CN108977579A - It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis - Google Patents
It is a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis Download PDFInfo
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Abstract
The invention discloses the PCR kit for fluorescence quantitative that a kind of detection leads to the novel duck reovirus of duck spleen necrosis.Contain probe and standard items and quantitative fluorescent PCR reaction reagent shown in primer shown in SEQ ID NO.1-SEQ ID NO.2 and SEQ ID NO.3 in the PCR kit for fluorescence quantitative.PCR kit for fluorescence quantitative of the invention can detect the novel duck reovirus, the high specificity of the kit, only specifically bind with novel duck reovirus, with other duck source viruses all without cross reaction;And the high sensitivity of detection, it is 4 × 10 in standard items1‑4×109There is fabulous linear relationship in copy range, detection range can be organized with efficient detection novel duck reovirus sick duck up to 9 orders of magnitude and the virus without clinical symptoms carries duck, improve detection efficiency.
Description
Technical field
The present invention relates to avian viruses detection technique fields, and in particular to a kind of detection causes the novel duck of duck spleen necrosis to be exhaled
The PCR kit for fluorescence quantitative of the lonely virus of intestines.
Background technique
Avianreovirus belongs to Hu Changgubing section (Reovirdae) Orthoreovirus (Orthoreovirus), can
To cause birds that a variety of diseases occur, clinical manifestation is different and variant because virus stain, virulence or infection host's.
From 2017, it is main that the ground such as China Shandong, Hebei, Henan and Jiangsu, Anhui duck, which is broken out on a large scale with spleen necrosis,
The communicable disease of symptom, the disease mainly cause each age level duck spleen enlargement, bleeding and necrosis, cause kind of a duck lay eggs and
There is the different necrosis region of several degree in meat duck appetite stimulator, syntexis, retarded growth, spleen, and enlargement, bleeding, edge increase
It is raw.Duck feedstuff-meat ratio increases, and meat duck delivers qualification rate significant decrease for sale, causes serious financial consequences to meat duck and kind duck aquaculture.
It has been investigated that the outburst of the disease is to there is no vaccine at present caused by a kind of novel duck reovirus infection
It can be prevented, existing commercially available duck reovirus vaccine can not carry out effective prevention and control to the disease;Conventional is antiviral
It is invalid with antibacterial therapy method.It is to the novel duck reovirus in the effective measures for currently in this case, controlling the disease
Infection is monitored, and isolation is taken to find infected duck group early to it or slaughters measure, exhales the lonely disease of intestines to reduce novel duck
Loss brought by poison infection.
Currently, mainly including that cytodiagnosis, immunology diagnosis and molecule are raw for the detection method of Avianreovirus
Object diagnoses three categories, wherein cytodiagnosis technology is mainly separately cultured using cell and is observed with Electronic Speculum.This method operation
Relatively complicated, detection cycle is long, and testing conditions are more demanding.Immunology diagnosis technology mainly includes enzyme linked immunosorbent assay (ELISA)
(ELISA) and immunofluorescence method, such method is sensitive higher, but also high to antibody preparation.Diagnostic technique in molecular biology master
It to include conventional polymeric enzyme chain reaction (PCR) method and fluorescence PCR method, and traditional PCR method can only be to virus infection
It is qualitative, it cannot quantify, sensitivity is relatively low;The advantages that fluorescent quantitative PCR technique is with its high sensitivity, speed is fast, high specificity
Gene expression dose analysis, pathogen qualitative and in terms of be widely used.But due to the novel duck
Reovirus system reports there are not the effective ways for detecting the novel duck reovirus yet so far for the first time.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of detections, and the novel duck of duck spleen necrosis to be caused to exhale intestines
The PCR kit for fluorescence quantitative of lonely virus.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides one group of primer and probe, the nucleotide sequence of the primer such as SEQ ID
Shown in NO.1-SEQ ID NO.2;The nucleotide sequence of the probe is as shown in SEQ ID NO.3.It is specific as follows:
Forward primer: M1-F:5 '-ATTAGCTCTAGCCACAGCCCTG-3 ';(SEQ ID NO.1)
Reverse primer: M1-R:5 '-CAATAGTGCATAGCATCAAGTAC-3 ';(SEQ ID NO.2)
Fluorescence probe: 5 '-FAM-TGCGAGTAGTCGGTTCTCGCA-TMARA-3 ';(SEQ ID NO.3)
The second aspect of the present invention, provide above-mentioned primer and probe causes the novel duck of duck spleen necrosis to be exhaled in preparation detection
Application in the reagent or kit of the lonely virus of intestines.
The novel duck reovirus is preserved in China typical culture collection center on July 18th, 2018, protects
Hiding number is CCTCC NO:V201843, address: Wuhan, China, Wuhan University, classification naming: novel duck reovirus N-
DRV-XT18 plants.
The third aspect of the present invention, providing a kind of detect causes the fluorescence of the novel duck reovirus of duck spleen necrosis fixed
P CR kit is measured, the PCR kit for fluorescence quantitative contains above-mentioned primer and probe.The novel duck reovirus
Deposit number is CCTCC NO:V201843.
Further, in the PCR kit for fluorescence quantitative further include: standard items and quantitative fluorescent PCR reaction reagent.
Preferably, the standard items are prepared by the following method:
Use primer amplification deposit number shown in SEQ ID NO.1-SEQ ID NO.2 for CCTCC NO:V20184 3
Duck reovirus genome, obtain amplified production, amplified production be connected on pMD18-T carrier, positive gram of screening
It is grand and extract Plasmid DNA, that is, standard items are prepared.
The quantitative fluorescent PCR reaction reagent includes: GoldStar TaqMan One Step Buffer, GoldStar
Ta qMan One Step EnzymeMix and ROX.
The fourth aspect of the present invention provides a kind of detection examination for detecting and leading to the novel duck reovirus of duck spleen necrosis
Agent, the detection reagent contain above-mentioned primer and probe.
The fifth aspect of the present invention provides above-mentioned PCR kit for fluorescence quantitative or above-mentioned detection reagent in following (1)-(3)
In it is any in application:
(1) the novel duck reovirus for being CCTCC NO:V201843 to duck infection deposit number carries out epidemiology tune
It looks into;
It (2) is the pollution of CCTCC NO:V201843 novel duck reovirus to the deposit number in blood or serum product
It is monitored;
(3) accurately detection deposit number is CCTCC NO:V201843 novel duck reovirus copy number, is specified novel
The progression of infection of duck reovirus.
The sixth aspect of the present invention, providing a kind of detect using above-mentioned PCR kit for fluorescence quantitative leads to duck spleen necrosis
Novel duck reovirus method, include the following steps:
(1) standard items are subjected to gradient dilution, carry out TaqMan Fluorescence PCR later, according to the concentration of standard items and
Ct value draws fluorescent quantitation standard curve;
(2) sample to be tested total serum IgE is extracted, TaqMan Fluorescence PCR is carried out, sample to be tested fluorescence signal is collected, to glimmering
Optical signal carries out data processing, obtains Ct value and amplification curve, carries out qualitative and quantitative detection to sample to be tested.
Preferably, in step (1) and step (2), the program of TaqMan Fluorescence PCR are as follows:
45℃10min;95℃10min;95 DEG C of 15s, 60 DEG C of 45s carry out 40 circulations later.
Result judgement method: if sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested containing novel duck reovirus.
Beneficial effects of the present invention:
(1) it is directed to the newfound novel duck reovirus that can result in duck spleen necrosis, the present invention devises can be with
To the PCR kit for fluorescence quantitative that the novel duck reovirus is detected, the high specificity of the kit, only and novel duck
Reovirus specific binding, with other duck sources virus, such as Duck parvovirus, duck tembusu virus, duck virus etc. is all
There is no cross reaction.
(2) high sensitivity detected is 4 × 10 in standard items1-4×109There is fabulous linear relationship in copy range,
Detection range, can be with efficient detection novel duck reovirus sick duck tissue and the disease without clinical symptoms up to 9 orders of magnitude
Poison carries duck, improves detection efficiency.
(3) novel duck reovirus infection can be monitored using kit of the invention, to find early
Infected duck group takes it isolation or slaughters measure, to reduce loss brought by novel duck reovirus infection;It can be with
To duck infection reovirus carry out epidemiological survey, and to the duck reovirus in blood or serum product pollute into
Row monitoring.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technique is introduced, from 2017, the big model of the ground such as China Shandong, Hebei, Henan and Jiangsu, Anhui duck
Outburst is enclosed using spleen necrosis as the communicable disease of cardinal symptom.It has been investigated that the communicable disease is by novel reovirus
Caused.It there is no the drug and method that can effectively control the novel duck reovirus infection at present.It is of the invention based on this
A kind of PCR kit for fluorescence quantitative for being able to detect the novel duck reovirus is proposed, to find infected duck early
Group takes it isolation or slaughters measure, to reduce loss brought by novel duck reovirus infection.
It is known that the host range of Avianreovirus is wider, generally existing this virus in various birds, from 1954
It has been separated in the birds of a variety of morbidities such as chicken, goose, pigeon, ostrich, duck, turkey and other pheasants or health since year
Reovirus, and the reovirus being separated to from different hosts its there are biggish variability, pathogenic, gene sequences
Column and the coding of major protein all have differences.
Present inventor isolates one plant of duck reovirus N-DRV-XT18 from morbidity duck spleen tissue, to above-mentioned
The duck reovirus N-DRV-XT18 newly separated has carried out genome sequencing, and respectively by 10 genetic fragments and existing report
The Avianreovirus in road has carried out sequence alignment and homology analysis, as a result, it has been found that, the 10 of new isolated strain N-DRV-XT18
L1, L2, L3, M1, M2, S3, S4 are respectively positioned in a relatively independent branch in a genetic fragment, and with uploaded at present
Reovirus-originated duck sequence variability is larger in Genbank, illustrates that the strain N-DRV-XT18 newly separated is different from it really
His Avianreovirus, it can be assumed that duck reovirus found in the present invention and it is existing it has been reported that duck exhale intestines lonely
Virus exists compared with Big mutation rate, is 1 new independent kind of Orthoreovirus.Therefore, which is exhaled intestines lonely by the present invention
For preservation in China typical culture collection center, deposit number is CCTCC NO:V201843.
