CN104894292A - Triple fluorogenic quantitative PCR detection primer, kit and method of DTMUV, EDSV and H9 subtype AIV - Google Patents
Triple fluorogenic quantitative PCR detection primer, kit and method of DTMUV, EDSV and H9 subtype AIV Download PDFInfo
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Abstract
The invention discloses a triple fluorogenic quantitative PCR detection primer, kit and method of the duck tembusu virus disease, the egg drop syndrome virus and the H9 subtype avian influenza virus. The detection primer for the duck tembusu virus disease, the egg drop syndrome virus and the H9 subtype avian influenza virus is shown in SEQ ID NO.1-6. The detection primer and kit can detect the EDSV, the H9 AIV and the DTMUV at the same time. No cross reaction with duck plague virus, goose parovovirus, muscovy duck parvovirus and escherichia coli genomic DNA and duck hepatitis A virus 1, duck hepatitis A virus 3, muscovy duck reovirus and newcastle disease virus RNA exists. The lower limits of detection for EDSV nucleic acid, H9 AIV nucleic acid and DTMUV nucleic acid are 8.0 copies, 4.8 copies and 1.3 copies respectively. The detection primer, kit and method can be used for qualitative and quantitative detection for the EDSV, the H9 AIV and the DTMUV.
Description
Technical field
The invention belongs to Molecular Detection field, the triple fluorescent quantitative PCR being specifically related to a kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus detects primer, test kit and method.
Background technology
Duck tembusu virus disease (Duck Tembusu Virus Disease) is a kind of acute infectious disease caused by duck tembusu virus (Duck Tembusu Virus, DTMUV).This disease can cause egg duck, plant goose and occur obvious egg drop reduction symptom at short notice, and the course of disease is several weeks.Recently, the report that laying hen and meat duck infect also is had.This disease pathogen belongs to flaviviridae (Flaviviridae), Flavivirus (Flavivirus), tembusu virus (Tembusu virus).Although this sick mortality ratio is not high, the course of disease is long, and potential hazard is huge.Duck tembusu virus genome is about 11 kb, and order is 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3', and wherein NS5 is very conservative.
Egg drop syndrome (Egg Drop Syndrome, EDS) is caused by aviadenovirus, causes a kind of transmissible disease that kind of egg fowl egg productivity declines.This virus only has a serotype, mainly causes the egg laying performance of kind of chicken to decline, and simultaneously its natural reservoir (of bird flu viruses) also comprises duck and goose, and also has and cause duck egg drop reduction, eggshell roughen, softening and thinning report.This disease can vertical transmission, and the band poison and the incidence that therefore accurately detect kind of fowl contribute to reducing the financial loss brought thus.
H9 subtype avian influenza virus (H9 Subtype AIV) can be carried by bird usually, generally clinical symptom is not attended after major part wild bird infects, but can cause duck egg drop reduction, chicken then can show the pathology (being usually expressed as egg drop reduction) of respiratory tract, digestive tube, urogenital organ.
Multiple fluorescence quantitative PCR technology is the new technology grown up on quantitative fluorescent PCR basis, and it emphasizes to increase multiple target in same reaction system simultaneously.Up to the present, there is no report and have the test kit simultaneously detecting duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus.
Summary of the invention
One object of the present invention is that the triple fluorescent quantitative PCR providing a kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus detects primer.
Another object of the present invention is the triple fluorescent quantitative PCR detection kit providing a kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus.
Another object of the present invention is the triple fluorescent quantitative PCR detection method providing a kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus.
The technical solution used in the present invention is:
A kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus triple fluorescent quantitative PCR detect Primer composition, and described detection primer is as follows:
Primer for duck tembusu virus:
DTMUV –U：5’-GCTTCAGCTGTCTGGGGATGCAGAAC-3’，
DTMUV –L：5’-AGCATAAGTTGCCTTGGGATTATGAG-3’；
Primer for egg-laying reduction syndrome virus:
EDS-U：5’-AGGTGTCTGATATTGGAGTG-3’，
EDS-L：5’-TGGATGAAACGCTTTATAAG-3’；
Primer for H9 subtype avian influenza virus:
H9-U：3’-GCCATAGCTGGATTCATAGA-5’，
H9-L：3’-AACTCTGCATTRTATGCCCA-5’。
A kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus triple fluorescent quantitative PCR detection kit, it contains respectively for the detection primer of duck tembusu virus, egg-laying reduction syndrome virus, H9 subtype avian influenza virus; The described detection primer for duck tembusu virus designs according to the E gene order of duck tembusu virus; Described is design according to the penton gene order of egg-laying reduction syndrome virus for the described detection primer for egg-laying reduction syndrome virus; The described detection primer for H9 subtype avian influenza virus designs according to the HA gene order of H9 subtype avian influenza virus.
