CN108642211A - One breeder Ankara disease LAMP primer group, kit and its detection method - Google Patents
One breeder Ankara disease LAMP primer group, kit and its detection method Download PDFInfo
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Abstract
The present invention provides breeder Ankara disease LAMP primer group, kit and its detection methods.Chicken Ankara disease LAMP kit includes 2 × reaction solution RM, 12.5 μ L, each 1.0 μ L of 40 μm of ol/L of inner primer FIP, BIP, each 1.0 μ L of 5 μm of ol/L of outer primer F3, B3,1.0 μ L of Bst archaeal dna polymerases, each 1.0 μ L of fluorescent dye I 0.5 μ L and 20 μm of ol/L of ring primer LF, LB.The detection method of the kit, including step：1) DNA profiling is extracted；2) LAMP reaction systems are mixed with the DNA profiling of 2.0 μ L, then the paraffin oil of 20.0 μ L is added in moisturizing to 25 μ L；3) 63 DEG C, 30s, 1 cycles of reaction condition are set by Real Time PCR System；60s, 40 cycles；Every time fluorescence signal is collected at the end of cycle.Its primer sets is designed according to the nucleotide sequence of the FADV 4 announced in GenBank, and reaction time ratio Taqman method can shorten 20min or more, and purchase Special experimental instrument or consumptive material without additional, and collocation degree is high.
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to breeder Ankara disease LAMP primer group, kit and
Its detection method.
Background technology
Hydropericardium syndrome (Hydropericardium syndrome, HPS) is also known as chicken Ankara disease, and cause of disease is chicken
Adenovirus (Fowl adenovirus, FAdV) belongs to the linear unimolecule distrand DNA virus of no cyst membrane.FAdV serotypes are many
More, respectively totally 12 kinds of 1~7,8a, 8b, 9 of serotype~11, different serotypes can lead to different degrees of inclusion body hepatitis.Its
4 type velogen strain of middle serum is stronger to the pathogenecity of chick, is the main reason for causing HPS to occur.Adenoviridae
(Adenoviridae) it is one of them to divide five categories, Aviadenovirus (adenovirus), and Aviadenovirus is divided into as fowl gland
8 totally kinds of viral A~E, goose adenovirus A, falcon adenovirus A and turkey adenovirus B.FAdV-4 and FAdV-10 belongs to fowl adenopathy
Malicious C.
FAdV is in worldwide distribution, chicken Ankara disease has occurred for the first time in Ankara town within 1987, also reports within 1989
In the generation of Mexico's disease, then successively in the ground such as India, Iraq, Peru, Chile, Ecuador, the Russia disease
It breaks out.Since in June, 2015, such symptom also has occurred in China's most laboratories.The clinical symptoms of the disease are that morbidity chicken often shows
Increasing for loss of appetite, anaemia, thick random feather, cockscomb lighter, drowsiness incidence, the death rate is up to 30%-90%,
Cause tremendous influence and economic loss.Morbidity chicken is mainly shown as that spirit is depressed, inverse vertical, the row's yellow loose stool of feather, falls suddenly
Two legs are drawn empty behind ground, dead in several minutes, and the visible pericardium product of dissect has the diffusate of pale yellow transparent；Liver swelling, hyperemia, matter
Ground becomes fragile, and color and luster is dimmed, and necrosis occurs；Kidney is pale or dark yellow.
Japanese scientist Notomi et al. has invented a kind of new nucleic acid amplification technologies ring mediated isothermal amplification within 2000
Technology (Loop-Mediated Isothermal Amplification, LAMP), LAMP are characterized on target dna chain
6 sections design 4 different primers (FIP, BIP, F3, B3), in constant temperature under the action of strand displacement Bst archaeal dna polymerases
60 DEG C of -65 DEG C of amplification 60min or so, you can carry out cyclic amplification, and efficiency is up to 109-1010A order of magnitude.LAMP reaction tools
There are higher specificity, 6 different zones of only 4 primers in target gene to exactly match and could be expanded.LAMP is anti-
It answers the detection means of product varied, there is agarose gel electrophoresis detection, restricted digestion identification, white precipitate to visually observe
Method, real-time turbidity quantitatively detect, calcein does fluorescence instruction, SYBR Green I dyeing viewing methods and nucleic acid test strip and examines
Survey etc..
Invention content
The present invention is intended to provide providing a kind of simple and efficient, sensitive to FAdV, special LAMP primer group.
The technical solution used in the present invention is：
One breeder Ankara disease LAMP primer group, including primers F IP, BIP, F3, B3, LF and LB, the sequence of each primer is such as
Shown in SEQ ID № 1~6.
Above-mentioned primer sets can be applied to chicken Ankara disease LAMP detection kit.
Chicken Ankara disease LAMP detection kit has LAMP reaction systems, including：
5 μm of ol/L of 2 × reaction solution RM 12.5 μ L, 40 μm of ol/L of inner primer FIP, BIP each 1.0 μ L, outer primer F3, B3
Each 1.0 μ L of 1.0 μ L, Bst archaeal dna polymerase, I 0.5 μ L of fluorescent dye.
