CN105483292A - FAdV-4 PCR detection kit and detection method - Google Patents
FAdV-4 PCR detection kit and detection method Download PDFInfo
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Abstract
The invention discloses an FAdV-4 PCR detection kit and detection method. The kit comprises protease K, a lysing solution, sodium acetate, saturated phenol, chloroform, isoamylol, anhydrous ethanol and sterile distilled water, and is characterized by comprising 2*TaqPCR Mix, a sense primer and a reverse primer, wherein the sequence of the sense primer is shown as SEQ ID No.1, and the sequence of the reverse primer is shown as SEQ ID No.2. The kit can be used for detecting FAdV-4. The method for detecting FAdV-4 by adopting the kit is also provided, and FAdV-4 can be detected quickly and specifically.
Description
Technical field
The present invention relates to the detection technique field of aviadenovirus 4 type.
Background technology
Since in June, 2015, the some areas such as China Henan, Shandong, Hebei, Liaoning, Anhui, Jilin, chicken group there occurs the disease being feature with pericardial effusion, liver enlargement of unknown cause, and according to pathology, this disease is called as " chicken pericardial effusion-hepatitis syndrome ".This disease mainly betides the broiler chicken of 20-30 age in days, and comprise 817, meat is assorted, within 4-8 days, be peak mortality after morbidity, course of disease 8-15 days, mortality ratio reaches 20 ~ 30%.Laying hen 20-70 age in days and 200-300 age in days also have generation simultaneously, but mortality ratio is lower than broiler chicken.At present, the diseased region of this disease constantly expands, and causes huge financial loss to China's poultry husbandry.Now confirm, the cause of disease of this disease is aviadenovirus 4 type (fowladenovirus4, FAdV-4).
Polymerase chain reaction (PCR) is widely used at animal epidemic detection field due to advantages such as specificity are good, susceptibility is high, reproducible.Domestic PCR detection kit and the detection method that there is no detection aviadenovirus 4 type at present.
Summary of the invention
The present invention is directed to new aviadenovirus 4 type, provide a kind of PCR kit for the detection of aviadenovirus 4 type and application thereof, detection aviadenovirus 4 type that this test kit can be quick, special.
The technical solution used in the present invention is: a kind of PCR kit detected for aviadenovirus 4 type, comprise Proteinase K, lysate, sodium-acetate, saturated phenol: chloroform: primary isoamyl alcohol, dehydrated alcohol, sterile distilled water, is characterized in that described test kit also comprises 2 × TaqPCRMix, upstream primer and downstream primer; The sequence of described upstream primer is for shown in SEQIDNo.1, and the sequence of described downstream primer is for shown in SEQIDNo.2.
Primer is the aviadenovirus 4 C-type virus C sequence announced according to GenBank, at a pair Auele Specific Primer of its Hexon gene conserved sequence region design.
Preferably, 2 × TaqPCRMix is made up of archaeal dna polymerase, 2 × TaqPCRbuffer and dNTPMixture.
Preferably, described test kit also comprises DNA extraction reagent: Proteinase K, lysate, sodium-acetate, saturated phenol: chloroform: primary isoamyl alcohol, dehydrated alcohol, sterile distilled water.
Preferably, described test kit also comprises positive control.
Wherein, positive control can be after aviadenovirus 4 type inoculation 9-11 day instar chicken embryo, collects the allantoic fluid of 96 ~ 144 h infection chicken embryos.
The invention allows for described test kit whether to detect in testing sample containing the application in the product of aviadenovirus 4 type in preparation.
