CN104561379A - Detection method and detection kit of genotype I grass carp reovirus - Google Patents
Detection method and detection kit of genotype I grass carp reovirus Download PDFInfo
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Abstract
The invention discloses a detection method of a genotype I grass carp reovirus. The detection method comprises steps as follows: 1), RNA (ribonucleic acid) of grass carp is extracted and added to a premixed reaction liquid, two pairs of primers are utilized to perform loop-mediated isothermal nucleic acid amplification on a conserved sequence of the genotype I grass carp reovirus, the primers are a primer GCRVI-F3 with a sequence represented as SEQ ID NO: 1 and a primer GCRVI-B3 with a sequence represented as SEQ ID NO: 2 as well as a primer GCRVI-FIP with a sequence represented as SEQ ID NO: 3 and a primer GCRVI-BIP with a sequence represented as SEQ ID NO: 4, an FITC (fluorescein isothiocyanate) probe is labeled, and a GCRVI-FITC-Probe sequence is represented as SEQ ID NO: 5; 2), an amplified product is dropwise added to the sample application position of an LFD (lateral flow detect) test paper, a buffer solution is dropped at the front end of the sample application position and moves to the other end of the test paper until the reaction is finished, and a detection result is determined according to a color developing strip on the test paper strip. The detection method and a kit of the genotype I grass carp reovirus combines isothermal amplification and nucleic acid test paper strip rapid detection technology, and have the characteristics of simplicity in 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 Genotype I 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 carpreovirus, 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-exists in three kinds of genotype GCRV at present clinically, wherein Genotype I and II type comparatively popular, Genotype I take GCRV873 as representative strains, and gene II type take HZ08 as representative strains.
Genotype I GCRV came into vogue from the eighties in last century, did not also have specific methods for the treatment of at present for this virus, and the healthy aquaculture carrying out grass carp is the most effective preventive measures, and this depends on vaccine immunity and early stage rapid detection.The detection method that it is reported at present has electron microscopy, enzyme linked immunosorbent assay (ELISA, Dot-ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) detection method, real time fluorescent quantitative (real-timePCR) detection 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, the applicable elements for concrete Viral diagnosis is still needed will exploration extensively and profoundly, not yet has the precedent successfully using above-mentioned technology at present for Genotype I GCRV.Once have studied GCRV RT-LAMP detection reagent box CN201210186709.6 in early days, but the design of primers that this technical scheme adopts cannot effective amplification gene I type GCRV, the experiment display that applicant carries out, adopt above-mentioned disclosed primer amplification Genotype I GCRV poor specificity, LFD ELISA test strip cannot be used for; Also once there was the RT-LAMP-LFD detection method of attempting research hiv virus this area, but because hiv virus and Genotype I GCRV exist huge sequence difference, its probe used cannot be combined on Genotype I GCRV amplified production completely.
Summary of the invention
For the disappearance of current this area detection technique, applicant discloses a kind of Genotype I GCRV detection method and based on the test kit manufactured by the method, the method disclosed in the present is based on conserved regions sequences Design two pairs of primers and the specific probe of Genotype I GCRV gene, thus set up GCRVRT-LAMP reaction system, and detect Genotype I 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 Genotype I GCRV in grass carp breeding process, avoid virus disseminating popular, improve scientific management efficiency.
