CN205741069U - Deer epidemic hemorrhage RT LAMP detection kit - Google Patents
Deer epidemic hemorrhage RT LAMP detection kit Download PDFInfo
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- CN205741069U CN205741069U CN201620686326.9U CN201620686326U CN205741069U CN 205741069 U CN205741069 U CN 205741069U CN 201620686326 U CN201620686326 U CN 201620686326U CN 205741069 U CN205741069 U CN 205741069U
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Abstract
This utility model purpose is to provide a kind of deer epidemic hemorrhage RT LAMP detection kit, and including box body 1, lid 2 and liner 3, described liner is provided with: reverse transcription container hole 4, built-in reverse transcription；10 × Bst polymerase buffer container hole 5, built-in 10 × Bst polymerase buffer；Inner primer container hole 6, built-in inner primer；Outer primer container hole 7, built-in outer primer；Ring primer container hole 8, built-in ring primer；DNTPs container hole 9, built-in dNTPs；Glycine betaine container hole 10, built-in glycine betaine；Bst archaeal dna polymerase container hole 11, built-in Bst archaeal dna polymerase；SYBR Green I dye reservoir hole 12, built-in SYBR Green I dyestuff；DEPC water container hole 13, built-in DEPC water；Positive control container hole 14, built-in positive control.This utility model is that to provide a species specificity good in deer epidemic hemorrhage detection, and sensitivity is high, quickly, inexpensively, and test kit simple to operate.
Description
Technical field
This utility model belongs to technical field of biological, is specifically related to a kind of deer epidemic hemorrhage detection kit.
Background technology
Deer epidemic hemorrhage (Epizootic hemorrhagic disease of deer, EHD) is that one causes deer
Body temperature rising mucosa and the serious lethal infectious diseases that serosal surface is the most hemorrhage and the lower death that often lies in a comatose condition is characterized, cattle
Can also infection morbidity EHD with sheep.Cause of disease is deer epizootic haemorrhagic disease virus (Epizootic hemorrhagic disease
Virus of deer, EHDV), the detection of cause of disease is mainly carried out by the amplification of viral nucleic acid, and the method set up has commonly
RT-PCR (reverse transcription-polymerase chain reaction) detection method and fluorescent RT-PCR method for detecting, but these two kinds of detection methods are equal
Need to carry out at the laboratory having specific apparatus, it is difficult to wide popularization and application in clinical diagnosis.
Ring mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technology is in recent years
A kind of new nucleic acid amplification method developed, its principle is to specific primer for 6 region designs 3 on target sequence,
Under there is the effect of Bst archaeal dna polymerase of strand-displacement activity, there is displacement and the cyclisation of DNA, carry out under isothermal conditions
The efficiently amplification purpose fragment of high sensitivity high specific, reaction need not the expensive instruments such as PCR instrument, its minimum sensitive detection
The limit is 10 copies, high several orders of magnitude than PCR.LAMP has high amplification efficiency, can be expanded by target sequence in one hour
Increase to 109～1010 times, after reactant liquor adds nucleic acid dye SYBR Green I, under uviol lamp or daylight, pass through naked eyes
Can judge, if containing amplified production, reactant mixture turns green；Otherwise, then keep SYBR Green I orange not
Becoming, this detection method is conducive to promoting at laboratories.
Summary of the invention
This utility model purpose is to provide a kind of deer epidemic hemorrhage RT-LAMP detection reagent box, this detectable
Box has easy and simple to handle, sensitive, specificity advantages of higher, it is possible to detect deer epizootic haemorrhagic disease virus efficiently.
Deer epizootic haemorrhagic disease virus real-time fluorescent RT-PCR detection reagent box according to this utility model embodiment, including
Box body 1, lid 2 and liner 3, described liner is provided with: reverse transcription container hole 4, built-in reverse transcription；10 × Bst polymerase
Buffer container hole 5, built-in 10 × Bst polymerase buffer；Inner primer container hole 6, built-in inner primer；Outer primer container hole 7,
Built-in outer primer；Ring primer container hole 8, built-in ring primer；DNTPs container hole 9, built-in dNTPs；Glycine betaine container hole 10, interior
Put glycine betaine；Bst archaeal dna polymerase container hole 11, built-in Bst archaeal dna polymerase；SYBR Green I dye reservoir hole 12, interior
Put SYBR Green I dyestuff；DEPC water container hole 13, built-in DEPC water；Positive control container hole 14, built-in positive control
Thing.
The cDNA of this utility model selection coding EHDV group specificity structural protein VP7 designs specificity for target sequence and draws
Thing.
