CN101323882B - Primer set for detecting pig influenza virus and blood serum subtype and reagent box thereof - Google Patents
Primer set for detecting pig influenza virus and blood serum subtype and reagent box thereof Download PDFInfo
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- CN101323882B CN101323882B CN2007100419848A CN200710041984A CN101323882B CN 101323882 B CN101323882 B CN 101323882B CN 2007100419848 A CN2007100419848 A CN 2007100419848A CN 200710041984 A CN200710041984 A CN 200710041984A CN 101323882 B CN101323882 B CN 101323882B
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Abstract
The invention discloses a primer set which comprises the primer pair of common NP gene of influenza virus and the primer pair of SIV subtypes. The primer set can be used for preparing an RT-PCR assay kit; the kit can be used for the differentiation of serological subtypes of swine influenza virus (SIV) H1/H3 and N1/N2 and can detect whether influenza virus of other subtypes apart from H1 and amp, H3, N1and amp and N2 exist in a sample.
Description
Technical field
The present invention relates to multiplex PCR and detect, relate in particular to the PCR that detects swine influenza virus (SIV) and detect primer.
Background technology
Swine influenza virus (Swine influenza virus, SIV) can cause the respiratory system disease of pig, but the pig of each kind, each age group is infection morbidity all, even more serious to piglet harm, this disease reports it is that the U.S. in 1918 swinery generation is popular the earliest, and nineteen thirty Shope etc. is separated to the H1N1 subtype virus first in the pig body.Fowl source H1N1 began in European swinery popular in 1979.Britain in 1994 reports at first and is separated to new reorganization H1N2 hypotype that then a plurality of countries report in succession and are separated to this novel strain.The U.S. was separated to the H3N2 strain in 1998, and ground such as the seventies in 20th century Hong-Kong, continent, Taiwan etc. is reported successively and is separated to SIV.Serology monitoring and pathogen separation are identified and are shown, the present SIV of China extensively exists, epidemic strain hemagglutinin (Hemagglut inin, HA) hypotype is mainly H1, H3, neuraminidase hypotype (Neuraminidase, NA) be N1, N2, therefore set up at these two kinds of hypotypes fast, the Sensitive Detection method is popular and distribute very necessary for timely monitor SIV.
The RT-PCR technology successfully is used for the differential diagnosis of animal influenza virus, mainly is according to influenza virus NP gene, M gene conserved sequence, and the design synthetic primer is used to detect viral nucleic acid.But this method can only identify whether contain the animal influenza virus in the sample, can't determine the blood serum subtype of virus, also needs further to adopt blood clotting or hemagglutination-inhibition test to identify.Utilize PCR or NASABA method that development in recent years is got up are differentiated the different blood serum subtype of influenza virus, also are able to successful Application, detect H5, H9 subtype virus nucleic acid etc. respectively as fluorescence quantitative PCR detection H5, H9 subtype virus nucleic acid, RT-PCR.But identify that a blood serum subtype needs a PCR system, testing process is loaded down with trivial details relatively, is unfavorable for quick diagnosis.
Multiplex PCR is a new developing technology on single PCR basis, make up by Oligonucleolide primers, a plurality of target sequences simultaneously increase in a PCR reaction system, the specificity of its amplification and efficient are suitable with single PCR, but can increase at a plurality of target sequences of different templates simultaneously, can save time and effort, be easy a, method fast in the differential diagnosis of sample, especially is suitable for clinical cause of disease somatotype and detects.Choi etc., Poddar adopt multiplex RT-PCR method that the detection of SIV somatotype, human influenza virus's somatotype are detected and study.But only limit to evaluation, can not determine the influenza virus that whether also has other hypotype in the test sample known H1, H3 and N1, N2 hypotype.
Therefore, this area presses for provides the diagnostic method that detects pig body influenza virus and differential diagnosis H1, H3 and N1, N2 blood serum subtype, for clinical monitoring SIV dynamically provides technical support.
Summary of the invention
The present invention aims to provide a kind of primer collection.
Another object of the present invention provides the test kit that contains above-mentioned primer collection.
A further object of the present invention provides a kind of method that detects pig body influenza virus and differential diagnosis blood serum subtype.
In a first aspect of the present invention, a kind of primer collection that same reaction system is carried out pcr amplification that is used for is provided, it comprises:
(1) primer of the universal NP gene of influenza virus is right; With
(2) primer of SIV hypotype is right.
In another preference, described SIV hypotype comprises hemagglutinin hypotype or neuraminidase hypotype.
In another preference, described hemagglutinin hypotype comprises H1 and/or H3; Described neuraminidase hypotype comprises N1 and/or N2.
In another preference, described primer collection comprises:
(1) primer of the universal NP of influenza virus is right; With
(2) primer of SIV hemagglutinin hypotype H1 and H3 is right; Or
(3) primer of SIV neuraminidase hypotype N1 and N2 is right.
In another preference, the primer of described NP is to being SEQ ID NO:11 and 12.
In another preference, the primer of described H1, H3 is to being selected from: SEQ ID NO:1 and 2, SEQ ID NO:3 and 4 or SEQID NO:17 and 18.
In another preference, the primer of described N1, N2 is to being selected from: SEQ ID NO:5 and 6, SEQ ID NO:25 and 6 or SEQ ID NO:7 and 8 or SEQ ID NO:9 and 10.
In a second aspect of the present invention, a kind of primer collection that same reaction system is carried out pcr amplification that is used for is provided, it comprises: the primer of SIV hypotype hemagglutinin hypotype H1 and H3 is right; The primer of described H1 and H3 is to being selected from SEQ ID NO:1-4 or SEQ ID NO:1,2,17 and 18.
