CN102321769A - Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof - Google Patents

Primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and application thereof Download PDF

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CN102321769A
CN102321769A CN201110303463A CN201110303463A CN102321769A CN 102321769 A CN102321769 A CN 102321769A CN 201110303463 A CN201110303463 A CN 201110303463A CN 201110303463 A CN201110303463 A CN 201110303463A CN 102321769 A CN102321769 A CN 102321769A
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primer
avian influenza
influenza virus
subtype avian
pcr
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CN102321769B (en
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蒲娟
唐庆冬
王金亮
包静楠
孙洪磊
刘金华
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China Agricultural University
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China Agricultural University
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Abstract

The invention discloses a primer pair for identifying newcastle disease virus and multi-subtype avian influenza virus and an application thereof. The PCR primer pair composition provided by the invention comprises primer pair NDV, primer pair H3V, primer pair H5V, and primer pair H9V; the PCR primer pair composition is applicable to the preparation of reagents for the diagnosis or auxiliary diagnosis of newcastle disease and avian influenza, or to the preparation of reagents for the identification or auxiliary identification of newcastle disease virus and multi-subtype avian influenza virus. When the PCR primer pair composition provided by the invention is used to simultaneously detect newcastle disease virus, H3, H5 and H9-subtype avian influenza virus, the specificity is strong; the sensitivity is 1EID50/100 microliters, 1EID50/100 microliters, 1*10-2EID50/100 microliters, and 1EID50/100 microliters respectively; compared with identification results of routine test methods such as virus isolation and hemagglutination inhibition tests, the results obtained by using the PCR primer pair composition of the invention has a coincidence rate of up to 100%; the PCR primer pair composition provided by the invention is applicable to the development of corresponding multiplex RT-PCR kits for the clinical diagnosis and epidemiological control of newcastle disease and avian influenza.

Description

The primer of identifying NDV and many subtype avian influenza virus to and use
Technical field
The present invention relates to a kind of primer of identifying NDV and many subtype avian influenza virus to and use.
Background technology
Bird flu and newcastle disease are respectively two kinds of viral infectious that caused by bird flu virus and NDV.Two kinds of infectious disease incidences are anxious, propagate soon, and sickness rate is high, can cause fatefulue strike to aquaculture.China chicken crowd's the hypotype of bird flu virus of being popular in is mainly H5 and H9 hypotype.Very easily producer variation of influenza virus, thus antigenic change caused.Therefore, though China takes immune measure comprehensively, H5 and H9 subtype influenza virus still take place in the chicken crowd repeatedly.The variation of H5 and H9 subtype avian influenza virus gene and antigenic characteristic causes conventional serological method and molecular biology monitoring method can't detect epidemic isolates, therefore need set up a kind of versatility good detection method that can detect the current popular strain.In addition; Epidemiology survey shows that there is the seropositivity antibody of H3 subtype influenza virus in China most of areas chicken crowd, and the report that from the chicken crowd, is separated to the H3 subtype avian influenza virus is arranged; Therefore, the H3 subtype avian influenza virus also should be as one of object of influenza virus monitoring.In recent years, NDV causes that with bird flu virus the sick clinical symptom of chicken mass-sending is tending towards similar, can't make a definite diagnosis with the routine clinical diagnostic method, presses for and sets up the differential diagnosis that a kind of detection method is used for newcastle disease and bird flu.
For traditional detection method, like virus separation, hemagglutination-inhibition test, neuraminic acid enzyme test etc., time-consuming, effort can not be made diagnosis fast and timely; And can not in same detection reaction, detect different objects, promptly can't carry out differential diagnosis simultaneously.The RT-PCR method has good, the characteristics such as save time of susceptibility height, specificity, and multiplex RT-PCR method can detect multiple cause of disease simultaneously in a reaction.At present, also there is not both at home and abroad to detect simultaneously the multiplex RT-PCR method of newcastle disease and H3, H5, H9 subtype avian influenza virus.Therefore; Clinically; Press for the detection method of a kind of ability rapid differential diagnosis newcastle disease and different subtype avian influenza viruses; Can confirm that not only whether virus is NDV or bird flu virus, can also confirm the hypotype of bird flu virus, and current epidemic isolates is had good detection spreadability.
Summary of the invention
The purpose of this invention is to provide a kind of primer of identifying NDV and many subtype avian influenza virus to and use.
The primer of identifying NDV and many subtype avian influenza virus provided by the present invention to for the PCR primer to compsn A, be following 1)-4) in any one:
1) said PCR primer to compsn A by following 4 primers to forming: primer to NDV, primer to H3V, primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus, H5 subtype avian influenza virus and the H9 subtype avian influenza virus;
Said primer is formed by two single stranded DNAs H9V H5V and primer H3V, primer NDV, primer;
Said primer is that the primer that the single stranded DNA shown in the sequence 2 is formed in the single stranded DNA shown in the sequence 1 and the sequence table in the sequence table is right to NDV;
Said primer is that the primer that the single stranded DNA shown in the sequence 4 is formed in the single stranded DNA shown in the sequence 3 and the sequence table in the sequence table is right to H3V;
Said primer is that the primer that the single stranded DNA shown in the sequence 6 is formed in the single stranded DNA shown in the sequence 5 and the sequence table in the sequence table is right to H5V;
Said primer is that the primer that the single stranded DNA shown in the sequence 8 is formed in the single stranded DNA shown in the sequence 7 and the sequence table in the sequence table is right to H9V;
2) said PCR primer to compsn A by following 3 primers to forming: said primer to NDV, primer to H3V and primer to H5V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H5 subtype avian influenza virus;
3) said PCR primer to compsn A by following 3 primers to forming: said primer to NDV, primer to H3V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H9 subtype avian influenza virus;
4) said PCR primer to compsn A by following 3 primers to forming: said primer to NDV, primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H5 subtype avian influenza virus and the H9 subtype avian influenza virus.
The present invention also provide a kind of identify or the PCR primer of the many subtype avian influenza virus of assistant identification to compsn B, be following 5)-8) in any one:
5) said PCR primer to compsn B by following 3 primers to forming: said primer to H3V, primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus, H5 subtype avian influenza virus and the H9 subtype avian influenza virus;
6) said PCR primer to compsn B by following 2 primers to forming: said primer to H3V and primer to H5V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H5 subtype avian influenza virus;
7) said PCR primer to compsn B by following 2 primers to forming: said primer to H3V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H9 subtype avian influenza virus;
8) said PCR primer to compsn B by following 2 primers to forming: said primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H5 subtype avian influenza virus and the H9 subtype avian influenza virus.
The present invention also protects the said primer of evaluation or assistant identification H5 subtype avian influenza virus to H5V.
Said 1) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,3,4,5,6,7 and 8: 1: 1: 1: 1: 1; Said 2) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,3,4,5 and 6: 1: 1: 1; Said 3) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,3,4,7 and 8: 1: 1: 1; Said 4) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,5,6,7 and 8: 1: 1: 1.
Said 5) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 3,4,5,6,7 and 8: 1: 1: 1; Said 6) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 3,4,5 and 6: 1; Said 7) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 3,4,7 and 8: 1; Said 8) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 5,6,7 and 8: 1.
The present invention protects following any one purposes in a)-f):
A) said PCR primer is to the application of compsn A in preparation diagnosis or auxiliary diagnosis newcastle disease and bird flu reagent or test kit;
B) said PCR primer is to the application of compsn B in preparation diagnosis or auxiliary diagnosis bird flu reagent or test kit;
C) said PCR primer is to the application of H5V in preparation diagnosis or auxiliary diagnosis H5 subtype avian influenza reagent or test kit.
