CN104673935A - Detection method and detection kit of gene II type grass carp reovirus - Google Patents
Detection method and detection kit of gene II type grass carp reovirus Download PDFInfo
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Abstract
The invention discloses a detection method of gene II type grass carp reovirus. The detection method comprises the following steps: (1) extracting the RNA of the grass carp sample, adding RNA into a premix reaction solution, and performing loop-mediated isothermal nucleic acid amplification on the conserved sequence of the gene II type grass carp reovirus by two pairs of primers, wherein the primers are respectively a primer GCRVII-F3 with the sequence of SEQ ID NO:1, a primer GCRVII-B3 with the sequence of SEQ ID NO:2, a primer GCRVII-F1P with the sequence of SEQ ID NO:3 and a primer GCRVII-BIP with the sequence of SEQ ID NO:4; labeling an FITC probe, wherein the sequence of the probe GCRVII-FITC-Probe is SEQ ID NO:5; and (2) dropwisely adding the amplified product on a sample spotting position of the LFD test paper, dropwisely adding a buffer solution in the front end of the sample position, finishing the reaction after the buffer solution is flown to the other end of the test paper, and judging the detection result according to the color development tape on the test paper. By combining the isothermal amplification and the nucleic acid test strip rapid detection technology, the detection method and the kit provided by the invention have the characteristics of simple operation, high detection reliability, good specificity and high sensitivity.
Description
Technical field
The present invention relates to the disease diagnosis technology in Aquatic animals virus field, be specifically related to RT-LAMP-LFD detection method and the detection kit thereof of gene II type GCRV.
Background technology
Hemorrhagic disease of grass carp (Hemorrhage of Grass Carp) is that a kind of serious viral hemorrhagic of grass carp aquaculture is sick, grass carp aquaculture industry is caused a significant threat, its epidemic season is between at the beginning of annual 5 months and by the end of October, water temperature comes into vogue at 20 DEG C, the suitableeest popular water temperature is 27 DEG C-30 DEG C, mortality ratio can reach 50%-80%, according to incompletely statistics, the financial loss caused due to hemorrhagic disease of grass carp every year reaches 21.26 hundred million yuan, and this disease is listed in two class animal epidemic diseases by the Ministry of Agriculture and planted register.This disease pathogen is GCRV (Grass carp reovirus, GCRV), this virus is the first strain fishes virus of Chinese isolation identification, be under the jurisdiction of Aquareovirus (Aquareovirus, ARV), the virus that in this genus member, virulence is the strongest, it is the first strain Aquareovirus that China completes full gene sequencing and analysis in the world first, also be study to obtain the deep Aquareovirus of most system so far, this virus is the segmented RNA viruses of double-strand, gene element 11 sections.Research shows, according to the feature of GCRV strain isolated gene order, co-exist in three kinds of genotype GCRV clinically at present, comprising with GCRV873 is the Genotype I of representative, the gene II type being representative with HZ08, GD108, the gene type III being representative with HGDRV (for GCRV104 in Genbank).
A large amount of sample detection result shows, clinical popular strain is I type and II type, because gene II type hemorrhagic disease of grass carp is dead fast among this, mortality ratio is high, morbidity state shows as enterorrhagia and hemorrhage of muscle, outward signs is not obvious, and cause the timely detection being difficult to realize this disease, high mortality brings huge financial loss.At present, the detection method for this genotype GCRV has electron microscopy, reverse transcriptase polymerase chain reaction (RT-PCR) detection method, multiplex RT-PCR method etc.But in actual testing, there is following problem in existing detection technique: one is insufficient sensitivity, latent infection sample cannot be detected; Two is easily produce false positive, requires high to template quality; Three is complex operations, high to requirement for experiment condition, is unfavorable for that raiser and line production technology personnel operate.
RT-LAMP is nucleic acid isothermal amplification detection technique, according to the principle of loop-mediated isothermal amplification (LAMP), 4 Auele Specific Primers are designed in specific region for target gene, utilize and there is the archaeal dna polymerase of strand displacement characteristic and AMV reversed transcriptive enzyme RT and LAMP is reacted carry out in same pipe simultaneously, simplify operation steps, under isothermal conditions just can efficiently, high amplified target sequence specifically.The detection of LAMP product generally adopts the methods such as the observation of agarose gel electrophoresis, turbidity, fluorescence dye observation.Horizontal effluent (Latteral flow detect, LFD) test strip is a kind of novel method detecting product, the probe utilizing the FITC in reaction system to mark and biotin labeled LAMP amplified production specific hybrid, decrease electrophoresis link, the artificial visual difference of dye colour and the false positive issue that caused by non-specific amplification.