In another preference, a kind ofly be used for the primer collection that same reaction system is carried out pcr amplification, it comprises: the primer of SIV neuraminidase hypotype N1 and N2 is right; The primer of described N1 and N2 is to being selected from SEQ ID NO:5-8, SEQ ID NO:5, and 6,9 and 10, SEQID NO:25,6,7 and 8 or SEQ ID NO:25,6,9 and 10.
In a third aspect of the present invention, a kind of test kit is provided, it comprises:
1. be used for same reaction system and carry out the primer collection of pcr amplification: the primer of the universal NP gene of influenza virus is to right with the primer of SIV hypotype; With
2. specification sheets.
In another preference, a kind of test kit comprises that the primer of SIV hypotype hemagglutinin hypotype H1 and H3 is right, and the primer of described H1 and H3 is to being selected from SEQ ID NO:1-4 or SEQ ID NO:1,2,17 and 18.
In another preference, a kind of test kit comprises that the primer of SIV neuraminidase hypotype N1 and N2 is right; The primer of described N1 and N2 is to being selected from SEQ ID NO:5-8, SEQ ID NO:5, and 6,9 and 10, SEQ IDNO:25,6,7 and 8 or SEQID NO:25,6,9 and 10.
In a fourth aspect of the present invention, provide a kind of SIV has been carried out the method for somatotype, it comprises step:
(a) in an individual system, add and be used for same reaction system and carry out the primer collection of pcr amplification (primer that comprises the universal NP gene of influenza virus to the primer of SIV hypotype to), carry out pcr amplification, check the SIV that whether contains a kind of hypotype in this system according to the electrophoretic band of amplified production;
(b) if electrophoresis result demonstrates corresponding band, then represent to exist in this system the SIV of this kind hypotype, thus the detected result of obtaining;
If electrophoresis result does not demonstrate corresponding band, represent not exist in this system the SIV of above-mentioned this hypotype, check whether contain influenza virus in this system according to the electrophoretic band of amplified production simultaneously;
(c) if electrophoresis result demonstrates the influenza virus band, the primer that then adds the SIV hypotype in another system is right, carries out pcr amplification, checks the SIV that whether contains another kind of hypotype in this system according to the electrophoretic band of amplified production;
(d), then represent to exist in this system described NA hypotype SIV if electrophoresis result demonstrates corresponding band;
If electrophoretic band does not demonstrate corresponding SIV hypotype band, represent not exist in this system described hypotype SIV.
In another preference, described SIV hypotype comprises hemagglutinin hypotype and neuraminidase hypotype.
In another preference, described detection method comprises the step mule:
(a) primer that adds the universal NP of influenza virus in an individual system carries out pcr amplification to right with the primer of SIV hemagglutinin hypotype H1 and H3, according to the electrophoretic band of amplified production, checks whether contain SIVH1 and/or H3 hemagglutinin hypotype in this system;
(b), then represent to exist in this system H1 and/or H3 hemagglutinin hypotype SIV if electrophoretic band demonstrates SIV hemagglutinin hypotype H1 and/or H3; If electrophoretic band does not show SIV hemagglutinin hypotype H1 and/or H3 band, then check whether contain influenza virus in this system according to the electrophoretic band of amplified production;
(c) if demonstrating, electrophoretic band contains influenza virus, the primer that then adds neuraminidase hypotype N1 and N2 in another system is right, carry out pcr amplification,, check whether contain neuraminidase N1 and/or N2 hypotype SIV in this system according to the electrophoretic band of amplified production;
(d), then represent to exist in this system neuraminidase N1 and/or N2 hypotype SIV if electrophoretic band demonstrates neuraminidase hypotype N1 and/or N2 respective strap; If electrophoretic band does not demonstrate N1 and N2 respective strap, represent not exist in this system neuraminidase N1 and/or N2 hypotype SIV.
In another preference, described detection method comprises step:
(a) in an individual system, add the primer of the universal NP of influenza virus to right with the primer of SIV neuraminidase hypotype N1 and N2, carry out pcr amplification, according to the electrophoretic band of amplified production, check whether contain SIV and whether be N1 and/or N2 neuraminidase subtype virus in this system;
(b), then represent to exist in this system N1 and/or N2 hypotype SIV neuraminidase if electrophoretic band demonstrates SIV neuraminidase hypotype N1 and/or N2; If electrophoretic band does not show SIV neuraminidase hypotype N1 and N2, then check whether contain influenza virus in this system according to the electrophoretic band of amplified production; (c) if electrophoretic band demonstrates influenza virus, the primer that then adds SIV hemagglutinin hypotype H1 and H3 in another system is right, carries out pcr amplification, according to the electrophoretic band of amplified production, checks whether contain H1 and/or H3 hemagglutinin hypotype SIV in this system;
(d), then represent to exist in this system hemagglutinin hypotype H1 and/or H3 if electrophoretic band demonstrates hemagglutinin hypotype H1 and/or H3; If electrophoretic band does not demonstrate hemagglutinin hypotype H1 and/or H3, represent not exist in this system H1 and/or H3 hemagglutinin hypotype.
In another preference, a kind of method that SIV is carried out somatotype comprises step:
(a) primer that adds SIV hypotype hemagglutinin hypotype H1 and H3 in an individual system carries out pcr amplification to right with the primer of the universal NP of influenza virus, checks whether contain H1 and/or H3 hemagglutinin hypotype SIV in this system according to the electrophoretic band of amplified production;
(b) if electrophoresis result demonstrates corresponding H1 and/or H3 band, then represent to exist in this system the SIV of H1 and/or H3 hypotype, thus the detected result of obtaining;
If electrophoresis result does not demonstrate corresponding H1 and H3 band, represent not exist in this system H1 and/or H3 hypotype SIV, check whether contain influenza virus in this system according to the electrophoretic band of amplified production;
(c) if electrophoresis result demonstrates the influenza virus band, the primer that then adds neuraminidase hypotype N1 and N2 in another system is right, carry out pcr amplification,, check whether contain neuraminidase N1 and/or N2 hypotype SIV in this system according to the electrophoretic band of amplified production;
(d) contain neuraminidase hypotype N1 and/or N2 if electrophoretic band shows, then represent to exist in this system neuraminidase N1 and/or N2 hypotype SIV; If electrophoretic band does not demonstrate neuraminidase hypotype N1 and N2, represent not exist in this system this two kinds of neuraminidase hypotype SIV.