The invention also discloses the detection methods for using above-mentioned chicken Ankara disease LAMP detection kit, including step：
1) DNA profiling is extracted；
2) LAMP reaction systems are mixed with the DNA profiling of 2.0 μ L, then the paraffin of 20.0 μ L is added in moisturizing to 25 μ L
Oil；
3) 63 DEG C, 30s, 1 cycles of reaction condition are set by Real-Time PCR System；60s, 40 cycles；
Every time fluorescence signal is collected at the end of cycle.
Wherein, 260/280 ratio of the step 1) DNA profiling is 1.8~2.0, and concentration is in 200~500ng/ μ L；Step
Fluorescent dye used in rapid LAMP reaction systems 2) is FAM.
The invention discloses breeder Ankara disease LAMP primer group, kit and its detection methods.Its primer sets according to
The nucleotide sequence of the FADV-4 announced in GenBank and design, rely on LAMP technology, this method can be such that the reaction time compares
Taqman methods can shorten 20min or more, provide technical support for quickly detection, and without additionally purchasing Special experimental instrument
Or consumptive material, collocation degree is high, can be selected according to specifically used environment.
Description of the drawings
Fig. 1 is chicken Ankara disease LAMP detection kit specific test result figure, FAdV-1, FAdV-2 in figure,
The curve overlapping of FAdV-10, negative control, newcastle disease infectiousness bronchitis bigeminy live vaccine；
Fig. 2 is chicken Ankara disease LAMP detection kit sensitivity test result figure, and figure label 1~9 distinguishes table
Show a concentration of 101To 1099 templates；
Fig. 3 is embodiment 4 first time stability test result；
Fig. 4 is second of the stability test result of embodiment 4.
Specific implementation mode
Below by specific implementation mode combination attached drawing, invention is further described in detail.But those skilled in the art
It will be understood that the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment
Particular technique or condition person, according to technology described in document in the art or condition (such as with reference to J. Pehanorm Brookers etc.
It writes, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Institute
Production firm person is not specified with reagent or instrument, being can be with conventional products that are commercially available.
Embodiment 1, chicken Ankara disease LAMP detection kit, including：
The primer and probe of LAMP primer group is as follows：
Use the detection method of above-mentioned chicken Ankara disease LAMP detection kit, including step：
1) it takes FAdV-4 synthetic plasmids to dilute 1000 times and is used as DNA profiling；
2) LAMP reaction systems are mixed with the DNA profiling of 2.0 μ L, then the paraffin of 20.0 μ L is added in moisturizing to 25 μ L
Oil；
3) 63 DEG C, 30s, 1 cycles of reaction condition are set by Real-Time PCR System；60s, 40 cycles；
Every time fluorescence signal is collected at the end of cycle.
Wherein, 260/280 ratio of the step 1) DNA profiling is 1.8~2.0, and concentration is in 200~500ng/ μ L；Step
Fluorescent dye used in rapid LAMP reaction systems 2) is FAM.
Embodiment 2, LAMP specific tests
According to the Fiber gene complete sequence synthetic plasmids of FAdV-1, FAdV-2, FAdV-4, FAdV-10；Newcastle disease, biography
Metachromia bronchitis bigeminy live vaccine, extracts its RNA and reverse transcription after cDNA at being tested.Above-mentioned plasmid and strain are inverted
Record product is used to be detected by the kit of embodiment 1.The results are shown in Figure 1, and FAdV-4 is occurred apparent by Successful amplification
Amplification curve, and other do not occur amplification curve.
Embodiment 3, LAMP sensitivity tests
It is positive criteria product by FAdV-4 synthetic plasmids.Plasmid concentration is measured with ultraviolet specrophotometer, according to Avobenzene gal
Moral sieve constant is scaled the copy number of target gene, with 10 times of gradients by 109Copy/μ L are diluted to 101Copy/μ L is dilute to gradient
The positive control released carries out LAMP reaction detections.To positive criteria product with 101Doubling dilution, the concentration that will be obtained are carried out for unit
It is 109To 1019 templates expanded using above-mentioned reaction system and reaction condition, to verify the sensitive of the detection method
Degree.The results are shown in Figure 2,102Still there is amplification curve, time≤20min, detectable minimum extension rate in copy grade
It is 102Copy/μ L.
Embodiment 4, LAMP stability tests：
Take FAdV-4 synthetic plasmids to dilute 1000 times and be used as DNA profiling, tested twice respectively, to the Ct values of acquisition into
Row analysis, as a result as shown in Figures 3 and 4, shows that 20 repeat samples results amplification curve occur in 9min or so in every group,
And two groups of test results essentially coincide, repeatability is good.