D) said PCR primer is to the application of compsn A in preparation evaluation or assistant identification NDV and many subtype avian influenza virus reagent or test kit;
E) said PCR primer is to the application of compsn B in preparation evaluation or many subtype avian influenza virus of assistant identification reagent or test kit;
F) said PCR primer is to the application of H5V in preparation evaluation or assistant identification H5 subtype avian influenza virus reagent or test kit.
The present invention also protects following A)-any one reagent in C):
A) reagent of evaluation or assistant identification NDV and many subtype avian influenza virus contains said PCR primer to compsn A in the said reagent;
B) reagent of evaluation or the many subtype avian influenza virus of assistant identification contains said PCR primer to compsn B in the said reagent;
C) reagent of evaluation or assistant identification H5 subtype avian influenza virus contains said PCR primer to H5V in the said reagent.
The present invention also protects following D)-any one PCR test kit in F):
D) the PCR test kit of evaluation or assistant identification NDV and many subtype avian influenza virus contains said A in the said PCR test kit) reagent;
E) the PCR test kit of evaluation or the many subtype avian influenza virus of assistant identification contains said B in the said PCR test kit) reagent;
F) the PCR test kit of evaluation or assistant identification H5 subtype avian influenza virus contains said C in the said PCR test kit) reagent.
The present invention also protects following G)-any one preparation method in I):
G) said preparation method comprises the step that said PCR primer is packed separately respectively the single stranded DNA of compsn A;
H) said preparation method comprises the step that said PCR primer is packed separately respectively the single stranded DNA of compsn B;
I) said preparation method comprises the step that said primer is packed separately respectively the single stranded DNA of H5V.
The present invention also protects the preparation method of said PCR test kit; Comprise the steps: said PCR primer compsn A, said PCR primer compsn B or said PCR primer to the single stranded DNA of H5V respectively separately after the packing; Be packaged in the same test kit with at least a material in the following substances: archaeal dna polymerase and following 4 kinds of dNTP:dATP; DTTP, dCTP and dGTP.
PCR primer provided by the present invention is to detectable viral species and branch thereof or genotype respectively as follows: the PCR primer specifically can be II type, III type, VII type or IX type NDV to the NDV that NDV can detect different genotype; The PCR primer can detect dissimilar H3 subtype avian influenza virus to H3V, specifically can be the H3N8 subtype avian influenza virus; The PCR primer can detect dissimilar and different ramose H5 subtype avian influenza virus to H5V, specifically can be 2.3.2 branch, 2.3.4 branch or 7 ramose H5N1 subtype avian influenza virus; The PCR primer can detect dissimilar and different ramose H9 subtype avian influenza virus to H9V, specifically can be G1-like branch or BJ/94-like ramose H9N2 subtype avian influenza virus; But be not limited to the above-mentioned type and the branch or the genotype of above-mentioned virus.
PCR primer provided by the present invention is distinguished as follows detectable viral source: the PCR primer can detect the NDV in Ji Yuan and duck source to NDV; The PCR primer can detect the H3 subtype avian influenza virus in duck source to H3V, and the PCR primer can detect the H5 subtype avian influenza virus in Ji Yuan, duck source and sparrow source to H5V; The PCR primer can detect the H9 subtype avian influenza virus of Ji Yuan, duck source or quail source to H9V; But be not limited to the above-mentioned source of above-mentioned virus.
Use 4 kinds of primers provided by the present invention to being used to detect the high specificity of NDV, H3, H5 and H9 subtype avian influenza virus simultaneously, sensitivity is respectively 1EID 50/ 100 μ l, 1EID 50/ 100 μ l, 1 * 10 -2EID 50/ 100 μ l and 1EID 50/ 100 μ l separate the qualification result that suppresses normal experiment methods such as experiment with blood clotting with virus and compare, and coincidence rate reaches 100%.The present invention compared with prior art; Highly sensitive, high specificity, accuracy rate with single RT-PCR technology are high; A RT-PCR can realize that to 4 kinds of viruses in the clinical sample be the detection of NDV, H3, H5 and H9 subtype avian influenza virus, and can detect the epidemic isolates among China fowl crowd in recent years, and cost is lower; Be fit to the corresponding multiple RT-PCR kit of exploitation, with clinical diagnosis and the prevailing disease control that is used for newcastle disease and bird flu.
Description of drawings
Fig. 1 is the specific detection of primer to NDV.Wherein, M is Maker; 1-6 is respectively the sample that infects NDV F48E9, Chicken/Henan/2009, Chicken/ShangDong/2010, Chicken/Hebei/2010, Duck/ShangDong/2011 or HG/Beijing/2009; 7-11 is respectively the sample that infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 2 is the specific detection of primer to H3V.Wherein, M is Maker; 1-6 is respectively the sample that infects H3 subtype avian influenza virus A/Duck/BeiJing/33/2004, A/Duck/BeiJing/40/2004, A/Duck/BeiJing/44/2004, A/Duck/BeiJing/56/2005, A/Duck/BeiJing/59/2005 or A/Duck/BeiJing/61/2005; 7-11 is respectively the sample that infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 3 is the specific detection of primer to H5V.Wherein, M is Maker; 1-6 is respectively the sample that infects H5 subtype avian influenza virus A/Chicken/Huabei/202/2010, A/Duck/Huabei/1/2011, A/treesparrow/Jiangsu/1/2008, A/Chicken/Huabei/204/2010, A/Duck/Huabei/7/2009 or A/Chicken/Huabei/7/2011; 7-11 is respectively the sample that infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 4 is the specific detection of primer to H9V.Wherein, M is Maker; 1-6 is respectively the sample that infects H9 subtype avian influenza virus A/Quail/HongKong/G1/1997, A/Chicken/Beijing/3/1999, A/Chicken/FX/01/2010, A/Duck/ShangDong/2CP/2010, A/Chicken/Sichuan/8/2011 or A/Duck/ZhuCheng/1/2011; 7-11 is respectively the sample that infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 5 is the specific detection of quadruple RT-PCR.Wherein, M is Maker; 1 for infecting the mixture of the cDNA sample of H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, H5 subtype avian influenza virus A/treesparrow/Jiangsu/1/2008, H3 subtype avian influenza virus A/Duck/Beijing/40/2004 and NDV HG/Beijing/2009 respectively; 2-5 is respectively the cDNA sample that infects H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008, NDV HG/Beijing/2009 and H3 subtype avian influenza virus A/Duck/Beijing/40/2004; 6-10 is respectively the sample that infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV, IBDV and reovirus, 11 negative chick embryo allantoic liquid contrasts.
Fig. 6 detects the chick embryo allantoic liquid sample result of the different virus strain infections of newcastle disease, H3, H5 or H9 subtype avian influenza virus respectively for quadruple RT-PCR.Wherein figure (a) is the H3 subtype avian influenza virus, and figure (b) is the H5 subtype avian influenza virus, and figure (c) is the H9 subtype avian influenza virus, and figure (d) is a NDV; The M of figure in (a)-(d) is molecular weight Maker, and 1 with 1 sample among Fig. 5; The sample of 1-6 among same respectively Fig. 2 of 2-7,3,4,1 among the figure (a)-(d); 8 negative chick embryo allantoic liquid contrasts.