In another preference, the primer of described H1 and H3 is to being selected from SEQ ID NO:1-4 or SEQ IDNO:1,2,17 and 18. The primer of described N1 and N2 is to being selected from SEQ ID NO:5-8, SEQID NO:5, and 6,9 and 10, SEQ ID NO:25,6,7 and 8 or SEQ ID NO:25,6,9 and 10.
In view of the above, the invention provides a kind of diagnostic method that detects pig body influenza virus and differential diagnosis H1, H3 and N1, N2 blood serum subtype, for clinical monitoring SIV dynamically provides technical support.
Description of drawings
Fig. 1 has shown that H1, H3 somatotype multiplex PCR detect the situation of H1/H3 hybrid template; Wherein
1 expression H1/H3/NP combination I; 2 expression H1/H3/NP combination II; 3 expression H1/H3/NP combination III; 4 expression H1/H3/NP combination IV; 5 expression H1/H3/NP combination V; M represents the DL2000 marker.
Fig. 2 has shown that the N1 primer is to H1N1, H5N1 template amplification result; Wherein
A represents the H1N1 template; B represents the H5N1 template;
1 expression N1 primer is to 495/855; 2 expression N1 primers are to 491/855; 3 expression N1 primers are to 710/1094; 4 expression N1 primers are to 824/1094; 5 expression N1 primers are to 802/1094; 6 expression N1 primers are to 721/931; 7 expression N1 primers are to 770/1201; 8 expression negative controls; M represents DL2000 molecular weight marker thing.
Fig. 3 has shown that the N2 serotype specific primer is to H3N2 template (A), the single PCR detected result of H9N2 template (B);
Wherein
1 expression N2 primer is to 273/432; 2 expression N2 primers are to 273/433; 3 expression N2 primers are to 818/1010; 4 expression N2 primers are to 411/628; 5 expression N2 primers are to 1036/1290; 6 expression N2 primers are to 566/836; 7 expression N2 primers are to 730/1013; 8 expression N2 primers are to 804/1132; 9 expression N2 primers are to 606/1012; 10 expression N2 primers are to 804/1290; M represents DL2000 molecular weight marker thing.
Fig. 4 has shown that N1/N2 hypotype combination of primers verifies the result to different templates; Wherein
1 expression H1 template; 2 expression templates; 3 expression H1/H3 templates; 4 expression H5 templates; 5 expression H9 templates; 6 expression H5/H9 templates.
Fig. 5 has shown H1, H3 hypotype combination of primers susceptibility detected result; Wherein
1-8 expression H1/H3 combination of primers I; 9-14 represents H1/H3 combination of primers II.
Fig. 6 has shown that H1/H3 combination I, II are to different subtype SIV template amplification result among the embodiment 2; Wherein
1,6 expression H1/H3 template; 2,7 expression H9 hypotype templates; 3,8 expression H5 hypotype templates; 4,9 expression H3 hypotype templates; 5.10 expression H1 hypotype template; M represents the DL2000 marker.
Fig. 7 has shown that NP/NA combination of primers I, II, III and VI are to different subtype SIV template amplification result among the embodiment 3; Wherein
1,5,9,13 is the H1N1 template; 2,6,10,14 is the H3N2 template; 3,7,11,15 is the H1N1H3N2 hybrid template; 4,8,12,16 negative contrasts; M represents DL2000 molecular weight marker thing.
Fig. 8 has shown that HA combination of primers I, II are to different subtype SIV template amplification result among the embodiment 3; Wherein
1,5 is the H1N1 template; 2,6 is the H3N2 template; 3,7 is the H1N1/H3N2 hybrid template; M represents DL2000 molecular weight marker thing.
Embodiment
The contriver has found a kind of primer collection that same reaction system is carried out pcr amplification that is used for through extensive and deep research, and described primer collection comprises that SIV hypotype primer is to right with the universal primer of influenza virus.
The contriver has also found a kind of primer collection that same reaction system is carried out pcr amplification that is used for, described primer collection comprises that the primer of SIV H1 and H3 hypotype is right, or the primer that comprises SIV N1 and N2 hypotype is right, the primer of described H1 and H3 is to being selected from SEQID NO:1-4 or SEQ ID NO:1,2,17 and 18; The primer of described N1 and N2 is to being selected from SEQ ID NO:5-8, SEQ ID NO:5, and 6,9 and 10, SEQ ID NO:25,6,7 and 8 or SEQ ID NO:25,6,9 and 10.
The contriver finds, if exist SIV hypotype primer to right with the universal primer of influenza virus, can whether have certain SIV hypotype in the checking system in a certain system.Even there is not certain SIV hypotype to exist in the system, also the multiplex PCR that can pass through to be carried out judges whether there is influenza virus in the system.
As used herein, " primer collection " is meant when carrying out pcr amplification in same system, and the used aggregate that contains many to primer wherein contains 1-10 to primer; Preferably, it is right to contain 1-5; More preferably, it is right to contain 2-3.
As used herein, " primer of the universal NP of influenza virus to " is meant the nucleotide sequence all conservative to all animal influenza viruses, such as but not limited to, SIV, avian influenza virus.
As used herein, " primer to " is meant at nucleotide sequence conservative relatively in the goal gene designed one group of forward and reverse primer.