Since Avianreovirus is RNA virus, there are multiple segmentations, in antigenic structure, pathogenic, cell between different strains
Cultural character and host specificity etc. have a certain difference, and variation is easy to happen in genetic evolution.Therefore, different
The coding of the antigenic, pathogenic of reovirus, gene order and major protein can all have differences.Using fluorescent quantitation
PCR detects reovirus, above all the sequence of hard objectives gene, is examined according to target gene sequence design primer
It surveys.But the present invention duck reovirus to be detected for leading to duck spleen necrosis is the strain newly separated, gene composition
It is unknown, and with it is existing it has been reported that duck reovirus exist compared with Big mutation rate, the lonely disease of intestines can not be also exhaled according to existing duck
The genome sequence design primer of poison.
In addition, for fluorescence quantitative PCR detection, how design primer and probe sequence are to guarantee detection validity
It is crucial.Although having software and design of primers principle of many design of primers etc. in the prior art, to design to obtain high specificity,
The primer and probe of high sensitivity combines, and being not can be simply obtained by primer-design software, this needs technology people
Member constantly selects optimization targeting regions using its professional knowledge, carries out design of primers further according to the targeting regions of optimization, and
Screening, optimization, redesign repeatedly are carried out to the primer of design.Specifically, since present invention novel duck to be detected is exhaled
The lonely virus of intestines is different from the reovirus-originated duck of existing report, and how the novel duck of Te Yixing detection the application exhales the lonely disease of intestines
Poison, and avoid that cross reaction occurs with other duck sources virus, it is the difficult point place of primer and probe design of the present invention.Exhale the lonely disease of intestines
There are 10 genetic fragments for the full-length genome of poison, and the conservative region of different genes segment is again different, therefore, lonely for exhaling intestines
For the detection of virus, how to select targeting regions to carry out primer and probe design there is no unified cognition at present.The present invention exists
The targeting regions for carrying out primer and probe design are optimized during test, as a result, it has been found that, exhale intestines lonely with the novel duck
The sequence conservation of the M1 gene of virus carries out primer and probe design, may be implemented to the special of the novel duck reovirus
Property detection.
Primer designed by the present invention, probe are used cooperatively, and the two complements each other, in the design process further
The reaction condition for having fully considered quantitative fluorescent PCR, avoid interfering with each other between primer, probe, final design the application
Primer, probe.Wherein:
Forward primer: M1-F:5 '-ATTAGCTCTAGCCACAGCCCTG-3 ';(SEQ ID NO.1)
Reverse primer: M1-R;5'-CAATAGTGCATAGCATCAAGTAC-3';(SEQ ID NO.2)
Fluorescence probe: 5 '-FAM-TGCGAGTAGTCGGTTCTCGCA-TMARA-3 ';(SEQ ID NO.3)
5 ' end mark fluorescent reporter group FAM of fluorescence probe, 3 ' end mark fluorescent quenching group TMARA, amplified fragments
Length 83bp.
The present invention during the test, also passes through other conserved region sequences of reovirus gene and other primers
Design principle devises the different primer of multiple groups, probe, and is respectively combined, and detects its specificity after reacted respectively and expands
Increasing Efficiency, to screen the optimal primer and probe combination that can be used for clinical detection.Such as:
First group:
Forward primer: F:5 '-TGCGCCTATCCTTGAGTTGA-3 ';
Reverse primer: R;5'-TTGCCAGGAAATACGGGTCT-3';
Fluorescence probe: 5 '-FAM-TCAAAATGGTGGACTTCAGTTTCGAT-TMARA-3 '.
Second group:
Forward primer: F:5 '-TGACAGGCCTGACTACGTTA-3 ';
Reverse primer: R;5'-ATGCTTGAAGTGAGACGTAC-3';
Fluorescence probe: 5 '-FAM-TGCGAGTAGTCGGTTCTCGCA-TMARA-3 '.
As a result, it has been found that with new to this using primer of the invention and probe combinations (SEQ ID NO.1-SEQ ID NO.3)
The specificity and sensitivity that type duck reovirus is detected are optimal.And other primer and probe combinations cannot then exhale duck
The lonely virus of intestines and other duck sources virus are effectively distinguished, and false positive or false negative are easy to appear.
The present invention also optimizes quantitative fluorescent PCR condition during the test, the maximum limit in the clinical application of kit
Degree reduce operation, not only guarantee sensitivity, but also reduce various pollutions to greatest extent, it is ensured that result it is scientific, accurate.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.Specific experiment condition and method are not specified in the embodiment of the present invention, usually according to routine
Condition, such as J. Pehanorm Brooker chief editor, Science Press, 2002, Molecular Cloning:A Laboratory guide (third edition);D.L. this peck
Top grade chief editor, Science Press, 2001, cell experiment guide;Or the condition suggested according to manufacturer.
Embodiment 1: the design of fluorescence quantification PCR primer and probe
The complete of novel duck reovirus (deposit number is CCTCC NO:V201843) is obtained according to two generation sequencing technologies
Genome sequence, the sequence point of this 10 genetic fragments of L1, L2, L3, M1, M2, M3, S1, S2, S3, S4 in whole genome sequence
Not as shown in SEQ ID NO.4-SEQ ID NO.13.
For the primer sequence and fluorescence probe sequence of the sequence conservation design specificity of M1 gene, sequence design is such as
Under:
Forward primer: M1-F:ATTAGCTCTAGCCACAGCCCTG;(SEQ ID NO.1)
Reverse primer: M1-R:CAATAGTGCATAGCATCAAGTAC;(SEQ ID NO.2)
Fluorescence probe: 5 '-FAM-TGCGAGTAGTCGGTTCTCGCA-TMARA-3 ';(SEQ ID NO.3)
5 ' end mark fluorescent reporter group FAM of fluorescence probe, 3 ' end mark fluorescent quenching group TMARA, amplified fragments
Shown in length 83bp (SEQ ID NO.14).
ATTAGCTCTAGCCACAGCCCTGACCTGTCCGTCCTTGATCTGTTCGACTGGTCGC GATTC
CAATAGTGCATAGCATCAAGTAC;(SEQ ID NO.14)
Embodiment 2: the foundation of fluorescent quantitative PCR detection method
(1) extraction of viral RNA:
Sample to be tested total serum IgE is extracted using classical Trizol method or commercial kit;
(2) Fluorescence PCR Establishing:
The GoldStar that it is CW2633S for the article No. of ShiJi Co., Ltd from health that one-step method fluorescence quantitative kit used, which is used,
Taqman One Step RT-qPCR Ki kit, sequentially adds 2 × GoldStar in 200 μ l Fluorescence PCR pipes
TaqMan One the Step μ of Buffer~12.5 l, Forward Primer (10 μM)~0.5 μ l, Reverse Primer (10
μM)~0.5 μ l, Probe (10 μM)~0.5 μ l, GoldStar TaqMan One Step μ of EnzymeMix~1.0 l, 50 ×
Low ROX or High ROX (optional)~0.5 μ l, the middle sample to be tested Total μ l of RNA~2 extracted of step (2),
Use RNase Free dH2O is supplemented to 25 μ l systems.It is placed in ABI 7300Fast fluorescence quantitative PCR instrument and is reacted, instead
Answering condition is 45 DEG C of 10min;95 DEG C of 15s, 60 DEG C of 45s carry out 40 circulations after 95 DEG C of 10min.
(3) preparation of standard curve:
For copy number viral in accurate quantitative analysis sample, preparation containing have the plasmid of purpose amplified fragments as standard items with
Draw standard curve.The forward and reverse primer (shown in SEQ ID NO.1 and SEQ ID NO.2) for designing M1 gene first, expands
Increase novel duck reovirus gene group and obtain the amplified production of 83bp (shown in SEQ ID NO.14).
It according to classical molecular cloning protocols method, is connected on pMD18-T carrier, is named as pMD-M1, screen
Positive colony simultaneously extracts Plasmid DNA, utilizes spectrophotometer measurement plasmid concentration, as standard items.10 times are carried out to standard items
Gradient dilution carries out TaqMan Fluorescence PCR later, and according to the concentration of standard items and Ct value, instrument software can be drawn automatically
Fluorescent quantitation standard curve out;Specifically: y=-3.3501x+45.561, coefficient R2=0.9997, good line is presented
Sexual intercourse;Wherein, the copy number logarithm of x representative sample, y represent Ct value.
(4) judgement of testing result:
According to quantitative fluorescent PCR react collect fluorescence signal, recycle instrument software handle data, obtain amplification curve,
Ct value.Using the corresponding Ct value of standard items minimum concentration and amplification curve as judgment basis.
Result judgement method: if sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested containing novel reovirus.
According to fluorescent quantitation standard curve, reovirus novel in sample can be quantitative determined.
Embodiment 3: the optimization of the composition, experiment parameter of kit and specificity, sensitivity and repeatability are investigated
1. the composition of kit:
The kit of the present embodiment is PCR kit for fluorescence quantitative, for detecting novel duck reovirus (deposit number
For CCTCC NO:V201843).Contain in kit: forward primer (10 μ shown in the SEQ ID NO.1 that embodiment 1 designs
M), reverse primer (10 μM) shown in the SEQ ID NO.2 that embodiment 1 designs, shown in the SEQ ID NO.1 that embodiment 1 designs
Probe, standard items and quantitative fluorescent PCR reaction reagent.