SEQUENCE LISTING
<110>Inspection ＆ Quarantine Technology Center of Zhuhai Entry-Exit Inspection ＆ Quarantine Bureau
<120>One breeder Ankara disease LAMP primer group, kit and its detection method
<130> 2018
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 38
<212> DNA
<213>Artificial sequence
<400> 1
agttccagca ctccgcttct agcagtggta tcgacctc 38
<210> 2
<211> 38
<212> DNA
<213>Artificial sequence
<400> 2
ggcatccaag ccgacagtac ttccagagtg ttgttgac 38
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
cgtacacctc aaccaacaa 19
<210> 4
<211> 19
<212> DNA
<213>Artificial sequence
<400> 4
ttgttgtcgt acttcagcc 19
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<400> 5
cgaggtgttg accgtgaa 18
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
ggtggatgaa agcctacaga t 21
Claims (5)
1. breeder Ankara disease LAMP primer group, including primers F IP, BIP, F3, B3, LF and LB, the sequence such as SEQ of each primer
Shown in ID № 1~6.
2. breeder Ankara disease LAMP detection kit, including LAMP reaction systems, which is characterized in that the LAMP reactants
System includes primer sets as described in claim 1.
3. chicken Ankara disease LAMP detection kit according to claim 2, which is characterized in that the LAMP reaction systems
Including 2 × reaction solution RM, 12.5 μ L, 5 μm of ol/L of 40 μm of ol/L of inner primer FIP, BIP each 1.0 μ L, outer primer F3, B3 each 1.0
μ L, Bst archaeal dna polymerase 1.0 μ L, I 0.5 μ L of fluorescent dye.
4. breeder Ankara disease LAMP detection method, which is characterized in that kit as claimed in claim 3 is used, including
Step：
1) DNA profiling is extracted；
2) LAMP reaction systems are mixed with the DNA profiling of 2.0 μ L, then the paraffin oil of 20.0 μ L is added in moisturizing to 25 μ L；
3) 63 DEG C, 30s, 1 cycles of reaction condition are set by Real-Time PCR System；60s, 40 cycles；Every time
Fluorescence signal is collected at the end of cycle.
5. chicken Ankara disease LAMP detection method according to claim 4, which is characterized in that the step 1) DNA profiling
260/280 ratio be 1.8~2.0, concentration is in 200~500ng/ μ L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810665619.2A CN108642211A (en) | 2018-06-26 | 2018-06-26 | One breeder Ankara disease LAMP primer group, kit and its detection method |
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| CN201810665619.2A CN108642211A (en) | 2018-06-26 | 2018-06-26 | One breeder Ankara disease LAMP primer group, kit and its detection method |
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| CN201810665619.2A Pending CN108642211A (en) | 2018-06-26 | 2018-06-26 | One breeder Ankara disease LAMP primer group, kit and its detection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706270A (en) * | 2019-02-19 | 2019-05-03 | 珠海出入境检验检疫局检验检疫技术中心 | One breeder Ankara disease detection method |
| CN110157840A (en) * | 2019-06-20 | 2019-08-23 | 四川农业大学 | A kind of Ankara rapid detection method and Primer composition based on LAMP technology |
| CN114990262A (en) * | 2022-06-20 | 2022-09-02 | 云南省畜牧兽医科学院 | Primer group for LAMP (loop-mediated isothermal amplification) detection of avian adenovirus serotype 4 and application of primer group |
| CN115261516A (en) * | 2022-08-02 | 2022-11-01 | 吉林正业生物制品股份有限公司 | Primer, kit and method for detecting avian adenovirus by fluorescent quantitative PCR |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571952A (en) * | 2013-10-24 | 2014-02-12 | 河南农业大学 | Primer, kit and method for detecting genetype on black skin locus of chick |
| CN106947833A (en) * | 2017-04-05 | 2017-07-14 | 天津市动物疫病预防控制中心 | Chicken anaemia virus LAMP LFD detection kits and its detection method and application |
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2018
- 2018-06-26 CN CN201810665619.2A patent/CN108642211A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571952A (en) * | 2013-10-24 | 2014-02-12 | 河南农业大学 | Primer, kit and method for detecting genetype on black skin locus of chick |
| CN106947833A (en) * | 2017-04-05 | 2017-07-14 | 天津市动物疫病预防控制中心 | Chicken anaemia virus LAMP LFD detection kits and its detection method and application |
Non-Patent Citations (1)
| Title |
|---|
| MASAJI MASE等: ""Characterization of Fowl adenovirus serotype 4 isolated from chickens with hydropericardium syndrome based on analysis of the short fiber protein gene"", 《J VET DIAGN INVEST》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706270A (en) * | 2019-02-19 | 2019-05-03 | 珠海出入境检验检疫局检验检疫技术中心 | One breeder Ankara disease detection method |
| CN110157840A (en) * | 2019-06-20 | 2019-08-23 | 四川农业大学 | A kind of Ankara rapid detection method and Primer composition based on LAMP technology |
| CN114990262A (en) * | 2022-06-20 | 2022-09-02 | 云南省畜牧兽医科学院 | Primer group for LAMP (loop-mediated isothermal amplification) detection of avian adenovirus serotype 4 and application of primer group |
| CN115261516A (en) * | 2022-08-02 | 2022-11-01 | 吉林正业生物制品股份有限公司 | Primer, kit and method for detecting avian adenovirus by fluorescent quantitative PCR |
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Application publication date: 20181012 |