtype avian influenza virus in the field of virus detection.
Background technology
High pathogenic avian influenza is by the caused bird deadly infectious disease of Highly Pathogenic Avian Influenza Virus (HPAIV), and morbidity is anxious, propagates soon, and the lethality rate height can cause fatefulue strike to aquaculture.The hypotype of Highly Pathogenic Avian Influenza Virus (HPAIV) is generally H5 and H7 hypotype; 2004, betide China and Asia other countries and geographic influenza virus sub-strain and be mainly the H5N1 hypotype, because this hypotype has not only caused serious economy loss to the aquaculture in Asia, and also caused people's infection and death in Thailand, Vietnam, thereby has important public hygienics meaning again.In the controlling unit of stream fowl sense, the most important thing is and will diagnose fast, in time delimit epidemic place, epidemic-stricken area, and take measure of control such as blockade, prevent the diffusion of epidemic situation.Therefore, set up a kind of sensitivity, special, diagnostic method has important practical significance fast.
Traditional influenza virus detection method is carried out the separation and Culture of virus for adopting the chicken embryo, adopts hemagglutination-inhibition test and neuraminidase inhibition test to carry out the evaluation of hypotype then, during test fee, effort, can not make diagnosis fast and timely.Therefore, clinically, press for a kind of detection method of energy quick diagnosis H5N1 subtype avian influenza virus, can determine not only whether virus is A type influenza virus, and can determine whether to be H5 hemagglutinin hypotype and N1 neuraminidase hypotype.
The innovation and creation content
The dedicated kit that the purpose of this invention is to provide a kind of energy rapid detection H5N1 subtype avian influenza virus.
The test kit of detection H5N1 subtype avian influenza virus provided by the present invention, comprise A type avian influenza virus universal primer NPF and NPL, the Auele Specific Primer H5F of H5 subtype avian influenza virus hemagglutinin and H5L and N1 subtype avian influenza virus neuraminidase Auele Specific Primer N1F and N1L; Described NPF has the nucleotide sequence of sequence 1 in the sequence table, described NPL has the nucleotide sequence of sequence 2 in the sequence table, described H5F has the nucleotide sequence of sequence 3 in the sequence table, described H5L has the nucleotide sequence of sequence 4 in the sequence table, described N1F has the nucleotide sequence of sequence 5 in the sequence table, and described N1L has the nucleotide sequence of sequence 6 in the sequence table.
Sequence 1 in the sequence table is by 20 based compositions, sequence 2 in the sequence table is by 20 based compositions, sequence 3 in the sequence table is by 19 based compositions, sequence 4 in the sequence table is by 19 based compositions, sequence 5 in the sequence table is by 20 based compositions, and the sequence 6 in the sequence table is by 21 based compositions; Wherein, m=a or c, y=t or c, r=a or g, k=g or t, w=a or t.
The pcr amplification product length of described A type avian influenza virus universal primer NPF and NPL is 467bp; The pcr amplification product length of described H5 hemagglutinin Auele Specific Primer H5F and H5L is 543bp; The pcr amplification product length of described N1 subtype avian influenza virus neuraminidase Auele Specific Primer N1F and N1L is 368bp.
Described primer NPF, NPL, H5F, H5L, N1F and N1L are contained in the multiple RT-PCR reaction solution, and the described multiple RT-PCR reaction solution of per 43.8 μ l contains RNase free water, 17.3 μ l; 10 * PCR Buffer, 7 μ l; 2.5mM each dNTPs, 7.5 μ l; 25mM MgCl
2, 5 μ l; Primer is totally 7 μ l (primer concentration is 20 μ M for each 1 μ l of NPF, NPL, H5F and H5L, each 1.5 μ l of N1F and N1L).
Detect for the ease of the scene, described test kit also comprises the RNA enzyme inhibitors, Taq archaeal dna polymerase, AMV ThermoScript II, extract the required reagent of the total RNA of sample (as lysate, chloroform, Virahol, ethanol, RNase free water) and electrophoresis detection reagent (as ethidium bromide, the TAE electrophoretic buffer, sample-loading buffer, Standard PC R product).
Described Standard PC R product is for extracting RNA from the H5N1 subtype influenza identified virus, is template with cDNA after the reverse transcription, is primer amplification and the 545bp pcr amplification product that reclaims with H5F and H5L; Be primer amplification and the 467bp pcr amplification product that reclaims and be the mix products of primer amplification and the 368bp pcr amplification product that reclaims with NPF and NPL with N1F and N1L.