Standard items are prepared by the following method:
Use primer amplification deposit number shown in SEQ ID NO.1-SEQ ID NO.2 for CCTCC NO:V201843
Duck reovirus genome, obtain the amplified production (shown in SEQ ID NO.14) of 83bp, amplified production be connected to
On pMD18-T carrier, screening positive clone simultaneously extracts Plasmid DNA, that is, standard items are prepared.
Quantitative fluorescent PCR reaction reagent includes: 2 × GoldStar TaqMan One Step Buffer, GoldStar
TaqMan One Step EnzymeMix, 50 × Low ROX or High ROX (optional), RNase Free dH2O。
2. the optimization of experiment parameter:
Exploration is optimized to the quantitative fluorescent PCR reaction condition of the primer (SEQ ID NO.1-2) in kit, is tied
Fruit shows that primer is 10 μM, and reaction condition is 45 DEG C of 10min;95℃10min;95 DEG C of 15s, 60 DEG C of 45s carry out 40 later
Circulation, kit sensibility, detection effect are best.
3. the specificity of kit, sensitivity and repeatability are investigated
(1) specific test
Using the kit, carry out the specific test of the kit, respectively with Duck parvovirus, duck tembusu virus,
Duck virus, NDRV-JM85 plants of duck reovirus, NDRV-TH11 plants of duck reovirus and the present invention are detected
The novel duck reovirus that deposit number is CCTCC NO:V201843 is template, carries out fluorescent quantitation using the kit
PCR, as a result, it has been found that, Duck parvovirus, duck tembusu virus, duck virus, RV-JM85 plants of duck reovirus ND, duck exhale
DRV-TH11 plants of intestines orphan's virus N cannot be expanded effectively;Only deposit number is that the novel duck of CCTCC N O:V201843 exhales intestines
Lonely virus can be expanded effectively.
The above test results show that the specificity of kit of the present invention is 100%, there is stronger specificity, kit
Middle primer and probe and novel reovirus are specifically bound, viral with other duck sources, such as Duck parvovirus, duck Tan Busu disease
Poison, duck virus etc. are all without cross reaction.
(2) repetitive test
Using the kit, the repetitive test of the kit is carried out.Take that clinical acquisitions arrive is main with spleen necrosis
The spleen tissue sample of the duckling of symptom carries out repeated detection using kit, and three repeat samples are corresponding in primary experiment
The coefficient of variation (CV) less than 0.5%, illustrate it with high repeatability.
(3) sensitivity is tested
Standard items are diluted to various concentration, are detected using the kit, at the same using regular-PCR detection as pair
Than.As a result, it has been found that fluorescent quantitative PCR result is shown, kit of the invention is able to detect 101The standard items of a copy number, and
And 101-109There is fabulous linear relationship in copy range, detection range is up to 9 orders of magnitude;And regular-PCR minimum
It can detect 104The standard items of a copy number.Show that the kit carries out quantitative fluorescent PCR sensitivity with higher.
Embodiment 4: the clinical application experiment 1 of kit of the present invention
(1) extraction of viral RNA:
The doubtful illness duck spleen tissue homogenate of 30mg is taken, is extracted using classical Trizol method or commercial kit
Total serum IgE.
(2) Fluorescence PCR Establishing:
The GoldStar that it is CW2633S for the article No. of ShiJi Co., Ltd from health that one-step method fluorescence quantitative kit used, which is used,
T aqman One Step RT-qPCR Ki kit, sequentially adds 2 × GoldStar in 200 μ l Fluorescence PCR pipes
TaqMan One the Step μ of Buffer~12.5 l, Forward Primer (10 μM)~0.5 μ l, Reverse Primer (10
μM)~0.5 μ l, Probe (10 μM)~0.5 μ l, GoldStar TaqMan One Step EnzymeMi μ of x~1.0 l, 50 ×
Low ROX or High ROX (optional)~0.5 μ l, the middle sample to be tested Total μ l of RNA~2 extracted of step (2), makes
With RNase Free dH2O is supplemented to 25 μ l systems.It is placed in ABI 7300Fast fluorescence quantitative PCR instrument and is reacted, reacted
Condition is 45 DEG C of 10min;95 DEG C of 15s, 60 DEG C of 45s carry out 40 circulations after 95 DEG C of 10min;Collect fluorescence.
Result judgement method: if sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested containing novel reovirus.It the results are shown in Table 1.
Table 1: morbidity duck inspection pathological material of disease fluorescence quantitative PCR detection result
Sample serial number | Sample message | Ct value |
1 | Shandong 2017.12.28 | 16.5450 |
2 | Jiangsu 2017.11.18 | 14.8971 |
3 | Hebei 2018.04.22 | 19.8565 |
4 | Shandong 2018.04.08 | 15.0195 |
5 | Shandong 2018.02.19 | 16.4998 |
6 | Henan 2018.01.14 | 16.8799 |
7 | Shandong 2018.06.01 | 21.9421 |
Exhale intestines lonely the result shows that can detect novel duck in 7 different regions duck pathological material of diseases using kit of the invention
Virus.
Moreover, detecting using kit of the invention to 40 parts of identified samples, there are 34 parts of pattern detections to contain
There is novel duck reovirus, consistent with qualification result, detection accuracy rate is 100%.
Embodiment 5: the clinical application experiment 2 of kit of the present invention
The spleen necrosis morbidity duck for the novel reovirus of infection that clinical acquisitions are arrived, takes the spleen tissue of necrosis to be added
It organizes to be homogenized after 5 times of physiological saline, after multigelation 3 times, takes supernatant to be inoculated with LMH cell, received after stable cytopathy to appear
Poison.
The extraction of above-mentioned viral RNA:
The cell sample for taking 400 μ l virus infections to be measured is extracted total using classical Trizol method or commercial kit
RNA。
Fluorescence PCR Establishing:
The GoldStar that it is CW2633S for the article No. of ShiJi Co., Ltd from health that one-step method fluorescence quantitative kit used, which is used,
T aqman One Step RT-qPCR Ki kit, sequentially adds 2 × GoldStar in 200 μ l Fluorescence PCR pipes
TaqMan One the Step μ of Buffer~12.5 l, Forward Primer (10 μM)~0.5 μ l, Reverse Primer (10
μM)~0.5 μ l, Probe (10 μM)~0.5 μ l, GoldStar TaqMan One Step EnzymeMi μ of x~1.0 l, 50 ×
Low ROX or High ROX (optional)~0.5 μ l, the middle sample to be tested Total μ l of RNA~2 extracted of step (2), makes
With RNase Free dH2O is supplemented to 25 μ l systems.It is placed in ABI 7300Fast fluorescence quantitative PCR instrument and is reacted, reacted
Condition is 45 DEG C of 10min;95 DEG C of 15s, 60 DEG C of 45s carry out 40 circulations after 95 DEG C of 10min;Collect fluorescence.
Result judgement method: if sample to be tested fluorescence signal is more than threshold value and Ct≤30.0, while amplification curve is smooth
" S " type, then can be determined that in sample to be tested containing novel duck reovirus.It the results are shown in Table 2.