Apply described test kit and detect the method whether containing aviadenovirus 4 type in testing sample, comprise the steps:
1, the extraction of viral DNA
Get 300 μ L and be placed in 1.5mL centrifuge tube through pretreated testing sample, add 10mg/ml Proteinase K 10 μ L, lysate 300 μ L, 50 DEG C hatch 2h after add the saturated phenol of 610 μ Ltris, fully shake 5min, the centrifugal 10min of 12000rpm; Supernatant is transferred in new 1.5mL centrifuge tube and adds equal-volume phenol: chloroform: primary isoamyl alcohol (1:24:25), fully the centrifugal 10min of mixing 12000rpm; Get supernatant, add phenol: chloroform: primary isoamyl alcohol repeats extracting once; After extracting, supernatant is transferred in new 1.5mL centrifuge tube, add dehydrated alcohol after 2 times of volume ice baths, 1/10 volume 3mol/L sodium-acetate, the centrifugal 10min of 12000rpm after-20 DEG C of precipitation 45min, abandon supernatant, the dry 5min of vacuum drying oven, with 20 μ L sterile distilled water dissolution precipitations ,-20 DEG C of storages are for subsequent use.
Testing sample can be the tissues such as chicken liver, spleen, lungs, kidney.Wherein, the pretreated method of testing sample is: after being shredded by testing sample, adds physiological saline and grinds evenly, get supernatant, carry out the extraction of DNA afterwards again after the centrifugal 5 ~ 10min of 3000 ~ 5000rpm according to the mass volume ratio of 1:5.
2, PCR reaction
In reaction tubes, add the downstream primer (SEQIDNo.2) of 10 μ L2 × TaqPCRMix, the above-mentioned DNA solution of 1 μ L, the upstream primer (SEQIDNo.1) of 0.5 μ L10 μM, 0.5 μ L10 μM, finally adding nuclease free water to cumulative volume is 20 μ L.
Undertaken by following response procedures after mixing: 94 DEG C of 3min, 1 circulation; 94 DEG C of 10s, 56 DEG C of 30s, 72 DEG C of 57s, totally 30 circulations; 72 DEG C 5min1 circulation.After reaction terminates, check pcr amplification product through 1% agarose gel electrophoresis, if there is the band of 954bp size, testing sample contains aviadenovirus 4 C-type virus C.
The invention provides the Auele Specific Primer of a pair detection aviadenovirus 4 type, utilize the PCR kit that it is made, detection aviadenovirus 4 type that can be quick, special.
Accompanying drawing explanation
Fig. 1 is aviadenovirus 4 type PCR detected result;
M:DL2000Marker; 1: positive control; 2: negative control;
Fig. 2 is aviadenovirus 4 type PCR sensitivity Detection result;
M:DL2000Marker; 1 ~ 6 is followed successively by 10
0~ 10
-5the amplified production of doubling dilution viral nucleic acid; 7: negative control;
Fig. 3 is aviadenovirus 4 type PCR specific detection result.
M:DL2000Marker; 1: aviadenovirus 4 type; 2: aviadenovirus 11 type; 3: infectious bronchitis virus; 4: Avian pneumo-encephalitis virus; 5: avian influenza virus; 6: negative control.
Embodiment
With reference to the accompanying drawings and the present invention is described in further detail in conjunction with the embodiments.But the invention is not restricted to given example.The experimental technique used in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1 one kinds detects the PCR kit most preferred embodiment of aviadenovirus 4 type
The present embodiment test kit comprises: (1) aviadenovirus 4 type DNA extraction reagent: Proteinase K, lysate, sodium-acetate, saturated phenol: chloroform: primary isoamyl alcohol, dehydrated alcohol, sterile distilled water.
(2) PCR reaction reagent: 2 × TaqPCRMix, upstream primer, downstream primer; (4) other: positive control and nuclease free water.Wherein, 2 × TaqPCRMix is made up of archaeal dna polymerase, 2 × TaqPCRbuffer and dNTPMixture, and positive control is that collect the allantoic fluid of 96 ~ 144 h infection chicken embryos, primer is lyophilized powder, and HPLC is pure by after aviadenovirus 4 type inoculation 9-11 day instar chicken embryo.The primer sequence that the present embodiment test kit comprises is in table 1.
