CN107858454A - H1N6 subtype avian influenza virus double RT PCR detection primers group, kit and its application - Google Patents

H1N6 subtype avian influenza virus double RT PCR detection primers group, kit and its application Download PDF

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CN107858454A
CN107858454A CN201711275853.6A CN201711275853A CN107858454A CN 107858454 A CN107858454 A CN 107858454A CN 201711275853 A CN201711275853 A CN 201711275853A CN 107858454 A CN107858454 A CN 107858454A
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influenza virus
avian influenza
subtype avian
primer
pcr
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熊文婕
曹国敏
覃海
凌小英
唐欣
徐雪梅
曹显永
裴幸彪
梁世谦
黄志琼
严小东
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Fangchenggang City Animal Epidemic Prevention And Control Center
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Abstract

The invention belongs to avian viral detection technique field, and the invention discloses a kind of H1N6 subtype avian influenza virus double RT PCR detection primers group, kit and its application.A kind of double RT PCR detection primer groups of H1N6 subtype avian influenza virus, including 2 pairs of specific primers, are primer pair H1 F and H1 R, primer pair N6 F and N6 R respectively, and it has the sequence as shown in SEQ ID No.1 to SEQ ID No.4 respectively.Inventor establishes the detection kit that can differentiate H1N6 subtype avian influenza virus based on double RT PCR methods.The present invention established the double RT PCR kits high specificity of H1N6 subtype avian influenza virus, high sensitivity, one pipe can detect H1 and N6 subtype avian influenza virus simultaneously, there is provided that a kind of easy, quickly and efficiently detection kit, the epidemiology survey and its antidiastole to H1N6 subtype avian influenza virus are significant for the detection of H1N6 subtype avian influenza virus.