Fig. 7 is a primer to H3V, H5V, H9V and NDV respectively to the sensitivity determination of H3, H5, H9 subtype avian influenza virus and NDV.Wherein, H3 represents the H3 subtype avian influenza virus, and H5 represents the H5 subtype avian influenza virus, and H9 represents the H9 subtype avian influenza virus, and ND represents NDV; 1-7 is respectively H3, H5, H9 subtype avian influenza virus or the NDV of different content among the figure, is followed successively by 1 * 10 4EID 50/ 100 μ l, 1 * 10 3EID 50/ 100 μ l, 1 * 10 2EID 50/ 100 μ l, 1 * 10EID 50/ 100 μ l, 1EID 50/ 100 μ l, 1 * 10 -1EID 50/ 100 μ l, 1 * 10 -2EID 50/ 100 μ l, 8 negative chick embryo allantoic liquid contrasts.
Fig. 8 is that the sensitivity of quadruple RT-PCR method detects.Wherein, Figure (a) be quadruple RT-PCR respectively to the sample detection of H3, H5, H9 subtype avian influenza virus or the NDV of different content, H3 wherein represents the H3 subtype avian influenza virus, H5 represents the H5 subtype avian influenza virus; H9 represents the H9 subtype avian influenza virus; ND represents NDV, and 1-7 is respectively the sample of H3, H5, H9 subtype avian influenza virus or the NDV of different content, and each viral level of sample is followed successively by 1 * 10 4EID 50/ 100 μ l, 1 * 10 3EID 50/ 100 μ l, 1 * 10 2EID 50/ 100 μ l, 1 * 10 1EID 50/ 100 μ l, 1EID 50/ 100 μ l, 1 * 10 -1EID 50/ 100 μ l, 1 * 10 -2EID 50/ 100 μ l, 8 negative chick embryo allantoic liquid contrasts; Figure (b) detects for the sensitivity of quadruple RT-PCR method to the mixture of the sample that contains H3, H5, H9 subtype avian influenza virus or NDV, and M is Maker; 1-5 is respectively different dilution viral sample mixtures, and each viral content is followed successively by 1 * 10 in every kind of sample mixture 3EID 50/ 100 μ l, 1 * 10 2EID 50/ 100 μ l, 1 * 10 1EID 50/ 100 μ l, 1EID 50/ 100 μ l, 1 * 10 -1EID 50/ 100 μ l, 6 negative chick embryo allantoic liquid contrasts.
Fig. 9 is the detections of the triple RT-PCR methods of double sum to bird flu virus and NDV.Wherein, M is Maker; 1 is that primer makes up H9V, H5V and NDV, and 2 is that primer makes up H9V, H3V and NDV, and 3 is that primer is to H5V, H3V and NDV combination; 4 is that primer makes up H9V, H5V and H3V, and 5 is that primer makes up H5V and H3V, and 6 is that primer is to H9V and H3V combination; 7 is that primer makes up H3V and NDV, and 8 is that primer makes up H9V and H5V, and 9 is that primer is to H5V and NDV combination; 10 is that primer makes up H9V and NDV, 11 negative chick embryo allantoic liquids contrasts.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The employed virus stain information of following embodiment is following:
Table 1 strain information used in the present invention
Embodiment 1, PCR primer are to design
Utilize NDV and the up-to-date sequence of bird flu virus among the GenBank, following to H3 hypotype, H5 hypotype and H9 subtype avian influenza virus HA gene and 4 pairs of primers of F gene of NDV strain design:
Be the target gene designed primer with the F gene of NDV strain to NDV:
Upstream primer NDV-275F:5 '-TCACTCCTCTTGGCGACTC-3 ' (sequence table sequence 1 sequence);
Downstream primer NDV-556R:5 '-CAAACTGCTGCATCTTCC-3 ' (sequence table sequence 2 sequences);
The primer that with H3 subtype avian influenza virus HA gene is target gene is to H3V:
Upstream primer H3-622F:5 '-TTCACCACCCGAGCACAA-3 ' (sequence table sequence 3 sequences);
Downstream primer H3-837R:5 '-GGGCGATTAGGTTTCCATTA-3 ' (sequence table sequence 4 sequences);
The primer that with H5 subtype avian influenza virus HA gene is target gene is to H5V:
Upstream primer H5-299F:5 '-ACCCAGCCAATGACCTCT-3 ' (sequence table sequence 5 sequences);
Downstream primer H5-718R:5 '-CACTTTGCCCGTTTACTT-3 ' (sequence table sequence 6 sequences);
The primer that with H9 subtype avian influenza virus HA gene is target gene is to H9V:
Upstream primer H9-544F:5 '-ATTCAAGACGCCCAATACAC-3 ' (sequence table sequence 7 sequences);
Downstream primer H9-1092R:5 '-TGACCAACCTCCCTCTATGA-3 ' (sequence table sequence 8 sequences).
The annealing temperature of above-mentioned 4 pairs of primers is 51 ℃.
Primer is 275-293 and the 539-556 position of Genbank HQ917083 to the target sequence of NDV.
Primer is 622-639 position and the 818-837 position of Genbank EU492531 to the target sequence of H3V.
Primer is 299-316 position and the 701-718 position of Genbank DQ993028 to the target sequence of H5V.
Primer is 544-563 position and the 1073-1092 position of Genbank HM773439 to the target sequence of H9V.
Embodiment 2, the right specific detection of PCR primer
One, primer is to the specific detection of NDV
1, total RNA extracts and quality control
Get respectively and infect NDV F48E9, Chicken/Henan/2009, Chicken/ShangDong/2010, Chicken/Hebei/2010, Duck/ShangDong/2011 or HG/Beijing/2009 (Rui, Z., Juan; P., Jingliang, S.; Jixun, Z., Xiaoting; W., Shouping, Z.; Xiaojiao; L.and Guozhong, Z., 2010a.Phylogenetic characterization of Newcastle disease virus isolatedin the mainland of China during 2001-2009.Vet Microbiol 141; 246-57.) chick embryo allantoic liquid and chick embryo allantoic liquid and the negative chick embryo allantoic liquid that infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or the reovirus (REO) of similar NDV, extract total RNA (method is with embodiment 4) respectively.Measure its OD value with nucleic acid-protein analyser BioPhotometer, draw its concentration and Reinheitszahl, and control the nucleic acid quality with this.
2, the synthetic cDNA of reverse transcription
(K1622 Fermentas) carries out reverse transcription with total RNA sample of step 1 respectively, obtains cDNA with the reverse transcription test kit; DEPC water is as the contrast of total RNA.
Reaction system (20 μ L): 1 μ L Oligo primer (20pmol/ μ L), 4 μ L Reaction (5 *), 1 μ LRibolock TMRnase Inhibitor (20U/ μ L), 2 μ L 10mM dNTPMix, 1 μ L RevertAid TMM-MuLVReverse Transcriptase (200U/ μ L), the total RNA of 650ng, DEPC water complement to 20 μ L.
Reaction conditions: after adding the DEPC water RNA of the extraction (dissolving wherein) and oligo primer, wink leaves, ice bath immediately behind 65 ℃ of 5min.After adding all the other five kinds, wink leaves, 37 ℃ of 1h, 70 ℃ of 5min again, 4 ℃ of preservations.
3, pcr amplification detects the right specificity of primer
Is primer with primer to NDV, and the cDNA sample that step 2 is obtained carries out pcr amplification.