Specifically, the present invention is achieved through the following technical solutions:
The detection method of Genotype I GCRV, comprise the steps: 1) extract the RNA of grass carp, join in premix reaction solution and utilize the conserved regions sequence of two pairs of primer pair Genotype I GCRVs to carry out ring mediated isothermal nucleic acid amplification, primer wherein used is respectively primer GCRVI-F3 sequence SEQ ID No:1, primer GCRVI-B3 sequence SEQ ID No:2; Primer GCRVI-FIP sequence SEQ ID No:3, primer GCRVI-BIP sequence SEQ ID No:4; And flag F ITC probe, probe GCRVI-FITC-Probe sequence SEQ ID No:5; Wherein GCRVI-BIP is 5 ' end vitamin H (biotin) labeled primer; GCRVI-FITC-Probe is 5 ' end FITC label probe; 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 adopted designs according to six different zones of the 6th gene sheet degree conserved regions of Genotype I GCRV, can combine with the specific probe of RNA, specificity and highly sensitive, 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 GCRVI-F3 and GCRVI-B3, 1-2 μM of GCRVI-FIP and GCRVI-BIP, 0.02-0.08 μM of GCRVI-FITC-Probe, 15-25mM pH8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-25mM ammonium sulfate, 5-15mM magnesium sulfate, 0.1-0.3%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-3mM dNTP, 1-3U reversed transcriptive enzyme AMV and 5-15U Bst archaeal dna polymerase large fragment, premixed liquid volume is 20-25 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 GCRVI-F3 and GCRVI-B3,1.6 μMs of GCRVI-FIP and GCRVI-BIP, 0.05 μM of GCRVI-FITC-Probe, 20mM Tris-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.
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 Genotype I 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 GCRVI-F3 and GCRVI-B3, 1-2 μM of GCRVI-FIP and GCRVI-BIP, 0.02-0.08 μM of GCRVI-FITC-Probe, 15-25mMpH8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-25mM ammonium sulfate, 5-15mM magnesium sulfate, 0.1-0.3%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-3mM dNTP, 1-3U reversed transcriptive enzyme AMV and 5-15U Bst archaeal dna polymerase large fragment, premixed liquid volume is 20-25 μ L.
Preferably, constant-temperature amplification premix reaction solution is made up of following component: 0.2 μM of GCRVI-F3 and GCRVI-B3,1.6 μMs of GCRVI-FIP and GCRVI-BIP, 0.05 μM of GCRVI-FITC-Probe, 20mM Tris-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, RNasefree water, nucleic acid absorption post, wherein sample dissociation liquid consists of 40-60mM pH7.4Tris-HCL, the guanidinium isothiocyanate of 3-6M, 8-15mM EDTA; Protein liquid removal consists of the Tris-HCL of 20-40mM pH7.4,0.3-0.5M guanidinium isothiocyanate and 15-30% 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 50mMpH7.4Tris-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 Genotype I GCRV RNA, and negative control sample is the RNase free water of free nucleic acid.
Detection kit disclosed in this invention, 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 DL2000DNA Marker (TaKaRa company); Wherein, 1 is amplification under 61 DEG C of constant temperatures; 2 is amplification under 63 DEG C of constant temperatures; 3 is amplification under 65 DEG C of constant temperatures.
Fig. 2 is Genotype I GCRV RT-LAMP specificity experiments result figure, M is DL2000DNA Marker (TaKaRa company); 1 is Genotype I GCRV; 2 is gene II type GCRV; 3 is carp simplexvirus II type (CyHV-2); 4 is SVCV (SVCV); 5 is Koi herpesvirus (KHV); 6 is negative control.
Fig. 3 is sensitivity comparison diagram and the LFD test paper color development comparison diagram that RT-LAMP-LFD method detects Genotype I GCRV, and M is DL2000DNA Marker (TaKaRa company); Left side swimming lane 1 ~ 6: template is respectively 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6the doubly geneome RNA of dilution; Right side 1 ~ 6: be the LFD detection paper figure of left side experimental result.
Fig. 4 is the sample result figure that in embodiment 1, RT-LAMP-LFD technology for detection grass carp infects Genotype I GCRV, and M is DL2000DNA Marker (TaKaRa company); Left side swimming lane 1: Genotype I 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.
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 Deqing County, Huzhou City of Zhejiang Province plant; 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, 12000rpm centrifugal (being generally 2min), 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 12000rpm centrifugal (being generally 1min), discards waste liquid;
(4) in adsorption column, add 600 μ L protein liquid removal RC, room temperature leaves standstill (being generally 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 RNasefree water in adsorption column, room temperature places (being generally 1min), 12000rpm centrifugal (being generally 1min).
Use primary elutriant to repeat this step once, increase RNA elution amount.