The primer of specific amplification animal influenza virus of the present invention or SIV hypotype can be synthesized into (for example can be synthetic with business-like automatic dna synthesizer) by the DNA synthetic method of routine.
These primers of the present invention can carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substances.
Primer collection
The universal NP of influenza virus has multiple natural variation form at nature.The primer of the universal NP of influenza virus provided by the invention is right, can be well known in the art at all conservative nucleotide sequence of all animal influenza viruses.Preferred primer is the primer that its sequence all exists in wild-type sequence and multiple variant sequence, for example work as primer to can in the reverse transcription dna profiling of 90% above animal influenza virus, going out product by specific amplification, and can't go out product by specific amplification in other viral reverse transcription dna profilings, this primer is to being exactly preferred so.Preferred primer is to comprising SEQ ID NO:11 and 12.
The SIV hypotype has multiple natural variation form at nature.The primer of SIV blood serum subtype provided by the invention is right, can be the nucleotide sequence at blood serum subtype nucleic acid conserved regions well known in the art.Preferred primer is the primer that its sequence all exists in wild-type sequence and multiple variant sequence, for example work as primer to can in the reverse transcription dna profiling of 90% above certain blood serum subtype of SIV, going out product by specific amplification, and can't go out product by specific amplification in the reverse transcription dna profiling of other hypotypes, this primer is to being exactly preferred so.
The primer of SIV hemagglutinin hypotype H1 provided by the invention is right, can be the nucleotide sequence at H3 blood serum subtype nucleic acid conserved regions well known in the art, include but not limited to SEQ ID NO:1, SEQID NO:2, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16.Preferred primer is to comprising SEQ ID NO:1 and 2.
The primer of SIV hemagglutinin hypotype H3 provided by the invention is right, can be the nucleotide sequence at H3 blood serum subtype nucleic acid conserved regions well known in the art.Preferred primer is to comprising SEQ ID NO:3 and 4 or SEQ IDNO:17 and 18.
The primer of SIV hemagglutinin hypotype N1 provided by the invention is right, can be the nucleotide sequence at N1 blood serum subtype nucleic acid conserved regions well known in the art, include but not limited to SEQID NO:5, SEQ IDNO:6, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:25.Preferred primer is to comprising SEQ IDNO:5 and 6 or SEQ ID NO:25 and 6.
The primer of SIV hemagglutinin hypotype N2 provided by the invention is right, can be the nucleotide sequence at N2 blood serum subtype nucleic acid conserved regions well known in the art, include but not limited to SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:23, SEQ ID NO:24.Preferred primer is to comprising SEQID NO:7 and 8 or SEQ ID NO:9 and 10.
It is right to contain 1-10 group primer in the primer collection provided by the invention; Preferably, contain the 1-5 group; More preferably, contain the 2-3 group.
Described primer is to including but not limited to, the primer of the universal NP of influenza virus is right to the primer of, SIV hypotype H1, H3, N1, N2.
Can contain in the primer collection provided by the invention.The primer that also can contain the universal NP of influenza virus is to right with the primer of SIV hypotype N1, N2.The primer that also can only contain SIV hypotype H1, H3 in the primer collection provided by the invention to, the primer of SIV hypotype N1, N2 to or the primer of the universal NP of influenza virus right.
Primer in the primer collection provided by the invention be to should be able to playing a role in same system, its molecular weight differ 100bp or more than, and each primer between should not disturb.
In a preference of the present invention, the primer that comprises the universal NP of influenza virus in the described primer collection is to right with the primer of SIV hypotype H1/H3, and the primer of the universal NP of described influenza virus is to being SEQ ID NO:11 and 12;
| NP-756NP-1200 | 5′GGATCAAGTGAGAGAAAGTCGAA-3′5′-GGCCCAGTATCTGCTTCTTAGTT-3′ | SEQ?ID?NO：11SEQ?ID?NO：12 |
The primer of described H1/H3 is to being selected from: SEQ ID NO:1 and 2/SEQ ID NO:3 and 4, or SEQ IDNO:1 and 2/SEQID NO:17 and 18.
| H1-494H1-679 | 5′-TTATGCTGGAGCAAACAGCTT-3′5′-ACATAGGCATCTGCATTTTGG-3′ | SEQ?ID?NO：1SEQ?ID?NO：2 |
| H3-111H3-335 | 5′-AAACGGAACGCTAGTGAAAACAATC-3′5′-TCCGGCACATCATAAGGGTAACAGT-3′ | SEQ?ID?NO：3SEQ?ID?NO：4 |
Or
| H1-496H1-679 | 5′-TTATGCTGGAGCAAACAGCTT-3′5′-ACATAGGCATCTGCATTTTGG-3′ | SEQ?ID?NO：1SEQ?ID?NO：2 |
| H3-958H3-1275 | 5.-GCCTGTCCCAGATATGTTAAGC-3.5.-GAGGTCCTGAATTCTCCCTTCT-3. | SEQ?ID?NO：17SEQ?ID?NO：18 |
In a preference of the present invention, the primer that comprises N1/N2 in the described primer collection is right, is selected from: SEQID NO:5 and 6/SEQID NO:7 and 8, SEQID NO:5 and 6/SEQ ID NO:9 and 10, SEQ ID NO:25 and 6/SEQ ID NO:7 and 8 or SEQ ID NO:25 and 6/SEQ ID NO:9 and 10.