Although independent above-mentioned two kinds of technology obtain Preliminary Applications in recent years, will exploration extensively and profoundly but the applicable elements for concrete Viral diagnosis is still needed, not yet there is the precedent using above-mentioned technology on gene II type GCRV detects current this area.The domestic GCRV RT-LAMP detection reagent box CN201210186709.6 that once have studied gene type III in early days, gene type III is clinical rarely found on the one hand, and this detection kit there is no the market requirement; On the other hand, the primer of the program cannot be used for increasing other GCRV, by too poor for above-mentioned disclosed primer amplification gene II type GCRV specificity, cannot be used for LFD ELISA test strip.Also once there was the RT-LAMP-LFD detection method of attempting research hiv virus this area, but there is huge sequence difference due to hiv virus and gene II type GCRV, its probe used to other detections without any reference value.
Summary of the invention
First object of the present invention is to provide a kind of detection method for gene II type GCRV, passes through two pairs of used primers and specific probe, efficient, the highly sensitive detection realizing gene II type GCRV.
Another object of the present invention is to provide a kind of detection kit of gene II type GCRV, the RT-LAMP-LFD that this test kit is built-in realizes gene II type GCRV detects premix reaction solution used, and the RNA sample adding gene II type GCRV in use can realize detecting fast, accurately.
For achieving the above object, the present invention is achieved through the following technical solutions:
The detection method of gene II type GCRV, comprises the steps: 1) extract the RNA of grass carp sample, join in premix reaction solution; Utilize the conserved regions sequence of two pairs of primer pair gene II type GCRVs to carry out ring mediated isothermal nucleic acid amplification, primer wherein used is respectively primer GCRVII-F3 sequence SEQ ID No:1, primer GCRVII-B3 sequence SEQ ID No:2; Primer GCRVII-FIP sequence SEQ ID No:3, primer GCRVII-BIP sequence SEQ ID No:4; And flag F ITC probe, probe GCRVII-FITC-Probe sequence SEQ ID No:5; 2) be added drop-wise to by amplified production on the point sample position of LFD test paper, then drip damping fluid in front end, point sample place, liquid to be buffered moves to the other end of test paper to reacting end, judges detected result according to the developed band in test strip.
Detection method disclosed in this invention, the primer sets of employing, according to the design of gene II type 6CRV the 6th gene fragment, can combine with the specific probe of RNA, specificity and highly sensitive, and it is highly sensitive in more than 1000 times of RT-PCR; Meanwhile, in conjunction with adopted LFD detection technique, to save time and plant and instrument requirement is low, can rapid detection be realized, in 1 hour, can detected result be obtained.
In the present invention, step 1) in amplified reaction constant-temperature amplification premix reaction solution used be made up of following component: 0.15-0.25 μM of GCRVII-F3 and GCRVII-B3, 1-2 μM of GCRVII-FIP and GCRVII-BIP, 0.03-0.08 μM of GCRVII-FITC-Probe, 15-25mM pH 8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-20mM ammonium sulfate, 5-12mM magnesium sulfate, 0.1-0.2%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-2mM dNTP, 0.8-1.5U reversed transcriptive enzyme AMV and 6-10U Bst archaeal dna polymerase large fragment, premixed liquid volume is 18-25 μ L.
Corresponding above-mentioned premix reaction solution, the consumption of measuring samples RNA to be measured used is 3-6 μ L.
Preferably, step 1) in be made up of following component for the constant-temperature amplification premix reaction solution of amplified reaction: 0.2 μM of GCRVII-F3 and GCRVII-B3,1.6 μMs of GCRVII-FIP and GCRVII-BIP, 0.05 μM of GCRVII-FITC-Probe, 20mM pH8.0 ~ 9.0Tris-HCl, 10mM Repone K, 15mM ammonium sulfate, 8mM magnesium sulfate, 0.1%Triton X-100,0.6M trimethyl-glycine, 1.4mM dNTP, 1U reversed transcriptive enzyme AMV and 8U Bst archaeal dna polymerase large fragment, premixed liquid volume is 21 μ L.
