CN105506177A - Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof - Google Patents
Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof Download PDFInfo
- Publication number
- CN105506177A CN105506177A CN201510951672.5A CN201510951672A CN105506177A CN 105506177 A CN105506177 A CN 105506177A CN 201510951672 A CN201510951672 A CN 201510951672A CN 105506177 A CN105506177 A CN 105506177A
- Authority
- CN
- China
- Prior art keywords
- lamp
- mitten crab
- esrv
- reovirus
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an eriocheir sinensis reovirus RT-LAMP detection reagent kit and a detection method thereof. According to the detection method, primers needed for an EsRV RT-LAMP reaction system are designed according to a conservative region of the sequence of an eriocheir sinensis reovirus(EsRV) disclosed in a GenBank, and the detection primers include a pair of outer primers and a pair of inner primers. The detection reagent kit comprises the detection primers, RT-LAMP amplified reaction liquid, DNA polymerase, AMV reverse transcriptase and a reference substance. The reagent kit further comprises a color developing agent. Compared with a common detection technology of the virus, the reagent kit is easy, convenient and fast to operate, high in specificity, high in detection sensitivity, suitable for on-site detection of front-line production technicians and farmers and favorable for diagnosis and prevention of eriocheir sinensis tremor diseases and has wide application prospects.
Description
Technical field
The present invention relates to hydrocoles detection field, particularly a kind of mitten crab reovirus RT-LAMP detection reagent box and detection method thereof.
Background technology
Appendage Shivering Disease " of Eriocheir Sinensis (tremordisease, TD), also known as Shiver disease, mitten crab reovirus (Eriocheirsinensisreovirus, EsRV) be the main pathogen of appendage Shivering Disease " of Eriocheir Sinensis, since 1994 since Jiangsu Province is by reported first, be transmitted to the main Eriocheir sinensis ground such as Anhui, Zhejiang, Hubei subsequently.The cardinal symptom of sick crab shows as step and occurs obvious jitter, and dactylus reddens and with features such as liver is rotten to the corn, the gill filament is slightly black, three corneal edemas, mortality ratio, up to more than 70%, causes great harm to mitten crab aquaculture.This disease is main to put prevention first, and finds this disease ahead of time and takes effective preventive measures to prevent big area from infecting.Therefore, the detection method setting up a kind of effective, quick, easy EsRV cause of disease is needed.
Research shows, mitten crab reovirus is diplornavirus, 9-12 genomic fragment, its sequence has measured and announced (accession number: KP638402-KP638413) on GenBank, the detection method of this virus has electron microscopy, RT-PCR detection method etc. at present, but these methods all exist a lot of problem in actually operating: one is that operating process is more loaded down with trivial details, required instrument compare is complicated, is unfavorable for big area generaI investigation and Site Detection; Two is that insufficient sensitivity is high cannot detect latent virus sample.
Summary of the invention
The object of the invention is to RT-LAMP detection primer sets and detection kit that a kind of mitten crab reovirus is provided to overcome above the deficiencies in the prior art.
Another object of the present invention is the RT-LAMP detection method providing a kind of mitten crab reovirus.
Technical scheme of the present invention is as follows:
For detecting a RT-LAMP amplimer group for mitten crab reovirus, this primer sets comprises following primer:
Primer EsRV-FIP:
CAAGAACCCACTTCGGAGCGT-GCTGCACCTGTAGTACTTGG；
Primer EsRV-BIP:
TGCGACTATCGAAGGCGTGC-GATAGCCCTGGATAAGCCAC；
Primer EsRV-F3:TGCTGTTCTACACTTTGCGC;
Primer EsRV-B3:CGTCGTGCCTTTTAGGTCC.
A kind of mitten crab reovirus RT-LAMP detection reagent box, comprises above-described RT-LAMP amplimer group.
Described mitten crab reovirus RT-LAMP detection reagent box, this test kit can also comprise viral RNA and extract reagent, RT-LAMP amplification reaction solution, BstDNA polysaccharase, AMV reversed transcriptive enzyme, negative control and positive control.
Described mitten crab reovirus RT-LAMP detection reagent box, described viral RNA extract reagent comprise Trizol, chloroform, Virahol, without RNase 75% dehydrated alcohol and DEPC water.