| N1-824N1-1094 | 5′-ACTACGAGGAATGCTCCTGTTATC-3′5′-GTGCTTTTAGTTCTCCCTATCCAA-3′ | SEQ?ID?NO：5SEQ?ID?NO：6 |
| N2-273N2-433 | 5′-TTGGGTGACGAGAGAACCTTAT-3′5′-GAAATGGAACACCCAACTCATT-3′ | SEQ?ID?NO：7SEQ?ID?NO：8 |
| N1-824N1-1094 | 5′-ACTACGAGGAATGCTCCTGTTATC-3′5′-GTGCTTTTAGTTCTCCCTATCCAA-3′ | SEQ?ID?NO：5SEQ?ID?NO：6 |
| N2-411N2-628 | 5′-GAATGAGTTGGGTGTTCCATTT-3′5′-TACAAACACATTCCGACTCCTG-3′ | SEQ?ID?NO：9SEQ?ID?NO：10 |
| N1802N1-1094 | 5′-GAGTTGAATGCCCCTAATTATCAC-3′5′-GTGCTTTTAGTTCTCCCTATCCAA-3′ | SEQ?ID?NO：25SEQ?ID?NO：6 |
| N2-273N2-433 | 5′-TTGGGTGACGAGAGAACCTTAT-3′5′-GAAATGGAACACCCAACTCATT-3′ | SEQ?ID?NO：7SEQ?ID?NO：8 |
Or
| N1802N1-1094 | 5′-GAGTTGAATGCCCCTAATTATCAC-3′5′-GTGCTTTTAGTTCTCCCTATCCAA-3′ | SEQ?ID?NO：25SEQ?ID?NO：6 |
| N2-411N2-628 | 5′-GAATGAGTTGGGTGTTCCATTT-3′5′-TACAAACACATTCCGACTCCTG-3′ | SEQ?ID?NO：9SEQ?ID?NO：10 |
Test kit
Can contain primer collection and specification sheets in the test kit provided by the invention.Can also contain and carry out the required reagent well known in the art of pcr amplification, such as but not limited to, the required reagent of RNA, ThermoScript II, amplification buffer, archaeal dna polymerase, template DNA etc. extracted.
In the primer collection that is contained in the test kit provided by the invention, the primer that can comprise the universal NP of influenza virus is to right with the primer of influenza virus sub-strain.
In the primer collection that is contained in the test kit provided by the invention, can comprise that also the primer of SIV hypotype is right, for example H1 and H3, or N1 and N2.The primer sequence of described H1 and H3 is preferably SEQ ID NO:1 and 2/SEQ ID NO:3 and 4, or SEQ ID NO:1 and 2/SEQ ID NO:17 and 18.The primer sequence of described N1 and N2 is preferably SEQ ID NO:5 and 6/SEQ ID NO:7 and 8, SEQ ID NO:5 and 6/SEQ ID NO:9 and 10, SEQ ID NO:25 and 6/SEQ ID NO:7 and 8 or SEQ ID NO:25 and 6/SEQ ID NO:9 and 10.
Detection method
The invention provides a kind of method of SIV somatotype, described method can be used for diagnostic uses, also can be used for non-diagnostic uses, for example is used for environment test and appraisal etc.Described method makes up the 1-5 individual system, preferably is the 1-3 individual system, more preferably is 2 individual system.
If 1 individual system, the primer that adds the universal NP of influenza virus in system, can detect and whether contain corresponding SIV hypotype in the system, or not contain influenza virus by pcr amplification right with the primer of SIV hypotype.
If 〉=2 individual system, the primer that adds the universal NP of influenza virus in an individual system, can detect and whether contain corresponding SIV hypotype in this system, or not contain influenza virus by pcr amplification right with the primer of SIV hypotype.If detect and do not have corresponding SIV hypotype in this system, but contain influenza virus, then in another system, add with first system in the primer of the different SIV hypotype that added right, by pcr amplification, detect whether contain corresponding SIV hypotype in this this system.
In a preference of the present invention, make up 2 multiple RT-PCR systems, system I is used for simultaneously whether identification system contains the animal influenza virus and whether be H1 or H3 hypotype, and can detect other hypotype SIV that whether contains in the sample beyond H1, the H3 hypotype.When system I determines to contain influenza virus particles, adopt system II to differentiate that this influenza virus is N1 or N2 hypotype.Utilize H1N1/H5N1 or H3N2/H9N2 for the accuracy of template checking system II to N1, the detection of N2 somatotype, all can amplify the expection band, promptly N1, the N2 hypotype at various sources all is suitable for.
Two multiple RT-PCR systems setting up all adopt Oligod (T) to do the reverse transcription primer, have avoided owing to adding the non-specific cross-reaction that Auele Specific Primer causes.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.
Major advantage of the present invention is:
1, the detection method of primer collection provided by the invention and foundation not only can be used for the discriminating of known SIV H1/H3, N1/N2 positive-virus blood serum subtype, and can detect the influenza virus that whether has other hypotype in the sample.
2, method provided by the invention can detect the influenza virus particles that contains in the sample tissue well, and H1/H3, N1/N2 hypotype are carried out accurate somatotype.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all per-cent and umber by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Experimental implementation program of the present invention:
A. reverse transcription reaction
A-1. by following composition preparation inverse transcription reaction liquid
| Magnesium chloride (MgCl 2) | 2ul |
| 10 * reverse transcription damping fluid (RT Buffer) | 1ul |
| DNTP mixture (Mixture) (each 10mM) | 1ul |
| RNA enzyme inhibitors (RNase Inhibitor) | 0.25ul |
| AMV ThermoScript II (AMV Reverse Transcriptase) | 0.5ul |
| Oligo T-adapter primer (Oligo T-Adaptor Primer) | 0.5ul |
| RNA(—500ng) | 4.75ul |
| Amount to | 10ul/ sample |
A-2. carry out reverse transcription reaction by following condition
| 42℃—55℃ | 30 minutes |
| 99 |
5 |
| 5 |
5 minutes |
The B.PCR amplification
B-1. by following set of dispense system reaction mixture.
The B-2.PCR amplification condition:
The result judges:
Reaction extracts reaction solution 5-10ul after finishing, and 2.0% agarose gel electrophoresis is judged reaction result.