Preferably, the consumption of the measuring samples RNA to be measured of corresponding premix reaction solution is 4 μ L.
For the reaction conditions of constant-temperature amplification, adopt the reaction conditions that this area is general, namely at 64 DEG C, reaction 30 ~ 60min.
In the present invention, step 2) amplified production consumption is 5 ~ 10 μ L, the consumption of LFD test paper damping fluid is 50 ~ 100 μ L, judges detected result after 5 ~ 10 minutes according to the developed band in LFD test strip.
Wherein judge that the standard of detected result is as showing that when only there is quality control band reaction result is negative, when quality control band and detection zone occur simultaneously, shows that reaction result is positive.
In the present invention, and unexposed damping fluid particular type used, as is known to the person skilled in the art, the damping fluid of LFD test paper and coupling thereof is that producer sells together, guidance according to commodity operation instructions uses, and usual this damping fluid is 1xTAE damping fluid.
In the present invention, be not particularly limited the extraction of RNA, the common grass carp RNA extraction method in this area all can be used for the present invention, and the extraction RNA operation of this area in disclosed similar detection method also can be used for the present invention.
For the ease of on a large scale, industrialization, the above-mentioned detection method of fast operating, the invention also discloses the detection kit of the gene II type GCRV realized based on aforesaid method, its core is to include above-mentioned constant-temperature amplification premix reaction solution, be made up of following component: 0.15-0.25 μM of GCRVII-F3 and GCRVII-B3, 1-2 μM of GCRVII-FIP and GCRVII-BIP, 0.03-0.08 μM of GCRVII-FITC-Probe, 15-25mM pH 8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-20mM ammonium sulfate, 5-12mM magnesium sulfate, 0.1-0.2%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-2mM dNTP, 0.8-1.5U reversed transcriptive enzyme AMV and 6-10U Bst archaeal dna polymerase large fragment, premixed liquid volume is 18-25 μ L.。
Preferably, constant-temperature amplification premix reaction solution is made up of following component: 0.2 μM of GCRVII-F3 and GCRVII-B3,1.6 μMs of GCRVII-FIP and GCRVII-BIP, 0.05 μM of GCRVII-FITC-Probe, 20mM pH8.0 ~ 9.0Tris-HCl, 10mM Repone K, 15mM ammonium sulfate, 8mM magnesium sulfate, 0.1%Triton X-100,0.6M trimethyl-glycine, 1.4mM dNTP, 1U reversed transcriptive enzyme AMV and 8U Bst archaeal dna polymerase large fragment, premixed liquid volume is 21 μ L.
It will be understood by those skilled in the art that extracting test kit based on above-mentioned contained constant-temperature amplification premix reaction solution in conjunction with other common RNA combines and can realize testing goal.In order to improve the integrated level of operation, easy operation, described detection kit also comprises sample dissociation liquid, protein liquid removal, rinsing liquid and 70% dehydrated alcohol, RNase free water, nucleic acid absorption post, wherein sample dissociation liquid consists of 40-60mM pH7.4Tris-HCL, the guanidinium isothiocyanate of 3-5M, 8-15mM EDTA; Protein liquid removal consists of the Tris-HCL of 20-40mM pH7.4,0.3-0.5M guanidinium isothiocyanate and 15-25% dehydrated alcohol; Rinsing liquid consists of the Tris-HCL of 20-40mM pH7.4, the dehydrated alcohol of 100-150mM NaCl and 75%; Preferably, sample dissociation liquid consists of 50mM pH7.4Tris-HCL, the guanidinium isothiocyanate of 4M, 10mM EDTA; Protein liquid removal consists of the Tris-HCL of 30mM pH7.4,0.4M guanidinium isothiocyanate and 20% dehydrated alcohol; Rinsing liquid consists of the Tris-HCL of 30mM pH7.4, the dehydrated alcohol of 130mM NaCl and 75%.
Further, detection kit also comprises negative controls, positive reference substance, LFD test strip, LFD test strip damping fluid.
Wherein, the damping fluid of LFD test paper is generally 1xTAE damping fluid, and positive control sample is gene II type GCRV RNA, and negative control sample is the RNase free water of free nucleic acid.