Described RNA enzyme inhibitors specification is 30U/ μ l.
Because false negative or false positive that manual operation caused, this test kit also comprises positive control solution (H5N1 hypotype inactivation of viruses liquid) and negative controls (RNase free water) in order to avoid in experiment.
Second purpose of the present invention provides a kind of method of rapid detection H5N1 subtype avian influenza virus.
The method of detection H5N1 subtype avian influenza virus provided by the present invention, be that total RNA with test sample is a template, utilize the dedicated kit of above-mentioned detection H5N1 subtype avian influenza virus to carry out the multiple RT-PCR reaction, test sample is if occur the amplified band of 467bp, 543bp and 368bp simultaneously, and test sample is the H5N1 subtype avian influenza virus positive; Test sample is if occur the amplified band of 467bp and 543bp simultaneously, and test sample is the H5 subtype avian influenza virus positive; Test sample is if occur the amplified band of 467bp and 368bp simultaneously, and test sample is the N1 subtype avian influenza virus positive; Test sample is if the amplified band of 467bp only occurs, and test sample is the A type avian influenza virus positive; Test sample is not if amplified band occurs, and test sample is an A type avian influenza virus feminine gender.
50 μ l reaction systems of described multiple RT-PCR reaction comprise that 43.8 μ l multiple RT-PCR reaction solutions (contain RNase free water, 17.3 μ l; 10 * PCR Buffer, 7 μ l; 2.5mM each dNTPs, 7.5 μ l; 25mMMgCl
2, 5 μ l; Primer is totally 7 μ l (primer concentration is 20 μ M for each 1 μ l of NPF, NPL, H5F and H5L, each 1.5 μ l of N1F and N1L); 0.8 μ l RNA enzyme inhibitors (specification is 30 U/ μ l, and Genview company produces), 0.8 μ l Taq archaeal dna polymerase, 0.6 μ l AMV ThermoScript II, the total RNA of 4.0 μ l sample to be checked.
The cycling program of described multiple RT-PCR reaction is 42 ℃ of 45min; 95 ℃ of 3min; 95 ℃ of 30s, 55 ℃ of 40s, 65 ℃ of 2min carry out 35 circulations; 65 ℃ are extended 10min.
The present invention has utilized multiplex RT-PCR method dexterously, has prepared the test kit of single stage method rapid detection H5N1 subtype avian influenza virus.This test kit can detect the infection of H5N1 subtype avian influenza virus special, delicately.And its preparation is easily, and is simple to operate, the susceptibility height, and reaction is (can finish detection and evaluation to the H5N1 subtype avian influenza virus) fast in 5 hours.Not only can be used for breadboard diagnosis and scientific research, also can be used for large-scale epidemiology survey, have a good application prospect.
Embodiment
Embodiment 1, sample collecting and processing
1, sample collecting
Tissue sample: die of illness or put out disease fowl, get tissues such as liver, brain, lung; Cotton swab: nasal cavity swab, cloaca swab.2-8 ℃ of preservation send the laboratory to detect.Require the censorship pathological material of disease fresh.
2, sample preparation: every duplicate samples is handled respectively
(1) tissue sample is handled: take by weighing pathological material of disease 100mg to be checked and put in the mill, add 0.8ml lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing) grind, ground pathological material of disease is moved in the 1.5ml centrifuge tube, 4 ℃ of centrifugal 5min of 8000rpm get supernatant 250 μ l, place 1.5ml sterilization centrifuge tube, add the above-mentioned lysate of 750 μ l, the thermal agitation mixing, room temperature is placed 5min.
(2) whole blood sample is handled: after treating blood clotting, get serum in 1.5ml sterilization centrifuge tube, 4 ℃ of centrifugal 5min of 8000rpm get supernatant 250 μ l, place 1.5ml sterilization centrifuge tube, add 750 μ l lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
(3) cotton swab is handled: the cotton swab in the immersion liquid is fully twisted, discard swab after wringing out, 4 ℃ of centrifugal 5min of 8000rpm get supernatant liquor 250 μ l, place 1.5ml sterilization centrifuge tube, add 750 μ l lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
3, positive control is handled: get positive control solution (H5N1 hypotype inactivation of viruses liquid) 250 μ l, place 1.5ml sterilization centrifuge tube, add 750 μ l lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
4, negative control (RNase free water): get sterilization distilled water 250 μ l, place 1.5ml sterilization centrifuge tube, add 750 μ l lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
Embodiment 2, detection H5N1 subtype avian influenza virus
1,10 part test kits of dedicated kit that detect the H5N1 subtype avian influenza virus are made up of the reagent shown in the table 1.
