CN102321769B - 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 PDFInfo
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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
Technical field
The present invention relates to a kind of primer of identifying Avian pneumo-encephalitis virus and many subtype avian influenza virus to and use.
Background technology
Bird flu and newcastle disease are respectively the two-strain sexually transmitted diseases that is caused by avian influenza virus and Avian pneumo-encephalitis virus.Two kinds of infectious disease incidences are anxious, propagate soon, and sickness rate is high, can cause fatefulue strike to aquaculture.China chicken group's the hypotype of avian influenza virus of being popular in is mainly H5 and H9 hypotype.Very easily producer variation of influenza virus, thus antigenic change caused.Therefore, although China takes immune measure comprehensively, H5 and H9 subtype influenza virus still occur in the chicken group repeatedly.The variation of H5 and H9 subtype avian influenza virus Genetic and antigenic characteristics causes conventional serological method and molecular biology monitoring method can't detect epidemic isolates, therefore need to set up a kind of good detection method of versatility 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 areas chicken group, and the report that is separated to the H3 subtype avian influenza virus from the chicken group is arranged, therefore, the H3 subtype avian influenza virus also should be as one of object of influenza virus monitoring.In recent years, Avian pneumo-encephalitis virus causes that with avian influenza 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, is used for the differential diagnosis of newcastle disease and bird flu in the urgent need to setting up a kind of detection method.
For traditional detection method, such as virus separation, hemagglutination-inhibition test, neuraminic acid enzyme test etc., time-consuming, effort can not be made fast and timely diagnosis; And can not in same detection reaction, detect different objects, namely can't carry out simultaneously differential diagnosis.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, detection method in the urgent need to a kind of energy rapid differential diagnosis newcastle disease and different subtype avian influenza viruses, can determine not only whether virus is Avian pneumo-encephalitis virus or avian influenza virus, can also determine the hypotype of avian influenza 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 Avian pneumo-encephalitis virus and many subtype avian influenza virus to and use.
The primer of identifying Avian pneumo-encephalitis virus and many subtype avian influenza virus provided by the present invention to for the PCR primer to composition A, be following 1)-4) in any one:
1) described PCR primer to composition A by following 4 primers to forming: primer to NDV, primer to H3V, primer to H5V and primer to H9V; Described 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;
Described primer forms by two single stranded DNAs H9V H5V and primer H3V, primer NDV, primer;
Described primer is the single stranded DNA shown in the sequence 2 forms in the single stranded DNA shown in the sequence 1 and the sequence table in the sequence table primer pair to NDV;
Described primer is the single stranded DNA shown in the sequence 4 forms in the single stranded DNA shown in the sequence 3 and the sequence table in the sequence table primer pair to H3V;
Described primer is the single stranded DNA shown in the sequence 6 forms in the single stranded DNA shown in the sequence 5 and the sequence table in the sequence table primer pair to H5V;
Described primer is the single stranded DNA shown in the sequence 8 forms in the single stranded DNA shown in the sequence 7 and the sequence table in the sequence table primer pair to H9V;
2) described PCR primer to composition A by following 3 primers to forming: described primer to NDV, primer to H3V and primer to H5V; Described many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H5 subtype avian influenza virus;
3) described PCR primer to composition A by following 3 primers to forming: described primer to NDV, primer to H3V and primer to H9V; Described many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H9 subtype avian influenza virus;
4) described PCR primer to composition A by following 3 primers to forming: described primer to NDV, primer to H5V and primer to H9V; Described 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 composition B, be following 5)-8) in any one:
5) described PCR primer to composition B by following 3 primers to forming: described primer to H3V, primer to H5V and primer to H9V; Described 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) described PCR primer to composition B by following 2 primers to forming: described primer to H3V and primer to H5V; Described many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H5 subtype avian influenza virus;
7) described PCR primer to composition B by following 2 primers to forming: described primer to H3V and primer to H9V; Described many subtype avian influenza virus are at least a in H3 subtype avian influenza virus and the H9 subtype avian influenza virus;
8) described PCR primer to composition B by following 2 primers to forming: described primer to H5V and primer to H9V; Described 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 described primer of evaluation or assistant identification H5 subtype avian influenza virus to H5V.