As preferably, the nucleotide sequence of described detection primer is as follows:
Primer for duck tembusu virus:
DTMUV –U：5’-GCTTCAGCTGTCTGGGGATGCAGAAC-3’，
DTMUV –L:5’-AGCATAAGTTGCCTTGGGATTATGAG-3’；
Primer for egg-laying reduction syndrome virus:
EDS-U：5’-AGGTGTCTGATATTGGAGTG-3’，
EDS-L：5’-TGGATGAAACGCTTTATAAG-3’；
Primer for H9 subtype avian influenza virus:
H9-U：3’-GCCATAGCTGGATTCATAGA-5’，
H9-L：3’-AACTCTGCATTRTATGCCCA-5’。
Also containing single stage method SYBR RT-PCR damping fluid and archaeal dna polymerase in described test kit.
A kind of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus triple fluorescent quantitative PCR detection method, comprise the steps:
(1) the detection primer for duck tembusu virus, egg-laying reduction syndrome virus, H9 subtype avian influenza virus is designed respectively;
(2) configure real-time fluorescence PCR reaction system, the final concentration of three kinds of Viral diagnosis primers is 20pmol, and often kind of DNA profiling amount is 1 μ L, carries out PCR reaction;
(3) whether the continuous acquisition fluorescent signal when each extension, come in judgement sample containing duck tembusu virus, egg-laying reduction syndrome virus or H9 subtype avian influenza virus according to melting curve.
The described detection primer for duck tembusu virus, egg-laying reduction syndrome virus, H9 subtype avian influenza virus is as follows:
Primer for duck tembusu virus:
DTMUV –U：5’-GCTTCAGCTGTCTGGGGATGCAGAAC-3’，
DTMUV –L:5’-AGCATAAGTTGCCTTGGGATTATGAG-3’；
Primer for egg-laying reduction syndrome virus:
EDS-U：5’-AGGTGTCTGATATTGGAGTG-3’，
EDS-L：5’-TGGATGAAACGCTTTATAAG-3’；
Primer for H9 subtype avian influenza virus:
H9-U：3’-GCCATAGCTGGATTCATAGA-5’，
H9-L：3’-AACTCTGCATTRTATGCCCA-5’。
Consisting of of described real-time fluorescence PCR reaction system:
Real-time fluorescence PCR response procedures is: 42 DEG C of 5min; 95 DEG C of 10sec; 95 DEG C of 5sec, 55 DEG C of 10sec, 72 DEG C of 10sec, 45cycles, gather fluorescent signal when each extension; Carry out 95 DEG C of 180sec again, the melt curve analysis routine analyzer of 40 DEG C of 240sec, 95 DEG C of 1sec, continuous acquisition fluorescent signal.
The invention has the beneficial effects as follows:
Detection primer of the present invention and test kit can detect EDSV, H9 AIV and DTMUV simultaneously, and with the RNA of duck plague virus, goose parvovirus, Muscovy duck parvovirus and genome of E.coli DNA and duck hepatitis A virus (HAV) 1 type, duck hepatitis A virus (HAV) 3 type, muscovy duck reovirus and Avian pneumo-encephalitis virus, cross reaction do not occur; Sensitivity test shows, the Monitoring lower-cut of the method to EDSV, H9 AIV and DTMUV nucleic acid is respectively 8.0,4.8 and 1.3 copies, more responsive than conventional PCR method 10 times of the method; The method repeatability is good, respond well to the sample amplification of different batches.Shown by the foundation of typical curve and the detection of clinical sample, the quantitative and qualitative analysis that the method may be used for EDSV, H9 AIV and DTMUV detects.