Description

H1N6 subtype avian influenza virus duplex RT-PCR detection primers group, kit and its Using
Technical field
The invention belongs to avian viral detection technique field, more particularly to a kind of double RT- of H1N6 subtype avian influenza virus PCR detection primers group, kit, and identification H1 subtype avian influenza virus, the primer pair or primer of N6 subtype avian influenza virus To group and its kit.
Background technology
Influenza type belongs to orthomyxoviridae family's Influenza Virus, is segmented sub-thread minus-stranded rna virus, containing 8 lists Body RNA chains codified is up to 17 kinds of albumen (HA, NA, NP, M1, M2, M42, NS1, NS2, NS3, PA, PA-X, PA-N155, PA- N182, PB1, PB1-F2, PB1-N40, PB2).By internal albumen (mainly NP and M1) serological reaction, influenza virus point For A types, Type B, c-type and newest D types.According to the variation situation of surface antigen HA and NA albumen, influenza A mesh at present It is preceding to be further divided into 18 kinds of HA hypotypes (H1-H18), 11 kinds of NA hypotypes (NA1-NA11).A types influenza disease infection people, birds, open country Bird, pig, marine mammal and horse etc., avian influenza virus (Avian Ifluenza Virus, AIV) belong to influenza A Category.
At present, most of H1 subtype avian influenzas are shown as to fowl low pathogenicity, but H1 hypotypes AIV can across species barrier, by Bird is broadcast to birds to mammal or by mammal, is circulated between wild bird, pig, the mankind.Sent out in human history Raw flu outbreak several times is all closely related with H1 subtype influenzas, such as the big influenza H1N1 of the mankind in 1918, nineteen fifty-seven people source The Asia influenza that H1N1 and fowl source H2N2 match somebody with somebody again, and the new H1N1 people of 2009, pig, the source recombinant influenza of fowl three, Therefore carrying out the monitoring of H1 subtype influenza virus has public health meaning.N6 hypotypes are the AIV hypotypes continuously emerged in recent years, various regions Also H4N6, H5N6 and H6N6 hypotype strain are separated to successively, illustrate that active restructuring just occurs with other HA hypotypes for N6 hypotypes.Invention Laboratory where people was once separated to one plant of H1N6 hypotype AIV in 2016, there is grinding for H1N1, H1N2 and H5N6 hypotype AIV at present Study carefully, reported but without H1N6 hypotypes AIV detection method.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of H1N6 subtype avian influenza virus duplex RT-PCR detection primer Group, kit and its application.
In order to solve the above technical problems, the present invention uses following technical scheme：
A kind of primer sets for being used to identify H1N6 subtype avian influenza virus, including 2 pairs of specific primers, are primer pair respectively H1-F and H1-R, primer pair N6-F and N6-R, it has the sequence as shown in SEQ ID No.1 to SEQ ID No.4 respectively.
A kind of primer pair for being used to identify H1 subtype avian influenza virus, including specific primer H1-F and H1-R, they divide Not Ju You sequence table SEQ ID No.1 to SEQ ID No.2 base sequence.
A kind of primer pair for being used to identify N6 subtype avian influenza virus, including specific primer N6-F and N6-R, they divide Not Ju You sequence table SEQ ID No.3 to SEQ ID No.4 base sequence.
A kind of H1N6 subtype avian influenza virus duplex RT-PCR detection kit, including Reverse Transcription, PCR reagent, DEPC water and the ultra-pure water without RNase, it is characterised in that：Contain 2 pairs of specific primers in described PCR reagent, draw respectively For thing to H1-F and H1-R, primer pair N6-F and N6-R, it has the sequence as shown in SEQ ID No.1 to SEQ ID No.4 respectively Row.
Preferably, the end of described specific primer H1-F and H1-R, N6-1 and N6-2 in described PCR reagent is dense Degree Wei not be 0.4 μM and 1 μM.
Present invention also offers described H1N6 subtype avian influenza virus duplex RT-PCR detection kits, its feature exists In：The every μ L of pipe 50 of described Reverse Transcription, wherein 5 × buffer solution 10,2 μ L of μ L, dNTP (10mmol/L), 12 base reverse transcriptions 1.5 μ L, AMV reverse transcriptase of primer, 1 μ L, RI inhibitor 0.5 μ L, the μ L of viral RNA 35.
Preferably, the volume of described PCR reagent is 25 μ L/ pipes, wherein comprising the μ L of 2 × PCR mix liquid 12.5, on Each μ L of 1 μ L, cDNA template 2 of anti-sense primer.
Present invention also offers it is described be used to identifying the primer sets of H1N6 subtype avian influenza virus, described primer pair or Described H1N6 subtype avian influenza virus duplex RT-PCR detection kits are in H1 hypotypes and N6 subtype avian influenza virus is differentiated Application.
Compared with prior art, the present invention has the advantages that：
1. the present invention establishes a RT-PCR reaction and just can simultaneously detect and differentiate H1 subtype avian influenza virus, N6 hypotypes The duplex RT-PCR detection kit of avian influenza virus or H1N6 subtype avian influenza virus.Pass through amplified conditions optimization, specificity And sensitivity tests, the duplex RT-PCR detection kit of H1 subtype avian influenza virus and N6 subtype avian influenza virus is established, should Kit has the advantages that high specificity, high sensitivity, low cost, high efficiency, available for clinical detection H1N6 subtype avian influenzas Virus.
2. because two pairs of primer expanding fragment lengths are different in the present invention, can directly by electrophoresis result of determination, it is easier, It is directly perceived and practical.Result of the test shows, this method it is amplifiable to H1N6 hypotypes AIV go out 581bp (H1 genetic fragments) and 296bp (N6 genetic fragments), 581bp fragments are only amplified to H1NX (X ≠ 6) hypotype AIV, HYN6 (Y ≠ 1) is only capable of amplifying 296bp Fragment, purpose fragment is not amplified to other hypotypes AIV.Sensitivity tests result shows that the method most low energy detects 103Copy Shellfish/μ L H1 hypotypes, N6 subtype avian influenza virus nucleic acid plasmids.