The composition of table 1. test kit (10 parts)
| Title | Quantity (10 parts) |
| Positive control solution | ????3.0ml |
| Lysate | ????30.0ml |
| Chloroform | ????8.0ml |
| Virahol | ????18.0ml |
| 75% ethanol | ????35.0ml |
| RNase free water | ????6.0μl |
| The RNA enzyme inhibitors | ????60.0μl |
| The multiple RT-PCR reaction solution | ????1.5ml |
| The TaqDNA polysaccharase | ????26.0μl |
| The AMV ThermoScript II | ????20.0μl |
| Ethidium bromide solution | ????30.0μl |
| 50 * TAE electrophoretic buffer (50 times of dilutions are used) | ????20.0ml |
| Sample-loading buffer | ????200μl |
| Standard PC R product | ????35.0μl |
Wherein, the composition of part reagent, compound method are as follows:
Lysate: guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing; RNase free water: 1mlDEPC is added in the 1L distilled water, shakes up autoclaving.
Per 43.8 μ l multiple RT-PCR reaction solutions contain RNase free water, 17.3 μ l; 10 * PCR Buffer, 7 μ l; 2.5mM each dNTPs, 7.5 μ l; 25mM MgCl
2, 5 μ l; Primer is totally 7 μ l (primer concentration is 20 μ M for each 1 μ l of NPF, NPL, H5F and H5L, each 1.5 μ l of N1F and N1L).
10mg/ml ethidium bromide (EB) stock solution: 100mg EB is dissolved in the 10ml distilled water, vigorous stirring, and after the dissolving, black out is preserved under the room temperature fully.
50 * TAE electrophoretic buffer (50 times of dilutions are used): Tris alkali, 121g; Glacial acetic acid, 28.55ml; 0.5M EDTA (pH8.0), 50ml.Add distilled water, mix, be settled to 500ml, working fluid concentration is 1 * TAE.
0.5M EDTA (pH8.0): 186.1g EDTA is dissolved in the 800ml water, and vigorous stirring on magnetic stirring apparatus is regulated pH value to 8.0 with 10M NaOH, is settled to 1L.
75% ethanol: 75ml ethanol adds 25ml water ,-20 ℃ of preservations.
6 * gel loading buffer: tetrabromophenol sulfonphthalein, 0.025g; The blue or green FF of dimethylbenzene, 0.025g; Glycerine, 3ml.Add a certain amount of deionized water dissolving, constant volume is to 10ml.
The RNA enzyme inhibitors: specification is 30U/ μ l, and Genview company produces.
Standard PC R product is for extracting RNA from the H5N1 subtype influenza virus of having identified, be template with cDNA after the reverse transcription, with H5F and H5L is primer amplification and the 545bp pcr amplification product that reclaims, is primer amplification and the 467bp pcr amplification product that reclaims and is the mix products of primer amplification and the 368bp pcr amplification product that reclaims with N1F and N1L with NPF and NPL.
2, detection method
(1) extraction of viral RNA
1) get doubtful be avian influenza fowl muscle or internal organs 100mg, carry out sample preparation according to the method for embodiment 1, every pipe adds 200 μ l chloroforms, acutely shakes up ice bath 10min, 4 ℃ of centrifugal 15min of 12000rpm.
2) get supernatant 500 μ l in new 1.5ml sterilization centrifuge tube, add isopyknic Virahol, put 3min in the liquid nitrogen, 4 ℃ of centrifugal 15min of 12000rpm.
3) abandon supernatant, slowly add 1ml 75% ethanol along tube wall, 4 ℃ of centrifugal 3min of 8000rpm outwell ethanol, with centrifuge tube back-off 1min on thieving paper, vacuum-drying 10min.
4) with 50 μ l RNase free water and 1 μ l RNA enzyme inhibitors (specification is 30U/ μ l, and Genview company produces) dissolution precipitation, promptly usefulness or-80 ℃ of preservations are standby.