When the appearance size is the 445bp fragment, is judged to be the SIV positive, otherwise is judged to be feminine gender;
When the appearance size is the 445bp/186bp fragment, be judged to be the SIV hemagglutinin H1 hypotype positive, otherwise judge SIV hemagglutinin H1 hypotype feminine gender;
When the appearance size is the 445bp/225bp amplified fragments, is judged to be the SIV hemagglutinin H3 hypotype positive, otherwise is judged to be SIV hemagglutinin H3 hypotype feminine gender.
(1) SIV viral nucleic acid, reproductive and respiratory syndrome virus (PRRSV), PCV-II (PCV), foot and mouth disease virus (FMDV), Pseudorabies virus (PRV) nucleic acid are respectively available from Animal Husbandary and Veterinary Inst., Shanghai Academy of Agricultural Science.
(2) design of primers and synthetic: SIV NP gene, H1N1 hypotype, the HA of H3N2 hypotype, the NA gene of downloading the Genbank login, carry out analogy analysis with Dnastar software, the conservative region of screening decision different subtype, software Primier Primer3.0 designs serotype specific primer, primer is used DEPC H available from Shen, Shanghai energy lottery industry bio-engineering corporation, Shanghai bio-engineering corporation
2O dilution primer working concentration is 20pmol/ μ L.
(3) RNA extracts reagent Trizol, is Invitrogen company product; ThermoScript II, rTaq enzyme and Buffer etc. are available from Dalian Bao Bio-Engineering Company (TakaRa Co.)
(4) RNA extracts and carries out to specifications.
(5) H1/H3 hemagglutinin hypotype, the single RT-PCR system of N1/N2 neuraminidase hypotype are set up
(6) reverse transcription and pcr amplification system, cycling condition optimization
(7) H1/H3HA hypotype, N1/N2NA hypotype multiplex PCR system combinations: according to the different subtype amplification, select NP/H1/H3 primer, N1/N2 primer to carry out the proportioning combination, make up the multiplex PCR system.Mix H1, H3 subtype virus RNA by a certain percentage, reverse transcription is done template with this reverse transcription product again, carries out multi-PRC reaction, adds different combination of primers in the system.Reaction finishes, sampling electrophoresis, observations.The good combination of primers of screening expanding effect.
(8) multiplex PCR system specificity, susceptibility
(9) set up the multiplex PCR system, the assembling test kit.
The result
One, the H1/H3 somatotype detects the foundation of multiplex PCR system
Vary in size according to single PCR result and amplified fragments, H1, H3 serotype specific primer carried out proportioning be combined into the multiplex PCR system:
Table 1H1/H3 hemagglutinin hypotype multiplex PCR system combination of primers
To the combination of primers that screens, adopt the checking of H1/H3 hybrid template, the results are shown in Figure 1.
The result shows that combination I, II detected result the best are selected as the test system.
Two, the single RT-PCR system of N1, N2 is set up and primer screening
Do template with SIV H1N1, AIV H5N1 RNA, checking N1 primer is seen Fig. 2 to the specificity of N1 gene amplification.
Be template with H3N2, H9N2 RNA respectively, checking N2 primer is seen Fig. 3 to the specificity to N2 gene amplification.
Three, the neuraminidase somatotype detects the foundation of multiplex PCR system
Vary in size according to single PCR result and amplified fragments, make up N1, N2 hypotype primer, and different templates is verified, see Fig. 4 (table 2).
Table 2N1/N2 neuraminidase hypotype multiplex PCR system combination of primers
Four, sensitivity analysis
Measure the RNA concentration of extracting, through the series concentration dilution, detect expanding effect, the minimum detectability amount is: H1 is 0.15ng, and H3 is 0.2ng.Promptly utilize the multiplex PCR system of setting up, detection H1, H3 hypotype template are limited the quantity of and are the ng level.
Five, specificity analyses
Be template with SIV, PRRSV, PRV, FMDV, PCV nucleic acid respectively, adopt the multiple system of setting up of RT-PCR to detect, the specificity of checking combination of primers.
The result shows that the multiplex PCR system of foundation can only detect the SIV specific nucleic acid, and can not amplify other viral nucleic acid, shows that multiplex PCR system specificity is good, can be used for SIV differential diagnosis.
Six, analog sample detects
With clinical detection is the swine excrement of SIV feminine gender, and the hybrid virus particle extracts RNA again, carries out the multiple RT-PCR augmentation detection, and detected result is with consistent with viral allantoic fluid detected result.
The result shows that it also is feasible that clinical sample is detected.
Seven, clinical sample detects
Directly detect 10 parts in clinical pig lung with respiratory symptom with multiplex PCR, 75 parts of cotton examination of respiratory tract, pathological material of disease is inoculated 10 age in days SPF chicken embryos simultaneously, virus and blood serum subtype thereof are differentiated in blood clotting/hemagglutination-inhibition test, and all samples multiple RT-PCR detection as a result separates back blood clotting/hemagglutination-inhibition test and identifies all negative with the chicken embryo.
The result shows that it is 100% that two kinds of methods detect coincidence rate to negative sample.
(1) SIV viral nucleic acid, reproductive and respiratory syndrome virus (PRRSV), PCV-II (PCV), foot and mouth disease virus (FMDV), Pseudorabies virus (PRV) nucleic acid are respectively available from Animal Husbandary and Veterinary Inst., Shanghai Academy of Agricultural Science.
(2) design of primers and synthetic: SIV NP gene, H1N1 hypotype, the HA of H3N2 hypotype, the NA gene of downloading the Genbank login, carry out analogy analysis with Dnastar software, the conservative region of screening decision different subtype, software Primi er Primer3 ' 0 design serotype specific primer, primer is used DEPC H available from Shen, Shanghai energy lottery industry bio-engineering corporation, Shanghai bio-engineering corporation
2O dilution primer working concentration is 20pmol/ μ L.