Described RT-LAMP amplification reaction solution is: 10 × Thermopolbuffer, 150mM magnesium sulfate, 10mMdNTP, 5M aqueous solutions of betaine be 8: 4: 5: 8 mixed mixed solutions by volume.
Described negative control is the RNasefreewater of free nucleic acid; Positive control is for containing mitten crab reovirus gene group RNA.
Above-described mitten crab reovirus RT-LAMP detection reagent box, can also comprise RT-LAMP nitrite ion, this nitrite ion is specially 7.5mMMnCl
2with 625 μMs of fluorexon aqueous solution by volume 1: 1 mixed mixed solution.
A detection method for described mitten crab reovirus RT-LAMP detection reagent box, when not comprising RT-LAMP nitrite ion, detection method comprises the following steps:
(1) measuring samples RNA extracts: get 30-50mg mitten crab muscle homogenate, extracts reagent extracting mitten crab total serum IgE, be placed in-20 DEG C and save backup with viral RNA;
(2) RT-LAMP amplified reaction: 25 μ l reaction systems comprise: 1.6 μm of ol/LEsRV-FIP and EsRV-BIP, 0.2 μm of ol/LEsRV-F3 and EsRV-B3, RT-LAMP reaction solution 12.5 μ l, 5UAMV reversed transcriptive enzyme, 8UBstDNA polysaccharase, measuring samples RNA2 μ l, insufficient section with ultrapure water polishing to 25 μ l; Positive control and negative control are set, centrifugal after the reaction tubes mixing prepared, 63-65 DEG C of reaction 30-60min on thermostat water bath;
(3) RT-LAMP reaction product detects: Turbidity measurement, and after having reacted, amplified production has magnesium pyrophosphate to generate, and reaction tubes presents muddiness then for positive, otherwise is negative; Detected through gel electrophoresis, after having reacted, gets 5 μ l amplified production gel electrophoresises, and what electrophoretic image had notch cuttype band is the positive, otherwise is negative.
The detection method of the mitten crab reovirus RT-LAMP detection reagent box described in more than one, when comprising RT-LAMP nitrite ion, detection method comprises the following steps:
(1) measuring samples RNA extracts: get 30-50mg mitten crab muscle homogenate, extracts reagent extracting mitten crab total serum IgE, be placed in-20 DEG C and save backup with viral RNA.
(2) RT-LAMP amplified reaction: 25 μ l reaction systems comprise: 1.6 μm of ol/LEsRV-FIP and EsRV-BIP, 0.2 μm of ol/LEsRV-F3 and EsRV-B3, RT-LAMP reaction solution 12.5 μ l, RT-LAMP nitrite ion 2 μ l, 5UAMV reversed transcriptive enzyme, 8UBstDNA polysaccharase, measuring samples RNA2 μ l, insufficient section with ultrapure water polishing to 25 μ l; Positive control and negative control are set, centrifugal after the reaction tubes mixing prepared, 63-65 DEG C of reaction 30-60min on thermostat water bath.
(3) RT-LAMP reaction product detects: after having reacted, naked-eye observation amplified production color presents green or yellow-green colour, and sample detection result is positive; Otherwise be negative.
The present invention adopts reverse transcription loop-mediated isothermal amplification (reversetranscriptionloop-mediatedisothermalamplification, RT-LAMP) technology, namely be the principle according to ring mediated isothermal amplification (LAMP) technology, be characterized in 6 zone design 4 species-specific primers for target gene, under utilizing a kind of strand displacement archaeal dna polymerase and the acting in conjunction of AMV reversed transcriptive enzyme, be incubated 30-60 minute in isothermal condition (about 63 DEG C), can nucleic acid amplification reaction be completed.
The invention has the beneficial effects as follows:
1, detection kit rapid sensitive of the present invention, 30-60min can obtain detected result, and time less than Standard PCR saving 3-4, sensitivity is 100-1000 times of Standard PCR.
2, detection kit of the present invention succinctly facilitates, and requires lower, do not need the PCR instrument needed for regular-PCR, electrophoresis apparatus and gel imaging instrument to plant and instrument.