Table 1 primer sequence
Embodiment 2 non-diagnostic object detects the method for aviadenovirus 4 type with PCR method
The present embodiment detection method adopts the test kit in embodiment 1.Get morbidity chicken liver, spleen, lungs, kidney etc. and be organized as testing sample.
The present embodiment detection method comprises the following steps:
1, viral DNA extraction concrete steps are as follows: after testing sample shreds by (1), and add physiological saline according to the mass volume ratio of 1:5 and grind evenly, 3000 ~ 5000rpm gets supernatant after centrifugal 5 ~ 10 minutes, gets supernatant.(2) get testing sample and positive control after the above-mentioned process of 300 μ L respectively, add Proteinase K 10 μ L, cell pyrolysis liquid 300 μ L, 50 DEG C hatch 2h after add 610 μ Ltris-balance phenols, fully shake 5min, the centrifugal 10min of 12000rpm.(3) supernatant is transferred in new 1.5mL centrifuge tube and adds equal-volume phenol: chloroform: primary isoamyl alcohol (1:24:25), fully the centrifugal 10min of mixing 12000rpm.(4) get supernatant, add phenol: chloroform: primary isoamyl alcohol repeats extracting once.(5) after extracting, supernatant is transferred in new 1.5mL centrifuge tube, add dehydrated alcohol after 2 times of volume ice baths, 1/10 volume 3mol/L sodium-acetate, the centrifugal 10min of 12000rpm after-20 DEG C of precipitation 45min, abandon supernatant, the dry 5min of vacuum drying oven, with 20 μ L sterile distilled water dissolution precipitations ,-20 DEG C of storages are for subsequent use.
2, PCR reaction: add 10 μ L2 × TaqPCRMix, the above-mentioned DNA solution of 1 μ L, 0.5 μ L upstream primer (10 μMs), 0.5 μ L downstream primer (10 μMs) respectively in reaction tubes, finally adding nuclease free water to volume is 20 μ L, mixing.
Response procedures is as follows: 94 DEG C of 3min, 1 circulation; 94 DEG C of 10s, 56 DEG C of 30s, 72 DEG C of 57s, totally 30 circulations; 72 DEG C 5min1 circulation.
3, electrophoresis detection: after reaction terminates, check pcr amplification product through 1% agarose gel electrophoresis, the stripe size of the positive control amplified production of aviadenovirus 4 type is 954bp, the results are shown in Figure 1.If there is the band of 954bp size after testing sample electrophoresis detection, containing aviadenovirus 4 type, otherwise then not containing aviadenovirus 4 type.
The sensitivity test of embodiment 3 aviadenovirus 4 type PCR detection method
Measured by ultramicrospectrophotometer, calculating aviadenovirus 4 type DNA concentration is that the nucleic acid-templated of 129.3ng/ μ L carries out sensitivity test.
Aviadenovirus 4 type PCR detects operating process to carry out according to embodiment 2.
Detect pcr amplification product with 1% agarose gel electrophoresis, result as shown in Figure 2.Visible, the most low energy of PCR detects that 0.0129ng/ μ L aviadenovirus 4 type is nucleic acid-templated.
The specific test of embodiment 4 aviadenovirus 4 type PCR detection method
According to the method for embodiment 2, respectively to aviadenovirus 4 type (fowladenovirus4, FAdV-4), aviadenovirus 11 type (fowladenovirus11, FAdV-11), infectious bronchitis virus (infectiousbronchitisvirus, IBV), avian influenza virus (avaininfluenzavirus, and Avian pneumo-encephalitis virus (newcastlediseasevirus AIV), NDV) canonical reference strain, and the negative control of sterilizing ultrapure water carries out PCR reaction, detects the specificity of PCR method.Above amplified production is all analyzed with the agarose gel electrophoresis of 1%.