The detection method of gene II type GCRV disclosed in this invention and test kit, two pairs of primers based on the conserved regions sequences Design of gene II type GCRV gene and specific probe, thus set up GCRV RT-LAMP reaction system, and detect gene II type GCRV in conjunction with horizontal lateral flow strip, it has high specificity, susceptibility is high, feature simple and efficient to handle, can be used for tracking monitor and the early diagnosis of gene II type GCRV in grass carp breeding process, avoid virus disseminating popular, improve scientific management efficiency.Disclosed test kit can make to be in the detection that the technician of production line and raiser conveniently realize corresponding virus, step explanation is used to operate according to simple, and test kit all ingredients used is not containing the objectionable impurities such as chloroform, mercaptoethanol, safer to human and environment.
Accompanying drawing explanation
Fig. 1 is the effect diagram that amplification temperature is reacted RT-LAMP.M:1DL2000DNA Marker (TaKaRa company); Swimming lane 1:61 DEG C; Swimming lane 2:62 DEG C; Swimming lane 3:63 DEG C; Swimming lane 4:64 DEG C; Swimming lane 5:65 DEG C.
Fig. 2 is gene II type GCRV RT-LAMP specificity experiments result figure.M:DL2000DNA Marker (TaKaRa company); Swimming lane 1: gene II type GCRV; Swimming lane 2: Genotype I GCRV; Swimming lane 3: carp simplexvirus II type (CyHV-2); Swimming lane 4: SVCV SVCV; Swimming lane 5: Aeromonas hydrophila; Swimming lane 6: negative control.
Fig. 3 is sensitivity comparison diagram and the LFD test paper color development comparison diagram that RT-LAMP-LFD method detects gene II type GCRV.M:100bp DNA Ladder Marker (TaKaRa company); Left side swimming lane 1 ~ 6: template is respectively 10-1, the geneome RNA that 10-2,10-3,10-4,10-5,10-6 doubly dilute; Swimming lane 7: without template; Right side 1 ~ 7: be the LFD detection paper figure of left side experimental result.
Fig. 4 is the sample result figure that in embodiment, RT-LAMP-LFD technology for detection grass carp infects gene II type GCRV.M:DL2000DNA Marker (TaKaRa company); Left side swimming lane 1: gene II type GCRV positive control; Left side swimming lane 2: grass carp nephridial tissue sample; Left side swimming lane 3: grass carp spleen tissue sample; 4: be negative control sample detected result; Right side is corresponding LFD detection paper colour developing result (C represents nature controlling line, and T represents detectability).
Embodiment
Below by specific embodiment, and by reference to the accompanying drawings, technical scheme of the present invention is described in further detail; In the following embodiments, the raw material adopted and equipment etc. all can be buied from market or this area is conventional.Detection method of the present invention is as described below:
1, material
The sick sample collection of hemorrhagic disease of grass carp is from plant of Jinhua, Zhejiang Province city; The primer and probe are synthesized by Nanjing Jin Sirui biotech firm; Bst archaeal dna polymerase large fragment is purchased from New England BIPolabs company; AMV enzyme, dNTP, purchased from Takara company; Trimethyl-glycine, magnesium sulfate etc. are purchased from sigma company.
2, the extraction of grass carp sample RNA:
(1) get 25mg grass carp spleen, kidney tissue to be measured respectively, grind after respectively adding 500 μ L lysate RA with disposable grinding rod, after waiting thorough homogenate, the centrifugal 2min of 12000rpm, draws supernatant to new centrifuge tube;
(2), after adding isopyknic 70% dehydrated alcohol RB in centrifuge tube, mix;
(3) proceeded to by mixing liquid in the adsorption column with glass fibre element film, adsorption column is placed in collection tube, and the centrifugal 1min of 12000rpm, discards waste liquid;
(4) in adsorption column, add 600 μ L protein liquid removal RC, room temperature leaves standstill 1min, and the centrifugal 30 ~ 60s of 12000rpm, discards waste liquid;
(5) add 600 μ L rinsing liquid RD, the centrifugal 30s of 12000rpm, discards waste liquid; Repeat this step once.
(6) adsorption column is placed in the new centrifuge tube without RNase, adds 50 μ L RNase free water in adsorption column, room temperature places the centrifugal 1min of 1min, 12000rpm.
Use primary elutriant to repeat this step once, increase RNA elution amount.