3, detection kit of the present invention has high specific, and four Auele Specific Primers of the present invention's design, 6 different zones of amplified target sequence, specificity is stronger than Standard PCR.The RNA of the present invention to nodavirus (nodavirus), Taura syndrome (Tauravirus) and yellow head virus (YellowHeadvirus) does not all increase.
4, detection kit result qualification of the present invention is easy, directly whether can present green fluorescence by observing amplified production, being applicable to Site Detection after having reacted.
Accompanying drawing explanation
Fig. 1 is that detection method of the present invention detects sample experiments figure; Wherein No. 1 pipe: the visual test result of positive (containing viral EsRV); No. 2 pipes: the visual test result of negative sample (RNasefreewater);
Fig. 2 is the effect diagram that amplification temperature is reacted RT-LAMP; Wherein M:500bpDNALadderMarker (TaKaRa company); Swimming lane 1: negative control; Amplification under swimming lane 2:55 DEG C constant temperature; Amplification under swimming lane 3:57 DEG C constant temperature; Amplification under swimming lane 4:59 DEG C constant temperature; Amplification under swimming lane 5:61 DEG C constant temperature; Amplification under swimming lane 6:63 DEG C constant temperature; Amplification under swimming lane 7:65 DEG C constant temperature;
Fig. 3 is EsRVRT-LAM specificity experiments result figure; Wherein M:500bpDNALadderMarker (TaKaRa company); Swimming lane 1: mitten crab reovirus; Swimming lane 2: Macrobrachium rosenbergii nodavirus; Swimming lane 3: Penaeus vannamei Taura syndrome; Swimming lane 4: prawn yellow head virus; Swimming lane N: negative control;
Fig. 4 is the sensitivity experiments result figure that detection method of the present invention detects EsRV; Wherein left figure is, M:500bpDNALadderMarker (TaKaRa company); Total serum IgE containing the mitten crab reovirus extracted in the reaction system of swimming lane 1-7, is respectively 1.5 μ g, 150ng, 15ng, 1.5ng, 150pg, 15pg, 1.5pg; Swimming lane N: negative control; Right figure is: add fluorescence dye visual test result.
Embodiment:
Below in conjunction with embodiment, and by reference to the accompanying drawings, the present invention is described in further detail.
Following examples are the present invention's preferably embodiment, but embodiments of the present invention are not by the restriction of following instance, and in the present invention, if not refer in particular to, the raw material adopted and equipment are all that this area is relatively more conventional.Embodiment 1: a kind of foundation of RT-LAMP detection reagent box of mitten crab reovirus
The RT-LAMP detection reagent box of mitten crab reovirus, comprises RT-LAMP primer sets, RT-LAMP amplification reaction solution, viral RNA extraction reagent, RT-LAMP nitrite ion, BstDNA polysaccharase, AMV reversed transcriptive enzyme, positive control and negative control.
(1) RT-LAMP primer sets: the primer comprising following sequence:
Primer EsRV-FIP:
CAAGAACCCACTTCGGAGCGT-GCTGCACCTGTAGTACTTGG。
(SEQIDNO.1)
Primer EsRV-BIP:
TGCGACTATCGAAGGCGTGC-GATAGCCCTGGATAAGCCAC。
(SEQIDNO.2)
Primer EsRV-F3:
TGCTGTTCTACACTTTGCGC。
(SEQIDNO.3)
Primer EsRV-B3:
CGTCGTGCCTTTTAGGTCC。
(SEQIDNO.4)
(2) RT-LAMP amplification reaction solution: comprise by volume 8: 4: 5: 8 mixing of 10 × Thermopolbuffer, 150mM magnesium sulfate, 10mMdNTP, 5M aqueous solutions of betaine.
(3) viral RNA extract reagent: comprise Trizol, chloroform, Virahol, without RNase 70% dehydrated alcohol and DEPC water.
(4) RT-LAMP nitrite ion: both 7.5mM Manganous chloride tetrahydrate and 625 μMs of fluorexon aqueous solution are 1: 1 mixing by volume.
(5) described positive control is the mitten crab reovirus RNA of purifying, it is for the ease of judging whether the sample detected infects mitten crab reovirus and gradient of infection, generally will arrange a positive control when detection one group of sample that test kit of the present invention arranges positive control.