(2) multiple RT-PCR reaction
Every tube reaction system is 50 μ l, and reaction system is as follows:
Multiple RT-PCR reaction solution 43.8 μ l
RNA enzyme inhibitors (available from Genview company) 0.8 μ l
Taq archaeal dna polymerase 0.8 μ l
AMV ThermoScript II 0.6 μ l
The total RNA 4.0 μ l of sample
Mixing, and perform mark, carrying out 42 ℃ of 45min on the PCR instrument automatically; 95 ℃ of 3min; 95 ℃ of 30s, 55 ℃ of 40s, 65 ℃ of 2min carry out 35 circulations; 65 ℃ are extended 10min.4 ℃ of preservations.
(3) electrophoresis
Prepare 1% sepharose, add EB, working concentration is 0.5 μ g/ml; 10 μ l PCR products, 3 μ l Standard PC R products respectively mix 3 μ l, 6 * gel loading buffer, go up sample respectively, and electrophoresis in 1 * TAE electrophoretic buffer is used the Ultraviolet Detector observations.When the result judged, 543bp, 467bp and 368bp amplified band should appear in positive control simultaneously, and negative control does not have band and (except the primer band) occur.The amplified band that occurs 543bp, 467bp and 368bp in the test sample simultaneously shows and contains the H5N1 subtype avian influenza virus in the sample.
Sequence table
<160>6
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(6)
＜223〉m=a or c
<400>1
gcacamagag?caatgatgga??????????????????????????????????????????????20
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<490>2
ataacaatag?agagaatggt??????????????????????????????????????????????20
<210>3
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(8)
＜223〉y=t or c
<220>
<221>misc-feature
<222>(11)
＜223〉r=a or g
<400>3
cacatgcyca?rgacatact???????????????????????????????????????????????19
<210>4
<211>19
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(2)
＜223〉y=t or c
<220>
<221>misc-feature
<222>(5)
＜223〉r=a or g
<400>4
tytgrttyag?tgttgatgt???????????????????????????????????????????????19
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(5)
＜223〉k=g or t
<220>
<221>misc-feature
<222>(11)
＜223〉r=a or g
<400>5
ctgtkgctgt?rttgaaatac??????????????????????????????????????????????20
<210>6
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(4)
＜223〉w=a or t
<220>
<221>misc-feature
<222>(13)
＜223〉y=t or c
<400>6
gtcwccgaaa?acyccactgc?a????????????????????????????????????????????21
Claims (7)
1, a kind of dedicated kit that detects the H5N1 subtype avian influenza virus, comprise A type avian influenza virus universal primer NPF and NPL, the Auele Specific Primer H5F of H5 subtype avian influenza virus hemagglutinin and H5L and N1 subtype avian influenza virus neuraminidase Auele Specific Primer N1F and N1L; Described NPF has the nucleotide sequence of sequence 1 in the sequence table, described NPL has the nucleotide sequence of sequence 2 in the sequence table, described H5F has the nucleotide sequence of sequence 3 in the sequence table, described H5L has the nucleotide sequence of sequence 4 in the sequence table, described N1F has the nucleotide sequence of sequence 5 in the sequence table, and described N1L has the nucleotide sequence of sequence 6 in the sequence table.
2, test kit according to claim 1 is characterized in that: described primer NPF, and NPL, H5F, H5L, N1F and N1L are contained in the multiple RT-PCR reaction solution, and the described multiple RT-PCR reaction solution of per 43.8 μ l contains RNasefree water, 17.3 μ l; 10 * PCR Buffer, 7 μ l; 2.5Mm dNTPs, 7.5 μ l; 25mM MgCl
2, 5 μ l; Concentration is each 1 μ l of NPF, NPL, H5F and H5L of 20 μ M, and concentration is N1F and each 1.5 μ l of N1L of 20 μ M.
3, test kit according to claim 1 and 2 is characterized in that: described test kit also comprises the RNA enzyme inhibitors, the TaqDNA polysaccharase, and the AMV ThermoScript II is extracted required reagent of the total RNA of sample and electrophoresis detection reagent.
4, test kit according to claim 3 is characterized in that: described test kit also comprises positive control solution and negative controls.
5, a kind of method that detects the H5N1 subtype avian influenza virus, be that total RNA with test sample is a template, utilize the dedicated kit of each described detection H5N1 subtype avian influenza virus in the claim 1 to 4 to carry out the multiple RT-PCR reaction, test sample is if occur the amplified band of 545bp, 467bp and 368bp simultaneously, and test sample is the H5N1 subtype avian influenza virus positive; Test sample is if occur the amplified band of 467bp and 545bp simultaneously, and test sample is the H5 subtype avian influenza virus positive; Test sample is if occur the amplified band of 467bp and 368bp simultaneously, and test sample is the N1 subtype avian influenza virus positive; Test sample is if the amplified band of 467bp only occurs, and test sample is the A type avian influenza virus positive; Test sample is not if amplified band occurs, and test sample is an A type avian influenza virus feminine gender.