(1) inner primer:
FIP (Flc+F2): 5 '-TCATGTTTACACCCCCCACGCAGCGGGTAATGCACAAG-3 '
BIP (Blc+B2): 5 '-GGTGTGATAATAGCTTATGATGGGCACTTCAATCTCGATCATACCTT-3 '
(2) outer primer:
F3:5 '-GAATTGGGACTTTTTCGAATG-3 '
B3:5 '-TCATGGTTTTGTCTAAACTCAA-3 '
(3) ring primer:
Floop:5 '-GTCACGCCCTGAGGTGTAT-3 '
Bloop:5 '-AAGCGCCGGTTAATGTGAAT-3 '
This utility model is according to above-mentioned primer sets exploitation deer epidemic hemorrhage RT-LAMP detection reagent box.
This utility model test kit also includes SYBR Green I fluorescence indicator and the application of nitrite ion.Mentioned reagent box
The reaction system that application in detection deer epidemic hemorrhage is used is as follows:
Reaction system is: 10 × polymerase buffer (pH 8.8,25 DEG C) each 3 μ L of 2.5 μ L, inner primer FIP and BIP, outward
The each 1 μ L of primers F 3 and B3, ring primers F loop and each 2 μ L of Bloop, dNTPs 3 μ L, glycine betaine 2 μ L, reverse transcription 0.5 μ L, Bst
Archaeal dna polymerase 1 μ L, RNA template 2 μ L, supplies 25 μ L with DEPC water.Detection all should set up positive control and negative control every time.
RT-LAMP amplification judge: experimental result can according to the color reaction of SYBR GREEN I nitrite ion,
1.5% agarose sugar gel electrophoresis and SYBR GREEN I fluorescence indicator fluorescence signal collection situation judge.Containing EHDV
The white precipitate of trace seen from naked eyes at the bottom of the amplification pipe of template, negative control is without precipitation；After adding dyestuff, containing EHDV template
Reactant liquor presents the most emerald green, and negative control is then greenish orange color；Each reaction tube after adding dyestuff is placed in uviol lamp
Lower irradiation, the reactant liquor containing EHDV template sends strong green fluorescence, and feminine gender is the most luminous.Agarose gel electrophoresis detects
After, the reactant liquor of EHDV template has specific scalariform band, and negative control then occurs without band.
This utility model is that to provide a species specificity good in deer epidemic hemorrhage detection, and sensitivity is high, quickly, inexpensively,
Test kit simple to operate.
Accompanying drawing explanation
Fig. 1: this utility model deer epidemic hemorrhage RT-LAMP detection reagent box structural representation.
Detailed description of the invention
Describe this utility model below in conjunction with the accompanying drawings with embodiment, example is served only for explaining this utility model, not
For limiting scope of the present utility model.
Embodiment 1: a kind of deer epidemic hemorrhage RT-LAMP detection reagent box.
The structure of this utility model test kit, as it is shown in figure 1, include box body 1, lid 2 and liner 3, described liner sets
Have: reverse transcription container hole 4, built-in reverse transcription；10 × Bst polymerase buffer container hole 5, built-in 10 × Bst polymerase
Buffer；Inner primer container hole 6, built-in inner primer；Outer primer container hole 7, built-in outer primer；Ring primer container hole 8, built-in ring
Primer；DNTPs container hole 9, built-in dNTPs；Glycine betaine container hole 10, built-in glycine betaine；Bst archaeal dna polymerase container hole 11,
Built-in Bst archaeal dna polymerase；SYBR Green I dye reservoir hole 12, built-in SYBR Green I dyestuff；DEPC water container hole
13, built-in DEPC water；Positive control container hole 14, built-in positive control.
The cDNA of this utility model selection coding EHDV group specificity structural protein VP7 designs specificity for target sequence and draws
Thing.
(1) inner primer:
FIP (Flc+F2): 5 '-TCATGTTTACACCCCCCACGCAGCGGGTAATGCACAAG-3 '
BIP (Blc+B2): 5 '-GGTGTGATAATAGCTTATGATGGGCACTTCAATCTCGATCATACCTT-3 '
(2) outer primer:
F3:5 '-GAATTGGGACTTTTTCGAATG-3 '
B3:5 '-TCATGGTTTTGTCTAAACTCAA-3 '
(3) ring primer:
Floop:5 '-GTCACGCCCTGAGGTGTAT-3 '
Bloop:5 '-AAGCGCCGGTTAATGTGAAT-3 '
This utility model is according to above-mentioned primer sets exploitation deer epidemic hemorrhage RT-LAMP detection reagent box.