(3) RNA extracts reagent Trizol, is Invitrogen company product; ThermoScript II, rTaq enzyme and Buffer etc. are available from Dalian Bao Bio-Engineering Company (TakaRa Co.)
(4) RNA extracts and carries out to specifications.
(5) H1/H3 hemagglutinin hypotype, the single RT-PCR system of N1/N2 neuraminidase hypotype are set up
(6) reverse transcription and pcr amplification system, cycling condition optimization
(7) H1/H3HA hypotype, N1/N2NA hypotype multiplex PCR system combinations: according to the different subtype amplification, select NP/H1/H3 primer, N1/N2 primer to carry out the proportioning combination, make up the multiplex PCR system.Mix H1, H3 subtype virus RNA by a certain percentage, reverse transcription is done template with this reverse transcription product again, carries out multi-PRC reaction, adds different combination of primers in the system.Reaction finishes, sampling electrophoresis, observations.The good combination of primers of screening expanding effect.
(8) multiplex PCR system specificity, susceptibility
(9) set up the multiplex PCR system, the assembling test kit.
Adopt H1/H3 combination of primers I, II to various hypotype template detection results:
Table 3H1/H3 hemagglutinin hypotype multiplex PCR system combination of primers
H5N1, H9N2 hypotype template influenza virus NP gene band only occurs, and H1 and/or H3 hypotype band do not occur.(accompanying drawing 6)
Utilize N1/N2 combination of primers I, II, III, VI then all can detect corresponding N 1 and/or N2 hypotype to various hypotype template detection (H1N1, H3N2, H5N1, H9N2).(accompanying drawing 5)
Table 4N1/N2 neuraminidase hypotype multiplex PCR system combination of primers
The result shows, animal influenza virus N1 and/or N2 neuraminidase hypotype that above-mentioned combination of primers all can effectively increase and contain in the sample.
(1) SIV viral nucleic acid, reproductive and respiratory syndrome virus (PRRSV), PCV-II (PCV), foot and mouth disease virus (FMDV), Pseudorabies virus (PRV) nucleic acid are respectively available from Animal Husbandary and Veterinary Inst., Shanghai Academy of Agricultural Science.
(2) design of primers and synthetic: SIV NP gene, H1N1 hypotype, the HA of H3N2 hypotype, the NA gene of downloading the Genbank login, carry out analogy analysis with Dnastar software, the conservative region of screening decision different subtype, software Primi er Primer3 ' 0 design serotype specific primer, primer is used DEPC H available from Shen, Shanghai energy lottery industry bio-engineering corporation, Shanghai bio-engineering corporation
2O dilution primer working concentration is 20pmol/ μ L.
(3) RNA extracts reagent Trizol, is Invitrogen company product; ThermoScript II, rTaq enzyme and Buffer etc. are available from Dalian Bao Bio-Engineering Company (TakaRa Co.)
(4) RNA extracts and carries out to specifications.
(5) H1/H3 hemagglutinin hypotype, the single RT-PCR system of N1/N2 neuraminidase hypotype are set up
(6) reverse transcription and pcr amplification system, cycling condition optimization
(7) H1/H3HA hypotype, N1/N2NA hypotype multiplex PCR system combinations: according to the different subtype amplification, select NP/N1/N2 primer, H1/H3 primer to carry out the proportioning combination, make up the multiplex PCR system.Mix N1, N2 subtype virus RNA by a certain percentage, reverse transcription is done template with this reverse transcription product again, carries out multi-PRC reaction, adds different combination of primers in the system.Reaction finishes, sampling electrophoresis, observations.The good combination of primers of screening expanding effect.
(8) multiplex PCR system specificity, susceptibility
(9) set up the multiplex PCR system, the assembling test kit.
Adopt N1/N2 combination of primers I, II, III, VI to various hypotype template detection results:
Table 5N1/N2 neuraminidase hypotype multiplex PCR system combination of primers (containing universal primer NP primer)
Utilize N1/N2/NP combination of primers I, II, III, VI all can detect corresponding N 1 and/or N2 hypotype band and animal influenza virus NP gene band to various hypotype template detection (H1N1, H3N2).(accompanying drawing 7)
Utilize H1/H3 combination of primers I, II can detect corresponding H1 and/or H3 hypotype purpose band to various hypotype template detection (H1N1, H3N2).(accompanying drawing 8)
Table 6H1/H3 hemagglutinin hypotype multiplex PCR system combination of primers (no universal primer NP)
The result shows, animal influenza virus H1 and/or H3 hemagglutinin hypotype that above-mentioned combination of primers all can effectively increase and contain in the sample.
Test kit
In order to help the commercialization of present method, special being equipped with, make things convenient for the test kit that uses, and contains following reagent and material in the box:
1. primer solution (20 μ mol/L): SEQ ID NO:1-26
2. working instructions (portion)
3. ThermoScript II AMV (5U/ul)
4.RNA enzyme inhibitors RNase Inhibitor (5U/ul)
5.0ligo?dT?Primer(2.5pmol/ul)
6.RNase?Free?dH
2O
7.Taq?DNA?polymerase(5U/ul)
8.10×RT?Buffer
9.5×PCR?Buffer
10.DNTP Mixture (each 10mM)
11.MgCl
2(25mM)
12.Primer?Mixture(20pmol/ul)
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Academy of Agricultural Sciences, Shanghai City
＜120〉a kind of primer collection and test kit thereof that detects swine influenza virus and blood serum subtype
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Claims (6)
1. one kind is used for the primer collection that same reaction system is carried out pcr amplification, it is characterized in that it comprises:
(1) primer of the universal NP gene of influenza virus is right; With
(2) primer of swine influenza virus hypotype is right;
Described swine influenza virus hypotype comprises hemagglutinin hypotype or neuraminidase hypotype;
Described hemagglutinin hypotype comprises H1 and/or H3; Described neuraminidase hypotype comprises N1 and/or N2;
The primer of described NP is to being SEQ ID NO:11 and 12.