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111; The Beijing Quanshijin Biotechnology Co., Ltd, its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer), concentration is 20pmol/ μ L sequence table sequence 1 primer 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 2 primers 0.5 μ L; CDNA2 μ L, DEPC water complement to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (Fig. 1); All obtain the PCR product band of a size between 200-300bp in the chick embryo allantoic liquid sample that F48E9, Chicken/Henan/2009, Chicken/ShangDong/2010, Chicken/Hebei/2010, HG/Beijing/2009 and Duck/ShangDong/2011 infect, learn that through order-checking the size of this band is 282bp; In the chick embryo allantoic liquid sample that negative chick embryo allantoic liquid and H4N6 subtype avian influenza virus, H6N1 subtype avian influenza virus, IBV, IBDV and REO infect between 200-300bp no PCR product band.The result shows that primer is fine to the specificity of NDV, can special detection NDV.
Two, primer is to the specific detection of H3V
1, total RNA extracts and quality control
Get respectively and infect H3 subtype avian influenza virus A/Duck/BeiJing/33/2004, A/Duck/BeiJing/40/2004, A/Duck/BeiJing/44/2004, A/Duck/BeiJing/56/2005, A/Duck/BeiJing/59/2005 or A/Duck/BeiJing/61/2005 (Pu; J., Liu, Q.F.; Xia; Y.J., Fan, Y.L.; Brown; E.G., Tian, F.L.and Liu; J.H.; 2009.Genetic analysis ofH3 subtype influenza viruses isolated from domestic ducks in northern Chinaduring 2004-2005.Virus Genes 38, the chick embryo allantoic liquid and the negative chick embryo allantoic liquid of chick embryo allantoic liquid 136-42.) and the H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or the reovirus (REO) that infect similar H3 subtype avian influenza virus extract total RNA (method is with embodiment 4) respectively.Measure its OD value with nucleic acid-protein analyser BioPhotometer, draw its concentration and Reinheitszahl, and control the nucleic acid quality with this.
2, the synthetic cDNA of reverse transcription
Method is with the step 2 in the step 1.
3, pcr amplification detects the right specificity of primer
Is primer with primer to H3V, and the cDNA sample that step 2 is obtained carries out pcr amplification.
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111; The Beijing Quanshijin Biotechnology Co., Ltd, its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer), concentration is 20pmol/ μ L sequence table sequence 3 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 4 primers 0.5 μ L; CDNA2 μ L, DEPC water complement to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (Fig. 2); All obtain the PCR product band of a size between 200-300bp in the chick embryo allantoic liquid sample that A/Duck/BeiJing/33/2004, A/Duck/BeiJing/40/2004, A/Duck/BeiJing/44/2004, A/Duck/BeiJing/56/2005, A/Duck/BeiJing/59/2005 or A/Duck/BeiJing/61/2005 infect, learn that through order-checking the size of this band is 216bp.In the chick embryo allantoic liquid sample that negative chick embryo allantoic liquid and H4N6 subtype avian influenza virus, H6N1 subtype avian influenza virus, IBV, IBDV or REO infect between 200-300bp no PCR product band.The result shows that primer is fine to the specificity of H3V, can specially detect the H3 subtype avian influenza virus.
Three, primer is to the specific detection of H5V
1, total RNA extracts and quality control
Get respectively and infect H5 subtype influenza virus of A/Chicken/Huabei/202/2010, A/Duck/Huabei/1/2011, A/Chicken/Huabei/204/2010, A/Duck/Huabei/7/2009, A/Chicken/Huabei/7/2011 or A/tree sparrow/Jiangsu/1/2008 (Liu; Q., Ma, J.; Kou; Z., Pu, J.; Lei; F., Li, T.and Liu; J.; 2010b.Characterization of a highlypathogenic avian influenza H5N1 clade 2.3.4 virus isolated from a tree sparrow.Virus Res 147, the chick embryo allantoic liquid and the negative chick embryo allantoic liquid of chick embryo allantoic liquid 25-9.) and the H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or the reovirus (REO) that infect similar H5 subtype avian influenza virus extract total RNA (method is with embodiment 4) respectively.Measure its OD value with nucleic acid-protein analyser BioPhotometer, draw its concentration and Reinheitszahl, and control the nucleic acid quality with this.
2, the synthetic cDNA of reverse transcription
Method is with the step 2 in the step 1.
3, pcr amplification detects the right specificity of primer
Is primer with primer to H5V, to step 2) the cDNA sample that obtains carries out pcr amplification.
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111; The Beijing Quanshijin Biotechnology Co., Ltd, its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer), concentration is 20pmol/ μ L sequence table sequence 5 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 6 primers 0.5 μ L; CDNA2 μ L, DEPC water complement to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (Fig. 3), all obtain the PCR product band of a size between 400-500bp in the chick embryo allantoic liquid sample that A/Chicken/Huabei/202/2010, A/Duck/Huabei/1/2011, A/tree sparrow/Jiangsu/1/2008, A/Chicken/Huabei/204/2010, A/Duck/Huabei/7/2009 or A/Chicken/Huabei/7/2011 infect and learn that through order-checking the size of this band is 420bp.In the chick embryo allantoic liquid sample that negative allantoic fluid and H4N6 subtype avian influenza virus, H6N1 subtype avian influenza virus, IBV, IBDV or REO infect between 400-500bp no PCR product band.The result shows that primer is fine to the specificity of H5V, can specially detect the H5 subtype avian influenza virus.
Four, primer is to the specific detection of H9V
1, total RNA extracts and quality control
Get respectively and infect H9 subtype avian influenza virus A/Chicken/FX/01/2010, A/Duck/ShangDong/2CP/2010, A/Chicken/Sichuan/8/2011, A/Duck/ZhuCheng/1/2011, A/Quail/HongKong/G1/1997 or A/Chicken/Beijing/3/1999 (Sun, Y., Pu, J.; Jiang, Z., Guan, T.; Xia, Y., Xu, Q.; Liu, L., Ma; B., Tian, F.; Brown, E.G.and Liu, J.; 2010.Genotypic evolution and antigenic drift ofH9N2 influenza viruses in China from 1994 to 2008.Vet Microbiol 146, the chick embryo allantoic liquid and the negative chick embryo allantoic liquid sample of chick embryo allantoic liquid 215-25.) and the H4N6 subtype avian influenza virus, H6N1 subtype avian influenza virus, IBV, IBDV or the reovirus that infect similar H9 subtype avian influenza virus extract total RNA (method for extracting total RNA is with embodiment 4) respectively.Measure its OD value with nucleic acid-protein analyser BioPhotometer, draw its concentration and Reinheitszahl, and control the nucleic acid quality with this.
2, the synthetic cDNA of reverse transcription
Method is with the step 2 in the step 1.
3, pcr amplification detects the right specificity of primer
Is primer with primer to H9V, and the cDNA sample that step 2 is obtained carries out pcr amplification.
Reaction system (25 μ L): reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111; The Beijing Quanshijin Biotechnology Co., Ltd, its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer), concentration is 20pmol/ μ L sequence table sequence 7 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 8 primers 0.5 μ L; CDNA2 μ L, DEPC water complement to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (Fig. 4), all obtain the PCR product band of a size between 500-600bp in the chick embryo allantoic liquid sample that A/Quail/HongKong/G1/1997, A/Chicken/Beijing/3/1999, A/Chicken/FX/01/2010, A/Duck/ShangDong/2CP/2010, A/Chicken/Sichuan/8/2011 or A/Duck/ZhuCheng/1/2011 infect and learn that through order-checking the size of this band is 549bp.In the chick embryo allantoic liquid sample that negative allantoic fluid and H4N6 subtype avian influenza virus, H6N1 subtype avian influenza virus, IBV, IBDV or REO infect between 500-600bp no PCR product band.The result shows that primer is fine to the specificity of H9V, can specially detect the H9 subtype avian influenza virus.