(6) negative control is RNasefreewater.
Embodiment 2: the concrete detection method of the RT-LAMP detection reagent box of mitten crab reovirus is as follows:
(1) RNA of measuring samples extracts:
1. get 30-50mg mitten crab muscle tissue, add 1mlTrizol (TaKaRaBio, Dalian, China) homogenate, then add 200 μ l chloroforms, the centrifugal 15min of 12000rpm;
2. the supernatant liquor getting the centrifugal gained of step 1 proceeds to new centrifuge tube, adds 500 μ l Virahol mixing, the centrifugal 10min of 12000rpm;
3. the liquid after step 2 is centrifugal is poured out, and adds the absolute ethanol washing precipitation of 75%, the centrifugal 5min of 7500rpm;
4. the liquid after step 3 is centrifugal is poured out, and adds 10-12 μ lDEPC hydroecium temperature dissolution precipitation;-20 DEG C save backup.
(2) RT-LAMP amplified reaction:
Isothermal amplification reactions: 25 μ l reaction systems comprise: 1.6 μm of ol/LEsRV-FIP and EsRV-BIP, 0.2 μm of ol/LEsRV-F3 and EsRV-B3, RT-LAMP amplification reaction solution 12.5 μ l, RT-LAMP nitrite ion 2 μ l, 5U reversed transcriptive enzyme AMV, 8UBstDNA polysaccharase, measuring samples RNA2 μ l, insufficient section with ultrapure water polishing to 25 μ l; Positive control and negative control are set, centrifugal after the reaction tubes mixing prepared, 63-65 DEG C of reaction 30-60min on thermostat water bath.
(3) RT-LAMP amplification judges:
1). amplified production fluoroscopic examination (carrying out when adding RT-LAMP nitrite ion)
After RT-LAMP amplified reaction completes, naked-eye observation amplified production color, if amplified production color presents green or yellow-green colour, then detected result is positive (containing viral EsRV); Otherwise detected result is negative (RNasefreewater), the results are shown in Figure 1.
2). amplified production detected through gel electrophoresis (carrying out when not adding RT-LAMP nitrite ion)
Mitten crab reovirus RT-LAMP amplified production carries out gel electrophoresis: the sepharose of preparation 2%, and each well adds the reaction product of 5 μ l; Electrophoresis 30-45min gel imaging, the results are shown in Figure 2, and electrophoresis result is the middle band occurring notch cuttype compared with negative control, shows that it is positive, if there is not notch cuttype band, shows that it is negative.
Embodiment 3:RT-LAMP optimization Test
For screening body series optimum temps, when other experiment condition is the same, several differing temps gradients such as 55 DEG C-65 DEG C are set and carry out RT-LAMP amplified reaction, reacted rear agarose gel electrophoresis, amplification the best (see Fig. 2) when result shows 65 DEG C.
Embodiment 4: specific test
In order to detect the specificity of test kit of the present invention, detection method, according to the detection method of embodiment 2, detects viral EsRV, nodavirus, Tauravirus and YellowHeadvirus respectively.
Detected result shows: after reaction terminates, get 5 μ l amplification product gel electrophoresises, only there is normal amplification in viral EsRV sample, the samples such as negative control (RNasefreewater) and nodavirus (nodavirus), Penaeus vannamei Taura syndrome (Tauravirus), prawn yellow head virus (YellowHeadvirus) all do not occur increasing (see Fig. 3), the above results show this test kit and detection method specificity higher, there is not false positive.
Embodiment 5: sensitivity experiment
In order to detect the sensitivity of test kit of the present invention, extract mitten crab reovirus total serum IgE, and measure virus total RNA concentration with ultraviolet spectrophotometer, ten times of dilution gradients are respectively 1.5 μ g/ μ l, 150ng/ μ l, 15ng/ μ l, 1.5ng/ μ l, 150pg/ μ l, 15pg/ μ l, 1.5pg/ μ l.Adopt the detection method in above-described embodiment 2, respectively the positive after dilution is detected.
