CN102876812B - Kit for detecting FADV (pigeon adenovirus) - Google Patents
Kit for detecting FADV (pigeon adenovirus) Download PDFInfo
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- CN102876812B CN102876812B CN201210404102.0A CN201210404102A CN102876812B CN 102876812 B CN102876812 B CN 102876812B CN 201210404102 A CN201210404102 A CN 201210404102A CN 102876812 B CN102876812 B CN 102876812B
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Abstract
The invention discloses a primer pair for detecting FADV (pigeon adenovirus). The primer pair is indicated as nucleotide sequence SEQ ID No.1 and SEQ ID No.2. The invention further discloses a kit containing the primer pair. Due to the fact that the primer used comes from a highly conservative area in FADV gene, whether pigeon circovirus is contained in disease material or not can be distinguished accurately, fast and conveniently by the primer, sensitivity can reach to mould DNA (deoxyribonucleic acid) of 0.04 microliter. The primer is designed for the highly conservative area in FADV gene, and a method of PCR (polymerase chain reaction) FADV detection method is built. Experiments show that the method is high in sensitivity and fine in specificity and is a method for detecting FADV fast and accurately, and a domestic gap is filled.
Description
Technical field
The invention belongs to the virus detection techniques field, be specifically related to a kind of detection kit of dove adenovirus.
Background technology
The dove adenovirus infection is to infect by adenovirus (Pigeon adenovirus, FADV) acute infectious disease caused, in the pigeon of healthy and morbidity, all can be separated to adenovirus
[1].Nineteen fifty-three, Rowe etc. isolate this virus first from tonsil piece culture.After this numerous biotechnology workers find the adenovirus of different organisms in succession
[2].1984, Cousement narrated Belgian dove adenovirus infection first
[3].
Dove adenovirus severe environment to external world has stronger resistibility, and fatsolvent is also had to resistibility, and the heat resistanceheat resistant acid-fast ability is strong, but not anti-formaldehyde
[4-5].Can preserve 70 days at 4 ℃, can preserve 10-20 minute for 50 ℃, can be very fast dead in 2.5-5 minute at 56 ℃
[6].The dove adenovirus must be induced and just can cause a disease down at its virulence factor, there is no the impact of virulence factor, can not fall ill by the pigeon of adenovirus infection.The dove adenovirus is generally not lethal when falling ill separately, but the sick dove that great majority infect also can be accompanied with other some diseases simultaneously
[7-9].At present the dove adenovirus does not have highly effective vaccine, and different biological adenovirus have specificity separately, can cross infection, so the adenovirus vaccine of different animals also can not cross-reference.This virus is mainly carried out self-replacation in dove digestive tube and respiratory tract, in time multiplexed cell system, also can form inclusion body.The visible pathology of naked eyes be take muco-enteritis as main symptom.Sick dove performance down in spirits, depressed, suffer from diarrhoea, arrange light yellow mucus shape ight soil, anaemia, jaundice, leg muscle hemorrhage serious even can sudden death.Slight respiratory symptom is arranged in early days
[10].For adult dove group, can cause dove group egg productivity to descend.Main pathological symptom is that kidney swelling, hemorrhage, hydropericardium, the fabricius bursa and atrophy of thymus gland diminish
[10-12].The circulation way of dove adenovirus is mainly vertical transmission, by parental generation, passes to filial generation.Ight soil is mainly passed through in horizontal transmission, feed, and the approach such as pigeon house are propagated.Once import the dove group into, can in the dove group, spread rapidly, after seeing this case first, in 2d, mass-sending is sick entirely usually.Clinical symptom can disappear usually in 1 week, because along with adenovirus is eliminated soon, gut epithelium is renewable after several days.But as the secondary infection due to Escherichia coli causes more serious and lasting disease
[3].Conventional diagnosis relies on Electronic Speculum, immunostaining or virus to separate, but these technology are time-consuming, effort, and susceptibility is also poor.