2. primer collection as claimed in claim 1 is characterized in that, the primer of described H1 is to being SEQ ID NO:1 and 2; The primer of described H3 is to being selected from: SEQ ID NO:3 and 4 or SEQ ID NO:17 and 18.
3. primer collection as claimed in claim 1 is characterized in that, the primer of described N1 is to being selected from: SEQ IDNO:5 and 6 or SEQ ID NO:25 and 6; The primer of described N2 is to being selected from: SEQ ID NO:7 and 8 or SEQ ID NO:9 and 10.
4. one kind is used for the primer collection that same reaction system is carried out pcr amplification, it is characterized in that it comprises: the primer of swine influenza virus hypotype hemagglutinin hypotype H1 and H3 is right; The primer of described H1 is to being SEQ ID NO:1 and 2; The primer of described H3 is to being selected from: SEQ ID NO:3 and 4 or SEQ ID NO:17 and 18.
5. test kit is characterized in that it comprises:
1. primer collection as claimed in claim 1; With
2. specification sheets.
A non-diagnostic uses swine influenza virus is carried out the method for somatotype, it is characterized in that it comprises step:
(a) primer that adds the universal NP of influenza virus in an individual system carries out pcr amplification to right with the primer of SIV hemagglutinin hypotype H1 and H3, according to the electrophoretic band of amplified production, checks whether contain SIVH1 and/or H3 hemagglutinin hypotype in this system;
(b), then represent to exist in this system H1 and/or H3 hemagglutinin hypotype SIV if electrophoretic band demonstrates SIV hemagglutinin hypotype H1 and/or H3; If electrophoretic band does not show SIV hemagglutinin hypotype H1 and/or H3 band, then check whether contain influenza virus in this system according to the electrophoretic band of amplified production;
(c) if demonstrating, electrophoretic band contains influenza virus, the primer that then adds neuraminidase hypotype N1 and N2 in another system is right, carry out pcr amplification,, check whether contain neuraminidase N1 and/or N2 hypotype SIV in this system according to the electrophoretic band of amplified production;
(d), then represent to exist in this system neuraminidase N1 and/or N2 hypotype SIV if electrophoretic band demonstrates neuraminidase hypotype N1 and/or N2 respective strap; If electrophoretic band does not demonstrate N1 and N2 respective strap, represent not exist in this system neuraminidase N1 and/or N2 hypotype SIV;
The primer of described NP is to being SEQ ID NO:11 and 12;
Perhaps comprise step:
(a) in an individual system, add the primer of the universal NP of influenza virus to right with the primer of SIV neuraminidase hypotype N1 and N2, carry out pcr amplification, according to the electrophoretic band of amplified production, check whether contain SIV and whether be N1 and/or N2 neuraminidase subtype virus in this system;
(b), then represent to exist in this system N1 and/or N2 hypotype SIV neuraminidase if electrophoretic band demonstrates SIV neuraminidase hypotype N1 and/or N2; If electrophoretic band does not show SIV neuraminidase hypotype N1 and N2, then check whether contain influenza virus in this system according to the electrophoretic band of amplified production;
(c) if electrophoretic band demonstrates influenza virus, the primer that then adds SIV hemagglutinin hypotype H1 and H3 in another system is right, carries out pcr amplification, according to the electrophoretic band of amplified production, checks whether contain H1 and/or H3 hemagglutinin hypotype SIV in this system;
(d), then represent to exist in this system hemagglutinin hypotype H1 and/or H3 if electrophoretic band demonstrates hemagglutinin hypotype H1 and/or H3; If electrophoretic band does not demonstrate hemagglutinin hypotype H1 and/or H3, represent not exist in this system H1 and/or H3 hemagglutinin hypotype;
The primer of described NP is to being SEQ ID NO:11 and 12.
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| CN2007100419848A CN101323882B (en) | 2007-06-14 | 2007-06-14 | Primer set for detecting pig influenza virus and blood serum subtype and reagent box thereof |
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| CN101323882B true CN101323882B (en) | 2011-03-23 |
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| CN101629217B (en) * | 2009-06-30 | 2012-03-28 | 中国兽医药品监察所 | RT-LAMP detection kit and detection method of swine influenza virus |
| CN102534052B (en) * | 2012-03-16 | 2013-06-05 | 天津市畜牧兽医研究所 | Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV) |
| CN103014176B (en) * | 2012-12-17 | 2018-04-03 | 北京大北农科技集团股份有限公司动物医学研究中心 | Primer collection for the detection of swine influenza virus real-time fluorescence quantitative PCR |
| CN103484572A (en) * | 2013-10-16 | 2014-01-01 | 天津市畜牧兽医研究所 | Kit for detecting swine influenza viruses and application thereof |
| CN107326103B (en) * | 2017-08-28 | 2021-06-15 | 聊城大学 | Triple RT-PCR specific amplification primer group and triple-identification RT-PCR detection method |
| CN109468416A (en) * | 2018-12-29 | 2019-03-15 | 博奥生物集团有限公司 | The RT-LAMP detection primer of specific detection swine influenza virus and its application, detection reagent and method |
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| CN1635163A (en) * | 2004-11-01 | 2005-07-06 | 中国农业大学 | Process for detecting H5N1 hypotype Avian influenza virus and special-purpose reagent kit |
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| CN1635163A (en) * | 2004-11-01 | 2005-07-06 | 中国农业大学 | Process for detecting H5N1 hypotype Avian influenza virus and special-purpose reagent kit |
Non-Patent Citations (1)
| Title |
|---|
| Poddar S K.Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis.《Journal of Virological Methods》.2002,(第99期),63-70. * |
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