Table 2: novel duck reovirus infection cell sample fluorescence quantitative PCR detection result
Sample serial number | Sample message | Ct value |
1 | Shandong 2017.12.28 | 17.7650 |
2 | Hebei 2018.04.22 | 22.1365 |
3 | Shandong 2018.04.08 | 17.6895 |
4 | Shandong 2018.02.19 | 18.7898 |
5 | Henan 2018.01.14 | 19.8321 |
Through detecting, novel duck reovirus is contained in sample, meets expection, show detection method of the invention it is accurate,
Reliably.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>a kind of to detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis
<130> 2018
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
attagctcta gccacagccc tg 22
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<400> 2
caatagtgca tagcatcaag tac 23
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
tgcgagtagt cggttctcgc a 21
<210> 4
<211> 3959
<212> DNA
<213>the L1 gene of strain N-DAV-XT18
<400> 4
gctttttctc cgaacgccga aatgagttcg cgcaaagtgg ctagacgtcg tcataaggat 60
gctaccgaca ctaaagattc taagtacact gataaagata agtcctcagc cacaactact 120
gaaacgaagt ccgtcgactc cactaacaag agtgtaagcg ctccaaaatc tgatactcca 180
gcagcatcaa caccatcctc tacagatggc gcttctcagg ttgcagtcgc taaacaaacc 240
aatgataatg acgcttcttc taaagattct tcccctaaac caactgtcac tcccgacgga 300
aaggatggaa tgcatggtgc ggttaaatct caagacgcta aagctaccat tgctgttgat 360
gataacaagg acagggacgt ggtcttcggt ggcaagggtt caggtgataa aaatgctatt 420
acaaagactg ggtccgtgga taatgatggt ggtgttaaaa cgataccggc taaggatact 480
actatcgcat ccgctaaagc catgatggaa cagaaacagt tagttgcggg tcttccgaag 540
cagccaaaac ctgccagcca tctatgcact gtttgcatgg ctcaattcgc ttcagctgat 600
gcattgacta tacaccaaac aactcattcg attggatcca acgctgcact gactagcttt 660
tcgatctcta ccgccgttga ggagttcatt caatcttggg cctcggctac ttccactgct 720
aatactaaaa cagcactaac tgtggcggac gttgattctt taatgatgac tgaagggata 780
cgtttaatca cttgggattc cggactctgt acctcctttg agcttgtgcc gatagtgcag 840
tctaacactg tccaagacgt tatctcatac tcttggttca catctagtta caatatcgct 900
actcctttcc ctcaagcgac tgtggtgcgt attgtgttac gtacgaactg ggctgctaga 960
ttggattccc catcttcctc tcgtgaatgc gatcttcggc ttgctcctcc gacagaaagt 1020
aatgcacgtt cttttgctat gctcctcaac actggtgcta ctcctgaagg tacatttaat 1080
cctaacactc tccgtatgaa cgtcttgcag atgtgcctcc agtatgtgtt agctaacctc 1140
catttaaaca ggagtactca atttacgatg gacctgaccg ctgccgctcc caatttatcc 1200
gcttctcaac ttcgaatcgt gcccgaagac aaggatggaa aatggttccc ggtcatgtac 1260
ccttctcgtg taaacatccc cctctttaat aaaacagccg atttcgttaa ccaatgtatt 1320
cgtgacagaa tcggtcgcta cgatcgcgct caaactttcg ctggtgctcc ttctgaatgg 1380
gctgatatgt gggagactgc agactcatta acactctcgg ttcgtgaaat gtggatgtct 1440
cggatctcac aaatgaacat ctctcccgcc gatatcgccg acgcaatctc gcgttgttcg 1500
cagtcgctcc tgacggtagc cgcacctact gctccctccg tagctcgtct cctgccgtgg 1560
cgtgttagtt ctgatgagcg ccaattatta caactgctga tgtatcttaa cgtaggtact 1620
agcgccgatt acatccagcc aattctatct gcgttcgctc gcactctctc gcgtgtttca 1680
cctttgcgta ttaaccccac tctcatcgct aacgcaatgt ctactattgt tgaaagcact 1740
actaacaccc agagtccagc cgctgccatc ttgtcaaagt tgaagccagt cgcatccgac 1800
ttctcagact tccgtctcgc atgtgctgct tggctgtata atggttgtgt ccagacgtac 1860
ttatctgaag actcttaccc cagtagcggc ggatctgtca ctagtattga cactctggtt 1920
gatatgtttg tctgtctctt ggctctcccg cttgtcactg atcctaatgc gccttgtcaa 1980
gctttcatgg tcgtggctaa tgctatggtc ggctatgaga atctgccgat ggatgatcct 2040
aactttaccc aacagagact ggctgccgcg tttaataacc ctaccacctg gccgcagtgc 2100
tttctacacc ctcagaacat cgatcgccgc caatgtccga tcctatcctg gtgggcacaa 2160
cagattcacc gcaactggcc tactccatca cagatcacct atggcgcgcc tgatattatc 2220
ggttcagcca acctattcac tccccctgat gttctgctgc tcccgctcca gcatcgcccc 2280
attcgcatta cgaatccaac tttgaacttc gacaacgagt taacaacctg gcgtaacacg 2340
gtagtagatt tgatcctacg catcattgac agtggcagat accaaccgaa ctggaatcag 2400
tccatccgcg cttcaatgcg aaacgctatg acgaacttta ggattattaa atcgtacact 2460
cccgcttaca tcgctgaatt gttacccgtt gagttggccg ctatcgctcc cacccttcct 2520
ttccagcctt tccaggttcc cttcgctcgt ctagaccgtg atgctattgt cacccatgtc 2580
aacgtgtcgc gtcaggcccc gaacgtgctt gctcagccag cgttaaacat gtccatgacg 2640
tatcaacgca ctggcgtgcc aatatcgttg agcgcccgtc ctcttgcagt tgctcttctg 2700
tccggccaat atcctactga gccacctctg cagacgaatg tgtggtatgt gaatacgctt 2760
acccctctgt attcaaatga tggattattt aataacgtcc agcatgctat ggtggccgct 2820
gaagcttacg ccactcttat cactatgtta gcacagtgta cggacatgca gtatcccgtg 2880
gaccgtccct taaactggtt acgtcaaatc aacctagcgg cgaatgaggc taccatcttt 2940
gggcgctcca tcaattctct cttccagacc gcctttgatc tttccccttc tactgttctc 3000
ctgcaaccgt ttttggagtc cgaccctcgc tctacacaac tcgccatctc ctacgtccgc 3060
tataatggtg atagcgaaac cttcgtgcct accgtccgcc cgtctatgat agctgaggct 3120
actctactcg tcgagcgcgt tctgtcacat gagtataact tatttggact ctgtcgcggt 3180
gacatcattt taggacagca catgacccct tccgcattta acccgttagc ccctcctcct 3240
tccgtcgtct ttagtcgcgg tgaccccgag gtctacgagt tcggccagcg tagtttcgcc 3300
aactttggta tgaatggtga agagatcctc gttatggacg cgaatggcgt acgtcgtccc 3360
ctgcttggta gatgggtgat gcccctacag ctactgatgg tgaacattgg cgtgttccct 3420
aagctcctgc tggaccggat tctgaaaggg cgtttgtaca ttaggctaga ggtcggtgcc 3480
tatccttaca ctgttcagta ctatcagggt cgagagttca ctgatggatt tacgctgttg 3540
gagcagtgga tgtcgaaagt gtcgcccatg ggcataccac ccgttccgtt cctgatgcca 3600
caatctgaag gtcataacat tacttctggc atggtgaccc actacatttg gtccactgag 3660
tataacgatg gctccctttt cgccacaaac acagacttgc ctgttactgt cttcggtcca 3720
gaccgcacta tccctattga gagataccgt gctttggtcg atccgggcgc tctgccagcc 3780
acaaaccaac tcccccacac tatcgatctc tactgttcgt tgagacgcta ctacttggaa 3840
acacctccga ttacagcgac ggtgacaact tatggtgatg gtctcccagc gttgaaccat 3900
tagagcggcg aggctagacg cgagctgatc gcgtcgactc tcgttggagg ttactcatc 3959
<210> 5
<211> 3830
<212> DNA
<213>the L2 gene of strain N-DAV-XT18
<400> 5
gctttttcct caccatgcat gtcaatgggt ttgatgatgc tactctcgca tacgcacgct 60
ccatatcggg tcttacttca atgtcaaatg atttgtttga gagagcttca ttgtctatgc 120
gctcattccc acgttctcat gcttatgaca tattagataa agtagaattt tctgtctctt 180
gtgtcattcc taatgccata tttcatcacc cggaccacgt tgagtacttt tatatcgatg 240
ctgtcaatag ggtcaggcgt aaacatgtca ttgattcgga tgatgtattt gtaccaaact 300
gtaattttca gggtctctta actcctatgg ataagttacc aaactatggt caactatctg 360
aggtcatctc ctcgaatgct cgcgacgggt tagcttcggc acgaatcgcc aatactttct 420
ataacattgc tgtctcacaa gctaggcaag tcaaagcgcc tcttgaatcg tttctccttc 480
ccctattact ttctgaaacc tgccctttat ctgatgaccc atgtggattg gatacttccc 540
cgtctcctcc gatcagtaga aatctggcgt tatgggtact gcgcgagtta agccgcacta 600
tttgtggatc gtcaggcgat cggtcaccat ggttgttgtt agattcaggt gtagcatggt 660
tcctttcacc actgatgtct tcagctatcc ctccccttat ggctgattta acgaatctgg 720
ctatctataa acaggtatgc tccgtctccg atgatatgca ctccctcgcc gttcaaatgg 780
tgttgcaagc agctgcctcg caatcctacg gacattatgt gctgcaaact aaggccgttt 840
ttccgcaaaa cactttgcat aacatgttta gaaccatcac tgacggtctg gtccctatga 900
tagactggct ggagcccagg tctaactacc gcttcatgct gcagggtgcg cgtaaggtga 960
catcagatga tgcgaatcaa tctccagata acacggaggc tgctgagaaa gttggccata 1020
agatgggttg tttagatgtt gttagatcct tacggaagat gtcttctgcc attacagtgc 1080
attcgcatga tgcgatgacg tttgttcggg acgccatgtc ttgtaccagt ggtattttca 1140
ttactcgcca acctactgaa actgtgttga aggaatatac gcaagctccg acgattgaag 1200
ttcccattcc tcaaactgat tggtctccac caattggatc ccttcgctac ttatctgatg 1260