3, Genotype I 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 Genotype I GCRV the 6th gene fragment, and its nucleotide sequence is as follows:
GCRVI-F3：CCCGTACTGCTACGTGAGA
GCRVI-B3：GCTAGTCGCGGAATCATCC
GCRVI-FIP：CGACCTCCTCAGACGTTTGGTT-GCGAAGTCGTTGACGCTA
GCRVI-BIP：CGACGCGATCGTGTTAGTGTCG-TCTTGAGGCGACGGGAAT
GCRVI-FITC-Probe：CGTACCAGCTACCGTCATGG
4, Genotype I 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 Genotype I 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.
Above experimental result shows, RT-LAMP-LFD detection kit provided by the present invention and detection method can be the Site Detection that Genotype I GCRV provides quick, easy, sensitive, have a good application prospect.
Claims (9)
1. the detection method of Genotype I GCRV, it is characterized in that comprising the steps: 1) extract the RNA of grass carp, join in premix reaction solution and utilize the conserved regions sequence of two pairs of primer pair Genotype I GCRVs to carry out ring mediated isothermal nucleic acid amplification, primer wherein used is respectively primer GCRVI-F3 sequence SEQID No:1, primer GCRVI-B3 sequence SEQ ID No:2; Primer GCRVI-FIP sequence SEQ ID No:3, primer GCRVI-BIP sequence SEQ ID No:4; And flag F ITC probe, probe GCRVI-FITC-Probe sequence SEQ ID No:5; Wherein GCRVI-BIP is 5 ' end vitamin H (biotin) labeled primer; GCRVI-FITC-Probe is 5 ' end FITC label probe; 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 Genotype I 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 GCRVI-F3 and GCRVI-B3, 1-2 μM of GCRVI-FIP and GCRVI-BIP, 0.02-0.08 μM of GCRVI-FITC-Probe, 15-25mM pH8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-25mM ammonium sulfate, 5-15mM magnesium sulfate, 0.1-0.3%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-3mM dNTP, 1-3U reversed transcriptive enzyme AMV and 5-15U Bst archaeal dna polymerase large fragment, premixed liquid volume is 20-25 μ L.
3. the detection method of Genotype I 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 GCRVI-F3 and GCRVI-B3, 1.6 μMs of GCRVI-FIP and GCRVI-BIP, 0.05 μM of GCRVI-FITC-Probe, 20mM Tris-HCl, 10mM Repone K, 15mM ammonium sulfate, 8mM magnesium sulfate, 0.1%TritonX-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.
4. the detection method of Genotype I 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 Genotype I 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 GCRVI-F3 and GCRVI-B3, 1-2 μM of GCRVI-FIP and GCRVI-BIP, 0.02-0.08 μM of GCRVI-FITC-Probe, 15-25mM pH8.0 ~ 9.0Tris-HCl, 8-15mM Repone K, 10-25mM ammonium sulfate, 5-15mM magnesium sulfate, 0.1-0.3%Triton X-100, 0.4-0.8M trimethyl-glycine, 1-3mM dNTP, 1-3U reversed transcriptive enzyme AMV and 5-15U Bst archaeal dna polymerase large fragment, premixed liquid volume is 20-25 μ L.
6. detection kit according to claim 5, is characterized in that constant-temperature amplification premix reaction solution is made up of following component: 0.2 μM of GCRVI-F3 and GCRVI-B3,1.6 μMs of GCRVI-FIP and GCRVI-BIP, 0.05 μM of GCRVI-FITC-Probe, 20mM Tris-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 8UBst 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 3-6M, 8-15mM EDTA; Protein liquid removal consists of the Tris-HCL of 20-40mMpH7.4,0.3-0.5M guanidinium isothiocyanate and 15-30% 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 130mMNaCl 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106811550A (en) * | 2017-03-11 | 2017-06-09 | 中国水产科学研究院珠江水产研究所 | A kind of type vaccine strain of GCRV II and street strain's diagnostic primerses and kit and diagnosis detecting method containing it |
| 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 |
| 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) |
| 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 |
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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) |
| 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 |
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