As shown in Figure 3, aviadenovirus 4 type nucleic acid amplification has gone out the band of the 954bp conformed to expection size to result, and negative control and other 4 kinds of viral nucleic acids all do not amplify any band.The above results shows PCR detection method energy specific amplification aviadenovirus 4 type nucleic acid of the present invention, and not with other viral nucleic acid generation cross reaction, PCR detection method of the present invention has good specificity.
Claims (5)
1., for the PCR kit that aviadenovirus 4 type detects, comprise Proteinase K, lysate, sodium-acetate, saturated phenol: chloroform: primary isoamyl alcohol, dehydrated alcohol, sterile distilled water, is characterized in that described test kit also comprises 2 × TaqPCRMix, upstream primer and downstream primer; The sequence of described upstream primer is for shown in SEQIDNo.1, and the sequence of described downstream primer is for shown in SEQIDNo.2.
2. the PCR kit detected for aviadenovirus 4 type according to claim 1, is characterized in that described 2 × TaqPCRMix is made up of archaeal dna polymerase, 2 × TaqPCRbuffer and dNTPMixture.
3. the PCR kit detected for aviadenovirus 4 type according to claim 1, is characterized in that described test kit also comprises DNA extraction reagent: Proteinase K, lysate, sodium-acetate, saturated phenol: chloroform: primary isoamyl alcohol, dehydrated alcohol, sterile distilled water.
4. the PCR kit detected for aviadenovirus 4 type according to claim 1, is characterized in that described test kit also comprises positive control.
5. whether the test kit as described in any one of Claims 1 to 4 detects in testing sample containing the application in the product of aviadenovirus 4 type in preparation.
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886502A (en) * | 2016-05-19 | 2016-08-24 | 浙江大学 | Primer pair for preparing kit for detecting type-4 avian adenovirus and application thereof |
| CN105907893A (en) * | 2016-06-20 | 2016-08-31 | 河南省动物疫病预防控制中心 | Fluorogenic quantitative PCR detection reagent and preparation method and application thereof |
| CN106018780A (en) * | 2016-05-17 | 2016-10-12 | 扬州大学 | F1-protein-based indirect immunofluorescence kit for detecting type 4 fowl adenovirus antibody |
| CN106811551A (en) * | 2017-03-22 | 2017-06-09 | 中国农业科学院上海兽医研究所 | The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method |
| CN106978510A (en) * | 2017-04-28 | 2017-07-25 | 福建省农业科学院畜牧兽医研究所 | A kind of primer of duck New-type adenovirus Eva Green real-time fluorescence quantitative PCRs detection |
| CN107083450A (en) * | 2017-05-27 | 2017-08-22 | 河北农业大学 | The type PCR detection kit of pig circular ring virus 3 and detection method |
| CN107385110A (en) * | 2017-08-02 | 2017-11-24 | 河南农业大学 | A kind of RPA primers and its detection method for being used to detect the type of aviadenovirus serum 4 |
| CN107841575A (en) * | 2017-11-06 | 2018-03-27 | 青岛农业大学 | A kind of nanometer multiple PCR method for distinguishing four kinds of serotype aviadenovirus I groups |
| CN109055610A (en) * | 2018-08-15 | 2018-12-21 | 安徽省农业科学院畜牧兽医研究所 | A kind of triple PCR detection method of Muscovy duck parvovirus, 4 type of goose parvovirus and aviadenovirus |
| CN109207637A (en) * | 2018-09-30 | 2019-01-15 | 河南牧业经济学院 | A kind of replaceable Ankara kit for detecting nucleic acid of reporting system and detection method |
| CN109609698A (en) * | 2019-01-07 | 2019-04-12 | 福建省农业科学院畜牧兽医研究所 | It is a kind of for detecting the nano PCR kit of aviadenovirus IV type |
| CN110564896A (en) * | 2019-09-26 | 2019-12-13 | 广西壮族自治区兽医研究所 | Primer group for identifying avian adenovirus type 4 and chicken infectious anemia viruses and application thereof |