Reference:
[1]De?Herdt?P，Ducatelle?R,Lepoudre?C,et?al.An?epidemic?of?fatal?hepatic?necrosis?of?viral?origin?in?racing?pigeons(Columba?livia)[J].Avian?Pathol,1995,24(3):475-483
[2] Zhang Junfeng, Wang Zhilei, Zhou Xiaoling. adenopathy viral disease [J] .2003.6.20:20-21 of fowl
[3] Hu Qinghai, Huang Jianfang. dove adenovirus infection [J] .1999,21(3): 67-68
[4]Freick?M,Muller?H,RaueR.Rapid?detection?of?pigeon?herpesvirus,fowl?adenovirus?and?pigeon?circovirus?in?young?racing?pigeons?by?multiplex?PCR[J].J?Virol?Methods,2008,148(1-2):226-231
[5]Vereecken?M,de?Herdt?P，Ducatelle?R.Adenovirus?infections?in?pigeons:A?review[J].Avian?Pathol,1998,27(4):333-338
[6] Wang Zhaoping, Shen Jianhua, Zhang Haixiao. dove adenopathy viral disease [J] .2008,9:41-43
[7]Goryo?M,Ueda?Y，Umemura?T,et?al.Inclusion?body?hepatitis?due?to?adenovirus?in?pigeons[J].Avian?Pathol,1988,17(2):391-401
[8]Hess?M,Prusas?C,Monreal?G.Growth?analysis?of?adenoviruses?isolated?from?pigeons?in?chicken?cells?and?serological?characterization?ofthe?isolates[J].Avian?Pathol,1998,27(2):196-199
[9]Hess?M,Prusas?C,Vereecken?M,et?al.Isolation?of?fowl?adenoviruses?serotype?4?from?pigeons?with?hepatic?necrosis[J].Berl?Munch?Tierarztl?Wochenschr,1998,111(4):140-142
[10] intelligence Hai Dong ，Wang Yun peak, Xie Shengliang, etc. aviadenovirus infects and diagnosis [J] .2010:33-35
[11]Wada?Y，Kondo?H,Nakazawa?M,et?al.Natural?infection?with?attaching?and?effacing?Escherichia?coli?and?adenovirus?in?the?intestine?of?a?pigeon?with?diarrhea[J].J?Vet?Med?Sci,1995,57(3):531-533
[12]Wang?CH,Chang?CM.Pathogenicity?and?gene?analysis?of?adenovirus?from?pigeons?with?inclusion?body?hepatitis[J].J?Vet?Med?Sci,2000,62(9):989-993
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of detection primer of dove adenovirus.
The technical problem that the present invention also will solve is to provide the detection kit of above-mentioned dove adenovirus, and to detect fast and accurately the dove adenovirus, isolation in time is subject to the disease dove, keeps off infection.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of primer pair for detection of the dove adenovirus, the nucleotide sequence as shown in SEQ ID NO:1 and SEQ ID NO:2.
A kind of dove adenovirus detection kit, comprise above-mentioned primer pair.
A kind of dove adenovirus detection kit comprises:
(1) the dove adenovirus positive and negative control;
(2) dove adenovirus Auele Specific Primer pair:
Upstream primer P1: as shown in SEQ ID NO:1;
Downstream primer P2: as shown in SEQ ID NO:2;
(3) DNA extraction reagent: adopt the viral nucleic acid that Geneaid company produces to extract test kit (Viral Nucleic Acid Extraction Kit II);
(4) PCR reaction reagent: by upstream primer P1 15 μ L (10mM), downstream primer P215 μ L (10mM), 2 * PCR Master Mix, 200 μ L, ddH
2the O(ultrapure water) 100 μ L form;
(5) electrophoresis detection reagent: by 50 * TAE electrophoretic buffer 20mL, GoldView 100 μ L form.
Adopt the detection method of mentioned reagent box, comprise the steps:
(1) extraction of DNA: adopt Geneaid company to produce viral nucleic acid extract test kit (Viral Nucleic Acid Extraction Kit II), extract viral DNA according to specification sheets;
(2) pcr amplification:
25 μ L systems: 2 * PCR Master Mix, 12.5 μ L; ddH
2o 6.5 μ L; Upstream primer P1 1 μ L; Downstream primer P21 μ L and template 2 μ L; ;
PCR reaction conditions: 94 ℃ of 5min, 90 ℃ of 1min, 61 ℃ of 1min, 72 ℃ of 1min, 36 circulations; 72 ℃ of 10min; 4 ℃ of preservations.