3, gene II type GCRV RT-LAMP constant-temperature amplification:
(1) negative and each 1 part of positive control is added according to measuring samples number 2 parts, get the nuclease assay reaction liquid (containing positive control, negative control) needed for 4 times of nuclease assay reaction liquid preparations, add each sample again and add 1U reversed transcriptive enzyme AMV and 8U Bst archaeal dna polymerase large fragment, often pipe detection pre-reaction liquid amasss is 21 μ L;
(2) the measuring samples RNA drawing 4 μ L different concns respectively adds in nuclease assay reaction pipe, mixes;
(3) after mark is known, detection reaction pipe is placed in 64 DEG C of water-baths, reaction 30min;
In above-mentioned, amplimer is the primer according to the design of gene II type GCRV the 6th gene fragment, and its nucleotide sequence is as follows:
GCRVII-F3：ACTCGCATGGATGAAAGTCG
GCRVII-B3：CAACGTAGGCACTGAACTCA
GCRVII-FIP：TACGGTGACCCGTCTGTTGC-CAGGATCAGGTATGGGACCA
GCRVII-BIP：TGGAAAAATCAGCAGGTGCCGT-CGTTCACTGTAGAGCAGGTT
GCRVII-FITC-Probe：CTCCGGACGCCATGTCTAGT
Wherein GCRVII-FIP is 5 ' end vitamin H (biotin) labeled primer; GCRVII-FITC-Probe is 5 ' end FITC label probe.
4, gene II type GCRV RT-LAMP amplified production carries out gel electrophoresis:
(1) configure the sepharose of 2%, each well adds the reaction product of 5 μ L;
(2) electrophoresis gel imaging after 30 minutes, the results are shown in Figure 4 left hand views.
5, the LFD detection paper of RT-LAMP amplified production:
(1) get RT-LAMP amplified production 6 μ L, be added in test strip sample pad; Drip 100 μ L damping fluids, liquid to be buffered moves to the other end of test paper, judges RT-LAMP-LFD detected result in 10 minutes, sees Fig. 4 right part of flg.
Detection kit of the present invention is utilized to detect gene II type GCRV, through gel electrophoresis, by the visible stair-stepping amplified band of Fig. 4 left hand view; Recycling LFD test strip inspection amplified production, by Fig. 4 right part of flg, visible and electrophoresis result is coincide.And Fig. 2 specificity experiments result and Fig. 3 sensitivity technique result show that this detection method has good specificity and sensitivity.
Claims (9)
1. the detection method of gene II type GCRV, is characterized in that comprising the steps: 1) extract the RNA of grass carp sample, join in premix reaction solution; Utilize the conserved regions sequence of two pairs of primer pair gene II type GCRVs to carry out ring mediated isothermal nucleic acid amplification, primer wherein used is respectively primer GCRVII-F3 sequence SEQ ID No:1, primer GCRVII-B3 sequence SEQ ID No:2; Primer GCRVII-FIP sequence SEQ ID No:3, primer GCRVII-BIP sequence SEQ ID No:4; And flag F ITC probe, probe GCRVII-FITC-Probe sequence SEQ ID No:5; 2) be added drop-wise to by amplified production on the point sample position of LFD test paper, then drip damping fluid in front end, point sample place, liquid to be buffered moves to the other end of test paper to reacting end, judges detected result according to the developed band in test strip.
2. the detection method of gene II type GCRV according to claim 1, it is characterized in that step 1) in be made up of following component for the constant-temperature amplification premix reaction solution of amplified reaction: 0.15-0.25 μM of GCRVII-F3 and GCRVII-B3, 1-2 μM of GCRVII-FIP and GCRVII-BIP, 0.03-0.08 μM of GCRVII-FITC-Probe, 15-25mM pH8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-20mM ammonium sulfate, 5-12mM magnesium sulfate, 0.1-0.2%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-2mM dNTP, 0.8-1.5U reversed transcriptive enzyme AMV and 6-10U Bst archaeal dna polymerase large fragment, premixed liquid volume is 18-25 μ L, the consumption of measuring samples RNA to be measured is 3-6 μ L.
3. the detection method of gene II type GCRV according to claim 2, it is characterized in that step 1) in be made up of following component for the constant-temperature amplification premix reaction solution of amplified reaction: 0.2 μM of GCRVII-F3 and GCRVII-B3, 1.6 μMs of GCRVII-FIP and GCRVII-BIP, 0.05 μM of GCRVII-FITC-Probe, 20mM pH8.0 ~ 9.0Tris-HCl, 10mM Repone K, 15mM ammonium sulfate, 8mM magnesium sulfate, 0.1%Triton X-100, 0.6M trimethyl-glycine, 1.4mM dNTP, 1U reversed transcriptive enzyme AMV and 8U Bst archaeal dna polymerase large fragment, premixed liquid volume is 21 μ L, the consumption of measuring samples RNA to be measured is 4 μ L.