Five, primer is to the mixed specific detection of NDV, H3V, H5V and H9V
Primer is added same PCR reaction system simultaneously to NDV, H3V, H5V and H9V, and the cDNA sample of the chick embryo allantoic liquid that respectively NDV HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/Beijing/40/2004, H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008, H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV (IBV), infectious bursa of Fabricius virus (IBDV) or reovirus (REO) is infected and the mixture of cDNA sample that infects the chick embryo allantoic liquid of above-mentioned preceding 4 virus stains respectively detect (result is as shown in Figure 5); And the cDNA sample in the above-mentioned steps one to four detected (result is shown in Fig. 6 (a)-(d)).
Reaction system (25 μ L): reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111; The Beijing Quanshijin Biotechnology Co., Ltd, its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer), concentration is 20pmol/ μ L sequence table sequence 1 primer 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 2 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 3 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 4 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 5 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 6 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 7 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 8 primers 0.5 μ L, 8 μ L cDNA (each 2 μ L of the cDNA of 4 kinds of samples mix) or 2 μ L cDNA (a kind of sample of virus strain infection); DEPC water complements to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis, result such as Fig. 5 and Fig. 6.In the biased sample that NDV HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/Beijing/40/2004, H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008 and H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infect respectively, amplify 4 PCR product bands simultaneously; Be respectively 216bp, 282bp, 420bp, 549bp through order-checking; All can amplify corresponding band in the step 1 to four in the sample that the different strains of 4 kinds of viruses infect respectively, no any specific band in the sample of negative allantoic fluid and H4N6 subtype avian influenza virus, H6N1 subtype avian influenza virus, IBV, IBDV and reovirus infection.
The above results shows: primer mixes the back to NDV, H3V, H5V and H9V does not have influence to the right specificity of each primer, can be in reaction of a sample special detection NDV, H3 hypotype, H5 hypotype and H9 subtype avian influenza virus simultaneously.
Embodiment 3, the right sensitivity of PCR primer detect
One, primer detects the sensitivity of NDV
1, the preparation of total RNA extraction and each diluent
Get the chick embryo allantoic liquid that infects NDV HG/Beijing/2009, adopt the Reed-Muench method to carry out viral EID 50Mensuration, obtain the toxic amount of this NDV in chick embryo allantoic liquid.According to this viral level, using aseptic PBS is 10 with this chick embryo allantoic liquid dilution as virus concentration 6EID 50(virus concentration like chick embryo allantoic liquid is 10 to/100 μ l 7EID 50/ 100 μ l then carry out 10 times of dilutions (10 7/ 10 6=10)), carry out 10 times of doubling dilutions then on this basis, in each diluent, viral level is respectively 10 5EID 50/ 100 μ l, 10 4EID 50/ 100 μ l, 10 3EID 50/ 100 μ l, 10 2EID 50/ 100 μ l, 10 1EID 50/ 100 μ l, 10 0EID 50/ 100 μ l, 10 -1EID 50/ 100 μ l, 10 -2EID 50/ 100 μ l.Measure virus concentration 10 4EID 50/ 100 μ l-10 -2EID 50The detection sensitivity of sample between/100 μ l.Get 10 4EID 50/ 100 μ l-10 -2EID 50Each dilution allantoic fluid sample between/100 μ l is extracted total RNA (method for extracting total RNA is with embodiment 4) respectively.
2, the synthetic cDNA of reverse transcription
(K1622 Fermentas) carries out reverse transcription with the total RNA that extracts in each diluent respectively, obtains cDNA with the reverse transcription test kit; The reverse transcription product of negative chick embryo allantoic liquid is as the contrast of total RNA.
Reaction system (20 μ L): 1 μ L Oligo primer (20pmol/ μ L), 4 μ L Reaction (5 *), 1 μ L Ribolock TMRnase Inhibitor (20U/ μ L), 2 μ L 10mM dNTP Mix, 1 μ L RevertAi TMM-MuLV ReverseTranscriptase (200U/ μ L), the total RNA of 11 μ L, DEPC water complements to 20 μ L.
Reaction conditions: after adding the DEPC water RNA of the extraction (dissolving wherein) and oligo primer, wink leaves, ice bath immediately behind 65 ℃ of 5min.After adding all the other five kinds, wink leaves, 37 ℃ of 1h, 70 ℃ of 5min again, 4 ℃ of preservations.
3, pcr amplification detects the right sensitivity of primer
Is primer with primer to NDV, and the cDNA sample that step 2 is obtained carries out pcr amplification.
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111; The Beijing Quanshijin Biotechnology Co., Ltd, its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer), concentration is 20pmol/ μ L sequence table sequence 1 primer 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 2 primers 0.5 μ L; CDNA 2 μ L, DEPC water complements to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (ND among result such as Fig. 7), 1EID 50Can be observed the amplified band of 282bp among total RNA of/100 μ l and above virus concentration sample.The result shows that primer can reach 1 EID to the detection sensitivity of HDV 50/ 100 μ l promptly detect under the viral level of sample and are limited to 1 EID 50/ 100 μ l.
Two, primer detects the sensitivity of H3V
1, the preparation of total RNA extraction and each diluent
Get the chick embryo allantoic liquid that infects H3 subtype avian influenza virus A/Duck/Beijing/40/2004, the preparation method is with 1 in the step 1.
2, the synthetic cDNA of reverse transcription
Method is with 2 in the step 1.
3, pcr amplification detects the right sensitivity of primer
Is primer with primer to H3V, and the cDNA sample that step 2 is obtained carries out pcr amplification, and method is with 3 in the step 1.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (H3 among result such as Fig. 7), 1EID 50Can be observed the amplified band of 216bp among total RNA of/100 μ l and above virus concentration sample.The result shows that primer can reach 1EID to the detection sensitivity of H3V 50/ 100 μ l promptly detect under the viral level of sample and are limited to 1EID 50/ 100 μ l.
Three, primer detects the sensitivity of H5V
1, the preparation of total RNA extraction and each diluent
Get the chick embryo allantoic liquid that infects H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008, the preparation method is with 1 in the step 1.
2, the synthetic cDNA of reverse transcription
Method is with 2 in the step 1.
3, pcr amplification detects the right sensitivity of primer
Is primer with primer to H5V, and the cDNA sample that step 2 is obtained carries out pcr amplification, and method is with 3 in the step 1.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (H5 among result such as Fig. 7), 1 * 10 -2EID 50Can be observed the amplified band of 420bp among total RNA of/100 μ l and above virus concentration sample.The result shows that primer can reach 1 * 10 to the detection sensitivity of H5V -2EID 50/ 100 μ l promptly detect under the viral level of sample and are limited to 1 * 10 -2EID 50/ 100 μ l.
Four, primer detects the sensitivity of H9V
1, the preparation of total RNA extraction and each diluent
Get the chick embryo allantoic liquid that infects H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, the preparation method is with 1 in the step 1.
2, the synthetic cDNA of reverse transcription
Method is with 2 in the step 1.
3, pcr amplification detects the right sensitivity of primer
Is primer with primer to H9V, and the cDNA sample that step 2 is obtained carries out pcr amplification, and method is with 3 in the step 1.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis (H9 among result such as Fig. 7), 1EID 50Can be observed the amplified band of 549bp among total RNA of/100 μ l and above virus concentration sample.The result shows that primer can reach 1EID to the detection sensitivity of H9V 50/ 100 μ l promptly detect under the viral level of sample and are limited to 1EID 50/ 100 μ l.
Five, primer detects the sensitivity that it is right that NDV, H3V, H5V and H9V mix each primer of back
1, primer is added same PCR reaction system simultaneously to NDV, H3V, H5V and H9V, the cDNA sample of the different virus content that respectively different virus in above-mentioned to four is infected detects separately respectively.