Accompanying drawing explanation
Fig. 1 is DTMUV, H9 AIV and EDSV nucleic acid PCR amplification electrophorogram;
Fig. 2 is melt curve analysis after EDSV nucleic acid fluorescent pcr amplification;
Fig. 3 is melt curve analysis after H9 AIV nucleic acid fluorescent pcr amplification;
Fig. 4 is melt curve analysis after DTMUV nucleic acid fluorescent pcr amplification;
Fig. 5 is melt curve analysis analysis after EDSV and H9 AIV nucleic acid fluorescent pcr amplification;
Fig. 6 is melt curve analysis analysis after EDSV and DTMUV nucleic acid fluorescent pcr amplification;
Fig. 7 is melt curve analysis analysis after H9AIV and DTMUV nucleic acid fluorescent pcr amplification;
Fig. 8 is melt curve analysis analysis after EDSV, H9AIV and DTMUV nucleic acid fluorescent pcr amplification;
Fig. 9 is the specificity of triple fluorescent PCR method amplification curve;
Figure 10 is the specificity of triple fluorescent PCR method amplified production melt curve analysis;
Figure 11 is fluorescent PCR and standard PCR amplification 8.0 × 10
4~ 8.0 × 10
-1copy pEDSV detection of nucleic acids lower limit electrophorogram (left side is fluorescence PCR products electrophorogram, and the right is Standard PCR product electrophorogram);
Figure 12 is fluorescent PCR and standard PCR amplification 4.8 × 10
4~ 4.8 × 10
-1copy pH9 detection of nucleic acids lower limit electrophorogram (left side is fluorescence PCR products electrophorogram, and the right is Standard PCR product electrophorogram);
Figure 13 is fluorescent PCR and standard PCR amplification 1.3 × 10
5~ 1.3 × 10
0copy pDTMUV detection of nucleic acids lower limit electrophorogram (left side is fluorescence PCR products electrophorogram, and the right is Standard PCR product electrophorogram);
Figure 14 is pEDS plasmid 8.0 × 10
5the fluorescent PCR amplification curve of ~ 8.0 copies;
Figure 15 is pH9 plasmid 4.8 × 10
5the fluorescent PCR amplification curve of ~ 4.8 copies;
Figure 16 is pDTMUV plasmid 1.3 × 10
6~ 1.3 × 10
1the fluorescent PCR amplification curve of copy.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.
1 materials and methods
1.1 viralduck tembusu virus JM strain, egg-laying reduction syndrome virus and H9 subtype avian influenza virus are preserved by this research department.
1.2 reagentone Step SYBR PrimeScript RT-PCR Kit II, DNA molecular amount standard, pMD18-T carrier, Takara MiniBEST DNA/RNA extract test kit, glue reclaims test kit purchased from the precious biotechnology company limited in Dalian, DH5 α competence is purchased from Tian Gen company, and other reagent is analytical pure.
1.3 design of primers and synthesisdownload duck tembusu virus complete genome sequence, egg-laying reduction syndrome virus and H9 subtype avian influenza virus gene order in GenBank, utilize Oligo 6.0 to design Auele Specific Primer for they conservative regions.Primer is synthesized by Invitrogen company, and sequence is in table 1.
Table 1 triple PCR Auele Specific Primer
1.4 viral nucleic acid amplifications and standard substance buildextract the nucleic acid of duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus according to Takara MiniBEST DNA/RNA test kit specification sheets respectively, utilize these three kinds of virus specific primers to increase to gene order respectively.PCR reaction system is in table 2.PCR reaction conditions is: 42 DEG C of 10min, 94 DEG C of 5min; 94 DEG C of 30sec, 55 DEG C of 30sec, 72 DEG C of 30sec, 30cycles; 72 DEG C of 10min.Through 1% agarose gel electrophoresis, glue reclaims, with pMD
18-carrier T connects, and after transforming DH5 α, PCR is accredited as positive bacterium liquid, carries out sequencing by Invitrogen company.The positive plasmid obtained is called after pDTMUV, pEDS and pH9 respectively, and measures the concentration of plasmid.
The reaction system of table 2 pcr amplification DTMUV, EDSV and H9 AIV nucleic acid
the foundation of 1.5 triple fluorescent PCR methodsafter the upstream and downstream primer (concentration of every bar primer is 10 μMs) of DTMUV, EDSV and H9 AIV respectively equal-volume mixing, in 25 μ L PCR reaction systems, the final concentration of three kinds of Viral diagnosis primers (upstream and downstream mixture) is 20pmol, often kind of viral nucleic acid amount is 1 μ L(reaction system in table 3), then react according to following condition: 42 DEG C of 5min; 95 DEG C of 10sec; 95 DEG C of 5sec, 55 DEG C of 10sec, 72 DEG C of 10sec, 45cycles(gather fluorescent signal when each extension); Carry out 95 DEG C of 180sec again, the melt curve analysis routine analyzer (continuous acquisition fluorescent signal) of 40 DEG C of 240sec, 95 DEG C of 1sec.These 3 kinds viral nucleic acid can be amplified under determining this reaction system.