3. the invention provides the detection side of rapid differential diagnosis H1 hypotypes, N6 hypotypes or H1N6 subtype avian influenza virus Method, it is the important supplement in terms of avian influenza virus classification diagnosis.The H1 hypotypes and N6 hypotype AIV duplex RT-PCRs that this research is established Detection method applicability is wider, can detect the HA genes in H1N1, H1N2, H1N3, H1N6 and H1N7, can also detect H4N6, NA genes in H5N6, H6N6, H13N6, there is important realistic meaning to monitoringof bird flu prevention and control, to safeguarding that public health is pacified Also it is significant in all directions.
Brief description of the drawings
Fig. 1 establishes the specific test result figure of H1N6 hypotype AIV duplex RT-PCR detection kits for the present invention；
Wherein：M-DNA Marker DL2000；1-H1N6；2-H1N1；3-H2N3；4-H3N2；5-H4N6；6-H5N6；7- H6N1；8-H7N9；9-H8N4；10-H9N2；11-H10N7；12-H11N9；13-H12N5；14-H13N6；15-H14N5；16- H15N9；17- blank；18-H1N6；19-H1N2；20-H1N3；21-H1N7；22,23-H1 separation strains；24- blank.
Fig. 2 establishes the sensitivity tests result figure of H1N6 hypotype AIV duplex RT-PCR detection kits for the present invention；
Wherein：M-DNA Marker DL2000；1-9 is followed successively by 108-100Copies/ μ L V-H1 and V-N6 plasmids.
Embodiment
With reference to specific embodiment, make further details of elaboration to the present invention, but embodiments of the present invention are not It is confined to the scope of embodiment expression.These embodiments are merely to illustrate the present invention, not for limitation the scope of the present invention.This Outside, after present disclosure is read, those skilled in the art can various modifications may be made to the present invention, and these equivalent variations are same Sample falls within appended claims limited range of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments The material that uses, reagent etc., unless otherwise specified, are commercially obtained.
AIV strains (H1N1, H1N2, H1N3, H1N6, H1N7, H1 separation strains, H3N2, H4N6, H6N6, H9N2 and H11N9) Prepared and preserved by Guangxi veterinary institute；AIV strains (H2N3, H8N4 and H12N5) are given by Hong Kong University；AIV strains CDNA (H10N7, H13N6, H14N5 and H15N9) is given by Connecticut, USA university；H5N6 and H7N9 subtype cDNAs are by Guangxi Give at Zhuang autonomous region Blight control center.
SPF chicken embryos are purchased from Beijing Cimmeria Wei Tong experimental animals Co., Ltd.
RNA extraction agents RNAIso plus, Reverse Transcription (AMV enzymes, RNA inhibitor), PCR reagent are purchased from Dalian TakaRa companies, connect reagent pEASY-Blunt Cloning Kit and glue reclaim kit is purchased from Beijing Quan Shi King Companies, draw Thing is synthesized by Dalian TakaRa companies.
Embodiment 1：The preparation of avian influenza virus duplex RT-PCR design of primers and kit
(1) design of primer
106 H1 hypotype AIV HA gene orders, 53 N6 hypotype AIV NA gene orders, fortune are downloaded in GenBank Sequence alignment is carried out with MEGA6.0, two pairs of primers, primer sequence are designed with Primer5.0 with respect to conservative region further according to sequence Row are as shown in table 1, the detection for H1 subtype avian influenza virus and N6 subtype avian influenza virus.
The primer information of table 1
Note：H1-F and H1-R primer pairs are used for detecting whether containing H1 subtype avian influenza virus；N6-F and N6-R primer pairs For detecting whether containing N6 subtype avian influenza virus.
(2) preparation of sample
With reference to Minibest viral RNA/DNA extraction kit ver.4.0 extracts kit specifications, divide Indescribably take the RNA of above-mentioned avian influenza virus.
(3) preparation of H1N6 avian influenza virus duplex RT-PCR detection kit and reaction condition
The specific often μ L of pipe 50 of Reverse Transcription：The 2 μ L of μ L, dNTP (10mmol/L) of wherein 5 × buffer solution 10, the reversion of 12 bases Record 1.5 μ L, AMV reverse transcriptase of primer, 1 μ L, RI inhibitor 0.5 μ L, the μ L of viral RNA 35.The optimum response pattern of reverse transcription is 42 DEG C of effect 90min.
25 μ L PCR reaction systems：The μ L of 2 × PCR mix liquid 12.5, each μ L of 1 μ L, cDNA template 4 of upstream and downstream primer.To RT- PCR primer concentration, reaction temperature, time and cycle-index etc. optimize, and finally determine H1-F and H1-R primers in RT-PCR Final concentration of 0.4 μM of best effort；Final concentration of 1 μM of N6-F and N6-R primer best efforts.Positive control is H1 and N6 bases Because of total length plasmid V-H1 and V-N6 mixture.PCR optimum response pattern is 94 DEG C of pre-degeneration 5min, subsequently into 94 DEG C of changes Property 45s, 54 DEG C annealing 45s, 72 DEG C extension 60s, altogether carry out 35 circulation, finally again through 72 DEG C extension 10min, terminate reaction.
PCR primer runs electrophoresis observation testing result.
Embodiment2：The specific detection of duplex RT-PCR kit
According to the reaction condition optimized, with the duplex RT-PCR established to different subtype AIV (H1N6, H1N1, H1N2, H1N3, H1N7, H1 separation strains, H2N3, H3N2, H4N6, H5N6, H6N6, H7N9, H8N4, H9N2, H10N7, H11N9, H12N5, H13N6, H14N5, H15N9) viral nucleic acid extract RNA detected, examine its specificity.
As shown in figure 1, there are 2 specific bands to H1+N6 hypotype AIV testing results using the kit, it is respectively 581bp and 296bp；Only there is 1 specific band, fragment to H1 hypotypes AIV (i.e. H1Ny hypotypes AIV, y ≠ 6) testing result Size is 581bp；Only there is 1 specific band to N6 hypotypes AIV (i.e. HxN6 hypotypes AIV, x ≠ 1) testing result, fragment is big Small is 296bp；Any band is not amplified to other hypotype AIV and common poultry diease pathogen, is consistent with desired design.
Embodiment3：The sensitivity tests of duplex RT-PCR detection kit
The plasmid V-H1 and V-N6 of doubling dilution concentration known, according to the reaction condition optimized, with the method for foundation to 9 Individual dilution factor (100-108Copies/ μ L) plasmid template expanded, examine its sensitiveness.
As shown in Fig. 2 with the kit to 100-108Copies/ μ L V-H1 and V-N6 plasmid amplification results are shown, dense Spend for 103-108Copies/ μ L V-H1 and V-N6 plasmids it is amplifiable go out 581bp and the band of 296bp two.Therefore, this method is most Low energy detects 103Copies/ μ L H1 and N6 hypotypes AIV.
Result above shows there is higher sensitivity using duplex RT-PCR kit detection H1N6 hypotypes AIV.