(3) agarose gel electrophoresis:
Pcr amplification product is loaded onto in 1% agarose, agarose is placed in electrophoresis apparatus and carries out electrophoresis about 40 minutes, use imaging system to record result;
(4) judged result:
If amplify a 600bp specific fragment, show the dove adenovirus positive of organizing to be checked;
If do not amplify fragment, show the dove adenovirus feminine gender of organizing to be checked.
Beneficial effect: the primer adopted due to the present invention, from the conservative zone of dove adenoviral gene camber, adopts this primer pair can differentiate accurately, fast and easily in pathological material of disease and whether contains pigeon circovirus, and sensitivity reaches the template DNA of 0.04 μ L; The zone design primer that this research is conservative for dove adenoviral gene camber, set up a kind of method that PCR detects the dove adenovirus.Proving that by experiment the method has the advantages that susceptibility is high, specificity is good, is a kind of method that quick and precisely detects the dove adenovirus, has filled up domestic blank.
The accompanying drawing explanation
The PCR detected result of Fig. 1: FADV.Wherein, 1:Marker, 2-4:PCR product.
Fig. 2: different annealing temperature detected results.Wherein, 1.Marker, 2.55 ℃, 3.61 ℃, 4.59 ℃, 5.63 ℃, 6.57 ℃, 7.65 ℃.
Fig. 3: specificity identification detected result.Wherein, 1.Marker, 2.PiCV, 3.PIHV, 4.FADV.
Fig. 4: sensitivity test detected result.Wherein, 1.Marker, 2.1:1,3.1:2,4.1:4,5.1:8,6.1:16,7.1:32,8.1:64,9.1:128,10.1:256,11.1:512.
Fig. 5: the clinical pathological material of disease detected result of part (5,6,7 is negative).Wherein, 1.Marker, 2-7:PCRproduct.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the preparation of test kit.
1, pathological material of disease.
From Zhejiang, the dove field on the ground such as Jiangsu, Anhui, Shandong, Shanghai, Fujian takes 135 parts of serum of disease dove; Pathological material of disease is preserved by Jinling School of Science and Technology Preventive Veterinary Medicine teaching and research room.
2, reagent.
DNA extraction test kit (Viral nucleic acid extraction kit II); 2xTaq Master Mix, Nanjing Bo Erdi bio tech ltd is produced; The Goldview nucleic acid dye, match Parkson company produces; Agarose, Beijing Baeyer enlightening bio tech ltd; 10xTBE; Marker, Shanghai Ke Xing Bioisystech Co., Ltd.
3, the design of primer and synthetic.
According to the gene order of the adenovirus of having delivered, utilize primer premier 5.0 design primers, synthetic by the handsome company in Shanghai.Estimate that size is 600bp.
Upstream primer pFADV15 '-CAATAGCATCTCCGGGGTGG-3 ';
Downstream primer pFADV25 '-TTCGTAGCCGTCGTTGTTGA-3 '.
4, the manufacture of other compositions in test kit.
4.1DNA extraction reagent: purchased from Geneaid company, produce viral nucleic acid extract test kit (Viral NucleicAcid Extraction Kit II).
4.2 ultrapure water (ddH
2o) preparation.
Purchased from Takara company, the 1mL/ pipe.
4.32 the preparation of * PCP master Mix.
Purchased from Nanjing Bo Erdi bio tech ltd, the 1mL/ pipe.
4.4 the preparation of 50 * TAE electrophoretic buffer.
0.5mo1/L second ammonium tetraacethyl disodium ((EDTA) solution (pH8.0) preparation:
The preparation of 50 * TAE electrophoretic buffer:
During use, the dilution of sterilizing distilled water is 50 times.
4.5Glodview the preparation of nucleic acid dye.
Purchased from match Parkson, Shanghai gene engineering company limited, the 1mL/ pipe.
4.6 the preparation of sample-loading buffer.
Purchased from Nanjing Bo Erdi bio tech ltd, the 1mL/ pipe.
4.7 the preparation of primer.
Primer is handsome synthetic by Shanghai, the 2OD/ pipe.
Embodiment 2: detection method.
