CN102925592A - Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection and application method thereof - Google Patents
Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection and application method thereof Download PDFInfo
- Publication number
- CN102925592A CN102925592A CN2012104931408A CN201210493140A CN102925592A CN 102925592 A CN102925592 A CN 102925592A CN 2012104931408 A CN2012104931408 A CN 2012104931408A CN 201210493140 A CN201210493140 A CN 201210493140A CN 102925592 A CN102925592 A CN 102925592A
- Authority
- CN
- China
- Prior art keywords
- dfv
- duck flavivirus
- duck
- fluorescent
- fluorescence quantitative
- 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.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a fluorescence quantitative PCR (polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection and an application method thereof. The kit comprises a) an RNA (ribonucleic acid) lysis solution; b) a fluorescence quantitative reaction solution; (c) a duck flavivirus strongly positive quality control product; and (d) a negative quality control product, wherein the fluorescence quantitative reaction solution contains an upstream primer DFV(duck flavivirus)-F, a downstream primer DFV-R and a fluorescent probe DFV-P, and the kit is suitable for rapid quantitative detection of duck flavivirus in clinical applications and scientific research. The kit disclosed by the invention has the following advantages that good specificity, very high accuracy due to double controls of high-specificity amplification of the primers and high-specificity hybridization of the fluorescent probe, and low false positive rate, high sensitivity, fast detection speed, and no post-treatment, no hybridization, electrophoresis, photographing and other processes, and no production of pollution, wherein the detection time is only 2 hours, and the whole process needs about 3 hours in total by considering the extraction of nucleic acid.
Description
Technical field
Fluorescent quantitative PCR rapid diagnosis reagent box and the application method thereof of a kind of specific detection duck flavivirus wild virus infection involved in the present invention belong to the viral nucleic acid detection field, are applicable in clinical and the scientific research fast qualitative of duck flavivirus is detected.
Background technology
Since 2010, the ground such as China Shanghai, Zhejiang and Jiangsu the duck flavivirus infection occurred first and have caused the death of lay eggs degradation and egg duck.The pest of duck feelings that this cause of disease causes also belong to first at world wide.The same year, the egg drop reduction of unknown cause also appearred in some duckeries in Hubei Province, and clinical manifestation is for laying eggs degradation even stop, and certain lethality rate is arranged, and cutd open the sick duck spleen of inspection and presented obvious enlargement, and ovary meat becomes, and ovarian follicle is seriously congested, hemorrhage.The outburst of this disease has brought huge financial loss to the raiser.
This seminar has carried out epidemiology survey, pathogen separation, the animal Orthogonal Rotational Regressive Tests of system, order-checking and the analysis of viral portion gene to this disease.Confirm that this disease is to be caused by a kind of new flavivirus, according to the passing rules of viral nomenclature, separate the place with reference to virus, with its called after HBJL strain.Preliminary the sequencing results shows that this virus belongs to the ntaya virus group of flaviviridae (Flaviviridae) Flavivirus (Flavivirus) carapuru virus class (Ntaya virus group, NTAV group).
The genome of duck flavivirus is the sub-thread positive chain RNA of non-segmented negative, approximately is comprised of 11000 Nucleotide.When our province egg duck cluster broke out the egg drop reduction epidemic situation in 2010, this seminar has carried out the massive epidemiology investigation and has carried out the isolation identification work of cause of disease, with reference to Tembusu virus NS5 gene order autonomous design specific detection primer, carry out SPF egg inoculation and isolate duck flavivirus Hubei strain HBJL detecting positive pathological material of disease.Finished at present the mensuration of duck flavivirus Hubei strain HBJL strain portion gene NS5, and be committed to the American National biomolecule information database (NCBI, accession number: JF423123), with reference to the NS5 gene design of HBJL strain primer:
DFV-F: atgaataaggtggtgaaggtaatgc；
DFV-R： cagattcgtgaaagtgttgagagc；
DFV-P: catgactgttttcccatcacgtcccgg。Product length is 130bp.
Because this disease is the new disease of animal, existing diagnostic method is very limited, mostly be to carry out making a definite diagnosis of this disease by traditional viral separation and Culture (Virus Isolation, VI), polymerase chain TRAP (Polymerase Chain Reaction, PCR).Therefore setting up a kind of quick diagnosis detection method is a problem in the urgent need to address.