| CN111218526A (en) * | 2019-12-09 | 2020-06-02 | 温氏食品集团股份有限公司 | PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof |
| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN111926116A (en) * | 2020-08-12 | 2020-11-13 | 广东省农业科学院动物卫生研究所 | Primer and probe for rapidly and quantitatively detecting duck adenovirus type 4, detection method and application thereof |
| CN112725532A (en) * | 2021-01-21 | 2021-04-30 | 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 identifying FAdV-4 and DAdV-4 and kit thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106018780A (en) * | 2016-05-17 | 2016-10-12 | 扬州大学 | F1-protein-based indirect immunofluorescence kit for detecting type 4 fowl adenovirus antibody |
| CN105886502B (en) * | 2016-05-19 | 2019-04-02 | 浙江大学 | A kind of primer pair and its application being used to prepare detection 4 type kit of aviadenovirus |
| CN105886502A (en) * | 2016-05-19 | 2016-08-24 | 浙江大学 | Primer pair for preparing kit for detecting type-4 avian adenovirus and application thereof |
| CN105907893A (en) * | 2016-06-20 | 2016-08-31 | 河南省动物疫病预防控制中心 | Fluorogenic quantitative PCR detection reagent and preparation method and application thereof |
| CN105907893B (en) * | 2016-06-20 | 2020-01-14 | 河南省动物疫病预防控制中心 | Fluorescent quantitative PCR detection reagent and preparation method and application thereof |
| CN106811551A (en) * | 2017-03-22 | 2017-06-09 | 中国农业科学院上海兽医研究所 | The primer pair of the type aviadenovirus of fluorescence quantitative PCR detection FAdV 4, probe, kit and method |
| CN106978510A (en) * | 2017-04-28 | 2017-07-25 | 福建省农业科学院畜牧兽医研究所 | A kind of primer of duck New-type adenovirus Eva Green real-time fluorescence quantitative PCRs detection |
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| CN107385110A (en) * | 2017-08-02 | 2017-11-24 | 河南农业大学 | A kind of RPA primers and its detection method for being used to detect the type of aviadenovirus serum 4 |
| CN107841575A (en) * | 2017-11-06 | 2018-03-27 | 青岛农业大学 | A kind of nanometer multiple PCR method for distinguishing four kinds of serotype aviadenovirus I groups |
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| CN109609698B (en) * | 2019-01-07 | 2022-08-16 | 福建省农业科学院畜牧兽医研究所 | Nano PCR kit for detecting avian adenovirus IV |
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| CN111218526B (en) * | 2019-12-09 | 2024-01-09 | 温氏食品集团股份有限公司 | PCR primer pair and probe for rapidly identifying I subgroup serum 11 type avian adenovirus and application thereof |
| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN111926116A (en) * | 2020-08-12 | 2020-11-13 | 广东省农业科学院动物卫生研究所 | Primer and probe for rapidly and quantitatively detecting duck adenovirus type 4, detection method and application thereof |
| CN112725532A (en) * | 2021-01-21 | 2021-04-30 | 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 identifying FAdV-4 and DAdV-4 and kit thereof |
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Effective date of registration: 20190926 Address after: 050000 South District of Shangzhuang Town Industrial and Trade District, Luquan District, Shijiazhuang City, Hebei Province Patentee after: SHIJIAZHUANG SHIMU ANIMAL PHARMACEUTICAL CO.,LTD. Address before: 071000, 2596 Kennan Avenue, Baoding, Hebei Patentee before: Agricultural University of Hebei |
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Address after: 050000 South District of industrial and trade community, Shangzhuang Town, Luquan District, Shijiazhuang City, Hebei Province Patentee after: Shijiazhuang animal husbandry Co.,Ltd. Address before: 050000 South District of industrial and trade community, Shangzhuang Town, Luquan District, Shijiazhuang City, Hebei Province Patentee before: SHIJIAZHUANG SHIMU ANIMAL PHARMACEUTICAL CO.,LTD. |
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