cctgtgctct acccgctgtg tacttggctc gttcttggcg tcgagctgcc tcagcagtcg 1320
tagataatcc tcacacttgg gatccactat atcaagctat tctccgctcc caatacgtca 1380
catcacgcgg tggttcgggc gctgcgttac gagacgcgtt aaaagctgct gaagtggagc 1440
ttccacagta tcctggtgtc agtattaagg tcgctacaaa gatataccag gcagctcaaa 1500
ctgctgatgt gccattcgat aagctctccc gtgccgtcct tgctcccttg tcgatggggc 1560
tgcgtaatca ggtccagcgt cgtcctcgca ccatcatgcc tatgaacgtc gtgcaacaac 1620
aggtctcagc agctcatact ctatctgccg actatatcaa ctatcacatg aacttatcca 1680
ccacatcggg tagtgccgtt atcgagaagg tcgttccgtt gggcatgtac gcctcctgtc 1740
cgcctgctca ggcagtcaat attgatatta aagcgtgtga cgcgtctatt acgtatcagt 1800
acttcctctc cgtcattgtt ggtgctattc atgaaggtgc tgctggacgt cgtgtatcct 1860
cctcgtttat gggtgttcct cccagcgtat tgtcggtcgt tgattccagt ggagtcacat 1920
cttcaatgcc catttccggt ttccaggtta tgtgtcaatg gttagcgaaa ctctatcagc 1980
gtggttttga gtatcaggtc acggacacat tttcacccgg caatatcttt acgcaccaca 2040
cgacgacttt tccttccgga tctaccgcca cttccaccga gcacactgcc aataatagca 2100
cgatgatgga tggatttcta agatcttggg tcccctcctc tgatgcatca gatattttga 2160
agaagttctg tcgctctatt tccatccagc gaaattatgt ctgtcagggt gacgatggcc 2220
taatgattgt cgatggtctc tcaacgggta agttatcggg cgaggtgatt gctgagtttg 2280
tcaaggagtt gcgagcgtac ggtaagtctt ttggatggaa ctatgacatc gagtttactg 2340
ggaacgctga gtatcttaag ttgtattttc tgaacggttg ccgggtacct aatgtgtcgc 2400
gacaccctat atgtggtaaa gagcgagctt ccggagataa gttagagatg tggccttcta 2460
ccatcgatat ttttaatggc atctttgtca acggtgtgca tgatggtctg ccttggcgtc 2520
gttggcttcg ctactgctgg gccttggcca cgttatattc tggaaagcgt gtgcgtcatg 2580
agaatgttga agtgtcaatc caatatccta tgtggtcatt tatctactgg ggcctgcctc 2640
ctgtcagcgt ctttggttcc gatccgtgga tattctctcc ctacatgccc actggcgatc 2700
acggattcta ttccatgcta actcttgtta gacccctaat taccgccctc tctccttcat 2760
cttcaatgga tggacttttt ggtcagtgcg atcacaacgc tctctttaac tccgagatgg 2820
tgtaccaggg ttactatatg gcgcagtgtc cgcgtcagcc atcgagatcc aaccgtcgtg 2880
atgacccgga gtctgtccaa cgatttgtta gggcgctcga atcgtactta tacatatccc 2940
cggagttgaa agcacgagtc aggcttggtc gtgaccgctg gcagaaatta gttgggtata 3000
ctgaaaaatc tccgccctct ctcgatgacg tcgctcataa atggttccgt agtgcgcagg 3060
aagccgactt acctaccacc tcggaaattc aggccatgga cttagcgtta atcgcggccc 3120
gccgtaaaac ataccaaggc ttctctaagc tacttaacac gtaccttcga gtaacttggg 3180
acctgaccga gccaatcgag catgccgtcg atccccgggt accactctgt gctggcattt 3240
ctccctcgaa tagtgagccc tttttgaagc tgtactcgat cggtcctatg atgcagtcga 3300
ctcggaaata tttcagtaac accttgttca ttcatcgtac cgtatctggc cttgacgtcg 3360
acgttgtaga tcgcgctctt ctacgtctga gagcccttaa tgctcctgat gaggtggtca 3420
ttgctcaact cctcatggtc ggcctgtccg aagcagaagc ggcgactttg gcagctaaga 3480
ttaggactat ggacatcaat gccgttcagt tggctcgagt ggtgaatctg tcaatacccg 3540
actcttggat gacgatggat ttcgatcgtt taatacgtga tattgtgtct actacccctt 3600
taactattcg ttctttgacg actgacctgc cctctggtgt cccatgggtt cgtgccatat 3660
tgcagttcct cggtgctgga gtcgccatga ccgccgttgg tcccgttaga cgcccatacc 3720
ttcactcagt cgctggtggc atgtcgtcat tcattaaaca gtttcgccgg tggatgcggg 3780
ctgagacgag gtagcgtccg tgcccagcat ggctcgagga attattcatc 3830
<210> 6
<211> 3906
<212> DNA
<213>the L3 gene of strain N-DAV-XT18
<400> 6
gcttttcacc catggctcag attagaggcc ttcgattgtc tacgacactc tcagcaccgc 60
cttcacgtcg atcgtacaca cctcagacat atgatgacct catttcagcg ctgaaattaa 120
ctgtcaagcc ctggcgaccg ttgcgatctg gcgctcaaga tgttatcacg gccgtgcaac 180
tgttctttcc actaaatggg tacgttgaac cactgttcat gctcgaaaag gaggtgagct 240
atgaagattt tgaagcatgg ttatcgccaa ttctatctgc cttggctgac cagttcttgc 300
gtcggtatcc catcgcctca taccatggcc gtttagtgaa tcctctgcta gcgaacgcca 360
ttgtagctgc gtttttgtca aatgcgccat atgcgcacgc tattgaacat ctcttcttag 420
tgcgtggagt gatagaagac attgtcgacg ctggtttatc cattcaaaat cacctgtggt 480
tcgatcgtgg cgctctcgtg tcacccgctg gtcagaaatt cattcagctt gacaagtact 540
ctttctcgtc tcgtgaccca tgtctcttcg ctaagcagtc tcgctactat ggcctggttt 600
actatttcct caacatatcg gactgcttgt cctattgcta tcgtcatctc tcaaacttca 660
cacctctgct gcactttgat cgaccatcca acggcgtgca ttgtctagta ccgtcggaat 720
ccacgactcc catagccggg tctttacctg tgtcctccct gagtgccatc ttattagaat 780
cttgcattca gcagtctatt ctgaacactc ggactccgac gggcgcacca tcgacgagac 840
agatagaggc tcttttgccc gtgtcctctc ccttctttga acgtcccacc actttggaat 900
actctttgtt ttctctttct aacgctttag taggtggtta ccagctatta gatctacctt 960
ctggtcaccc tgattgttct accgttgctg ctttgttggc tcgattaacg gatttttcca 1020
aggagatcac agtcattcaa cctattccgt cgcgttttac tttgtacgct gacagtccat 1080
tgaattacag tggtgagaac gccatgtttc taagccgttt gccatgtgaa tctggtaaat 1140
ccgtcggtcc tgtctttact ggtaaacctg ttaataggag tattggttgg atgcctcagt 1200
tcgatcctgc tacgtcatat gatcctgaaa tggcagctga gtccctagcc aaagcgacca 1260
ctctcccatt gcgtgcaaaa ttttcatcat tctggtccgg acccgcactt ttctctgccg 1320
cctcatgtga cagacacaat ggtgtgtatg acctgcgctt tatgccacct tttccatcca 1380
catactttga tgaggatgac gtgttttctc gctctcggtt ttcatcttat cgtgctgtcc 1440
gggaccgttc gcttttgaag gataccgcca atctcatgta tgtctccaat ttgtcgaatt 1500
cacatgacca gcgtcttctt cctgaatcta aaactatggt gtatgtgggc gcttctggta 1560
ctcacactga taatcagcca tcaattatca agcccttgtt agacggcact cttcctggcg 1620
tcttcaagcc cctttcggtt aaacaggttg gttgggaggt taccaacggt actatctgtg 1680
atatcgagtt acccttggct actggaacat tcttcttcgt atacagtgat gtcgatcagg 1740
tacagtcggg cgattccgat ctcgtctcct cttctcgacg tttctgtctt caattagaca 1800
tgctgatgaa gctgacgtat acctctggtt ctgtgatcgt gaagtgcaat ttccctactg 1860
acttagtgtg gcgtcacatt ttctcaacta tctctccata tttcatgtcg atccatctta 1920
tgaagccgct tgtctccaat aacttagaac tgtacattct atttgctgaa aagttatccg 1980
tccctgacgc gcctttcaat ccctccgctg acgtggtgac attctggcgc tctcaacttc 2040
agcgctacag agttctccgg gattcttttt cctccgtccc tcagattgac tcagctctta 2100
cgctggacga cccactaacc gtgtcggtct taaatttcgt agatgtcaca tcactttctt 2160
cgctggatga tcagcgtgca ctggctgcct tctctgttct aacatctttg gggtcacaaa 2220
agttgtctat tcatccatat tttgacagtt accgcacaca gctgaccggc atcgtgacac 2280
ctcattctcg caacctcgtt gatcgcttag cgtacgtgcc gcgcgtgttc ccttctacca 2340
ttgacgtcca acatcgcact atcgctcctt ctgatcctga gatattcggt tttcgttcca 2400
acgcgtggac gcagctctct ttcttctacg atcacgcact cacgttgatg gattttactg 2460
atgttaagca ttggctcgac ctcggcacag gtcctgaagc gcgtccctta tctttcttgc 2520
cttccgacct tccagtgact ctttgtgacg tacgcccctt cgtcctgccc accggatgtt 2580
ggtccacatt taccgatatg ctgaattatg attatttaac aactaatgtg gtgttgtcca 2640
ctggtgcgga tgttgtctcg tgtgttctct ctctgggtgc ggcttgcgct gatgcaaaca 2700
tgtcccttca tgatggtgta cgccagttag tctctcagtg tgttgaagct aatgtccgca 2760
cacttttcct ccaacttaac tgtcctctcc cttccgcggg cgatgtatct cgtgatgttt 2820
tggagttggt ccagtctaac tcaacttatg tcttccattc ccttggacgc gtcgagccct 2880
tccttcctta ctcagctttg cttgaattga ttgaagatat ctgccctggc gttgtggttg 2940
agattaaaac catggatcct tccctagcat ggctaagtta cgcggtgcaa gccaacgctt 3000
cggtaacatc cgatgacatg gcattagcga tgcggctgtc ccacttctgc ccgttatttg 3060
ttttccgttt cgatcaaaga tctgcccaat ttccggacga cgctcgcgtc ggcgttccgt 3120
tctccgtgat tgtgtctaac tatgatgcta cacactctta tgaggtcact cttgacaacg 3180
ttaccatcgc gaccgtcact gctggttcct taataggctt ctcttctggt gtgactgttt 3240
cagttcaagg tactctactg actctctcta ttaatccgtc tagccctggt gttctctccg 3300
tagttcagac attgccagtt cgcatctctc ttggcagttg cgttatagac gctccggatc 3360
catcgctgtc tcttgtgttc ccttcagtcc tagatacttc tttgtcgggg acgaacttag 3420
agttgtactt gtccgactgg tacgatgttg cactcttcta catcgacgag gtcaactctc 3480
gactgattcc tctatccgat tcaaaatatg aaatctttcg acgtgatcaa gcccctaata 3540
gccgcaccct caactatatc ttcgaccgat cggatgtttt ctttaaaatc gtgttgtgtg 3600
acgtctcatc ctctggtgta ggccgattca tctatcgtga acttcctgag ctgagttctc 3660
cggtatggcc tgacaacatg cgcaccttct tgtcattgcc ttttgatgct cctatggtga 3720
ttatctcccc ggatggtccc gtgaattacg atggtgctgt aatcacccct cctacttcat 3780
ggctgacggt ggatggtagt acctgcgtca ttgatggacg tccatcattt tacgttcctc 3840
ctggtcgata cggtctggtg agagtctaaa cgatcgcggg cctccagtag aagggtgtta 3900
ctcatc 3906
<210> 7
<211> 2264
<212> DNA
<213>the M1 gene of strain N-DAV-XT18
<400> 7
atctagccac accggtgcta ggagttggtt ctcgtattac cgcattggat cgcactattg 60
acgcacttac tttgaaacca cgtaccgaac tgcacgatgt ctacactctt gatccatctt 120
taactctacg ccagattgag ttgatttcat ctggtacttc aatggacgat atcgctcgcg 180
gtttgcttca tcgggattgg cgtcgtcaaa ctgtcattac tctgcttcct tctcgtcgga 240
ctttactcga ctatctactc tctaatcctt cgttgtgtcc tgatggttta gatcgttcca 300
ggctcaaagg gttcaagaag cgtcccaatg acttccgcat cagtgacttc ttttctccgt 360
taatcactga gtccacttct attagcacat actcacgctg gctcaatgcc catcctgtta 420
tattctccat cactcacaag gtagtcggcg ctcgcttacg cctattcggt ccgtctaaat 480
tatatacatt atctcccgac gttcttcgag agctatcgat tttgaagtca actgatcgtg 540
ttcttgtcac acccactgcg cgtgtgtacg tgagttgttt ccctagtgct tctacgagta 600
actgcgtcct ctctgcccgc gaacgttgga acgctcccga tgtccatccc gtcgttaaag 660
ccatacaact ggtctacgat caccagtacc gcgtcaccgc gcgttatctc tccgatccct 720
tagcttcggc tttattgctt gggactcagt cggtacgctc tctgaaggtt ccccccatcg 780
aggctagagc tgctcgttca gttggcgttc gtgtacaggc aatgacccct cctcgtggta 840
tcaacacctc catcatacaa gtcatcgatc tgcgtttgca gtgccgtcac tcacttatcc 900
ctgttgagag accatttcca ttaactctcg taggcttgcc gtcctgcttg cttcagcatt 960
tggagttgac tttatccgat agctggacgc ctattcgaga ctcaactggt atgtttgaga 1020
tgtggttcat ggtactcact cttacgtgcg acaagattgt ggatggacgt ggtaatgctg 1080
tctttctaac ccctggttct actgcctctt cttcaattaa ttatgttcag ctcgtgtcta 1140
cttcttctcc tcggccgcaa tcgctggcat ctaacgcgtc tggtcgtatt gactccgtag 1200
gtttgtgtat gccaaagggt tcattcaaat cgactatgat taagttcctg actggcttag 1260
aaatatgtgg cacgcgcgta atgtactctg atgtcgtcat ggacagcgac gatgttggtg 1320
attcattgga ccctaccttt gagactactc tctttgatga gttgatggcg ttggatcctc 1380
ctttcgactt agataagtta gccagttcaa ctgaccttgt cgatcagtct tacatcgcct 1440
ctcacatgta tcccaccttt ctgaggcttg ttaacgaact gttgactcct cgagcttctg 1500
agctatactc agaacacagt gctgagttta ggtctttgac ttacgcccat gcagactctg 1560
aatttcttaa cgcttgttgg acagcacgat tgatgcgctg ctttattaac tactatgagg 1620
agcagaacat cctgctacgt cctgggagag ttggaggtgt attgtttcaa gttgcgctca 1680
gtcgttgcta taagatgttc gcgacatcca ctccttcagt tccattgtca ttattcctaa 1740
aatcactgtt cgtcccttgg attgaatcgg caccccttct cgccccactg actctcaacg 1800
aatcctcacg cgtactcgct tggtatatac ccgagtcaca atggacggaa aatggatggt 1860
gcatgtgtga taagcatcgt catgtgacct tttcttttat tcgtggtttg cctgctgacc 1920
tgtccgtcct tgatctgttc gactggtcgc gattccgcgc aactatcgga gtagacacaa 1980
ctctcgtgga gctgggtacc tccattcgtg ccgttcgcgt ctccgttttc tggacttccc 2040
aaaaacccac tgtggacgtg tttgataacc gcgctctctt caccccattc aaacactatc 2100
atcttagtct tcactgtcgg tgcgcccttg gtcgaccatt caccgcgaag aacatgaaat 2160
tgtatttgtc cacggtagga aacgagaact gacgggccgt ggggcggtga cacccaggga 2220
gggtatgctg gtaaccctgg gttagtcgtc ttgagatact catc 2264
<210> 8
<211> 2158
<212> DNA
<213>the M2 gene of strain N-DAV-XT18
<220>
<221> misc_feature
<222> (1)..(4)
<223> n is a, c, g, or t
<400> 8
nnnntttgag tgctaacctt tctcacacga tgggtaacgc gacgtctgtg gttcagaact 60
tcaacataca aggtgatggt aaccattttt ctccttctgc cgagactacc tcctccgctg 120
taccgtcgct atcattaaat ccaggtctgc ttaatcctgg cggtaaagca tggaccctta 180
tcaacccaac gctcaatgca tccgatccct cgtcgttacg attgatgaca tccgctgact 240
tgtccacctt gtcccaatcc gcgattggta actccactgg ccttctccca acgtcaggca 300
tgtataccgt ggctaataaa gagacgttga gtgtggtgac gaatcatgca ctgtctcaat 360
ttgagaagct tcaaatggcc tgcgaactgg atcgtgacta tctggatgct cgaggcgtgt 420
caccagaatc cgtggatatc cacaattaca tagtctacgt ggactgcttt gttggtgtgt 480
ccgctcgaca agcggcttct aattttcagc agcacgttcc agtcatcacc aagtcaagaa 540
tgacacagtt catgacgtcc gcgcagaata tgcttcaggt ccttggccca tgggaacatg 600
acgttcgtga gcttctcacg atattgccga cttctaccac tgcaggcaag atcacttgtg 660
acatgaaatc cgtggttaac tttgtcaacg accagctctc tgacacaaat ttatgccgtc 720
tctatcctga atgtgctgct tcgtctgtag ctaaacgtaa tggcgggata cgatggaagc 780
aacctgacac tgacgaggct ccctcccttg caactaacga tatcgccgct tccacgatgg 840
gtgcgcttgc taacaccacc cccctggctg agaagtctaa ttctggtgaa gagtctatgc 900
gtttagtcaa cgacgtaggt gtggatatca tctgttctag agcgccagtc agctcctcag 960
tgtggtcacg tactgttgag ccaaaatcgt ataacatcag gacactccgc gtggaagaag 1020
cgctttggtt gcgggaatgt cagacatcta atggttttga cgttcagtac actctgccag 1080
atcagacaac tcataagcat ttctggcttc aacaaggttc cacagtgatt aacttggagc 1140
agacgggcgg catgatgttt gaggtgaata tctctggtaa agactacaag aagggcactt 1200
ttgatccaga taaccagaag ttggtgctcc tagttatgca atccaaaata ccttttgagt 1260
catggacttc cgcagcacag attactggta tagctcaagt ggctgaagtc actgtacacg 1320
ccgccgacag ctcgacacct ggtcggaaaa tcattggtga gacatcgtta tcgtatctgt 1380
ttgaaagaga gaccgttact acagctaaca ccgaggtgaa cacctacctc ttttgcacgt 1440
ggcaacttga cgatgctcag agtaatggcg acaatgcttg gcaggatgcg tgggatggta 1500
ttaccacttt gaccccactc acttcaggca ccgtgacggt taagggtacg tctgtggact 1560
ctgtcgtgcc gactgactta gtgggctctt atactcctga agctctggct gctgcccttc 1620
caaacgacgc cggacgtatt ctcgccaata aggctattaa gctggctgat gctatcaaga 1680
aagaggatga ttctgtcatt gacgagtctt ctccattcag cacccccatt cagggagtcc 1740
tcgccgtcca acagctagat accgttggga cacgtggtgt gcgtatcatc caacctccct 1800
cctttctgaa acgtgtcgct tcacgtgctc ttcacatgtt cctcggcgac cctcagtcca 1860
tcttgaagca ggcgacaccc gttcttcgag acccggacgt ttggactggc ttcatccaag 1920
gagttcgtga tgggatccgc accaagtctc tttccgctgg agttagatct gtctacaaca 1980
acgtcaccgc aactcaatca gtgcagacct ggaagcaggg atttctgact aagatacaga 2040
cgttgttcag accatagtga ggtgctaagg cctctctgcg cggcgggtcg gtgggcacgt 2100
cgcgttgatg ctgaatgcac ggggaggtga cgctctctgg gttagcacgt tactcatc 2158
<210> 9
<211> 1996
<212> DNA
<213>the M3 gene of strain N-DAV-XT18
<220>
<221> misc_feature
<222> (1)..(4)
<223> n is a, c, g, or t
<400> 9
nnnntttgag tcctagcgtg gatcatgtcg tcaaccaagt ggggagacaa accgatgtcg 60
ctctcaatgt ctcatgatgg gtcgtccatc cgttctgctg catcacaatt tttgtcggtt 120
ccattgtccc actccactcc tattccacct caacggaaga ctgtgcttct gaagttcatg 180
ctcggcgaag atctggttac cgttcagggt actctggctc cgtttgatga ctactggttc 240
gataatcaac ccctgctttc ccaggctgtg gaactgttgg catctgagga ccgtctacgt 300
aattttgagc attatgagaa gtttcttctc aagaagggtc atcagctacc ggagattatg 360
aacagactgc gtttattctt cactgatgtg ttgaaggtga aaatggaagc tgacaatcta 420
ccgtcccttg ctcaatattt gatggctgga accatggatg ccgtttctac tggtcaccct 480
cccggcgcct ctgtgccaga cgtctctaaa gtggtagcta agcagcaaac gatctctaaa 540
tcgcccggtc gtttggatga ggaagagtat aacgtaatac gttcacgctt cttaactcac 600
gaggtatttg acctgtcatc cgatctccca ggtgtccaac cctttctgga catgtattat 660
gctaccgttc cacgtgctga tgctactggc tggtgcgctt cacgacgcaa agggttactt 720
gttcacgctc ctggcgaacg tttcgaggat ctaaccatat ttgccactga tacctctcta 780
ccaaatgagc tcatattaac ggcaggtgat gtcactgtag cgcgctttga ccttcttgac 840
gtgtccggga tagcccctca acatcacgct tcagtccaag aagagcgtac tgttggaacg 900
agtcggtatt cagccattac tgcaaacgat caccctttgg tgttcttctc tccctcagcc 960
attcgttggg cgattgatca ttcgtgcact gattcgctca tttctcctcg gaatatcaga 1020
gtgtgcgttg gcatcgaccc gttgattacc cgttggacgc gcgatggtgt gcaagaagct 1080
gctattctga tggatgacaa gctcccgtct gtcggtcgcg ctcgcatggc tctcaggaca 1140
ttgactctcg ctcgtcggtc gccgatgatt tctttcatga ttggagcgtt gaagcactca 1200
ggtggtcaac tcatggaaca ctatcgatgt gacgcagcta atcgatacgg ttcgcccacg 1260
gtgccagcct ctcaccctcc accctgcgct aagtgcccgg agcttagaga acaaatcacg 1320
aggctatctt ctcaacccgt cgccaattca caacctctgg ccggtccagc tgctctgctg 1380
tcgaagatct ctgaattgca acgcctcaac cgggaattgt ctctgaagtt ggcagatgtt 1440
caaccggcac gtgaagacca tctcctagcg tatttgaacg agcatgtgtg cgtgaacgcc 1500
aaggatcatg agaaggacct ccttgcccgt tgcaatgtgt ctggtgactc agtcaatacc 1560
gtcgtcgccc agagagctaa gaatcgtgac aggtttgagg cccgcttgcg tcaggaagcc 1620
aacgctgaat gggagccccg catggctgca ctaaatcaag agctcacaca atcacgcgct 1680
gaacagcagg agatgatgac gcaggcgctg caatacctca acgagcgtga cgaattggct 1740
caagagttgg atgagttgaa acgggagatc actactctga gatctgcgaa cgtgaggttg 1800
aatgctgaga accaccgaat gagtcgagcg accagagtgg gtgattcttt tgtcagtgat 1860
gtcctctcga cgccttccga cgttccacga acttctgcac cttccatgga tgacctggtg 1920
gacgacctgt gagctttgac ctgtgactcg gtttctctct gactccatga ccccacggcg 1980
gactcggtta tccatc 1996
<210> 10
<211> 1568
<212> DNA
<213>the S1 gene of strain N-DAV-XT18
<400> 10
gcttttttct tctctgccca tggctgacgg tgcatgcaat cacgctactt ctatttttgg 60
agccgtatat tgtcaaatat cacagaatat tgcacacgga aatattgatt cttacacctc 120
ttggacttct tatttacctc ctattctagg cggtggtttt ggtcttattg ttcttctcgt 180
gcttgtggtt ttgatcgtgt attgttgtaa gcattcaaaa attctttctg ccgttaaggc 240
gactgattct gccgtgacaa ctttgcttcg tgatgtcact cccgccaacc ccgatcctgt 300
tcaagtcgtt taaagtccac tcgtggcgtc ttttgtccca atccccgttc cacgttcagt 360
tctgcgacgc aggactccaa tcctacgacg tctattcacg tttcccttct gtgtgtgacc 420
tttcgctctg ttattatctt aatacacctt tcgagtttgg aattgctgct atagaggggc 480
gtcccggtga ctattatttg ctgttcgctg gcaagtccag tgactctaac tcacgagtat 540
ctttgtacgc tacgcggcaa gccggtgacg atggatcgca acgaggtgat acgcctgata 600
ctttccctcc tcccctacca gtcaagcgac gtcgatcatt tgacgacaca gatcaaatcc 660
ctccaaagcg ccgtcgactc actgaaagaa tcacaagtgg tagtgttgag acgcctgact 720
acgattacgt cgacggtggc ggatctacaa tcaacaactg aattgttgac ctcacaggtg 780
gcaggactta gttcccgtgt ggcttcagtg actgatgagg tagtccgtgt aaattcagtg 840
attggaacta cgatcactaa tcttgacaat gtccggtccg agctatcctc tctctcctcc 900
caagtctcgt cgcagacgtc cactctaacg aatcttacat caaccgtttc atcccagtct 960
cttgcgattt ctgatctcca gcgacgagtt acggccttag aacgatcggg tggtgcgccg 1020
acacaatttg aagctccctt gcacctacaa aacggagtcg tctcactcca agcatctccc 1080
tctttctgtt ctttgtctcc gatcctctcc ggacctgctg atgctgctgt tttcaaggtt 1140
ggtgagtggc tgggaactgt catatctggt caaagtcagt catctgcaat catgaacgtg 1200
cggattcatt catttgggca gcggaccatg ttgcttatgt cttcgcaaaa tgtattcact 1260
attccgccag gttcgggtgc gtctttgcag ctagatgtga atcgtataac gacccctgcc 1320
attgacgctg ctatggtaac tccttccgct gcttttgctt ctgcttcctt tatggctgac 1380
atagctttca aagactctaa gacaggagaa gtccatgctt tacacactac tggctctttt 1440
cgatcacctt ctttctctat cgtttgggtc ccggttgctt cggaaactcg taattatcaa 1500
ataatggcgt tacgcttcac cgtcgccacg ggctaggctg tggcgcgcaa tgagaagagc 1560
tattcatc 1568
<210> 11
<211> 1324
<212> DNA
<213>the S2 gene of strain N-DAV-XT18
<220>
<221> misc_feature
<222> (1)..(1)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (1323)..(1324)
<223> n is a, c, g, or t
<400> 11
nctttttctt ccacgatggc gcgtgccgtg tacgactttt tatctacgcc tttcggtaac 60
cgtggtctag caactaaccg tactcaactg tcgtcactac tgtcaagttc aaattcgcca 120
tggcaacgct ttttgtccgc cttaactcca ctgaccgctc caggcattgt ttcaacccct 180
gaggcaccct acccgggttc atcgttatac caggagtcca tgcttcacag cgctactatc 240
cctgggattc taggtaatag agacgcgtgg cgcaatttca acgtattcgg cttttcatgg 300
acagatgaag gtttgtcagg acttgtcgct gctcaggacc ctcctccagc tccaccgtac 360
caaccagctt cgggacagtg gtctgatctg cttcagtatc ctcgatgggc taatcgtcaa 420
cgtgagttgc agtctaaata tcccattctg ttgagatcta ctttgctttc agctatgcgc 480
gctggaccgg tgttgtacgt tgagacttgg cccaacatga tttctggtcg acttgctgac 540
tggttcatgt cgcagtatgg aaacaacttc gttgacatgt gtgcacgatt aactcagtcc 600
tgcatgaaca tgccagttga gccggatggt aattatgacc agcagatgcg agcattaatt 660
agtctgtggc tcctctcgta tattggcgtt gtgaatcaat ctaacactat taacggcttc 720
tactttgctt ccaagacgcg tgggcaggct atggataact ggacgctgtt ctatgctacc 780
aacaccaatc gggtgcagat tactcagcgc cactttgcct acgtttgcgc tcgctccccc 840
gactggaatg tcgataagtc ctggatcagc gctgccaatt tgactgccat tatcatggct 900
tgccgccagc cgccagcctt tgccaaccag ggggtaatca accaggccca gaatcgaccc 960
ggattctcga tgaatggtgg aacgcctgtg cacgagctga acctgctgac taccgctcaa 1020
gcttgtatcc agcagtgggt cgtagctggt ttgatctcag cagctaaggg tcagtccatg 1080
acgcaggagg cgaatgactt ctcgaacctc atccaagccg atcttgggcg gatcaaagcg 1140
caagatgacg cgttgtacaa ccaacaacct ggttacgctc gtcgcattaa gccgtttgcc 1200
aacggagatt ggacgcctgg aatgactgcg caagcattag ctctactagc cacttttacc 1260
gtctaggcgt agggtcgtac gctgcctgag tccagccctc cggcagcacg tggacgtact 1320
cann 1324
<210> 12
<211> 1202
<212> DNA
<213>the S3 gene of strain N-DAV-XT18
<400> 12
gctttttgag tccttagcgt gcaagccgca atggaggtgc gtgtgccaaa ctttcactcc 60
tttatcgaag gtattactac tagttacttg tgttctcctg cgtgctggaa ttcgaagacg 120
ttatgggata ttgaagaatt tcacacacct gacgttatca gggtcggcaa cgcttattgt 180
tgcactcagt gctgtggtgt tctgtactat ggtgcccctc cctctgatgg aaactgtttt 240
ccacatcaca agtgtcatca acagcaatat cgtactgaga ctccgctcat gagatatatt 300
aaggtgggtc gcactacaga gcaactgctt gatcaatatg ccattgctct gcatgtcatt 360
gcagattact atgatgaggc gagtaagcaa cctcatgata ttgctgaagc tgagtcaatc 420
gcaccatttg atatcgtaac caggactgaa tctattcgca gtgaccgtgc cgttgacccg 480
gaattctgga cttatccgtt agagaggcga ggatacgacg cgcgacatga gattgctaga 540
gcgggttgga agatgatcga tgcttcatcg cgaagtcaca ctcttcctga atgtctggtg 600
tcaaatatgc tacatactag gcatgtcttc agccaaatgt tgaccacgac aaccatctat 660
gatgtcgctg tcacgggtaa ggccgttaaa ttcagtccga tggtagcaac catgccaact 720
cgaggagatg gtgctgtggc tctgtcaaga ggtaacttgg atcatgatgt cgaggactgt 780
tggatggatg gttttgcatt ctcccccctc atcggcggtg ttggcatcac tggtcaattt 840
gagcgtggtt cctgccataa ttttgggcac cccatgattg ggagcggtaa gaaagcttct 900
cactaccgca atttgttcat ggaatcctgg cgtggatggt caaagtcgtg ctttacatgt 960
gctgcaggga tggagcccgc ggagtgcgaa tctaggctgc gaggccacgc cagaactatg 1020
ttcggacgtt ctcttccgga tatctgtgac ttcgaggaga ctacccacgt tggccagtcg 1080
tccgcgccat taaagaaggc cacgaaattg tccttcctgg agtgtaggtg gtaagcacct 1140
ctgggtcaaa atgcacatag gctcccacct atgtgacggt tagcgggact cacctattca 1200
tc 1202
<210> 13
<211> 1191
<212> DNA
<213>the S4 gene of strain N-DAV-XT18
<220>
<221> misc_feature
<222> (1)..(4)
<223> n is a, c, g, or t
<400> 13
nnnntttgag tccttgttca gccatggaca acaccgtgcg tgttggagtt tcccgcaaca 60
catccgtcgc cgctggtcag actgttttca agaacttcta cttactccga tgcaacattt 120
cagctgacgg tcgaaatgcc acgaaagcgg tccagtctca cttcccatac ttgtctcgcg 180
ctgtccgttg cttgtccccg cttgctgctc actgtgctga tagaactctt cgtcgtgata 240
acgtcaagaa cattttgacg cgtgatctgc cctttccatc cgatctcatt aactacgctc 300
accacgtgaa ctcgtcctcc ctcaccactt cccaaggcgt tgaagccgct cgtctcgtgt 360
cccaggttta cggggagcaa gttcctttcg accacgttta tccatctggt tcagcaacct 420
actgtcccgg tgccgtcgcc aacgctatct ctcggcttat ggcaggtttt gtgcctcagg 480
aaggtgacga tttcatgccg actggtccaa tcgattatct cgcagctgat cttgtggcgt 540
acaagtttgt tttaccgtat atgttagata tggtggatgg acgtccccaa attgtgctac 600
cctctcacac cgttgaggaa atgttgactg acactggcct gctgaatgct attgatgcct 660
catttggcat tgagtctaaa agtgatcaac gcatgacgcg tgatgctgct gagatgagct 720
ctcgttcgct caatgagtta gagaaccatg aacatcgcgg gcgcatgccg tggaagatta 780
tgcttgctat gatggccgcg caactgaagc ttgaactgga cgcattagca gacgaacgca 840
ctgagtctca ggctaacgct catgttacat ccttcggctc acgtctattc aatcagatgt 900
cagcctttgt gcctatcgat cgtgagttga tggagcttgc tctcctaatc aaagaacaag 960
gctttgccat gaatcctggt caggttgcgg ccaaatggtc gtctattagg aggtccagtg 1020
cagtacgttc cctggcgagt gcgcgtcttg agattcgaaa tgggaactgg atgatccgcg 1080
aaggcgacca gacgctgctg tctgtctctc cagctaggat ggcgtagacg ggacccatgg 1140
tgtgggtgag gggggccacg acctctgcca cgacttggac tcttattcat c 1191
<210> 14
<211> 83
<212> DNA
<213>artificial sequence
<400> 14
attagctcta gccacagccc tgacctgtcc gtccttgatc tgttcgactg gtcgcgattc 60
caatagtgca tagcatcaag tac 83
Claims (10)
1. one group of primer and probe, which is characterized in that the nucleotide sequence of the primer such as SEQ ID NO.1-SEQ ID NO.2
It is shown;The nucleotide sequence of the probe is as shown in SEQ ID NO.3.
2. the examination that primer and probe described in claim 1 leads to the novel duck reovirus of duck spleen necrosis in preparation detection
Application in agent or kit;The deposit number of the novel duck reovirus is CCTCC NO:V201843.
3. a kind of detect the PCR kit for fluorescence quantitative for leading to the novel duck reovirus of duck spleen necrosis, which is characterized in that
The PCR kit for fluorescence quantitative contains primer and probe described in claim 1.
4. PCR kit for fluorescence quantitative according to claim 3, which is characterized in that in the PCR kit for fluorescence quantitative
Further include: standard items and quantitative fluorescent PCR reaction reagent.
5. PCR kit for fluorescence quantitative according to claim 4, which is characterized in that the standard items are made by the following method
It is standby to form:
Primer amplification deposit number shown in SEQ ID NO.1-SEQ ID NO.2 is used to exhale for the duck of CCTCC NO:V201843
The genome of the lonely virus of intestines, obtains amplified production, amplified production is connected on pMD18-T carrier, screening positive clone simultaneously mentions
Plasmid DNA is taken, that is, standard items are prepared.
6. PCR kit for fluorescence quantitative according to claim 4, which is characterized in that the quantitative fluorescent PCR reaction reagent
It include: GoldStar TaqMan One Step Buffer, GoldStar TaqMan One Step EnzymeMix and ROX.
7. a kind of detect the detection reagent for leading to the novel duck reovirus of duck spleen necrosis, which is characterized in that the detection
Reagent contains primer and probe described in claim 1;The deposit number of the novel duck reovirus is CCTCC NO:
V201843。
8. the described in any item PCR kit for fluorescence quantitative of claim 3-6 or detection reagent as claimed in claim 7 are as follows
(1) in-(3) it is any in application:
(1) the novel duck reovirus for being CCTCC NO:V201843 to duck infection deposit number carries out epidemiological survey;
It (2) is that the pollution of CCTCC NO:V201843 novel duck reovirus carries out to the deposit number in blood or serum product
Monitoring;
(3) accurately detection deposit number is CCTCC NO:V201843 novel duck reovirus copy number, specifies novel duck and exhales
The progression of infection of the lonely virus of intestines.
9. a kind of lead to the new of duck spleen necrosis using the described in any item PCR kit for fluorescence quantitative detections of claim 3-6
The method of type duck reovirus, includes the following steps:
(1) standard items are subjected to gradient dilution, carry out TaqMan Fluorescence PCR later, according to the concentration of positive criteria product and
Ct value draws fluorescent quantitation standard curve;
(2) sample to be tested total serum IgE is extracted, TaqMan Fluorescence PCR is carried out, collects sample to be tested fluorescence signal, fluorescence is believed
Number carry out data processing, obtain Ct value and amplification curve, to sample to be tested carry out qualitative and quantitative detection.
10. according to the method described in claim 9, it is characterized by: TaqMan fluorescent PCR is anti-in step (1) and step (2)
The program answered are as follows:
45℃10min;95℃10min;95 DEG C of 15s, 60 DEG C of 45s carry out 40 circulations later.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110669870A (en) * | 2019-10-15 | 2020-01-10 | 云南省畜牧兽医科学院 | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for serotype of Palima serogroup virus |
CN111235321A (en) * | 2020-04-15 | 2020-06-05 | 福建省农业科学院畜牧兽医研究所 | Dual TaqMan fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for Muscovy duck reovirus and novel duck reovirus |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102352346A (en) * | 2011-10-24 | 2012-02-15 | 福建省农业科学院畜牧兽医研究所 | Novel-pathotype duck reovirus and attenuated vaccine thereof |
CN103451157A (en) * | 2013-07-12 | 2013-12-18 | 广东温氏食品集团股份有限公司 | Method for separating new type duck reovirus from hybrid virus sample |
CN105854011A (en) * | 2016-04-26 | 2016-08-17 | 中国农业科学院上海兽医研究所 | Preparation and application of efficient cell inactivation vaccine against new duck reovirus disease |
-
2018
- 2018-08-10 CN CN201810911066.4A patent/CN108977579A/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102352346A (en) * | 2011-10-24 | 2012-02-15 | 福建省农业科学院畜牧兽医研究所 | Novel-pathotype duck reovirus and attenuated vaccine thereof |
CN103451157A (en) * | 2013-07-12 | 2013-12-18 | 广东温氏食品集团股份有限公司 | Method for separating new type duck reovirus from hybrid virus sample |
CN105854011A (en) * | 2016-04-26 | 2016-08-17 | 中国农业科学院上海兽医研究所 | Preparation and application of efficient cell inactivation vaccine against new duck reovirus disease |
Non-Patent Citations (2)
Title |
---|
YI TANG等: "Whole genome alignment based one-step real-time RT-PCR for universal detection of avian orthoreoviruses of chicken, pheasant and turkey origins", 《INFECTION,GENETICS AND EVOLUTION》 * |
袁远华: "新型鸭呼肠孤病毒SYBR Green I实时荧光定量RT-PCR检测方法的建立", 《中国预防兽医学报》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110669870A (en) * | 2019-10-15 | 2020-01-10 | 云南省畜牧兽医科学院 | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for serotype of Palima serogroup virus |
CN110669870B (en) * | 2019-10-15 | 2023-08-04 | 云南省畜牧兽医科学院 | Real-time fluorescent quantitative RT-PCR detection primer, probe and detection kit for Pariemam serogroup virus serotypes |
CN111235321A (en) * | 2020-04-15 | 2020-06-05 | 福建省农业科学院畜牧兽医研究所 | Dual TaqMan fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for Muscovy duck reovirus and novel duck reovirus |
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