Detected result as shown in Figure 4, detected result not only can pass through visual inspection (the left figure as in Fig. 4), can also be verified by detected through gel electrophoresis (the right figure as in Fig. 4), it is the same that result shows two kinds of detection method detected results.Therefrom can find out that the sensitivity that RT-LAMP test kit of the present invention detects can reach 15pg/ μ l, susceptibility, higher than conventional RT-PCR 100 times, illustrates the susceptibility that this test kit and the diagnosis of detection method to viral EsRV have nearly.
Embodiment 6: clinical practice application evaluation
Adopt test kit of the present invention to detect 20 parts of mitten crab samples, adopt the detection method in above-described embodiment 2, use the method for RT-PCR conventional at present to the detection of 20 parts of mitten crab samples as a comparison simultaneously.Result shows, and detection kit Positive rate of the present invention is 55% (having 11 parts to detect as the positive in 20 parts); The positive rate that the method for RT-PCR detects is 45% (having 9 parts to detect as the positive in 20 parts), 9 parts of positive test symbol in two kinds of detected results are identical, the two parts of positive findings differences existed, the RNA of these two increments product is carried out to RT-PCR amplification and checks order, and sequencing result show sample is positive.Illustrate that detection kit of the present invention has higher accuracy rate.
Show according to the above results, mitten crab reovirus RT-LAMP detection reagent box and detection method can provide quick for EsRV, easy and reliable Site Detection.
Sequence table
<110> China Aquatic Science Research Academy Fresh Water Fishery Research Center
<120> mitten crab reovirus RT-LAMP detection reagent box and detection method thereof
<130>1
<160>4
<170>PatentInversion3.3
<210>1
<211>41
<212>DNA
<213> artificial sequence
<400>1
caagaacccacttcggagcgtgctgcacctgtagtacttgg41
<210>2
<211>40
<212>DNA
<213> artificial sequence
<400>2
tgcgactatcgaaggcgtgcgatagccctggataagccac40
<210>3
<211>20
<212>DNA
<213> artificial sequence
<400>3
tgctgttctacactttgcgc20
<210>4
<211>19
<212>DNA
<213> artificial sequence
<400>4
cgtcgtgccttttaggtcc19
Claims (9)
1., for detecting a RT-LAMP amplimer group for mitten crab reovirus, it is characterized in that, this primer sets comprises following primer:
Primer EsRV-FIP:CAAGAACCCACTTCGGAGCGT-GCTGCACCTGTAGTACTTGG;
Primer EsRV-BIP:TGCGACTATCGAAGGCGTGC-GATAGCCCTGGATAAGCCAC;
Primer EsRV-F3:TGCTGTTCTACACTTTGCGC;
Primer EsRV-B3:CGTCGTGCCTTTTAGGTCC.
2. a mitten crab reovirus RT-LAMP detection reagent box, is characterized in that, comprises RT-LAMP amplimer group according to claim 1.
3. mitten crab reovirus RT-LAMP detection reagent box according to claim 2, it is characterized in that, this test kit also comprises viral RNA and extracts reagent, RT-LAMP amplification reaction solution, BstDNA polysaccharase, AMV reversed transcriptive enzyme, negative control and positive control.
4. mitten crab reovirus RT-LAMP detection reagent box according to claim 3, is characterized in that, described viral RNA extract reagent comprise Trizol, chloroform, Virahol, without RNase 75% dehydrated alcohol and DEPC water.
5. mitten crab reovirus RT-LAMP detection reagent box according to claim 3, it is characterized in that, described RT-LAMP amplification reaction solution is: 10 × Thermopolbuffer, 150mM magnesium sulfate, 10mMdNTP, 5M aqueous solutions of betaine be 8: 4: 5: 8 mixed mixed solutions by volume.
6. mitten crab reovirus RT-LAMP detection reagent box according to claim 3, it is characterized in that, described negative control is the RNasefreewater of free nucleic acid; Positive control is for containing mitten crab reovirus gene group RNA.
7. mitten crab reovirus RT-LAMP detection reagent box according to claim 3, it is characterized in that, also comprise RT-LAMP nitrite ion, this nitrite ion is specially 7.5mMMnCl
2with 625 μMs of fluorexon aqueous solution by volume 1: 1 mixed mixed solution.