Key instrument is as follows:
The PCR instrument, Xi'an Tianlong Science & Technology Co., Ltd. manufactures;
Electrophoresis chamber, commerce and trade difficult to understand company limited of the prosperous section in Beijing;
Imaging system, Xiamen Yi Chen Science and Technology Ltd..
Concrete grammar is as follows:
(1) extraction of DNA: adopt DNA extraction test kit (Viral nucleic acid extraction kit II), according to specification sheets, extract viral DNA:
(1a) pipette 200 μ l serum samples in the 1.5mlEP pipe, if sample less than 200 μ l are adjusted to 200 μ l with the PBS damping fluid.The VB lysis buffer that adds 400 μ l mixes in sample.At room temperature place 10min.
(1b) the AD buffer that adds 450 μ l mixes immediately in sample.Pipette the mixed solution of top 600 μ l to VBcolumn.With the centrifugal 1min of 13000rpm rotating speed.Discard following liquid after centrifugal, add remaining mixed solution in same VB column.With the centrifugal 1min of 13000rpm rotating speed, discard the liquid in collection tube, then VBcolumn is transferred to
In the collection tube of new 2ml.
(1c) W1buffer that adds 400 μ l is in VB column, with the centrifugal 1min of 13000rpm rotating speed.Discard the liquid in collection tube.Add the Wash buffer of 600 μ l in VB column, with the centrifugal 1min of 13000rpm rotating speed, discard the liquid in collection tube, with the centrifugal 1min of 13000rpm rotating speed.
(1d) dry VB column is put in the EP pipe of a clean 1.5ml.Add 50 μ l without RNA enzyme water to VB column periosteum center, standing 3min, until water is completely absorbed, with the centrifugal 1min of 13000rpm rotating speed.
(2) pcr amplification:
25 μ L systems: 2 * PCR Master Mix, 12.5 μ L; ddH
2o 6.5 μ L; Upstream primer P1 1 μ L; Downstream primer P21 μ L and template 2 μ L;
PCR reaction conditions: 94 ℃ of 5min, 90 ℃ of 1min, 61 ℃ of 1min, 72 ℃ of 1min, 36 circulations; 72 ℃ of 10min; 4 ℃ of preservations.
(3) agarose gel electrophoresis:
Pcr amplification product is loaded onto in 1% agarose, agarose is placed in electrophoresis apparatus and carries out electrophoresis about 40 minutes, use imaging system to record result;
(4) judged result:
If amplify a 600bp specific fragment, show the dove adenovirus positive of organizing to be checked;
If do not amplify fragment, show the dove adenovirus feminine gender of organizing to be checked.
The optimization of embodiment 3:PCR amplification condition.
Annealing temperature being optimized, all under consistent situation, having set 6 different annealing temperatures in other condition respectively, is respectively 55 ℃, 57 ℃, 59 ℃, 61 ℃, 63 ℃, 65 ℃.
By the condition of optimizing, the DNA of three positive dove adenovirus is carried out to pcr amplification, 1% agarose gel electrophoresis, amplifying a size is the specific fragment of 600bp left and right, and size conforms to expection, and result is as Fig. 1.
Optimization to the PCR condition is to realize by six different annealing temperatures, the result of six different annealing temperature gained is all very desirable, and result is as Fig. 2, because in checking repeatedly, 61 ℃ have stronger stability, so now using 61 ℃ as the net result of optimizing.
Embodiment 4: specific test.
Extract the DNA of columbid herpesvirus (PIHV), pigeon circovirus by identical method, and and the DNA of dove adenovirus (FADV) together with as template, carry out pcr amplification with the primer designed, the specificity of the PCR condition of optimization is detected.And the PCR product of PiAV is delivered to order-checking company and checked order, the gene order recorded is compared with the gene order of the PiAV delivered.
Extract the DNA of PIHV, PiCV by identical method, and, as template, with the primer of design, carry out pcr amplification, 1% agarose gel electrophoresis, result is as Fig. 3.
Embodiment 5: sensitivity test.
The positive DNA of dove adenovirus is carried out to doubling dilution, be altogether 10 pipes: 1:2
0, 1:2
1, 1:2
2, 1:2
3, 1:2
4, 1:2
5, 1:2
6, 1:2
7, 1:2
8, 1:2
9, then with the primer of design, increased, the susceptibility of the PCR condition optimized is detected.