Quantitative fluorescent PCR (Fluorogenetic Quatitative PCR, FQ-PCR) method is the technology that the real-time quantitative that grows up the mid-90 in 20th century detects specific nucleic acid, to have increased a fluorescent label DNA probe with high specific on the basis of regular-PCR, by initial point quantitatively and the fluorescence detecting system Real-Time Monitoring accumulate fluorescence intensity and realize nucleic acid is carried out detection by quantitative.To be used in the world at present virus to detect one of state-of-the-art detection method, compare with the regular-PCR amplification technique have advantages of incomparable.But this technology has short, high specificity highly sensitive, consuming time, need not gel electrophoresis high-throughput high-level efficiency, the advantage such as detection by quantitative.
Summary of the invention
The fluorescent quantitative PCR rapid diagnosis reagent box and the application method thereof that the object of the present invention is to provide specific detection duck flavivirus that a kind of utilization should fluorescent quantitative PCR technique to infect, this test kit can specially detect duck flavivirus antigen delicately, thereby reaches the purpose of quick diagnosis.
The present invention is that the technical scheme that solves the problems of the technologies described above employing is: the fluorescence quantitative RT-RCR quick diagnosis reagent kit that a kind of specific detection duck flavivirus infects, it is characterized in that this test kit comprises: a) RNA lysate, b) fluorescent quantitation reaction solution, c) duck flavivirus strong positive quality control product, d) negative quality control product, the fluorescent quantitation reaction solution contains upstream primer DFV-F and downstream primer DFV-R and fluorescent probe DFV-P, and upstream primer DFV-F sequence is: atgaataaggtggtgaaggtaatgc 25nt; Downstream primer DFV-R sequence is: cagattcgtgaaagtgttgagagc 24nt; Fluorescent probe DFV-P sequence is: catgactgttttcccatcacgtcccgg 27nt, and that fluorescent probe DFV-P 5 ＇ hold mark is fluorescence report group FAM, that 3 ＇ hold mark is fluorescent quenching group TAMRA.
Press such scheme, the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, described upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, described fluorescent probe DFV-P is 0.2 μ M.
Press such scheme, described duck flavivirus strong positive quality control product is the duck flavivirus Jianli strain of high titre, and it is bred at the duck embryo, obtains after deactivation, through measuring ELD
50Be 10
3.7/ mL.
Press such scheme, described negative quality control product is to gather healthy and without the duck tissue of cause of disease, 1:5 adds PBS liquid by volume, and fully homogenate is broken, and centrifugal removal precipitates, and supernatant saves backup.
The application method of the fluorescence quantitative RT-RCR quick diagnosis reagent kit that a kind of specific detection duck flavivirus infects is characterized in that may further comprise the steps:
1) extraction of duck flavivirus geneome RNA: in 200 μ l sample to be detected, duck flavivirus strong positive quality control product and negative quality control product, add the 1mlRNA lysate respectively, after leaving standstill 5min, add 200 μ l chloroforms, draw supernatant behind the high speed centrifugation, add isopyknic Virahol in the supernatant, carry out ice bath and high speed centrifugation precipitation again, gained precipitates with after 75% washing with alcohol, with the dissolving of 20 μ l aqua sterilisas, gained solution is the RNA template;
2) getting respectively the RNA template that step 1) obtains joins in the fluorescent quantitation reaction solution, carry out PCR with fluorescent quantitative detector and react the fluorescence intensity that discharges in the also real-time detection reaction process, the PCR reaction obtains duck flavivirus specific amplification goal gene, and described duck flavivirus specific amplification goal gene sequence is: atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt.
Press such scheme, the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, described upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, described fluorescent probe DFV-P is 0.2 μ M.
Press such scheme, the reaction conditions that carries out the PCR reaction for the fluorescence quantitative RT-RCR detector is:
42 ℃ of reverse transcription 20min
94 ℃ of denaturation 5min
℃ annealing of 94 ℃ of sex change 10s ← → 60, extend 50s, 50 circulations.
The present invention compared with prior art has the following advantages:
1 specificity is good, and the dual control of high specific hybridization by the amplification of Auele Specific Primer high specific and fluorescent probe has very high accuracy, and false positive is low;
2 is highly sensitive, and reaction process is by the fluorescence detecting system Real Time Monitoring, and fluorescence detecting system has very high detection sensitivity to fluorescent signal;
3 detection speeds are fast, do not need to prepare in addition cDNA, are single stage method, only need 2 hours, add the extraction of nucleic acid, need 3 hours altogether;
4 do not have post-processed, need not hybridization, electrophoresis, the process such as take pictures, and all reagent all are once to increase, and need not uncap, and do not produce pollution.