This utility model test kit also includes SYBR Green I fluorescence indicator and the application of nitrite ion.Mentioned reagent box
The reaction system that application in detection deer epidemic hemorrhage is used is as follows:
Reaction system is: 10 × polymerase buffer (pH 8.8,25 DEG C) each 3 μ L of 2.5 μ L, inner primer FIP and BIP, outward
The each 1 μ L of primers F 3 and B3, ring primers F loop and each 2 μ L of Bloop, dNTPs 3 μ L, glycine betaine 2 μ L, reverse transcription 0.5 μ L, Bst
Archaeal dna polymerase 1 μ L, RNA template 2 μ L, supplies 25 μ L with DEPC water.Detection all should set up positive control and negative control every time.
RT-LAMP amplification judge: experimental result can according to the color reaction of SYBR GREEN I nitrite ion,
1.5% agarose sugar gel electrophoresis and SYBR GREEN I fluorescence indicator fluorescence signal collection situation judge.Containing EHDV
The white precipitate of trace seen from naked eyes at the bottom of the amplification pipe of template, negative control is without precipitation；After adding dyestuff, containing EHDV template
Reactant liquor presents the most emerald green, and negative control is then greenish orange color；Each reaction tube after adding dyestuff is placed in uviol lamp
Lower irradiation, the reactant liquor containing EHDV template sends strong green fluorescence, and feminine gender is the most luminous.Agarose gel electrophoresis detects
After, the reactant liquor of EHDV template has specific scalariform band, and negative control then occurs without band.
Embodiment 2: deer epidemic hemorrhage RT-LAMP detection reagent box specific assay.
With deer epidemic hemorrhage RT-LAMP detection reagent box to EHDV-1,2 types, bluetongue (BTV), Akabane Disease
(AKV), vesicular stomatitis (VSV) detect, result EHDV-1,2 types in substantially positive, and negative control, blank,
AKV, BTV, VSV virus is the most negative, no cross reaction, and the method high specificity is described.
Claims (1)
1. a deer epidemic hemorrhage RT-LAMP detection reagent box, it is characterised in that include box body (1), lid (2) and lining
Pad (3), described liner is provided with:
Reverse transcription container hole (4), built-in reverse transcription；
10 × Bst polymerase buffer container hole (5), built-in 10 × Bst polymerase buffer；
Inner primer container hole (6), built-in inner primer；
Outer primer container hole (7), built-in outer primer；
Ring primer container hole (8), built-in ring primer；
DNTPs container hole (9), built-in dNTPs；
Glycine betaine container hole (10), built-in glycine betaine；
Bst archaeal dna polymerase container hole (11), built-in Bst archaeal dna polymerase；
SYBR Green I dye reservoir hole (12), built-in SYBR Green I dyestuff；
DEPC water container hole (13), built-in DEPC water；
Positive control container hole (14), built-in positive control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620686326.9U CN205741069U (en) | 2016-07-01 | 2016-07-01 | Deer epidemic hemorrhage RT LAMP detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620686326.9U CN205741069U (en) | 2016-07-01 | 2016-07-01 | Deer epidemic hemorrhage RT LAMP detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN205741069U true CN205741069U (en) | 2016-11-30 |
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ID=57380437
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201620686326.9U Expired - Fee Related CN205741069U (en) | 2016-07-01 | 2016-07-01 | Deer epidemic hemorrhage RT LAMP detection kit |
Country Status (1)
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| CN (1) | CN205741069U (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257561A (en) * | 2019-07-23 | 2019-09-20 | 深圳海关动植物检验检疫技术中心 | Reagent, detection method and application for epizootic hemorrhagic disease virus of deer (EHDV) detection |
| CN113337643A (en) * | 2021-06-28 | 2021-09-03 | 云南省畜牧兽医科学院 | RT-LAMP primer group and kit for detecting bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus |
-
2016
- 2016-07-01 CN CN201620686326.9U patent/CN205741069U/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257561A (en) * | 2019-07-23 | 2019-09-20 | 深圳海关动植物检验检疫技术中心 | Reagent, detection method and application for epizootic hemorrhagic disease virus of deer (EHDV) detection |
| CN110257561B (en) * | 2019-07-23 | 2023-09-05 | 深圳海关动植物检验检疫技术中心 | Reagent for detecting deer epidemic hemorrhagic fever virus, detection method and application |
| CN113337643A (en) * | 2021-06-28 | 2021-09-03 | 云南省畜牧兽医科学院 | RT-LAMP primer group and kit for detecting bluetongue virus, animal epidemic hemorrhagic disease virus and paliyama virus |
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| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20161130 |
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| CF01 | Termination of patent right due to non-payment of annual fee |