The reaction system of table 3 triple fluorescent pcr amplification DTMUV, EDSV and H9 AIV nucleic acid
the optimization of 1.6 primer concentrations
Will after the mixing of often kind of virus-specific upstream and downstream primer (10 μMs) equal-volume, according to 0.4 μ L, 0.8 μ L and 1.6 μ L totally 3 injection volume, namely these three kinds of different contents of 4pmol, 8pmol and 16pmol combine, and determine best primer content.Subsequently under primer content optimal conditions, single or Multiple detection is carried out to the nucleic acid of single virus nucleic acid and various combination, to verify the effect of primer.
1.7 specificity
The duck plague virus that this laboratory is preserved, goose parvovirus, Muscovy duck parvovirus and genome of E.coli DNA, and duck hepatitis A virus (HAV) 1 type, duck hepatitis A virus (HAV) 3 type, muscovy duck reovirus and newcastle disease virus RNA carry out multiple fluorescence PCR reaction, whether and between the nucleic acid of these viruses to verify there is cross reaction in establishment method.
1.8 susceptibility and repeatability
Extract pDTMUV, pEDS and pH9 plasmid built in 1.4, after DNA concentration determination, according to plasmid copy number calculation formula.That is, copy number (copies/ μ L)=6.02 × 10
23(copies/mol) × plasmid concentration (g/ μ L)/(base number) × 660 (g/mol).Subsequently, plasmid is used respectively ultrapure water 10 times of doubling dilutions, namely from 10
-1be diluted to 10 always
-11after, get 10
-6-10
-11the plasmid of these 6 gradients carries out sensitivity test.Subsequently, 10 are got
-5-10
-10the plasmid of these 6 gradients carries out 3 repetitions, the repeatability of defining method.Meanwhile, single primer is utilized to carry out standard PCR amplification, the sensitivity differences of more triple quantifying PCR method and conventional PCR method.
1.9 clinical samples detect
The nucleic acid of the 40 parts of DTMUV cloacal swab preserved this research department and 2 parts of positive pathological material of diseases of EDSV, H9 AIV, carries out triple fluorescent PCR checking.
2 results
the amplification of 2.1 DTMUV, H9 AIV and EDSV
Through pcr amplification, 1% agarose gel electrophoresis and determined dna sequence, confirm that the primer used can each self-corresponding viral nucleic acid (as shown in Figure 1) of specific amplification.
the foundation of 2.2 triple fluorescent PCR methods
Through Fluorescence PCR and melt curve analysis analysis, can find that the method can increase the nucleic acid of EDSV, H9 AIV and DTMUV, the Tm value of melt curve analysis analysis display institute amplified production is respectively close to 81 DEG C, 84 DEG C and 87 DEG C (Fig. 2, Fig. 3, Fig. 4).Through 10 increment product repeated authentication, and determine the Tm value scope of three kinds of products according to Tm mean value ± 3 times standard deviation, result display EDSV, H9 AIV and DTMUV amplified production Tm value scope is respectively: 81.2 ± 0.5,83.8 ± 0.4,86.9 ± 0.5.
2.3 primer optimum concn screenings
Under the permutation and combination of these three kinds of different primers content of 4pmol, 8pmol and 16pmol, finally determine that the best primer content of DTMUV be the best primer content of 4pmol, EDSV be the best primer content of 8pmol, H9 AIV is 16pmol.In the case, the biased sample (Fig. 5, Fig. 6, Fig. 7, Fig. 8) of any two kinds or 3 kinds viral nucleic acids can be detected.Through 1% agarose gel electrophoresis confirm, all melt curve analysis analytical resultss and electrophoresis result completely the same.
the specificity of 2.4 triple fluorescent PCR method
Verify through triple fluorescent PCR, duck plague virus, goose parvovirus, Muscovy duck parvovirus and genome of E.coli DNA, and duck hepatitis A virus (HAV) 1 type, duck hepatitis A virus (HAV) 3 type, muscovy duck reovirus and newcastle disease virus RNA not with EDSV, H9 AIV and DTMUV Auele Specific Primer generation cross reaction, namely there is not obvious S type amplification curve (Fig. 9); Melt curve analysis analysis display simultaneously, EDSV, H9 AIV and DTMUV significantly melts peak (Figure 10) at 81 DEG C, 84 DEG C and about 87 DEG C appearance respectively.Therefore the result of comprehensive amplification curve and melt curve analysis is determined, the specificity of triple fluorescent PCR method is good.