Description of drawings
Fig. 1 is that 10 times of gradient dilutions of standard substance carry out the resulting amplification curve of quantitative fluorescent PCR, X-coordinate represents cycle number, and ordinate zou represents fluorescence intensity, and the straight line of parallel principle X-coordinate represents the fluorescence threshold values among the figure, wherein, 1. ~ 7. be followed successively by the standard substance of 10 times of dilutions;
Fig. 2 is that the as a result typical curve of made of fluorescent quantitation is carried out in 10 times of dilutions according to standard substance, and X-coordinate representative sample extension rate, ordinate zou represent fluorescence ct value.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these examples only are used for the present invention being described and being not used in restriction the scope of protection of present invention.
The fluorescent quantitative PCR rapid diagnosis reagent box that the specific detection duck flavivirus infects
1. reagent forms:
Trizol RNA lysate is Invitrogen company product; Taq enzyme (5U/ μ L), dNTPs(10mM), MgCI
2(25mM), M-MLV enzyme (50U/ μ L) is all available from Promega company; PCR primer pair DFV-F and DFV-R give birth to worker bio-engineering corporation by Shanghai and synthesize; Fluorescent probe is synthetic by upper sea base health bio-engineering corporation; The strain of duck flavivirus Jianli is preserved by this laboratory;
2. reagent preparation:
A) fluorescent quantitation reaction solution: 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, each 0.2 μ M of upstream primer DFV-F and downstream primer DFV-R and fluorescent probe DFV-P 0.2 μ M form; Wherein upstream primer DFV-F sequence is: atgaataaggtggtgaaggtaatgc 25nt; Downstream primer DFV-R sequence is: cagattcgtgaaagtgttgagagc 24nt; Fluorescent probe DFV-P sequence is: catgactgttttcccatcacgtcccgg 27nt, and that fluorescent probe DFV-P 5 ＇ hold mark is fluorescence report group FAM, that 3 ＇ hold mark is fluorescent quenching group TAMRA.
B) duck flavivirus strong positive quality control product is the duck flavivirus Jianli strain of high titre, and it is bred at the duck embryo, obtains after deactivation, through measuring ELD
50Be 10
3.7/ mL.
C) negative quality control product: gather healthy and without each tissue of duck of cause of disease, 1:5 adds PBS liquid by volume, fully homogenate is broken, centrifugal removal precipitation, and supernatant saves backup.
D) provide reagent for oneself: 75% ethanol of the sterilization distilled water preparation that chloroform, Virahol, DEPC process.
Embodiment 2
The using method of the fluorescent quantitative PCR rapid diagnosis reagent box that the specific detection duck flavivirus infects
1, the processing of sample
A) tested sample is tissue sample: get sample 50-100mg to be checked in the homogenizer of cleaning, sterilization and oven dry, add PBS liquid with the 1:5 volume ratio, fully homogenate, the centrifugal 10min of 4000rpm, get 100 μ L supernatant liquors (each reaction) and change in the aseptic 1.5mL centrifuge tube, number for subsequent use;
B) tested sample is liquid sample (as: whole blood, serum, nose swab etc.), directly gets 100 μ L to change in the aseptic 1.5mI centrifuge tube, numbers for subsequent use.
2, the extraction of tested sample rna
Get the 1.5ml centrifuge tube of n sterilization, wherein n be the test sample number with the contrast of duck flavivirus Jianli strain strong positive and the contrast of negative quality control product with; Every pipe adds respectively tested sample, duck flavivirus Jianli strain positive control and each 100 μ L of negative quality control product contrast, adds respectively 500 μ L RNA lysates again, and fully mixing leaves standstill 5min; Add again 100 μ L chloroforms, vibration mixing, 4 ℃ of centrifugal 15min of 12000rpm; Draw supernatant to another centrifuge tube, add isopyknic Virahol, ice bath 15min, 4 ℃ of centrifugal 10min of 12000rpm; Abandon most supernatant, add 1ml 75% ethanol, mixing, 4 ℃ of centrifugal 10min of 12000rpm; Thoroughly abandon supernatant.Behind the precipitation Air drying 5min, be dissolved in the sterilization distilled water that 20 μ L DEPC processed, namely get the RNA template;
3, quantitative fluorescent PCR reaction
Get respectively each 19 μ L of fluorescent quantitation reaction solution, get each 1 μ L of RNA template of the 3rd step gained, add respectively different PCR reaction tubess, detect at the enterprising performing PCR of quantitative fluorescent PCR reaction instrument.Cycling condition is: 94 ℃ of 10s of 94 ℃ of 5min of 42 ℃ of 20min
← →50 circulations of 60 ℃ of 50s amplifications.The program setting of fluoroscopic examination is carried out when the second step of each circulation finishes, and the detection wavelength is 530nm;
The PCR reaction obtains duck flavivirus specific amplification goal gene, and described duck flavivirus specific amplification goal gene sequence is: atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt.