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer for AS111, Beijing Quanshijin Biotechnology Co., Ltd); Concentration is 20pmol/ μ L sequence table sequence 1 primer 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 2 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 3 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 4 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 5 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 6 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 7 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 8 primers 0.5 μ L; 2 μ L cDNA, DEPC water complements to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 45s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 10h.
Electrophoresis: get PCR product 1% agarose gel electrophoresis (result such as Fig. 8 (a)), in the detection of each extent of dilution sample of chick embryo allantoic liquid that the H3 subtype avian influenza virus is infected, 10 4EID 50/ 100 μ l-10 1EID 50Dilution sample can be observed the PCR product band of 216bp, 10 between/100 μ l 0EID 50/ 100 μ l-10 -2EID 50Dilution sample is not observed the PCR product band of 216bp between/100 μ l; In the detection of each extent of dilution sample of chick embryo allantoic liquid that the H5 subtype avian influenza virus is infected, 10 4EID 50/ 100 μ l-10 -1EID 50Dilution sample can be observed the PCR product band of 420bp, 10 between/100 μ l -2EID 50The dilution sample of/100 μ l is not observed the PCR product band of 420bp; In the detection of each extent of dilution sample of chick embryo allantoic liquid that the H9 subtype avian influenza virus is infected, 10 4EID 50/ 100 μ l-10 0EID 50Dilution sample can be observed the PCR product band of 549bp, 10 between/100 μ l -1EID 50/ 100 μ l-10 -2EID 50The dilution sample of/100 μ l is not observed the PCR product band of 549bp; In the detection to each extent of dilution sample of chick embryo allantoic liquid of newcastle disease virus infection, 10 4EID 50/ 100 μ l-10 0EID 50Dilution sample can be observed the PCR product band of 282bp, 10 between/100 μ l -1EID 50/ 100 μ l-10 -2EID 50The dilution sample of/100 μ l is not observed the PCR product band (size of above-mentioned 4 kinds of product bands is all learnt through order-checking) of 282bp.
The result shows that primer mixes the right sensitivity of each primer of back to H3V, H5V, H9V and NDV and is respectively: 10 1EID 50/ 100 μ, 10 -1EID 50/ 100 μ l, 1 EID 50/ 100 μ l and 1 EID 50/ 100 μ l.
2, primer is added same PCR reaction system simultaneously to NDV, H3V, H5V and H9V, the biased sample to different concns detects respectively, and method and result are following:
At first according to each viral level that records in the step 1 in the step 1 to four, through calculating, each toxic allantoic fluid is diluted respectively, and contain each viral chick embryo allantoic liquid mixing respectively after will dilute, make each virus content in mixed solution all reach 10 6EID 50/ 100 μ l carry out 10 times of doubling dilutions then on this basis, obtain 8 kinds of mixed solutions, and each viral level in every kind of mixed solution is 10 5EID 50/ 100 μ l, 10 4EID 50/ 100 μ l, 10 3EID 50/ 100 μ l, 10 2EID 50/ 100 μ l, 10 1EID 50/ 100 μ l, 10 0EID 50/ 100 μ l, 10 -1EID 50/ 100 μ l or 10 -2EID 50/ 100 μ l's.Measure the blended primer to virus concentration 10 3EID 50/ 100 μ l-10 -1EID 50The susceptibility of the sample between/100 μ l.At first, get 10 3EID 50/ 100 μ l-10 -1EID 50Each dilution allantoic fluid sample between/100 μ l is extracted total RNA (method for extracting total RNA is with embodiment 4) respectively.
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer for AS111, Beijing Quanshijin Biotechnology Co., Ltd); Concentration is 20pmol/ μ L sequence table sequence 1 primer 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 2 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 3 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 4 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 5 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 6 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 7 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 8 primers 0.5 μ L; 2 μ L cDNA, DEPC water complements to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 45s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 10h.
Electrophoresis: get PCR product 1% agarose gel electrophoresis (result such as Fig. 8 (b)), detect in each extent of dilution biased sample of chick embryo allantoic liquid of H3 subtype avian influenza virus, H5 subtype avian influenza virus, H9 subtype avian influenza virus or newcastle disease virus infection 10 simultaneously 3EID 50/ 100 μ l-10 1EID 50Dilution sample all can be observed the PCR product band of 216bp, 282bp, 420bp, 549bp between/100 μ l, at 1EID 50Only observe the PCR product band of 420bp in the dilution sample of/100 μ l, negative chick embryo allantoic liquid and 10 -1EID 50The dilution sample of/100 μ l is not all observed any one (size of above-mentioned 4 kinds of bands is all learnt through order-checking) of above-mentioned 4 kinds of PCR product bands.
The result shows that the sensitivity that detects the mixture of H3 subtype avian influenza virus, H5 subtype avian influenza virus, H9 subtype avian influenza virus, NDV after primer mixes H3V, H5V, H9V and NDV simultaneously is 10EID 50/ 100 μ l.
Embodiment 4, the accuracy rate of primer to detecting
One, four kinds of primers are to mixing the accuracy rate that detects
With 380 parts of healthy chickens of each 10 in lung tissue sample being accredited as the chicken that NDV HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/BeiJing/40/2004, H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008 or H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infect through normal experiment methods such as virus separations, hemagglutination-inhibition tests, the collection from live-bird market and throat swab and the ight soil cotton swab sample of duck is test sample; Tissue sample and the negative contrast of PBS with healthy chicken; The positive contrast of infecting respectively with above-mentioned 4 kinds of viruses of chick embryo allantoic liquid; Extract total RNA; After the reverse transcription; Primer is added same PCR reaction system simultaneously to NDV, H3V, H5V and H9V, carry out the PCR reaction, process is following:
1, sample collecting and processing
The tissue sample of chicken: the tissue of getting lung; The cotton swab sample of chicken and duck: get throat swab and cloaca swab; 2-8 ℃ of preservation send the laboratory to detect.The requirement submitted sample is fresh.
2, the preparation of sample liquid:
(1) tissue sample: take by weighing the 100mg tissue sample and put in the mill, add 0.8ml lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanide, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing) grind, move to ground tissue sample 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) cotton swab sample: the cotton swab in the immersion liquid is fully twisted, discard swab after wringing out, 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 thiocyanide, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
(3) negative control (PBS): get sterilization distilled water 250 μ l, place 1.5ml sterilization centrifuge tube, add 750 μ l lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanide, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
3, total RNA extracts
1) get sample liquid (chick embryo allantoic liquid need not to handle and promptly can be used as sample liquid) 300 μ l after the processing, (15596-018 Invitrogen), puts upside down mixing for several times, ice bath 10min gently to add 900ulTRIZOL.
2) add 200 μ l chloroforms, put upside down mixing 15s, ice bath 5min, 4 ℃ are centrifugal, 13000r/m, 15min.
3) get the 700ul supernatant and move into new centrifuge tube, add the Virahol of equivalent, put upside down mixing gently, behind the mixing, 4 ℃ centrifugal, 13000r/m, 15min.
4) abandon supernatant, adherently slowly add the alcohol that 1ml 75%DEPC handled, gently take two turns after adding.
5)4℃，13000r/m，5min。
6) fall supernatant, place air-dry 5-10min on ice.
7) with 15 μ L DEPC water and 1 μ L RNA enzyme inhibitors (200U/ μ L, #k1622, Fermentas) dissolution precipitation, promptly with or-80 ℃ of preservations subsequent use.
4, reverse transcription
With the reverse transcription test kit (K1622, the total RNA sample that Fermentas) respectively step 3 is obtained carries out reverse transcription, obtains cDNA; DEPC water is as the contrast of total RNA.
Reaction system (20 μ L): 1 μ L Oligo primer (20pmol/ μ L), 4 μ L Reaction (5 *), 1 μ L Ribolock TMRnase Inhibitor (20U/ μ L), 2 μ L 10mM dNTP Mix, 1 μ L RevertAid TMM-MuLV ReverseTranscriptase (200U/ μ L), the total RNA of 11 μ L, DEPC water complements to 20 μ L.
Reaction conditions: after adding the DEPC water RNA of the extraction (dissolving wherein) and oligo primer, wink leaves, ice bath immediately behind 65 ℃ of 5min.After adding all the other five kinds, wink leaves, 37 ℃ of 1h, 70 ℃ of 5min again, 4 ℃ of preservations.
5, quadruple RT-PCR detects
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (its composition is EasyTaq archaeal dna polymerase, dNTPs and reaction buffer for AS111, Beijing Quanshijin Biotechnology Co., Ltd); Concentration is 20pmol/ μ L sequence table sequence 1 primer 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 2 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 3 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 4 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 5 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 6 primers 0.5 μ L, and concentration is 20pmol/ μ L sequence table sequence 7 primers 0.5 μ L; Concentration is 20pmol/ μ L sequence table sequence 8 primers 0.5 μ L; 2 μ L cDNA, DEPC water complements to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 45s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
6, electrophoresis
Get the PCR product and carry out 1% agarose gel electrophoresis.The result: 216bp, 282bp, 420bp and 549bp amplified band appear in the chick embryo allantois liquid mixture that NDV HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/BeiJing/40/2004, H5 subtype avian influenza virus A/treesparrow/Jiangsu/1/2008 or H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infect; The sample of blank PBS, healthy chicken and duck does not have above-mentioned 4 kinds of amplified bands; 549bp, 420bp, 282bp or 216bp amplified band appear in part in the test sample; Show and contain H9 subtype avian influenza virus, H5 subtype avian influenza virus, NDV or H3 subtype avian influenza virus in the sample, the detected result of each test sample is following:
10 in the lung tissue sample of the chicken that the H3 subtype avian influenza virus infects all is positive; 10 in the lung tissue sample of the chicken that the H5 subtype avian influenza infects all is positive; 10 in the lung tissue sample of the chicken that the H9 subtype avian influenza infects all is positive; 10 in the lung tissue sample of the chicken of newcastle disease virus infection all is positive.In the throat swab and ight soil swab sample of 380 chickens and duck, detect 1 of H3 subtype avian influenza virus; 4 of H5 subtype avian influenza virus; 2 of H9 subtype avian influenza virus; 2 of NDVs; Above-mentioned detected result is separated the coincidence rate that waits the qualification result of normal experiment methods to compare with blood clotting inhibition experiment with virus be 100%.
Two, primer is to the accuracy rate of H5V
With 380 parts of healthy chickens of the tissue sample that is accredited as the chicken that H5 subtype avian influenza virus A/treesparrow/Jiangsu/1/2008 infects through normal experiment methods such as virus separations, hemagglutination-inhibition tests and the collection from live-bird market and throat swab and the ight soil cotton swab sample of duck is test sample; With the negative contrast of the tissue sample of healthy chicken; PBS is a blank; The positive contrast of chick embryo allantoic liquid with H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008 infection; Is that primer carries out PCR reaction, the same step 1 of detection method with primer to H5V.
The result: positive control can be observed the 420bp amplified band; Blank and negative control do not have this amplified band; The lung tissue sample of the chicken that 10 H5 subtype avian influenza virus infect all is positive, and detects 4 in throat swab of 380 chickens and duck and the ight soil swab sample and is positive; Above-mentioned detected result is separated the coincidence rate that waits the qualification result of normal experiment methods to compare with blood clotting inhibition experiment with virus be 100%.
Three, primer is to the accuracy rate of NDV
With 380 parts of healthy chickens of the tissue sample that is accredited as the chicken that NDV HG/Beijing/2009 infects through normal experiment methods such as virus separations, hemagglutination-inhibition tests and the collection from live-bird market and throat swab and the ight soil cotton swab sample of duck is test sample; With the negative contrast of the tissue sample of healthy chicken; PBS is a blank; The positive contrast of chick embryo allantoic liquid with NDV HG/Beijing/2009 infection; Is that primer carries out PCR reaction, the same step 1 of detection method with primer to NDV.
The result: positive control can be observed the 282bp amplified band, and blank and negative control do not have this amplified band, and the lung tissue sample of the chicken of 10 newcastle disease virus infections all is positive; Detecting 2 in throat swab of 380 chickens and duck and the ight soil swab sample is positive; Above-mentioned detected result is separated the coincidence rate that waits the qualification result of normal experiment methods to compare with blood clotting inhibition experiment with virus be 100%.
Four, primer is to the accuracy rate of H9V
With 380 parts of healthy chickens of the tissue sample that is accredited as the chicken that H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infects through normal experiment methods such as virus separations, hemagglutination-inhibition tests and the collection from live-bird market and throat swab and the ight soil cotton swab sample of duck is test sample; The negative contrast of lung tissue sample with healthy chicken; PBS is a blank; The positive contrast of chick embryo allantoic liquid with H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infection; Is that primer carries out PCR reaction, the same step 1 of detection method with primer to H9V.
The result: positive control can be observed the 549bp amplified band, and blank and negative control do not have this amplified band, and the tissue sample of the chicken of 10 H9 subtype avian influenza virus infection all is positive; Detecting 2 in throat swab of 380 chickens and duck and the ight soil swab sample is positive; Above-mentioned detected result is separated the coincidence rate that waits the qualification result of normal experiment methods to compare with blood clotting inhibition experiment with virus be 100%.
Five, primer is to the accuracy rate of H3V
To identify the tissue sample of the chicken that H3 subtype avian influenza virus A/Duck/Beijing/40/2004 infects through normal experiment methods such as virus separations, hemagglutination-inhibition tests and throat swab and the ight soil cotton swab sample of 380 parts of healthy chickens of the collection from live-bird market and duck are test sample; The negative contrast of lung tissue sample with healthy chicken; PBS is a blank; The positive contrast of chick embryo allantoic liquid with H3 subtype avian influenza virus A/Duck/Beijing/40/2004 infection; Is that primer carries out PCR reaction, the same step 1 of detection method with primer to H3V.
The result: positive control can be observed the 216bp amplified band, and blank and negative control do not have this amplified band, and the tissue sample of the chicken of 10 H3 subtype avian influenza virus infection all is positive; Detecting 1 in throat swab of 380 chickens and duck and the ight soil swab sample is positive; Above-mentioned detected result is separated the coincidence rate that waits the qualification result of normal experiment methods to compare with blood clotting inhibition experiment with virus be 100%.
Embodiment 5, of the detection of different primer to making up
With primer among H9V, H5V, H3V and the NDV any three kinds or any two kinds the combination primers be primer; In the cDNA sample of the chick embryo allantoic liquid that infects respectively with H9, H5, H3 subtype avian influenza virus or NDV three kinds or two kinds of mixtures are template; Carry out triple or double PCR reaction, primer, template and result in each reaction see table 2 and Fig. 9.
The different primers of table 2 are to the triple or double PCR reaction result of combination
Primer is to combination The blended sample Result's (swimming lane of correspondence among Fig. 9)
H9V, H5V and NDV H9, H5 and ND ?1
H9V, H3V and NDV H9, H3 and ND ?2
H5V, H3V and NDV H5, H3 and ND ?3
H9V, H5V and H3V H9, H5 and H3 ?4
H5V and H3V H5 and H3 ?5
H9V and H3V H9 and H3 ?6
H3V and NDV H3 and ND ?7
H9V and H5V H9 and H5 ?8
H5V and NDV H5 and ND ?9
H9V and NDV H9 and ND ?10
Annotate: H9, H5, H3, ND represent the cDNA sample of the chick embryo allantoic liquid that H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008, H3 subtype avian influenza virus A/Duck/Beijing/40/2004, NDV HG/Beijing/2009 infect respectively respectively.
Pcr amplification system (25 μ L): 12.5 μ L, 2 * MIX buffer, each primer be to making up each 0.5 μ L of pairing primer (20pmol/ μ L), cDNA (every kind of each 2 μ L of cDNA), and DEPC water complements to 25 μ L, and each cDNA is provided with 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s carry out 30 circulations, 72 ℃ of 10min since second step; 4 ℃ of 1h.
Get the PCR product and carry out 1% agarose gel electrophoresis (as shown in Figure 9); All in twos or the primer of three or three combinations in the mixture (table 2) of counter sample cDNA detects, all obtain expecting the PCR product band of size, no PCR product band in the negative chick embryo allantoic liquid sample detection.
The result shows, primer can be in reaction of a sample to NDV, H3V, H5V and H9V three or three or after mixing in twos the corresponding NDV of special detection simultaneously, H3 hypotype, H5 hypotype or H9 subtype avian influenza virus.
Figure IDA0000097122720000011
Figure IDA0000097122720000021
Figure IDA0000097122720000031
Figure IDA0000097122720000041

Claims (10)

1. identify or the PCR primer of assistant identification NDV and many subtype avian influenza virus to compsn A, be following 1)-4) in any one:
1) said PCR primer to compsn A by following 4 primers to forming: primer to NDV, primer to H3V, primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus, H5 subtype avian influenza virus and the H9 subtype avian influenza virus;
Said primer is formed by two single stranded DNAs H9V H5V and primer H3V, primer NDV, primer;
Said primer is that the primer that the single stranded DNA shown in the sequence 2 is formed in the single stranded DNA shown in the sequence 1 and the sequence table in the sequence table is right to NDV;
Said primer is that the primer that the single stranded DNA shown in the sequence 4 is formed in the single stranded DNA shown in the sequence 3 and the sequence table in the sequence table is right to H3V;
Said primer is that the primer that the single stranded DNA shown in the sequence 6 is formed in the single stranded DNA shown in the sequence 5 and the sequence table in the sequence table is right to H5V;
Said primer is that the primer that the single stranded DNA shown in the sequence 8 is formed in the single stranded DNA shown in the sequence 7 and the sequence table in the sequence table is right to H9V;
2) said PCR primer to compsn A by following 3 primers to forming: said primer to NDV, primer to H3V and primer to H5V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H5 subtype avian influenza virus;
3) said PCR primer to compsn A by following 3 primers to forming: said primer to NDV, primer to H3V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H9 subtype avian influenza virus;
4) said PCR primer to compsn A by following 3 primers to forming: said primer to NDV, primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H5 subtype avian influenza virus and the H9 subtype avian influenza virus.
2. identify or the PCR primer of the many subtype avian influenza virus of assistant identification to compsn B, be following 5)-8) in any one:
5) said PCR primer to compsn B by following 3 primers to forming: said primer to H3V, primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus, H5 subtype avian influenza virus and the H9 subtype avian influenza virus;
6) said PCR primer to compsn B by following 2 primers to forming: said primer to H3V and primer to H5V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H5 subtype avian influenza virus;
7) said PCR primer to compsn B by following 2 primers to forming: said primer to H3V and primer to H9V; Said many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H9 subtype avian influenza virus;
8) said PCR primer to compsn B by following 2 primers to forming: said primer to H5V and primer to H9V; Said many subtype avian influenza virus are at least a in H5 subtype avian influenza virus and the H9 subtype avian influenza virus.
3. PCR primer according to claim 1 is characterized in that compsn A: PCR primer said 1) is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,3,4,5,6,7 and 8: 1: 1: 1: 1: 1; Said 2) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,3,4,5 and 6: 1: 1: 1; Said 3) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,3,4,7 and 8: 1: 1: 1; Said 4) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn A 1,2,5,6,7 and 8: 1: 1: 1.
4. PCR primer according to claim 2 is characterized in that compsn B: PCR primer said 5) is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 3,4,5,6,7 and 8: 1: 1: 1; Said 6) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 3,4,5 and 6: 1; Said 7) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 3,4,7 and 8: 1; Said 8) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in sequence table sequence among the compsn B 5,6,7 and 8: 1.
5. the said primer of evaluation or assistant identification H5 subtype avian influenza virus is to H5V.
6. following any one purposes in a)-f):
A) claim 1 or 3 described PCR primers are to the application of compsn A in preparation diagnosis or auxiliary diagnosis newcastle disease and bird flu reagent or test kit;
B) claim 2 or 4 described PCR primers are to the application of compsn B in preparation diagnosis or auxiliary diagnosis bird flu reagent or test kit;
C) the described primer of claim 5 is to the application of H5V in preparation diagnosis or auxiliary diagnosis H5 subtype avian influenza reagent or test kit;
D) claim 1 or 3 described PCR primers are to the application of compsn A in preparation evaluation or assistant identification NDV and many subtype avian influenza virus reagent or test kit;
E) claim 2 or 4 described PCR primers are to the application of compsn B in preparation evaluation or many subtype avian influenza virus of assistant identification reagent or test kit;
F) the described primer of claim 5 is to the application of H5V in preparation evaluation or assistant identification H5 subtype avian influenza virus reagent or test kit.
7. any one reagent following A)-C):
A) reagent of evaluation or assistant identification NDV and many subtype avian influenza virus contains claim 1 or 3 described PCR primers to compsn A in the said reagent;
B) reagent of evaluation or the many subtype avian influenza virus of assistant identification contains claim 2 or 4 described PCR primers to compsn B in the said reagent;
C) reagent of evaluation or assistant identification H5 subtype avian influenza virus contains the described primer of claim 5 to H5V in the said reagent.
8. any one PCR test kit following D)-F):
D) the PCR test kit of evaluation or assistant identification NDV and many subtype avian influenza virus contains A in the claim 7 in the said PCR test kit) described reagent;
E) the PCR test kit of evaluation or the many subtype avian influenza virus of assistant identification contains B in the claim 7 in the said PCR test kit) described reagent;
F) the PCR test kit of evaluation or assistant identification H5 subtype avian influenza virus contains C in the claim 7 in the said PCR test kit) described reagent.
9. any one preparation method following G)-I):
G) said preparation method comprises claim 1 or 3 described PCR primers the single stranded DNA of the compsn A step of packing separately respectively;
H) said preparation method comprises claim 2 or 4 described PCR primers the single stranded DNA of the compsn B step of packing separately respectively;
I) said preparation method comprises the step that the described primer of claim 5 is packed separately respectively the single stranded DNA of H5V.
10. the preparation method of the described PCR test kit of claim 8; Comprise the steps: claim 1 or 3 described PCR primers compsn A, claim 2 or 4 described PCR primers compsn B or the described primer of claim 5 to the single stranded DNA of H5V respectively separately after the packing; Be packaged in the same test kit with at least a material in the following substances: archaeal dna polymerase and following 4 kinds of dNTP:dATP; DTTP, dCTP and dGTP.
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