the susceptibility of 2.5 triple fluorescent PCR method and repeatability
Through DNA concentration determination, pEDS plasmid concentration is 247ng/ μ L, and pH9 plasmid concentration is 156ng/ μ L, and pDTMUV plasmid concentration is 425ng/ μ L, determines that the copy number of these three plasmids is respectively 8.0 × 10 as calculated
10copies/ μ L, 4.8 × 10
10copies/ μ L, 1.3 × 10
11copies/ μ L.By triple fluorescent PCR method and conventional PCR method to above-mentioned plasmid 10
-5-10
-10nucleic acid after extent of dilution carries out Monitoring lower-cut and compares discovery, the triple fluorescent PCR method that this institute sets up is 8.0 copies (Figure 11) to the Monitoring lower-cut of EDSV nucleic acid, be 4.8 copies (Figure 12) to the Monitoring lower-cut of H9 AIV nucleic acid, be 1.3 copies (Figure 13) to the Monitoring lower-cut of DTMUV nucleic acid, these more responsive than conventional PCR method 10 times.Get 10
-5-10
-10these 6 dilution plasmids carry out 3 repetition PCR and to react and after Criterion curve, result shows, in Figure 14 8.0 × 10
5the average Cq value of pEDS plasmid of ~ 8.0 copies is respectively 15.12 ± 0.11,18.17 ± 0.14,21.42 ± 0.12,24.63 ± 0.08,28.15 ± 0.34 and 31.64 ± 0.27.In Figure 15 4.8 × 10
5the average Cq value of pH9 plasmid (or claiming Ct value) of ~ 4.8 copies is respectively: 15.94 ± 0.24,19.30 ± 0.21,22.86 ± 0.20,26.62 ± 0.76,29.95 ± 0.10 and 33.84 ± 0.60; In Figure 16 1.3 × 10
6~ 1.3 × 10
1the average Cq value of pDTMUV plasmid of copy is respectively: 13.35 ± 0.19,16.65 ± 0.10,20.13 ± 0.04,23.55 ± 0.11,26.90 ± 0.14 and 30.80 ± 0.41; Confirmed by linear regression, the amplification efficiency of typical curve is respectively 2.08,1.87 and 2.03; R
2be respectively 0.99,0.98 and 1.00; The slope of linear regression equation is respectively-3.14 ,-3.68 and-3.26, and the intercept of Y-axis is respectively 37.13,36.55 and 36.87.
2.6 clinical samples detect
40 parts of DTMUV cloacal swab find after triple fluorescent PCR detects and melt curve analysis is analyzed, and positive number is 31 parts, and negative sample number is 9 parts; Standard PCR detects and electrophoresis result display positive number is 26 parts, and negative sample number is 14 parts.Nucleic acid and the Standard PCR coincidence rate of 2 parts of positive pathological material of diseases of EDSV, H9 AIV are 100%.
More than experiment shows: detection primer of the present invention and test kit can detect EDSV, H9 AIV and DTMUV simultaneously, and with the RNA of duck plague virus, goose parvovirus, Muscovy duck parvovirus and genome of E.coli DNA and duck hepatitis A virus (HAV) 1 type, duck hepatitis A virus (HAV) 3 type, muscovy duck reovirus and Avian pneumo-encephalitis virus, cross reaction do not occur; Sensitivity test shows, the Monitoring lower-cut of the method to EDSV, H9 AIV and DTMUV nucleic acid is respectively 8.0,4.8 and 1.3 copies, more responsive than conventional PCR method 10 times of the method; The method repeatability is good, respond well to the sample amplification of different batches.Shown by the foundation of typical curve and the detection of clinical sample, the quantitative and qualitative analysis that the method may be used for EDSV, H9 AIV and DTMUV detects.