4, the result judges
A) the interpretation of result condition is set
After the loop ends, use instrument to carry the software analysis detected result.Circulation thresholding (Threshold Cycle, Ct) setting principle is adjusted according to the noise of instrument situation, is as the criterion with the vertex of Ct value line just above normal negative sample amplification curve;
B) quality control standard
Negative control is without the Ct value and without amplification curve;
Typical amplification curve should and appear about 20 in the Ct value of positive control;
C) result judges
Negative findings is judged: without the Ct value, and without typical amplification curve;
Positive findings is judged: the Ct value is less than 30, and typical amplification curve occurs;
The experiment gray area: Ct value is greater than 35, and the sample suggestion that typical amplification curve occurs is reformed.The result that reforms the Ct value occurs and typical amplification curve person is positive, otherwise negative.
Experimental example 3
The application of the fluorescent quantitative PCR rapid diagnosis reagent box that the specific detection duck flavivirus infects
1, sensitivity test
Carry out (10 of 10 times of gradient dilutions with standard substance
3.7~ 10
-7.7ELD
50/ mL) as template, detect at quantitative real time PCR Instrument, obtain PCR in real time amplification curve and typical curve and see respectively accompanying drawing 1 and accompanying drawing 2.Detecting minimum virus quantity is 10
-2.7ELD
50/ mL.
2, specific test
The fluorescent quantitative PCR rapid diagnosis reagent box that adopts the specific detection duck flavivirus to infect carries out specific test to bird flu, avian leukosis, avian escherichia coli, Salmonellas, riemerella anatipestifer, newcastle disease, egg-decreasing syndrome etc., and is all negative to above-mentioned 7 kinds of Detecting results.In addition, all negative to the various tissue detection results of water and duck.Above this test kit of as a result sufficient proof has good specificity in the detection of clinical sample.
3, replica test
Repeatability detected between the duck flavivirus sample of getting 3 parts of different virus levels was organized, and obtained table 1, calculated to such an extent that the between-group variation factor beta value of repeated detection is respectively 4.4%, 4.8%, 3.7%, all less than 5%, proved that this detection method has good specificity.
Repeatability detects the virus titer result between the group of table 1 sample
A: circulation thresholding
B: mean CT-number.
Sequence table
＜110〉Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences (C
＜120〉a kind of fluorescence quantitative RT-RCR quick diagnosis reagent kit and application method thereof of specific detection duck flavivirus infection
<160>4
<210>1
<211>130
<212>DNA
＜213〉duck flavivirus (Duck Flavivirus)
<400>1
atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt 70
<210>2
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the design of duck flavivirus nucleotide sequence comparison, for detection of the specific primer sequence of duck flavivirus wild virus infection
<400>2
<210>3
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the design of duck flavivirus nucleotide sequence comparison, for detection of the specific primer sequence of duck flavivirus wild virus infection
<400>3
cagattcgtgaaagtgttgagagc 24
<210>4
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the design of duck flavivirus nucleotide sequence comparison, for detection of the specificity fluorescent probe of duck flavivirus wild virus infection, that 5' holds mark is fluorescence report group FAM, and that 3' holds mark is fluorescent quenching group TAMRA;
<400>4
catgactgttttcccatcacgtcccgg 27
Claims (7)
1. the fluorescence quantitative RT-RCR quick diagnosis reagent kit that infects of a specific detection duck flavivirus, it is characterized in that this test kit comprises: a) RNA lysate, b) fluorescent quantitation reaction solution, c) duck flavivirus strong positive quality control product, d) negative quality control product, the fluorescent quantitation reaction solution contains upstream primer DFV-F and downstream primer DFV-R and fluorescent probe DFV-P, and upstream primer DFV-F sequence is: atgaataaggtggtgaaggtaatgc 25nt; Downstream primer DFV-R sequence is: cagattcgtgaaagtgttgagagc 24nt; Fluorescent probe DFV-P sequence is: catgactgttttcccatcacgtcccgg 27nt, and that fluorescent probe DFV-P 5 ＇ hold mark is fluorescence report group FAM, that 3 ＇ hold mark is fluorescent quenching group TAMRA.