Described 1) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 1,2,3,4,5,6,7 and 8 among the composition A: 1: 1: 1: 1: 1; Described 2) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 1,2,3,4,5 and 6 among the composition A: 1: 1: 1; Described 3) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 1,2,3,4,7 and 8 among the composition A: 1: 1: 1; Described 4) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 1,2,5,6,7 and 8 among the composition A: 1: 1: 1.
Described 5) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 3,4,5,6,7 and 8 among the composition B: 1: 1: 1; Described 6) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 3,4,5 and 6 among the composition B: 1; Described 7) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 3,4,7 and 8 among the composition B: 1; Described 8) PCR primer is 1: 1: 1 to the mol ratio of single stranded DNA shown in the sequence table sequence 5,6,7 and 8 among the composition B: 1.
The present invention protects following any one purposes in a)-f):
A) described PCR primer is to the application of composition A in preparation diagnosis or auxiliary diagnosis newcastle disease and bird flu reagent or test kit;
B) described PCR primer is to the application of composition B in preparation diagnosis or auxiliary diagnosis bird flu reagent or test kit;
C) described PCR primer is to the application of H5V in preparation diagnosis or auxiliary diagnosis H5 subtype avian influenza reagent or test kit.
D) described PCR primer is to the application of composition A in characterization or assistant identification Avian pneumo-encephalitis virus and many subtype avian influenza virus reagent or test kit;
E) described PCR primer is to the application of composition B in characterization or many subtype avian influenza virus of assistant identification reagent or test kit;
F) described PCR primer is to the application of H5V in characterization 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 Avian pneumo-encephalitis virus and many subtype avian influenza virus contains described PCR primer to composition A in the described reagent;
B) reagent of evaluation or the many subtype avian influenza virus of assistant identification contains described PCR primer to composition B in the described reagent;
C) reagent of evaluation or assistant identification H5 subtype avian influenza virus contains described PCR primer to H5V in the described 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 Avian pneumo-encephalitis virus and many subtype avian influenza virus contains described A in the described PCR test kit) reagent;
E) the PCR test kit of evaluation or the many subtype avian influenza virus of assistant identification contains described B in the described PCR test kit) reagent;
F) the PCR test kit of evaluation or assistant identification H5 subtype avian influenza virus contains described C in the described PCR test kit) reagent.
The present invention also protects following G)-any one preparation method in I):
G) described preparation method comprises the step that described PCR primer is packed separately respectively the single stranded DNA of composition A;
H) described preparation method comprises the step that described PCR primer is packed separately respectively the single stranded DNA of composition B;
I) described preparation method comprises the step that described primer is packed separately respectively the single stranded DNA of H5V.
The present invention also protects the preparation method of described PCR test kit; comprise the steps: described PCR primer composition A, described PCR primer composition B or described PCR primer to the single stranded DNA of H5V respectively separately after the packing; be packaged in the same reagent box 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 as follows respectively to detectable viral species and branch thereof or genotype: the PCR primer specifically can be II type, III type, VII type or IX type Avian pneumo-encephalitis virus to the Avian pneumo-encephalitis virus 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 the H5 subtype avian influenza virus of dissimilar and different branches to H5V, specifically can be the H5N1 subtype avian influenza virus of 2.3.2 branch, 2.3.4 branch or 7 branches; The PCR primer can detect the H9 subtype avian influenza virus of dissimilar and different branches to H9V, specifically can be the H9N2 subtype avian influenza virus of G1-like branch or BJ/94-like branch; But be not limited to the above-mentioned type and branch or the genotype of above-mentioned virus.
PCR primer provided by the present invention is as follows respectively to detectable viral source: the PCR primer can detect the Avian pneumo-encephalitis virus 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 the high specificity of while for detection of Avian pneumo-encephalitis virus, H3, H5 and H9 subtype avian influenza virus, 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 the 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, one time RT-PCR can realize 4 kinds of detections that virus is Avian pneumo-encephalitis virus, H3, H5 and H9 subtype avian influenza virus in the clinical sample, and can detect the epidemic isolates among China fowl group in recent years, 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 that primer is to the specific detection of NDV.Wherein, M is Maker, 1-6 is respectively and infects Avian pneumo-encephalitis virus F48E9, Chicken/Henan/2009, Chicken/ShangDong/2010, Chicken/Hebei/2010, the sample of Duck/ShangDong/2011 or HG/Beijing/2009,7-11 is respectively and infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the sample of infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 2 is that primer is to the specific detection of H3V.Wherein, M is Maker, 1-6 is respectively and 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, the sample of A/Duck/BeiJing/59/2005 or A/Duck/BeiJing/61/2005,7-11 is respectively and infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the sample of infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 3 is that primer is to the specific detection of H5V.Wherein, M is Maker, 1-6 is respectively and 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, the sample of A/Duck/Huabei/7/2009 or A/Chicken/Huabei/7/2011,7-11 is respectively and infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the sample of infectious bursa of Fabricius virus (IBDV) or reovirus (REO), 12 negative chick embryo allantoic liquid contrasts.
Fig. 4 is that primer is to the specific detection of H9V.Wherein, M is Maker, 1-6 is respectively and 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, the sample of A/Chicken/Sichuan/8/2011 or A/Duck/ZhuCheng/1/2011,7-11 is respectively and infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the sample of 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 respectively H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, H5 subtype avian influenza virus A/treesparrow/Jiangsu/1/2008, the mixture of the cDNA sample of H3 subtype avian influenza virus A/Duck/Beijing/40/2004 and Avian pneumo-encephalitis virus HG/Beijing/2009,2-5 is respectively and infects H9 subtype avian influenza virus A/Chicken/Beijing/3/1999, H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008, the cDNA sample of Avian pneumo-encephalitis virus HG/Beijing/2009 and H3 subtype avian influenza virus A/Duck/Beijing/40/2004,6-10 is respectively and infects H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, IBV, the sample of IBDV and reovirus, 11 negative chick embryo allantoic liquid contrasts.
Fig. 6 is the chick embryo allantoic liquid sample result that quadruple RT-PCR detects respectively the different virus strain infections of newcastle disease, H3, H5 or H9 subtype avian influenza virus.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 Avian pneumo-encephalitis virus; The M of figure in (a)-(d) is molecular weight Maker, and 1 with 1 sample among Fig. 5; The 2-7 of figure in (a)-(d) be the sample of 1-6 among same Fig. 2,3,4,1 respectively; 8 negative chick embryo allantoic liquid contrasts.
Fig. 7 is primer to H3V, H5V, H9V and NDV respectively to the sensitivity determination of H3, H5, H9 subtype avian influenza virus and Avian pneumo-encephalitis virus.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 Avian pneumo-encephalitis virus; 1-7 is respectively H3, H5, H9 subtype avian influenza virus or the Avian pneumo-encephalitis virus 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) is that quadruple RT-PCR is respectively to the sample detection of H3, H5, H9 subtype avian influenza virus or the Avian pneumo-encephalitis virus 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 Avian pneumo-encephalitis virus, and 1-7 is respectively the sample of H3, H5, H9 subtype avian influenza virus or the Avian pneumo-encephalitis virus 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 Avian pneumo-encephalitis virus, 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 that the triple RT-PCR methods of double sum are to the detection of avian influenza virus and Avian pneumo-encephalitis virus.Wherein, M is Maker; 1 is that primer is to H9V, H5V and NDV combination, 2 is that primer is to H9V, H3V and NDV combination, 3 is that primer makes up H5V, H3V and NDV, and 4 is that primer makes up H9V, H5V and H3V, and 5 is that primer is to H5V and H3V combination, 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 if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The employed virus stain information of following embodiment is as follows:
Table 1 strain information used in the present invention
Utilize Avian pneumo-encephalitis virus and the up-to-date sequence of avian influenza virus among the GenBank, as follows for H3 hypotype, H5 hypotype and H9 HA Gene of H 9 Subtype AIV and 4 pairs of primers of F gene of NDV strain design:
The primer that designs for target gene with F gene of NDV strain is 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);
Primer take the H3 HA Gene of H 9 Subtype AIV as 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);
Primer take the H5 HA Gene of H 9 Subtype AIV as 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);
Primer take the H9 HA Gene of H 9 Subtype AIV as 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.
One, primer is to the specific detection of NDV
1, total RNA extracts and quality control
Get respectively and infect Avian pneumo-encephalitis virus 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 the H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010 that infects similar Avian pneumo-encephalitis virus, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the chick embryo allantoic liquid of infectious bursa of Fabricius virus (IBDV) or reovirus (REO) and negative chick embryo allantoic liquid extract respectively total RNA (method is with embodiment 4).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
Respectively total RNA sample of step 1 is carried out reverse transcription with reverse transcription test kit (K1622, Fermentas), obtain 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 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 is from, ice bath immediately behind 65 ℃ of 5min.After adding all the other five samples, wink is from, 37 ℃ of 1h, 70 ℃ of 5min again, 4 ℃ of preservations.
3, pcr amplification detects the right specificity of primer
Take primer to NDV as primer, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 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 without PCR product band.The result shows that primer is fine to the specificity of NDV, can the specific detection Avian pneumo-encephalitis virus.
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 38,136-42.) chick embryo allantoic liquid and infect the H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010 of similar H3 subtype avian influenza virus, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the chick embryo allantoic liquid of infectious bursa of Fabricius virus (IBDV) or reovirus (REO) and negative chick embryo allantoic liquid extract respectively total RNA (method is with embodiment 4).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
Take primer to H3V as primer, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 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 without 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 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 147,25-9.) chick embryo allantoic liquid and infect the H4N6 subtype avian influenza virus A/Duck/ShangDong/1/2010 of similar H5 subtype avian influenza virus, H6N1 subtype avian influenza virus A/Duck/Beijing/1/2003, infectious bronchitis virus (IBV), the chick embryo allantoic liquid of infectious bursa of Fabricius virus (IBDV) or reovirus (REO) and negative chick embryo allantoic liquid extract respectively total RNA (method is with embodiment 4).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
Take primer to H5V as primer, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 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 without 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 H4N6 subtype avian influenza virus of chick embryo allantoic liquid 215-25.) and the similar H9 subtype avian influenza virus of infection, the H6N1 subtype avian influenza virus, IBV, IBDV, or the chick embryo allantoic liquid of reovirus and negative chick embryo allantoic liquid sample, extract respectively total RNA (method for extracting total RNA is with embodiment 4).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
Take primer to H9V as primer, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 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 without 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
With primer to NDV, H3V, H5V and H9V add same PCR reaction system simultaneously, respectively to Avian pneumo-encephalitis virus 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, infectious bronchitis virus (IBV), the cDNA sample of the chick embryo allantoic liquid that infectious bursa of Fabricius virus (IBDV) or reovirus (REO) infect and the mixture of cDNA sample that infects respectively the chick embryo allantoic liquid of above-mentioned front 4 virus stains detect (result 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 the 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, 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, 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, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 4 ℃ of 1h.
Electrophoresis: get the PCR product and carry out 1% agarose gel electrophoresis, result such as Fig. 5 and Fig. 6.At Avian pneumo-encephalitis virus HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/Beijing/40/2004, amplify simultaneously 4 PCR product bands in the biased sample that H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008 and H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infect respectively, be respectively 216bp through order-checking, 282bp, 420bp, 549bp, 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, at negative allantoic fluid and H4N6 subtype avian influenza virus, the H6N1 subtype avian influenza virus, IBV, in the sample of IBDV and reovirus infection without any specific band.
The above results shows: after primer mixes NDV, H3V, H5V and H9V on each primer on specificity without impact, can be in reaction of a sample simultaneously specific detection Avian pneumo-encephalitis virus, H3 hypotype, H5 hypotype and H9 subtype avian influenza virus.
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 Avian pneumo-encephalitis virus HG/Beijing/2009, adopt the Reed-Muench method to carry out viral EID
50Mensuration, obtain the toxic amount of this Avian pneumo-encephalitis virus in chick embryo allantoic liquid.According to this viral level, be that virus concentration is 10 with aseptic PBS with this chick embryo allantoic liquid dilution
6EID
50(virus concentration such as 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)), then carry out on this basis 10 times of doubling dilutions, 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
50The dilution allantoic fluid sample of/100 μ l amongs is extracted respectively total RNA (method for extracting total RNA is with embodiment 4).
2, the synthetic cDNA of reverse transcription
Respectively the total RNA that extracts in each diluent is carried out reverse transcription with reverse transcription test kit (K1622, Fermentas), obtain cDNA; 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 is from, ice bath immediately behind 65 ℃ of 5min.After adding all the other five samples, wink is from, 37 ℃ of 1h, 70 ℃ of 5min again, 4 ℃ of preservations.
3, pcr amplification detects the right sensitivity of primer
Take primer to NDV as primer, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 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 namely 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
Take primer to H3V as primer, the cDNA sample that step 2 is obtained carries out pcr amplification, 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 namely 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
Take primer to H5V as primer, the cDNA sample that step 2 is obtained carries out pcr amplification, 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 namely 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
Take primer to H9V as primer, the cDNA sample that step 2 is obtained carries out pcr amplification, 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 namely detect under the viral level of sample and are limited to 1EID
50/ 100 μ l.
Five, the right sensitivity of each primer detected after primer mixed NDV, H3V, H5V and H9V
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 (AS111, the Beijing Quanshijin Biotechnology Co., Ltd, its composition is the 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, 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, 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, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 45s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 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 the right sensitivity of each primer was respectively after primer mixed H3V, H5V, H9V and NDV: 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, respectively the biased sample of different concns detected, method and result are as follows:
At first according to each viral level that records in the step 1 in the step 1 to four, by calculating, each toxic allantoic fluid is diluted respectively, and contain respectively each viral chick embryo allantoic liquid mixing after will dilute, make each virus content in mixed solution all reach 10
6EID
50Then/100 μ l carry out 10 times of doubling dilutions 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.The primer that measure to mix 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
50The dilution allantoic fluid sample of/100 μ l amongs is extracted respectively total RNA (method for extracting total RNA is with embodiment 4).
Reaction system (25 μ L): 12.5 μ L, 2 * MIX buffer (AS111, the Beijing Quanshijin Biotechnology Co., Ltd, its composition is the 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, 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, 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, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 45s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 4 ℃ of 10h.
Electrophoresis: get PCR product 1% agarose gel electrophoresis (result such as Fig. 8 (b)), detect simultaneously 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
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 simultaneously the mixture of H3 subtype avian influenza virus, H5 subtype avian influenza virus, H9 subtype avian influenza virus, Avian pneumo-encephalitis virus after primer mixes H3V, H5V, H9V and NDV is 10EID
50/ 100 μ l.
One, four kinds of primers are to mixing the accuracy rate that detects
To separate through virus, the normal experiment methods such as hemagglutination-inhibition test are accredited as Avian pneumo-encephalitis virus HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/BeiJing/40/2004, each 10 in the lung tissue sample of the chicken that H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008 or H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infect, 380 parts of healthy chickens that gather from live-bird market and throat swab and the ight soil cotton swab sample of duck are test sample, tissue sample and the negative contrast of PBS with healthy chicken, the positive contrast of chick embryo allantoic liquid of infecting respectively with above-mentioned 4 kinds of viruses, extract total RNA, after the reverse transcription, with primer to NDV, H3V, H5V and H9V add same PCR reaction system simultaneously, carry out the PCR reaction, process is as follows:
1, the acquisition and processing of sample
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 thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing) grind, ground tissue sample is moved in the 1.5ml centrifuge tube, 4 ℃ of centrifugal 5min of 8000rpm get supernatant 250 μ l, place 1.5ml sterilization centrifuge tube, add the above-mentioned lysate of 750 μ l, the thermal agitation mixing, room temperature is placed 5min.
(2) 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 liquor 250 μ l, place 1.5ml sterilization centrifuge tube, add 750 μ l lysate (guanidine thiocyanate, 0.8M; Ammonium thiocyanate, 0.4M; Sodium-acetate buffer, 0.1M; Glycerine 5% and phenol 38%, mixing), the thermal agitation mixing, room temperature is placed 5min.
(3) 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 thiocyanate, 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) gets sample liquid (chick embryo allantoic liquid need not to process and namely can be used as sample liquid) 300 μ l after the processing, add 900ulTRIZOL (15596-018, Invitrogen), put upside down gently mixing for several times, ice bath 10min.
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 gently mixing, behind the mixing, 4 ℃ centrifugal, 13000r/m, 15min.
4) abandon supernatant, adherently slowly add the alcohol that 1ml 75%DEPC processed, add and gently take two turns afterwards.
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, namely with or-80 ℃ save backup.
4, reverse transcription
Respectively total RNA sample that step 3 obtains is carried out reverse transcription with reverse transcription test kit (K1622, Fermentas), obtain 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 is from, ice bath immediately behind 65 ℃ of 5min.After adding all the other five samples, wink is from, 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 (AS111, the Beijing Quanshijin Biotechnology Co., Ltd, its composition is the 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, 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, 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, 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 arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 45s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 4 ℃ of 1h.
6, electrophoresis
Get the PCR product and carry out 1% agarose gel electrophoresis.Result: Avian pneumo-encephalitis virus HG/Beijing/2009, H3 subtype avian influenza virus A/Duck/BeiJing/40/2004,216bp appears in the chick embryo allantois liquid mixture that H5 subtype avian influenza virus A/treesparrow/Jiangsu/1/2008 or H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infect, 282bp, 420bp and 549bp amplified band, blank PBS, the sample of healthy chicken and duck is without above-mentioned 4 kinds of amplified bands, 549bp appears in part in the test sample, 420bp, 282bp or 216bp amplified band, show and contain the H9 subtype avian influenza virus in the sample, the H5 subtype avian influenza virus, Avian pneumo-encephalitis virus or H3 subtype avian influenza virus, the detected result of each test sample is as follows:
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 Avian pneumo-encephalitis virus; 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
Take 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 from the throat swab of 380 parts of healthy chickens of live-bird market collection and duck and ight soil cotton swab sample as test sample, with the negative contrast of the tissue sample of healthy chicken, PBS is blank, the positive contrast of chick embryo allantoic liquid with H5 subtype avian influenza virus A/tree sparrow/Jiangsu/1/2008 infection, take primer H5V as carrying out PCR, primer is reacted the same step 1 of detection method.
The result: positive control can be observed the 420bp amplified band, blank and negative control are without 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 the 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
Take the tissue sample that is accredited as the chicken that Avian pneumo-encephalitis virus HG/Beijing/2009 infects through normal experiment methods such as virus separations, hemagglutination-inhibition tests and from the throat swab of 380 parts of healthy chickens of live-bird market collection and duck and ight soil cotton swab sample as test sample, with the negative contrast of the tissue sample of healthy chicken, PBS is blank, the positive contrast of chick embryo allantoic liquid with Avian pneumo-encephalitis virus HG/Beijing/2009 infection, take primer NDV as carrying out PCR, primer is reacted the same step 1 of detection method.
The result: positive control can be observed the 282bp amplified band, and blank and negative control are without this amplified band, and the lung tissue sample of the chicken of 10 newcastle disease virus infections all is positive; Detecting 2 in the 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
Take 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 from the throat swab of 380 parts of healthy chickens of live-bird market collection and duck and ight soil cotton swab sample as test sample, the negative contrast of lung tissue sample with healthy chicken, PBS is blank, the positive contrast of chick embryo allantoic liquid with H9 subtype avian influenza virus A/Chicken/Beijing/3/1999 infection, take primer H9V as carrying out PCR, primer is reacted the same step 1 of detection method.
The result: positive control can be observed the 549bp amplified band, and blank and negative control are without this amplified band, and the tissue sample of the chicken of 10 H9 subtype avian influenza virus infection all is positive; Detecting 2 in the 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
Take the tissue sample of identifying 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 from the throat swab of 380 parts of healthy chickens of live-bird market collection and duck and ight soil cotton swab sample as test sample, the negative contrast of lung tissue sample with healthy chicken, PBS is blank, the positive contrast of chick embryo allantoic liquid with H3 subtype avian influenza virus A/Duck/Beijing/40/2004 infection, take primer H3V as carrying out PCR, primer is reacted the same step 1 of detection method.
The result: positive control can be observed the 216bp amplified band, and blank and negative control are without this amplified band, and the tissue sample of the chicken of 10 H3 subtype avian influenza virus infection all is positive; Detecting 1 in the 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%.
Make up primers as primer take primer to any three kinds or any two kinds among H9V, H5V, H3V and the NDV, in the cDNA sample of the chick embryo allantoic liquid that infects respectively take H9, H5, H3 subtype avian influenza virus or Avian pneumo-encephalitis virus three kinds or two kinds of mixtures are as template, carry out triple or double PCR reaction, primer, the template in each reaction and the results are shown in Table 2 and Fig. 9.