8. a detection method for mitten crab reovirus RT-LAMP detection reagent box according to claim 3, is characterized in that, comprise the following steps:
(1) measuring samples RNA extracts: get 30-50mg mitten crab muscle homogenate, extracts reagent extracting mitten crab total serum IgE, be placed in-20 DEG C and save backup with viral RNA;
(2) RT-LAMP amplified reaction: 25 μ l reaction systems comprise: 1.6 μm of ol/LEsRV-FIP and EsRV-BIP, 0.2 μm of ol/LEsRV-F3 and EsRV-B3, RT-LAMP reaction solution 12.5 μ l, 5UAMV reversed transcriptive enzyme, 8UBstDNA polysaccharase, measuring samples RNA2 μ l, insufficient section with ultrapure water polishing to 25 μ l; Positive control and negative control are set, centrifugal after the reaction tubes mixing prepared, 63-65 DEG C of reaction 30-60min on thermostat water bath;
(3) RT-LAMP reaction product detects: Turbidity measurement, and after having reacted, amplified production has magnesium pyrophosphate to generate, and reaction tubes presents muddiness then for positive, otherwise is negative; Detected through gel electrophoresis, after having reacted, gets 5 μ l amplified production gel electrophoresises, and what electrophoretic image had notch cuttype band is the positive, otherwise is negative.
9. a detection method for mitten crab reovirus RT-LAMP detection reagent box according to claim 7, is characterized in that, comprise the following steps:
(1) measuring samples RNA extracts: get 30-50mg mitten crab muscle homogenate, extracts reagent extracting mitten crab total serum IgE, be placed in-20 DEG C and save backup with viral RNA;
(2) RT-LAMP amplified reaction: 25 μ l reaction systems comprise: 1.6 μm of ol/LEsRV-FIP and EsRV-BIP, 0.2 μm of ol/LEsRV-F3 and EsRV-B3, RT-LAMP reaction solution 12.5 μ l, RT-LAMP nitrite ion 2 μ l, 5UAMV reversed transcriptive enzyme, 8UBstDNA polysaccharase, measuring samples RNA2 μ l, insufficient section with ultrapure water polishing to 25 μ l; Positive control and negative control are set, centrifugal after the reaction tubes mixing prepared, 63-65 DEG C of reaction 30-60min on thermostat water bath;
(3) RT-LAMP reaction product detects: after having reacted, naked-eye observation amplified production color presents green or yellow-green colour, and sample detection result is positive; Otherwise be negative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510951672.5A CN105506177A (en) | 2015-12-17 | 2015-12-17 | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510951672.5A CN105506177A (en) | 2015-12-17 | 2015-12-17 | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105506177A true CN105506177A (en) | 2016-04-20 |
Family
ID=55714467
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510951672.5A Pending CN105506177A (en) | 2015-12-17 | 2015-12-17 | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105506177A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111455110A (en) * | 2020-04-21 | 2020-07-28 | 浙江省淡水水产研究所 | Double rapid diagnosis kit for scylla paramamosain reovirus-bicistronic virus |
| CN114058738A (en) * | 2021-11-25 | 2022-02-18 | 广州双螺旋基因技术有限公司 | Fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus |
| CN114107528A (en) * | 2021-12-10 | 2022-03-01 | 广州双螺旋基因技术有限公司 | Constant-temperature rapid detection kit for detecting eriocheir sinensis spiroplasma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703608A (en) * | 2012-06-07 | 2012-10-03 | 中国水产科学研究院长江水产研究所 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses |
| CN104450961A (en) * | 2014-11-25 | 2015-03-25 | 浙江省淡水水产研究所 | Kit for detecting grass carp reovirus types I, II and III based on RT-LAMP visualization technology and method for detecting grass carp reovirus types I, II and III |
-
2015
- 2015-12-17 CN CN201510951672.5A patent/CN105506177A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703608A (en) * | 2012-06-07 | 2012-10-03 | 中国水产科学研究院长江水产研究所 | Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay kit and detection method for grass carp reoviruses |
| CN104450961A (en) * | 2014-11-25 | 2015-03-25 | 浙江省淡水水产研究所 | Kit for detecting grass carp reovirus types I, II and III based on RT-LAMP visualization technology and method for detecting grass carp reovirus types I, II and III |