4. the detection method of gene II type GCRV according to claim 1, it is characterized in that step 2) amplified production consumption is 5 ~ 10 μ L, the consumption of LFD test paper damping fluid is 50 ~ 100 μ L, judges detected result after 5 ~ 10 minutes according to the developed band in LFD test strip.
5. the detection kit of gene II type GCRV, it is characterized in that comprising constant-temperature amplification premix reaction solution, be made up of following component: 0.15-0.25 μM of GCRVII-F3 and GCRVII-B3, 1-2 μM of GCRVII-FIP and GCRVII-BIP, 0.03-0.08 μM of GCRVII-FITC-Probe, 15-25mM pH 8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-20mM ammonium sulfate, 5-12mM magnesium sulfate, 0.1-0.2%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-2mM dNTP, 0.8-1.5U reversed transcriptive enzyme AMV and 6-10U Bst archaeal dna polymerase large fragment, premixed liquid volume is 18-25 μ L.
6. detection kit according to claim 5, it is characterized in that constant-temperature amplification premix reaction solution is made up of following component: 0.2 μM of GCRVII-F3 and GCRVII-B3,1.6 μMs of GCRVII-FIP and GCRVII-BIP, 0.05 μM of GCRVII-FITC-Probe, 20mM pH8.0 ~ 9.0Tris-HCl, 10mM Repone K, 15mM ammonium sulfate, 8mM magnesium sulfate, 0.1%Triton X-100,0.6M trimethyl-glycine, 1.4mM dNTP, 1U reversed transcriptive enzyme AMV and 8U Bst archaeal dna polymerase large fragment, premixed liquid volume is 21 μ L.
7. detection kit according to claim 5, characterized by further comprising sample dissociation liquid, protein liquid removal, rinsing liquid and 70% dehydrated alcohol, RNase free water, nucleic acid absorption post, wherein sample dissociation liquid consists of 40-60mMpH7.4Tris-HCL, the guanidinium isothiocyanate of 3-5M, 8-15mM EDTA; Protein liquid removal consists of the Tris-HCL of 20-40mMpH7.4,0.3-0.5M guanidinium isothiocyanate and 15-25% dehydrated alcohol; Rinsing liquid consists of the Tris-HCL of 20-40mMpH7.4, the dehydrated alcohol of 100-150mM NaCl and 75%.
8. detection kit according to claim 7, is characterized in that sample dissociation liquid consists of 50mMpH7.4Tris-HCL, the guanidinium isothiocyanate of 4M, 10mM EDTA; Protein liquid removal consists of the Tris-HCL of 30mMpH7.4,0.4M guanidinium isothiocyanate and 20% dehydrated alcohol; Rinsing liquid consists of the Tris-HCL of 30mMpH7.4, the dehydrated alcohol of 130mM NaCl and 75%.
9. detection kit according to claim 5, characterized by further comprising negative controls, positive reference substance, LFD test strip, LFD test strip damping fluid.
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| CN108330216A (en) * | 2018-05-22 | 2018-07-27 | 北京科弘生物技术有限公司 | Ring mediated isothermal amplification combination lateral flow ELISA test strip grass carp reovirus |
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| CN110592269A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) |
| CN110894554A (en) * | 2019-11-18 | 2020-03-20 | 浙江省淡水水产研究所 | Primer probe set for detecting carp herpesvirus II, kit and application |
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| CN109971890A (en) * | 2019-04-30 | 2019-07-05 | 上海海洋大学 | The droplet type digital pcr detection kit and its primer special and probe of II type grass carp reovirus |
| CN110894554A (en) * | 2019-11-18 | 2020-03-20 | 浙江省淡水水产研究所 | Primer probe set for detecting carp herpesvirus II, kit and application |
| CN110894554B (en) * | 2019-11-18 | 2023-05-02 | 浙江省淡水水产研究所 | Primer probe group for detecting cyprinid herpesvirus II, kit and application |
| 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 |
| CN112680544A (en) * | 2020-12-28 | 2021-04-20 | 中国水产科学研究院珠江水产研究所 | RT-RPA primer, probe, kit and detection method for detecting II-type grass carp reovirus |
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