Table 2 different primers is to the triple or double PCR reaction result of combination
| Primer is to combination | The sample that mixes | 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 respectively 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, Avian pneumo-encephalitis virus HG/Beijing/2009 infect respectively.
Pcr amplification system (25 μ L): 12.5 μ L, 2 * MIX buffer, each primer is to making up each 0.5 μ L of corresponding primer (20pmol/ μ L), cDNA (every kind of each 2 μ L of cDNA), DEPC water complements to 25 μ L, and each cDNA arranges 2 repetitions.
Response procedures: 94 ℃ of 5min; 94 ℃ of 30s; 51 ℃ of 45s; 72 ℃ of 45s begin to carry out 30 circulations from second step, 72 ℃ of 10min; 4 ℃ of 1h.
Get the PCR product and carry out 1% agarose gel electrophoresis (as shown in Figure 9), all in twos or the primers of three or three combinations in the mixture (table 2) of counter sample cDNA detects, all obtain expecting the PCR product band of size, in the negative chick embryo allantoic liquid sample detection without PCR product band.
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 simultaneously corresponding Avian pneumo-encephalitis virus of specific detection, H3 hypotype, H5 hypotype or H9 subtype avian influenza virus.
Claims (6)
1. identify or the PCR primer of assistant identification Avian pneumo-encephalitis virus and many subtype avian influenza virus to composition A, by following 4 primers to forming: primer to NDV, primer to H3V, primer to H5V and primer to H9V; Described 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;
Described primer forms by two single stranded DNAs H9V H5V and primer H3V, primer NDV, primer;
Described primer is the single stranded DNA shown in the sequence 2 forms in the single stranded DNA shown in the sequence 1 and the sequence table in the sequence table primer pair to NDV;
Described primer is the single stranded DNA shown in the sequence 4 forms in the single stranded DNA shown in the sequence 3 and the sequence table in the sequence table primer pair to H3V;
Described primer is the single stranded DNA shown in the sequence 6 forms in the single stranded DNA shown in the sequence 5 and the sequence table in the sequence table primer pair to H5V;
Described primer is the single stranded DNA shown in the sequence 8 forms in the single stranded DNA shown in the sequence 7 and the sequence table in the sequence table primer pair to H9V.
2. identify or the PCR primer of the many subtype avian influenza virus of assistant identification to composition B, by following 3 primers to forming: described primer to H3V, primer to H5V and primer to H9V; Described 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;
Described primer is the single stranded DNA shown in the sequence 4 forms in the single stranded DNA shown in the sequence 3 and the sequence table in the sequence table primer pair to H3V;
Described primer is the single stranded DNA shown in the sequence 6 forms in the single stranded DNA shown in the sequence 5 and the sequence table in the sequence table primer pair to H5V;
Described primer is the single stranded DNA shown in the sequence 8 forms in the single stranded DNA shown in the sequence 7 and the sequence table in the sequence table primer pair to H9V.
3. PCR primer according to claim 1 is characterized in that composition A: the mol ratio of single stranded DNA is 1:1:1:1:1:1:1:1 shown in the described sequence table sequence 1,2,3,4,5,6,7 and 8.
4. PCR primer according to claim 2 is characterized in that composition B: the mol ratio of single stranded DNA is 1:1:1:1:1:1 shown in the described sequence table sequence 3,4,5,6,7 and 8.
5. claim 1 or 3 described PCR primers are to the application of composition A in characterization or assistant identification Avian pneumo-encephalitis virus and many subtype avian influenza virus reagent or test kit.
6. claim 2 or 4 described PCR primers are to the application of composition B in characterization or many subtype avian influenza virus of assistant identification reagent or test kit.
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| CN103320540B (en) * | 2013-07-11 | 2016-08-10 | 广西壮族自治区兽医研究所 | Duck tembusu virus, egg-decreasing syndrome virus and the Triplex RT-PCR kit of Avian pneumo-encephalitis virus |
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| CN106498092B (en) * | 2016-10-28 | 2019-05-07 | 扬州大学 | A kind of one-step method real-time fluorescent quantitative PCR kit of joint-detection H5 subtype avian influenza virus and Virulent Newcastle Disease Virus |
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| CN101717830A (en) * | 2009-03-20 | 2010-06-02 | 山东艾克韦生物技术有限公司 | Method for detecting influenza virus by liquid phase chip |
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