<110> Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
The triple fluorescent quantitative P of <120> duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus
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Claims (8)
1. duck tembusu virus, egg-laying reduction syndrome virus and H9 subtype avian influenza virus triple fluorescent quantitative PCR detect a Primer composition, and described detection primer is as follows:
Primer for duck tembusu virus:
DTMUV –U：5’-GCTTCAGCTGTCTGGGGATGCAGAAC-3’，
DTMUV –L：5’-AGCATAAGTTGCCTTGGGATTATGAG-3’；
Primer for egg-laying reduction syndrome virus:
EDS-U：5’-AGGTGTCTGATATTGGAGTG-3’，
EDS-L：5’-TGGATGAAACGCTTTATAAG-3’；
Primer for H9 subtype avian influenza virus:
H9-U：3’-GCCATAGCTGGATTCATAGA-5’，
H9-L：3’-AACTCTGCATTRTATGCCCA-5’。
2. duck tembusu virus, egg-laying reduction syndrome virus and a H9 subtype avian influenza virus triple fluorescent quantitative PCR detection kit, it contains respectively for the detection primer of duck tembusu virus, egg-laying reduction syndrome virus, H9 subtype avian influenza virus; The described detection primer for duck tembusu virus designs according to the E gene order of duck tembusu virus; Described is design according to the penton gene order of egg-laying reduction syndrome virus for the described detection primer for egg-laying reduction syndrome virus; The described detection primer for H9 subtype avian influenza virus designs according to the HA gene order of H9 subtype avian influenza virus.
3. detection kit according to claim 2, is characterized in that, the nucleotide sequence of described detection primer is as follows:
Primer for duck tembusu virus:
DTMUV –U：5’-GCTTCAGCTGTCTGGGGATGCAGAAC-3’，
DTMUV –L:5’-AGCATAAGTTGCCTTGGGATTATGAG-3’；
Primer for egg-laying reduction syndrome virus:
EDS-U：5’-AGGTGTCTGATATTGGAGTG-3’，
EDS-L：5’-TGGATGAAACGCTTTATAAG-3’；
Primer for H9 subtype avian influenza virus:
H9-U：3’-GCCATAGCTGGATTCATAGA-5’，
H9-L：3’-AACTCTGCATTRTATGCCCA-5’。
4. detection kit according to claim 2, is characterized in that, also containing single stage method SYBR RT-PCR damping fluid and archaeal dna polymerase in described test kit.
5. duck tembusu virus, egg-laying reduction syndrome virus and a H9 subtype avian influenza virus triple fluorescent quantitative PCR detection method, comprise the steps:
(1) the detection primer for duck tembusu virus, egg-laying reduction syndrome virus, H9 subtype avian influenza virus is designed respectively;
(2) configure real-time fluorescence PCR reaction system, the final concentration of three kinds of Viral diagnosis primers is 20pmol, and often kind of DNA profiling amount is 1 μ L, carries out PCR reaction;
(3) whether the continuous acquisition fluorescent signal when each extension, come in judgement sample containing duck tembusu virus, egg-laying reduction syndrome virus or H9 subtype avian influenza virus according to melting curve.
6. detection method according to claim 5, is characterized in that, the described detection primer for duck tembusu virus, egg-laying reduction syndrome virus, H9 subtype avian influenza virus is as follows:
Primer for duck tembusu virus:
DTMUV –U：5’-GCTTCAGCTGTCTGGGGATGCAGAAC-3’，
DTMUV –L:5’-AGCATAAGTTGCCTTGGGATTATGAG-3’；
Primer for egg-laying reduction syndrome virus:
EDS-U：5’-AGGTGTCTGATATTGGAGTG-3’，
EDS-L：5’-TGGATGAAACGCTTTATAAG-3’；
Primer for H9 subtype avian influenza virus:
H9-U：3’-GCCATAGCTGGATTCATAGA-5’，
H9-L：3’-AACTCTGCATTRTATGCCCA-5’。
7. detection method according to claim 5, is characterized in that, consisting of of described real-time fluorescence PCR reaction system:
。
8. detection method according to claim 5, is characterized in that, real-time fluorescence PCR response procedures is: 42 DEG C of 5min; 95 DEG C of 10sec; 95 DEG C of 5sec, 55 DEG C of 10sec, 72 DEG C of 10sec, 45cycles, gather fluorescent signal when each extension; Carry out 95 DEG C of 180sec again, the melt curve analysis routine analyzer of 40 DEG C of 240sec, 95 DEG C of 1sec, continuous acquisition fluorescent signal.
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| CN111378782A (en) * | 2018-12-27 | 2020-07-07 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Duck egg laying-reduction syndrome virus detection method and kit thereof |
| CN111378782B (en) * | 2018-12-27 | 2022-11-29 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Duck egg laying-reduction syndrome virus detection method and kit thereof |
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