6, method according to claim 5, it is characterized in that: 50 μ l reaction systems of described multiple RT-PCR reaction comprise 43.8 μ l multiple RT-PCR reaction solutions, 0.8 μ l RNA enzyme inhibitors, 0.8 μ l Taq archaeal dna polymerase, 0.6 μ l AMV ThermoScript II, the total RNA of 4.0 μ l sample to be checked.
7, according to claim 5 or 6 described methods, it is characterized in that: the cycling program of described multiple RT-PCR reaction is 42 ℃ of 45min; 95 ℃ of 3min; 95 ℃ of 30s, 55 ℃ of 40s, 65 ℃ of 2min carry out 35 circulations; 65 ℃ are extended 10min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410088716 CN1274851C (en) | 2004-11-01 | 2004-11-01 | Process for detecting H5N1 hypotype Avian influenza virus and special-purpose reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410088716 CN1274851C (en) | 2004-11-01 | 2004-11-01 | Process for detecting H5N1 hypotype Avian influenza virus and special-purpose reagent kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1635163A true CN1635163A (en) | 2005-07-06 |
| CN1274851C CN1274851C (en) | 2006-09-13 |
Family
ID=34847515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410088716 Expired - Fee Related CN1274851C (en) | 2004-11-01 | 2004-11-01 | Process for detecting H5N1 hypotype Avian influenza virus and special-purpose reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1274851C (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1313605C (en) * | 2005-07-08 | 2007-05-02 | 扬州大学 | Gene recombinant fowl influenza virus D3/F-R2/6 and its construction method |
| WO2007060366A1 (en) * | 2005-11-25 | 2007-05-31 | bioMérieux | Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of h5 and n1 genes of the influenza a virus |
| CN101070347B (en) * | 2006-05-12 | 2010-06-30 | 青岛宝麦德生物医药科技有限公司 | Bird-flu H5N1 novel mucous membrane immunization vaccine and its use |
| CN1904068B (en) * | 2006-05-10 | 2010-12-15 | 浙江省疾病预防控制中心 | H5N1 type poultry grippal virus fluorescent augmentation detection kit and detection method |
| CN101323882B (en) * | 2007-06-14 | 2011-03-23 | 上海市农业科学院 | Primer set for detecting pig influenza virus and blood serum subtype and reagent box thereof |
| CN102140453A (en) * | 2011-01-25 | 2011-08-03 | 中国医学科学院医学实验动物研究所 | Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method |
| CN104131009A (en) * | 2014-07-28 | 2014-11-05 | 中国农业大学 | Primer composition for detecting different influenza viruses and application thereof |
| CN106676103A (en) * | 2017-03-01 | 2017-05-17 | 中国农业大学 | Primer group for detecting North American H3N1 subtype swine influenza virus and application thereof |
| JP2019144268A (en) * | 2006-05-10 | 2019-08-29 | セラノス アイピー カンパニー エルエルシー | Influenza virus real-time detection |
-
2004
- 2004-11-01 CN CN 200410088716 patent/CN1274851C/en not_active Expired - Fee Related
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1313605C (en) * | 2005-07-08 | 2007-05-02 | 扬州大学 | Gene recombinant fowl influenza virus D3/F-R2/6 and its construction method |
| WO2007060366A1 (en) * | 2005-11-25 | 2007-05-31 | bioMérieux | Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of h5 and n1 genes of the influenza a virus |
| FR2902430A1 (en) * | 2005-11-25 | 2007-12-21 | Biomerieux Sa | OLIGONUCLEOTIDES, USE, DETECTION METHOD AND KIT FOR DIAGNOSING THE PRESENCE OF INFLUENZA VIRUS H5 AND N1 GENES |
| US8168387B2 (en) | 2005-11-25 | 2012-05-01 | Biomerieux | Oligonucleotides, use thereof, detecting method and kit for diagnosing the presence of H5 and N1 genes of the Influenza A virus |
| JP2019144268A (en) * | 2006-05-10 | 2019-08-29 | セラノス アイピー カンパニー エルエルシー | Influenza virus real-time detection |
| CN1904068B (en) * | 2006-05-10 | 2010-12-15 | 浙江省疾病预防控制中心 | H5N1 type poultry grippal virus fluorescent augmentation detection kit and detection method |
| JP7050847B2 (en) | 2006-05-10 | 2022-04-08 | ラブラドール ダイアグノスティクス エルエルシー | Real-time detection of influenza virus |
| JP2020124202A (en) * | 2006-05-10 | 2020-08-20 | セラノス アイピー カンパニー エルエルシー | Influenza virus real-time detection |
| CN101070347B (en) * | 2006-05-12 | 2010-06-30 | 青岛宝麦德生物医药科技有限公司 | Bird-flu H5N1 novel mucous membrane immunization vaccine and its use |
| CN101323882B (en) * | 2007-06-14 | 2011-03-23 | 上海市农业科学院 | Primer set for detecting pig influenza virus and blood serum subtype and reagent box thereof |
| CN102140453A (en) * | 2011-01-25 | 2011-08-03 | 中国医学科学院医学实验动物研究所 | Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method |
| CN102140453B (en) * | 2011-01-25 | 2013-03-20 | 中国医学科学院医学实验动物研究所 | Primers used for amplifying nucleotide segment of nucleocapsid protein gene of H5N1 influenza virus and detection method |
| CN104131009A (en) * | 2014-07-28 | 2014-11-05 | 中国农业大学 | Primer composition for detecting different influenza viruses and application thereof |
| CN106676103A (en) * | 2017-03-01 | 2017-05-17 | 中国农业大学 | Primer group for detecting North American H3N1 subtype swine influenza virus and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1274851C (en) | 2006-09-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1476485A (en) | Kit for detecting non-pathogenic or pathogenic influenza A subtype H5 virus | |
| CN1274851C (en) | Process for detecting H5N1 hypotype Avian influenza virus and special-purpose reagent kit | |
| CN1814805A (en) | H5, H7, H9 subtype auian flu virus real-time fluo rescent quantixative PCR detecting method | |
| CN1164767C (en) | Fluorescent quantitative PCR kit and application for rapid quantitative detection of classical swine fever virus and classical swine fever rabbitized attenuated vaccine | |
| CN1289692C (en) | Magnetic capture of SARS coronary virus and PCR detection therewith | |
| CN108085413B (en) | High-throughput kit for simultaneously detecting three avian viral diseases and detection method and application thereof | |
| CN1900314A (en) | Fluorescence labelling ABO gene typing method and its reagent kit | |
| CN1690220A (en) | Method and kit for testing chain reaction of reverse transcription polymerase of avian influenza virus | |
| CN102329891B (en) | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) primer group for detecting H9 subtype avian influenza virus as well as detection method and application thereof | |
| CN1904068A (en) | H5N1 type poultry grippal virus fluorescent augmentation detection kit and detection method | |
| CN101045939A (en) | HBV DNA gene subtype detecting method and kit | |
| CN104805217A (en) | MRT-PCR (multiple reverse transcription-polymerase chain reaction) kit for discriminating and detecting important avian economic animal viruses and application of mRT-PCR kit | |
| CN1294277C (en) | Methods for identifying animal hide and skin | |
| CN1710101A (en) | Fluorescent quantitative PCR kit for detecting porcine pseudorabies virus and its application | |
| CN102304591A (en) | PCR (polymerase chain reaction) primer pair for identifying H3 subtype avian influenza virus and application thereof | |
| CN102559484B (en) | Prawn infectivity muscle necrosis virus fluorescent quantificationally PCR detecting kit and detection method | |
| CN1693479A (en) | Kit with fluorescent quantitative RT-PCR detection technique used for cell keratin 19(Ck19)mRNA | |
| CN106435030B (en) | Four-channel fluorescent quantitative PCR detection method and kit for combined detection of avian influenza H7N9 and H9N2 | |
| CN1858255A (en) | Two step method multiple RT-PCR solidified detection reagent kit for potato virus disease and its detecting method | |
| CN1233843C (en) | Kit of testing garlic virus and testing method | |
| CN1282750C (en) | FQ-PCR diagnosis kit of WSSV and test method | |
| CN1712547A (en) | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA | |
| CN1752214A (en) | A molecular marker method for predicting and identifying chicken body fat traits | |
| CN102586489B (en) | Detection of infection of avian anemia viruses in pathologic material by combination of polymerase chain reaction (PCR) and nucleic acid probe and dot blot hybridization technology | |
| CN1618982A (en) | Fluorescene quantitative PCR reagant box for detecting SARS-Cov virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060913 Termination date: 20091201 |