Non-Patent Citations (3)
| Title |
|---|
| 《2010年中国水产学会学术年会论文摘要集》 * |
| 《NCBI GENBANK》 * |
| 《中国优秀硕士学位论文全文数据库，农业科技辑，华中农业大学硕士学位论文》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111455110A (en) * | 2020-04-21 | 2020-07-28 | 浙江省淡水水产研究所 | Double rapid diagnosis kit for scylla paramamosain reovirus-bicistronic virus |
| CN114058738A (en) * | 2021-11-25 | 2022-02-18 | 广州双螺旋基因技术有限公司 | Fluorescence quantitative PCR detection kit for detecting eriocheir sinensis reovirus |
| CN114058738B (en) * | 2021-11-25 | 2023-12-22 | 广州双螺旋基因技术有限公司 | Fluorescent quantitative PCR detection kit for detecting Eriocheir sinensis reovirus |
| CN114107528A (en) * | 2021-12-10 | 2022-03-01 | 广州双螺旋基因技术有限公司 | Constant-temperature rapid detection kit for detecting eriocheir sinensis spiroplasma |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103773861B (en) | Clonorchis sinensis and angiostrongylus cantonensis dual real-time fluorescence PCR detection reagent, test kit and detection method thereof | |
| CN103966358B (en) | A kind of mandarin fish infectious spleen and kidney necrosis virus fluorescent quantificationally PCR detecting kit and detection method | |
| CN102002531B (en) | Toxoplasma gondii detection kit and application thereof | |
| CN107828914B (en) | RAA constant temperature fluorescence detection method and reagent for Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) | |
| CN103409555B (en) | Macrobrachium rosenbergii Nodavirus RT-LAMP-LFD detection method and detection kit thereof | |
| CN109517927A (en) | A kind of A type, influenza B virus rapid typing detection reagent box and its application | |
| CN105441586A (en) | A-type H5N6 subtype avian influenza virus dual-channel real-time fluorescence PCR (polymerase chain reaction) detection kit and detection method | |
| CN105543409A (en) | Double-target-gene real-time fluorescent PCR detection method for Middle East respiratory syndrome coronavirus | |
| CN105506177A (en) | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof | |
| CN105441589A (en) | Human parainfluenza virus I, II, III, and IV-type quadruple-PCR detection kit, and detection method thereof | |
| CN102534051B (en) | Kit for detecting enterovirus | |
| CN110117678A (en) | EHP and SHIV dual real-time fluorescence quantitative PCR detection primer combination of probe and kit | |
| CN101724712B (en) | Animal insect-borne disease multi-RT-PCR distinguishing and detecting reagent as well as preparation method and application | |
| CN103789450B (en) | A kind of fluorescence quantitative kit detecting CA 2, A5 type | |
| CN105296669A (en) | RT-LAMP detection primer pairs, kit and detection method for infectious haematopoietic necrosis virus (IHNV) | |
| CN105331739A (en) | Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method | |
| CN105039324A (en) | Specific amplification primers for detecting trionyx sinensis hemorrhagic disease viruses and detection kit using the same | |
| CN102071263A (en) | Nested fluorescence reverse transcription-polymerase chain reaction (RT-PCR) detection method for avian influenza virus (AIV) H5 subtype and detection kit | |
| CN102649985B (en) | Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) visual detection kit for H1N2 avian influenza viruses | |
| CN104342501B (en) | A kind of primer, detection kit and preparation method who detects swine vesicular disease virus | |
| CN107190103A (en) | Multiple PCR primer group, kit and the method for three kinds of fishes virus are detected simultaneously | |
| CN103409563B (en) | RT-LAMP (reverse transcription-loop-mediated isothermal amplification) detection kit for infectious pancreatic necrosis virus of rainbow trout | |
| CN104059994B (en) | A kind of test kit based on serum miRNA and using method thereof and application | |
| CN102925592B (en) | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection | |
| CN102876811A (en) | RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and RT-LAMP detection method for SVCV (spring viremia of carp virus) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160420 |