Claims (8)

  1. A kind of 1. primer sets for being used to identify H1N6 subtype avian influenza virus, it is characterised in that：Including 2 pairs of specific primers, divide It is not primer pair H1-F and H1-R, primer pair N6-F and N6-R, it has such as SEQ ID No.1 to SEQ ID No.4 institutes respectively The sequence shown.
  2. 2. a kind of primer pair for being used to identify H1 subtype avian influenza virus, including specific primer H1-F and H1-R, they distinguish Base sequence with sequence table SEQ ID No.1 to SEQ ID No.2.
  3. 3. a kind of primer pair for being used to identify N6 subtype avian influenza virus, including specific primer N6-F and N6-R, they distinguish Base sequence with sequence table SEQ ID No.3 to SEQ ID No.4.
  4. 4. a kind of H1N6 subtype avian influenza virus duplex RT-PCR detection kit, including Reverse Transcription, PCR reagent, DEPC Water and the ultra-pure water without RNase, it is characterised in that：Contain 2 pairs of specific primers in described PCR reagent, be primer pair respectively H1-F and H1-R, primer pair N6-F and N6-R, it has the sequence as shown in SEQ ID No.1 to SEQ ID No.4 respectively.
  5. 5. H1N6 subtype avian influenza virus duplex RT-PCR detection kit according to claim 4, it is characterised in that：Institute Final concentrations of the specific primer H1-F and H1-R, N6-1 and N6-2 stated in described PCR reagent is respectively 0.4 μM and 1 μM.
  6. 6. H1N6 subtype avian influenza virus duplex RT-PCR detection kit according to claim 4, it is characterised in that：Institute The every μ L of pipe 50 of the Reverse Transcription stated, wherein 5 × buffer solution 10,2 μ L of μ L, dNTP (10mmol/L), 12 base reverse transcription primers 1.5 μ L, AMV reverse transcriptase, 1 μ L, RI inhibitor 0.5 μ L, the μ L of viral RNA 35.
  7. 7. H1N6 subtype avian influenza virus duplex RT-PCR detection kit according to claim 4, it is characterised in that：Institute The volume for the PCR reagent stated is managed for 25 μ L/, wherein including the μ L of 2 × PCR mix liquid 12.5, each 1 μ L, cDNA mould of upstream and downstream primer The μ L of plate 2.
  8. 8. according to claim 1 be used to identify any institute of the primer sets of H1N6 subtype avian influenza virus, claim 2-3 Any described H1N6 subtype avian influenza virus duplex RT-PCR detection kits of primer pair or claim 4-7 stated are differentiating Application in H1 subtype avian influenzas and N6 hypotypes fowl stream virus.
CN201711275853.6A 2017-12-06 2017-12-06 H1N6 subtype avian influenza virus double RT PCR detection primers group, kit and its application Pending CN107858454A (en)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN110735005A (en) * 2019-11-26 2020-01-31 广西大学 SIV and PRRSV multiple RT-PCR rapid detection kit and primer
CN111117970A (en) * 2020-01-20 2020-05-08 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Monoclonal antibody for recognizing N6 subtype avian influenza virus neuraminidase protein and application thereof

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110735005A (en) * 2019-11-26 2020-01-31 广西大学 SIV and PRRSV multiple RT-PCR rapid detection kit and primer
CN110735005B (en) * 2019-11-26 2023-01-10 广西大学 SIV and PRRSV multiple RT-PCR rapid detection kit and primer
CN111117970A (en) * 2020-01-20 2020-05-08 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Monoclonal antibody for recognizing N6 subtype avian influenza virus neuraminidase protein and application thereof
CN111117970B (en) * 2020-01-20 2020-11-17 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Monoclonal antibody for recognizing N6 subtype avian influenza virus neuraminidase protein and application thereof

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Application publication date: 20180330