The DNA extracted of take is template, since 20 μ lDNA doubling dilutions, is altogether 10 pipes: 1:1,1:2,1:4,1:8,1:16,1:32,1:64,1:128,1:256,1:512, with the primer of design, carries out pcr amplification, 1% agarose gel electrophoresis.Result shows that PCR method can detect the template DNA of 0.04 μ l, has very high susceptibility.Because pathological material of disease comes from six different provinces, illustrate that its sickness rate is higher, lethality rate is also higher simultaneously, and result is as Fig. 4.
Embodiment 6: the research of test kit stability.
Test kit is stored in-20 ℃, takes out at quarterly intervals, and the positive DNA of the dove adenovirus of again take carries out the PCR detection as template, usings electrophoresis result as basis for estimation, does continuously the research one-year age.
After being mixed, the PCR reaction reagent is stored in-20 ℃.After the preservation and multigelation of 1 year, by the FADV template, detected, result shows, and the stability of this test kit is better, after 1 year, still has very high recall rate, and explanation can prolonged preservation.
Embodiment 7: clinical pathological material of disease detects.
Use the method for embodiment 2 to be detected 135 parts of clinical pathological material of diseases.
In the DNA that 135 parts of pigeon serum is carried, 61 parts of test positive are wherein arranged, infection rate is very high, comes from the dove field in six different provinces due to pathological material of disease simultaneously, illustrates that its sickness rate is higher, and lethality rate is also higher, and partial results is as Fig. 5.
Claims (3)
1. the primer pair for detection of the dove adenovirus, is characterized in that the nucleotide sequence as shown in SEQ ID NO:1 and SEQ ID NO:2.
2. the detection kit of a dove adenovirus, is characterized in that, it comprises primer pair claimed in claim 1.
3. the detection kit of dove adenovirus according to claim 2 is characterized in that comprising:
(1) the dove adenovirus positive and negative control;
(2) dove adenovirus Auele Specific Primer pair:
Upstream primer P1: as shown in SEQ ID NO:1;
Downstream primer P2: as shown in SEQ ID NO:2;
(3) DNA extraction reagent: adopt the viral nucleic acid that Geneaid company produces to extract test kit;
(4) PCR reaction reagent: by upstream primer P1 10mM, 15 μ L, downstream primer P2 10mM, 15 μ L, 2 * PCR Master Mix200 μ L, ddH
2o100 μ L forms;
(5) electrophoresis detection reagent: by 50 * TAE electrophoretic buffer 20mL, GoldView 100 μ L form.
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| CN103667536B (en) * | 2013-12-13 | 2015-08-05 | 金陵科技学院 | The LAMP detection kit of pigeon circovirus |
| CN107586889B (en) * | 2017-11-03 | 2020-10-30 | 福建省农业科学院畜牧兽医研究所 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer for pigeon adenovirus EvaGreen |
| CN107604099B (en) * | 2017-11-03 | 2020-11-03 | 福建省农业科学院畜牧兽医研究所 | LAMP (Loop-mediated isothermal amplification) detection primer group and kit for novel pigeon adenovirus |
| CN107604100B (en) * | 2017-11-03 | 2021-06-08 | 福建省农业科学院畜牧兽医研究所 | double-EvaGreen real-time fluorescent quantitative PCR (polymerase chain reaction) detection kit for pigeon TTV (total tatami virus) and novel pigeon adenovirus |
| CN107604102B (en) * | 2017-11-03 | 2021-04-20 | 福建省农业科学院畜牧兽医研究所 | Double Real time PCR detection kit for pigeon TTV and novel pigeon adenovirus |
| CN107604101B (en) * | 2017-11-03 | 2020-08-14 | 福建省农业科学院畜牧兽医研究所 | Novel pigeon adenovirus real-time fluorescent quantitative PCR detection kit |
| CN111088399B (en) * | 2019-12-31 | 2023-03-28 | 金陵科技学院 | Primer group, kit and method for simultaneously detecting pigeon circovirus, pigeon adenovirus and pigeon herpesvirus type 1 |
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| CN113073147B (en) * | 2021-03-30 | 2023-10-10 | 福建省农业科学院畜牧兽医研究所 | Primer and kit for B-type fluorescence quantitative detection of pigeon adenovirus |
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