2. the fluorescence quantitative RT-RCR quick diagnosis reagent kit that infects by specific detection duck flavivirus claimed in claim 1 is characterized in that the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, described upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, described fluorescent probe DFV-P is 0.2 μ M.
3. the fluorescence quantitative RT-RCR quick diagnosis reagent kit that infects by specific detection duck flavivirus claimed in claim 1, it is characterized in that described duck flavivirus strong positive quality control product is the duck flavivirus Jianli strain of high titre, it is bred at the duck embryo, obtains after deactivation, through measuring ELD
50Be 10
3.7/ mL.
4. the fluorescence quantitative RT-RCR quick diagnosis reagent kit that infects by specific detection duck flavivirus claimed in claim 1, it is characterized in that described negative quality control product is to gather healthy and without the duck tissue of cause of disease, 1:5 adds PBS liquid by volume, fully homogenate is broken, centrifugal removal precipitation, supernatant saves backup.
5. the application method of the fluorescence quantitative RT-RCR quick diagnosis reagent kit that infects of specific detection duck flavivirus claimed in claim 1 is characterized in that may further comprise the steps:
1) extraction of duck flavivirus geneome RNA: in 200 μ l sample to be detected, duck flavivirus strong positive quality control product and negative quality control product, add the 1mlRNA lysate respectively, after leaving standstill 5min, add 200 μ l chloroforms, draw supernatant behind the high speed centrifugation, add isopyknic Virahol in the supernatant, carry out ice bath and high speed centrifugation precipitation again, gained precipitates with after 75% washing with alcohol, with the dissolving of 20 μ l aqua sterilisas, gained solution is the RNA template;
2) getting respectively the RNA template that step 1) obtains joins in the fluorescent quantitation reaction solution, carry out PCR with fluorescent quantitative detector and react the fluorescence intensity that discharges in the also real-time detection reaction process, the PCR reaction obtains duck flavivirus specific amplification goal gene, and described duck flavivirus specific amplification goal gene sequence is: atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt.
6. press the application method of the fluorescence quantitative RT-RCR quick diagnosis reagent kit of a kind of specific detection duck flavivirus infection claimed in claim 5, it is characterized in that the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, described upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, described fluorescent probe DFV-P is 0.2 μ M.
7. press the application method of the fluorescent quantitative PCR rapid diagnosis reagent box of claim 5 or 6 described specific detection duck flavivirus infection, it is characterized in that, the reaction conditions that carries out the PCR reaction for the fluorescence quantitative RT-RCR detector is:
42 ℃ of reverse transcription 20min
94 ℃ of denaturation 5min
℃ annealing of 94 ℃ of sex change 10s ← → 60, extend 50s, 50 circulations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210493140 CN102925592B (en) | 2012-11-28 | 2012-11-28 | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210493140 CN102925592B (en) | 2012-11-28 | 2012-11-28 | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102925592A true CN102925592A (en) | 2013-02-13 |
| CN102925592B CN102925592B (en) | 2013-12-25 |
Family
ID=47640515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210493140 Expired - Fee Related CN102925592B (en) | 2012-11-28 | 2012-11-28 | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102925592B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320541A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR kit |
| CN106399584A (en) * | 2016-08-23 | 2017-02-15 | 中国水产科学研究院珠江水产研究所 | Siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) inactivation rapid detection kit and detection method |
| CN110846441A (en) * | 2019-12-20 | 2020-02-28 | 浙江省淡水水产研究所 | Specific primer, probe and rapid detection kit for detecting macrobrachium flavivirus-1 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
| CN102397539A (en) * | 2011-11-25 | 2012-04-04 | 广东省农业科学院兽医研究所 | Duckling flavivirus disease inactivated vaccine and preparation method thereof |
| CN102533668A (en) * | 2010-12-20 | 2012-07-04 | 中国农业科学院上海兽医研究所 | Duck flavivirus, and vaccine and kit thereof |
-
2012
- 2012-11-28 CN CN 201210493140 patent/CN102925592B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533668A (en) * | 2010-12-20 | 2012-07-04 | 中国农业科学院上海兽医研究所 | Duck flavivirus, and vaccine and kit thereof |
| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
| CN102397539A (en) * | 2011-11-25 | 2012-04-04 | 广东省农业科学院兽医研究所 | Duckling flavivirus disease inactivated vaccine and preparation method thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320541A (en) * | 2013-07-11 | 2013-09-25 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR kit |
| CN103320541B (en) * | 2013-07-11 | 2016-06-08 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR detection kit |
| CN106399584A (en) * | 2016-08-23 | 2017-02-15 | 中国水产科学研究院珠江水产研究所 | Siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) inactivation rapid detection kit and detection method |
| CN106399584B (en) * | 2016-08-23 | 2019-11-19 | 中国水产科学研究院珠江水产研究所 | A kind of mandarin fish infectious spleen and kidney necrosis virus inactivation quick testing reagent kit and detection method |
| CN110846441A (en) * | 2019-12-20 | 2020-02-28 | 浙江省淡水水产研究所 | Specific primer, probe and rapid detection kit for detecting macrobrachium flavivirus-1 |
| CN110846441B (en) * | 2019-12-20 | 2023-04-11 | 浙江省淡水水产研究所 | Specific primer, probe and rapid detection kit for detecting macrobrachium flaviviridae-1 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102925592B (en) | 2013-12-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107513584B (en) | A kind of five heavy fluorescence quantitative kits detecting enterovirus | |
| CN103966358B (en) | A kind of mandarin fish infectious spleen and kidney necrosis virus fluorescent quantificationally PCR detecting kit and detection method | |
| CN103757134A (en) | Fluorescent quantitative PCR (Polymerase Chain Reaction) detection reagent, kit and detection method for African swine fever virus (ASFV) | |
| CN103627816A (en) | Multiplex RT-PCR detection kit for porcine reproductive and respiratory syndrome virus and application thereof | |
| CN101328506B (en) | Fluorescent quantitative PCR rapid diagnosis reagent kit for specific detection of classical swine fever virus wild virus infection and its application method | |
| CN107828914A (en) | Infectious subcutaneous and haematopoietic necrosis virus（IHHNV）RAA constant temperature fluorescence detection method and reagent | |
| CN115478120A (en) | Method for simultaneously detecting nodavirus and decapod iridovirus 1 of macrobrachium rosenbergii | |
| CN102925592B (en) | Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection | |
| CN106435031A (en) | PCR detection kit for specific detection of fowl adenovirus group I and detection method thereof | |
| CN105463132A (en) | Genetic marker of canine parvovirus and specific primers as well as probe thereof | |
| CN110373496A (en) | Kit and detection method for the detection of 8 type aviadenovirus of I subgroup serum | |
| Xu et al. | Simultaneous detection of novel H7N9 and other influenza A viruses in poultry by multiplex real-time RT-PCR | |
| CN105950785A (en) | Ternary fluorescence RT-PCR detection kit of avian influenza virus, new castle disease virus and infectious bronchitis virus, primers and probes | |
| CN110343784A (en) | The composition and kit of quadruple influenza nucleic acids detection based on melting curve | |
| CN106282414B (en) | Reagent for detecting H5N6 avian influenza virus, detection method and application | |
| CN102676697B (en) | Primers and probe for detecting peste des petits ruminants virus and kit | |
| CN109722492B (en) | Method for detecting H5 and H7N9 subtype highly pathogenic avian influenza virus and H9 subtype avian influenza virus | |
| CN101575649A (en) | Quick detection technology for H9 type avian influenza virus | |
| CN105506177A (en) | Eriocheir sinensis reovirus RT-LAMP detection reagent kit and detection method thereof | |
| CN104278106B (en) | Duplex fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection kit for duck tembusu virus and egg drop syndrome virus | |
| CN107574263A (en) | A kind of kit and method for the type of PCR quick detections pig circular ring virus 3 | |
| CN102154523B (en) | Primer for detecting human BK viral nucleic acid, fluorescent probe and application thereof | |
| CN114381551A (en) | Real-time fluorescent RAA primer, probe and kit for detecting iridovirus of micropterus salmoides | |
| CN106011248A (en) | Method for detecting amyotrophy and kit | |
| CN103320528B (en) | Primer pair and probe for detecting avian influenza virus in sample by fluorescence RT-PCR and kit containing primer pair and probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| 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: 20131225 Termination date: 20181128 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |