CN103484568A - Method and kit for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II - Google Patents
Method and kit for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II Download PDFInfo
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Abstract
The invention discloses a method for dual fluorescence quantitative PCR detection of grass carp reovirus types I and II. The method can realize that the grass carp reovirus types I and II can be simultaneously detected in one to-be-detected sample, and the load levels of the two reovirus types can be quantified. The invention further provides a kit for dual fluorescence quantitative PCR detection of the grass carp reovirus types I and II. According to the detection method and the kit which are provided by the invention, the operation is simple and convenient, the specificity is strong, the detection sensitivity and accuracy rate are high and reliable, and a wide application prospect is achieved.
Description
Technical field
The present invention relates to a kind of double fluorescent quantitative PCR detection kit, be specifically related to the double fluorescent quantitative PCR detection kit of a kind of GCRV I type and II type, further related to the detection method of the double fluorescent quantitative PCR of a kind of GCRV I type and II type.
Background technology
GCRV (Grass carp reovirus, GCRV) is subordinate to Aquareovirus, is Reoviridae one newcomer, is the first strain fishes virus that separate China's Mainland.This virus mainly causes that the grass carp kind in Asian countries's freshwater aquicultures such as China, Vietnam, Burma, in the fingerling stage, hemorrhagic disease occurs, and mortality ratio can be up to more than 60%.This viral prevalence is wide, harm is large, mortality ratio is high, morbidity season is long, the serious threat fish production.GCRV tool double capsid, the virus particle mean diameter is 60 nm～70nm, the icosahedron symmetry, without cyst membrane, genome is comprised of 11 segmented double-stranded RNAs.Oneself is through having reported more than 20 strain isolated at present, comprise GCRV854, GCRV861, GCRV873, GCRV875, GCRV876, GCRV991, GCRV H962, ZV-8802, GCRV-854, GCRV HZ08, GCRV JX09-01, GCRV JX09-02, GCRV GD10, GCRV GCRV104 strain etc., different isolates is at genome sequence, genome banding pattern, cytopathy, differ greatly to the aspects such as virulence of grass carp.Up to the present, the strain that has completed complete genome sequencing has GCRV 873 strains, GCRV HZ08, GCRV GD10, GCRV 104 etc., the examining order of also had many strains only to complete part sections or partial sequence.The GCRV more complicated, each gene segment of different isolates exists reprovision and antigenic drift phenomenon, makes at present and also on serology or genotype, it is not carried out to Subtypes.But by existing strain isolated sequence information, at least three classes should be arranged, all kinds of representative strains respectively: the first kind is 873 strains and JX09-01 strain, and Equations of The Second Kind is HZ08 strain and GD10 strain, and the 3rd type is 104 strains.Have and mention the discussion of it being carried out to gene type in relevant research report, respectively one, two and three classes are divided into to I, II and III type (Zeng Weiwei according to gene order difference, Wang Qing, Liu Yongkui etc. the separation of a strain GCRV low virulent strain, evaluation and immunogenicity initial analysis. hydrobiont journal, 2011.35 (5): 790-795; Zeng Weiwei, Wang Qing, Wang Yingying, Zhang Lesheng, Liu Baoqin, Shi Cunbin, Wu Shuqin. foundation and the Preliminary Applications thereof of the triple RT-PCR detection methods of GCRV. Chinese aquatic science .2013,20 (2): 329-335).The strain of same hypotype, its corresponding each sections DNA homolog is all more than 95%.
Current, in the epidemic strain that all parts of the country are separated to, three class hypotypes all have report, and the independent infection had, also have polyinfection.According to hemorrhagic disease of grass carp monitoring and epidemiological survey and analysis in recent years, show, cause at present the main for GCRV II type of the popular and outburst of hemorrhagic disease of grass carp, next is GCRV I type, and GCRV III type does not almost detect and is separated to therefore, epidemic characteristic and GCRV molecular biological characteristic according to hemorrhagic disease of grass carp, design respectively primer according to total conserved sequence between the different strains of same hypotype, foundation all can be diagnosed for the genotypic GCRV of current Major Epidemic simultaneously, and the Multiplex real-time PCR that can identify its hypotype is quick, the specific detection technology.
The detection method of GCRV has multiple, each tool advantage and defect, virus separation, electron microscopic observation, nucleic acid belt type analysis are still the method that GCRV generally adopts that detects so far, but these methods waste time and energy, operate more complicated, can not carry out quick diagnosis to virus, be unfavorable for the epidemiology survey work of hemorrhagic disease of grass carp.The totivirus diagnosis relates to virus culture, purifying reaches the processes such as concentrated, and not only complex manufacturing process, consuming time, and cost is higher, but also potential loose poison is dangerous, therefore is unfavorable for promoting the use of.Polymerase chain reaction (PCR) can be enlarged millions of times to certain fragment gene within a few hours.This technology high specificity, susceptibility is high, detection is quick, has been widely used in each field of the subjects such as medical science, microbiology, veterinary science.The RT-PCR detection method of the different strains of GCRV is extensively set up, the method has the advantages such as rapidity, accuracy and high sensitivity, can infect and make early diagnosis GCRV, giving the real-time analysis of epidemic situation and controlling provides strong technical support.Real-time quantitative RT-PCR detection platform wherein is as simple and direct and reliable GCRV detection method, not only can qualitative detection virus have or not, and number that can the quantitative analysis viral level, for the diagnosis of hemorrhagic disease of grass carp and the fundamental research of GCRV, all have great importance.Multiple fluorescence quantitative PCR is a kind of special quantitative fluorescent PCR form, and its outstanding feature is that a PCR can detect multiple cause of disease simultaneously, is particularly useful for the more cause of disease of cause of disease complexity or genotype and detects.
Therefore, foundation can be simultaneously carried out the technology of rapid detection to GCRV I type and II type, not only can accomplish the GCRV of different subtype is carried out to differential diagnosis and quantitative analysis, and can simplify procedures, cost-saving, the epidemiology survey of hemorrhagic disease of grass carp, cause of disease monitoring and early stage early warning are all had great importance.
Summary of the invention
The object of the present invention is to provide the double fluorescent quantitative PCR detection kit of a kind of GCRV I type and II type.
Another object of the present invention is to provide the double fluorescent quantitative PCR detection method of a kind of GCRV I type and II type, the method can realize detecting the existence of GCRV I type and II type in same sample to be tested simultaneously, and can carry out quantitatively the load level of two-strain.
The technical solution used in the present invention is:
The double fluorescent quantitative PCR detection kit of a kind of GCRV I type and II type, comprise following composition: double fluorescent quantitative reaction solution, enzyme mixation, RT-PCR reaction solution, positive quality control product and without RNA enzyme deionized water, contain GCRV I type and II type specificity primer and dual probe in described double fluorescent quantitative reaction solution, sequence is respectively:
GCRV I type:
Upstream primer: GCRV-I-F:5 '-CTCTCTGGCAGAAACACTTAGAC-3 ' (SEQ ID NO.1);
Downstream primer: GCRV-I-R:5 '-CGTTGTAGAACTCATTTGGGTAGG-3 ' (SEQ ID NO.2);
Fluorescent probe: GCRV-I-P:5 '-FAM-CCGCCATGACCATGCTAACACCTGACA-BHQ1-3 ' (SEQ ID NO.3)
GCRV II type:
Upstream primer: GCRV-II-F:5 '-TGATGAGTTCAGTGCCTACG-3 ' (SEQ ID NO.4);
Downstream primer: GCRV-II-R:5 '-GAGCCATGAGGTGTGTCTAC-3 ' (SEQ ID NO.5);
Fluorescent probe: GCRV-II-P:5 '-CY5-ATCATAGCAGCCGCCGTCCGTAT-BHQ1-3 ' (SEQ ID NO.6)
The fluorescent probe reporter group of GCRV I type is FAM, and the fluorescent probe reporter group of GCRV II type is CY5, and quenching group is BHQ1.
Further, described double fluorescent quantitative reaction solution is composed as follows:
GCRV-Ⅰ-F（100μM） 5μL
GCRV-Ⅰ-R（100μM） 5μL
GCRV-Ⅰ-P（100μM） 4μL
GCRV-Ⅱ-F（100μM） 6μL
GCRV-Ⅱ-R（100μM） 6μL
GCRV-Ⅱ-P（100μM） 5μL
Without RNA enzyme deionized water 169 μ L
Total 200μL。
Further, described enzyme mixation comprises reversed transcriptive enzyme and Taq enzyme.
Further, the GCRV I type GCRV09-01 strain that described positive quality control product is deactivation and the nutrient solution of II type GCRV-HZ08 strain.
The double fluorescent quantitative PCR detection method of a kind of GCRV I type and II type, comprise the steps:
1) extract the viral RNA in testing sample;
2) to take the RNA extracted be template, uses GCRV I type
Upstream primer: GCRV-I-F:5 '-CTCTCTGGCAGAAACACTTAGAC-3 ' (SEQ ID NO.1);
Downstream primer: GCRV-I-R:5 '-CGTTGTAGAACTCATTTGGGTAGG-3 ' (SEQ ID NO.2);
Fluorescent probe: GCRV-I-P:5 '-FAM-CCGCCATGACCATGCTAACACCTGACA-BHQ1-3 ' (SEQ ID NO.3) and GCRV II type
Upstream primer: GCRV-II-F:5 '-TGATGAGTTCAGTGCCTACG-3 ' (SEQ ID NO.4);
Downstream primer: GCRV-II-R:5 '-GAGCCATGAGGTGTGTCTAC-3 ' (SEQ ID NO.5);
Fluorescent probe: GCRV-II-P:5 '-CY5-ATCATAGCAGCCGCCGTCCGTAT-BHQ1-3 ' (SEQ ID NO.6)
Carry out the double fluorescent quantitative PCR detection, wherein, the fluorescent probe reporter group of GCRV I type is FAM, and the fluorescent probe reporter group of GCRV II type is CY5, and quenching group is BHQ1;
3) interpretation of result: reaction judges according to the fluorescence Ct value of testing sample whether testing sample is GCRV I type, the GCRV II type positive after finishing.
Further, the reaction system of described double fluorescent quantitative PCR comprise viral RNA template, double fluorescent quantitative reaction solution, enzyme mixation, RT-PCR reaction solution, without RNA enzyme deionized water.
Further, the reaction conditions of described double fluorescent quantitative PCR is 42 ℃ of 30min, and then 94～96 ℃ of 3～8min gather 40 circulations of fluorescent signal through 94～96 ℃ of 7～10s and 58～62 ℃ of 32 ~ 38s.
The invention has the beneficial effects as follows:
Test kit provided by the invention can carry out rapid detection to GCRV I type and II type two-strain simultaneously, and can carry out the nucleic acid of pathogenic agent in sample accurately detecting in real time and quantitative analysis, accuracy rate is high, easy and simple to handle, cost is low, is with a wide range of applications.
Test kit provided by the invention has been introduced specificity amplification primer and fluorescent probe, make sensitivity and the specificity of detection be improved significantly, thereby avoided the not high problem of easily failing to pinpoint a disease in diagnosis with mistaken diagnosis of other detection method specificitys, based on above-mentioned advantage, this GCRV I type and II detection kit all have great importance to epidemiology survey, cause of disease monitoring and the early stage early warning of hemorrhagic disease of grass carp.
The accompanying drawing explanation
The double fluorescent quantitative PCR detection kit that Fig. 1 is GCRV I type and II type detects the figure as a result of hemorrhagic disease of grass carp clinical sample;
Fig. 2 detects the sensitivity experiment figure of I type GCRV in double fluorescent PCR system, from left to right be followed successively by 1 * 10
8, 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3, 1 * 10
2, 1 * 10 copy/μ L the amplification of standard substance;
Fig. 3 detects the sensitivity experiment figure of II type GCRV in double fluorescent PCR system, from left to right be followed successively by 1 * 10
8, 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3,, the amplification of the standard substance of 1 * 10 copy/μ L;
The specificity lab diagram that Fig. 4 is GCRV I C-type virus C in double fluorescent PCR detection method;
The specificity lab diagram that Fig. 5 is GCRV II C-type virus C in double fluorescent PCR detection method;
The specificity lab diagram that Fig. 6 is GCRV I type and II C-type virus C in double fluorescent PCR detection method.
Embodiment
Below in conjunction with tool the present embodiment, the invention will be further described, but be not limited to this.
embodiment 1:
1, the design of Auele Specific Primer and probe
According to the nucleotide sequence of the GCRV I type retrieved from Genbank and II type, be designed for the primer and the probe that detect GCRV I type and II type, its sequence is as follows:
Fish reovirus I type:
Upstream primer: GCRV-I-F:5 '-CTCTCTGGCAGAAACACTTAGAC-3 ' (SEQ ID NO.1);
Downstream primer: GCRV-I-R:5 '-CGTTGTAGAACTCATTTGGGTAGG-3 ' (SEQ ID NO.2);
Fluorescent probe: GCRV-I-P:5 '-FAM-CCGCCATGACCATGCTAACACCTGACA-BHQ1-3 ' (SEQ ID NO.3), amplified fragments 159bp;
GCRV II type:
Upstream primer: GCRV-II-F:5 '-TGATGAGTTCAGTGCCTACG-3 ' (SEQ ID NO.4);
Downstream primer: GCRV-II-R:5 '-GAGCCATGAGGTGTGTCTAC-3 ' (SEQ ID NO.5);
Fluorescent probe: GCRV-II-P:5 '-CY5-ATCATAGCAGCCGCCGTCCGTAT-BHQ1-3 ' (SEQ ID NO.6) amplified fragments 198bp.
Fluorescent probe reporter group for detection of GCRV I type is FAM, for detection of the fluorescent probe reporter group of GCRV II type, is CY5, and quenching group is BHQ1.
2, the preparation of positive quality control product
The nutrient solution of the GCRV09-01 strain of GCRV I type and II type GCRV-HZ08 strain is carried out to boiling water bath 30min, carry out inactivation treatment, the nutrient solution of the GCRV I type GCRV09-01 strain after deactivation and II type GCRV-HZ08 strain is positive quality control product.
3, the extraction of viral RNA
Press the Trizol test kit (purchased from Invitrogen company, article No.: specification sheets 15596-026) or extract test kit (purchased from Qiagen company according to Qiagen RNA, article No.: specification sheets step 74104), extract RNA from the fish body viscera tissue that infects corresponding virus, as the template of RT-PCR reaction.
4, double fluorescent quantitative PCR amplification
Each test reaction system is formulated as follows, the RNA template 5 μ L of RT-PCR reaction solution 12.5 μ L, detected sample, double fluorescent quantitative reaction solution 4 μ L, enzyme mixation 1 μ L, without RNA enzyme deionized water 2.5 μ L, according to above-mentioned system, the positive and negative control are set equally, add positive quality control product or increased without the deionized water 5 μ L of RNA.Wherein the RT-PCR reaction solution comprises 500mmol/L KCl, 100mmol/L Tris-HCl, 1.5mmol/L MgCl
2, 10mmol/L dNTPs and 1% (v/v) TritonX-100.
Each reaction tubes is put into to the reactive tank of quantitative PCR instrument, each title detected and fluorophor kind are set, and (reporter group that detects the GCRV-I is selected FAM, detect the reporter group of GCRV-II and select CY5, quenching group is selected BHQ1), setting reaction conditions is 42 ℃ of 30min, 95 ℃ of 5min, then gather 40 circulations of fluorescent signal through 95 ℃ of 8s and 60 ℃ of 35s.
5, interpretation of result and judgement
After reaction finishes, in the FAM passage, the S-type curve of fluorescence curve and Ct value≤34.0, be judged to be the GCRV I type positive; If without typical S type curve or Ct value>34.0, be judged to be GCRV I type feminine gender.In CY 5 passages, the S-type curve of fluorescence curve and Ct value≤34.0, be judged to be the GCRV II type positive; If without typical S type curve or Ct value>34.0, be judged to be GCRV II type feminine gender.The two is all positive, is judged as type GCRV I type and II common anode type.
According to above-mentioned detection method, to get 15 parts of clinical samples and detected, concrete detected result is shown in Fig. 1, as can be seen from Figure 1, in 15 duplicate samples, have 2 parts of I type signal detected, have 5 parts of II type signal detected.In addition, the clinical sample that all participations detect detects by cellular segregation and conventional RT-PCR method respectively simultaneously, and its detected result is consistent with the detected result of double fluorescent quantitative PCR of the present invention.
6, sensitivity experiment
Extract respectively I type and II type GCRV RNA(1.0 * 10
8copy/μ L), dilution is 1 * 10 successively
8, 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3, 1.0 * 10
2, 1 * 10 copy/μ L, carry out sensitivity experiment.
The sensitivity experiment that in the double fluorescent quantitative PCR system, I type GCRV detects is shown in Fig. 2, from left to right is followed successively by 1 * 10
8, 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3, 1.0 * 10
2, 1 * 10 copy/μ L the amplification of standard substance.The sensitivity experiment that in the double fluorescent quantitative PCR system, II type GCRV detects is shown in Fig. 3, from left to right is followed successively by 1 * 10
8, 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3, 1.0 * 10
2, 1 * 10 copy/μ L the amplification of standard substance.
Detected result shows, the sensitivity that test kit of the present invention detects can reach 1.0 * 10 copy/μ L, and the Ct value reduces in gradient and changes with concentration, accuracy is better than the regular-PCR method, shows that test kit of the present invention and detection method have the susceptibility of height to the diagnosis of GCRV I type/II type.
7, specificity experiment
In order to detect the specificity of test kit of the present invention, detect KHV, IHNV, LMBV, ISKNV, GCRV HZ08 strain, GCRV JX09-01 strain and negative control group (without the RNA deionized water) with GCRV I type of the present invention/II type dual fluorescence quantification RT-PCR detection kit, analyze the detection case of this test kit to other common virus of fish and GCRV.
Detected result shows:
The FAM passage is only increased (as shown in Figure 4) to I type GCRV, and as can be seen from Figure 4, GCRV JX09-01 strain is positive, and KHV, IHNV, LMBV, ISKNV and negative control group are all negative.
The CY5 passage is only increased (as shown in Figure 5) to II type GCRV, and as can be seen from Figure 5, GCRV HZ08 strain is positive, and KHV, IHNV, LMBV, ISKNV and negative control group are all negative.
While having GCRV I type and II type two viroid in sample, FAM passage and CY5 passage all can be increased (as shown in Figure 6).As can be seen from Figure 6, GCRV JX09-01 strain and GCRV HZ08 strain are positive, and KHV, IHNV, LMBV, ISKNV and negative control group are all negative.
The above results explanation, detection kit energy specific amplification of the present invention goes out corresponding genotypic one or both GCRVs, and not with other viral nucleic acid generation cross reaction.Illustrate that the inventive method and test kit specificity are good, false positive do not occur.
Test kit of the present invention confirms to have good repeatability and stability through repeatedly different repeated experiments.
<110 > China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
<120 > double fluorescent quantitative PCR detection method and the test kit thereof of a kind of GCRV I type and II type
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213 > artificial primer
<400> 1
ctctctggca gaaacactta gac 23
<210> 2
<211> 24
<212> DNA
<213 > artificial primer
<400> 2
cgttgtagaa ctcatttggg tagg 24
<210> 3
<211> 27
<212> DNA
<213 > artificial primer
<400> 3
ccgccatgac catgctaaca cctgaca 27
<210> 4
<211> 20
<212> DNA
<213 > artificial primer
<400> 4
tgatgagttc agtgcctacg 20
<210> 5
<211> 20
<212> DNA
<213 > artificial primer
<400> 5
<210> 6
<211> 23
<212> DNA
<213 > artificial primer
<400> 6
atcatagcag ccgccgtccg tat 23
Claims (7)
1. the double fluorescent quantitative PCR detection kit of a GCRV I type and II type comprises following composition: double fluorescent quantitative reaction solution, enzyme mixation, RT-PCR reaction solution, positive quality control product and without RNA enzyme deionized water, it is characterized in that: contain GCRV I type and II type specificity primer and dual probe in described double fluorescent quantitative reaction solution, sequence is respectively:
GCRV I type:
Upstream primer: GCRV-I-F:5 '-CTCTCTGGCAGAAACACTTAGAC-3 ';
Downstream primer: GCRV-I-R:5 '-CGTTGTAGAACTCATTTGGGTAGG-3 ';
Fluorescent probe:
GCRV-Ⅰ-P：5’-FAM-CCGCCATGACCATGCTAACACCTGACA-BHQ1-3’；
GCRV II type:
Upstream primer: GCRV-II-F:5 '-TGATGAGTTCAGTGCCTACG-3 ';
Downstream primer: GCRV-II-R:5 '-GAGCCATGAGGTGTGTCTAC-3 ';
Fluorescent probe:
GCRV-Ⅱ-P：5’-CY5-ATCATAGCAGCCGCCGTCCGTAT-BHQ1-3’；
The fluorescent probe reporter group of GCRV I type is FAM, and the fluorescent probe reporter group of GCRV II type is CY5, and quenching group is BHQ1.
2. the double fluorescent quantitative PCR detection kit of a kind of GCRV I type according to claim 1 and II type is characterized in that: described double fluorescent quantitative reaction solution composed as follows:
GCRV-Ⅰ-F（100μM） 5μL
GCRV-Ⅰ-R（100μM） 5μL
GCRV-Ⅰ-P（100μM） 4μL
GCRV-Ⅱ-F（100μM） 6μL
GCRV-Ⅱ-R（100μM） 6μL
GCRV-Ⅱ-P（100μM） 5μL
Without RNA enzyme deionized water 169 μ L
Total 200μL。
3. the double fluorescent quantitative PCR detection kit of a kind of GCRV I type according to claim 1 and II type, it is characterized in that: described enzyme mixation comprises reversed transcriptive enzyme and Taq enzyme.
4. the double fluorescent quantitative PCR detection kit of a kind of GCRV I type according to claim 1 and II type, is characterized in that: the nutrient solution of the GCRV I type GCRV09-01 strain that described positive quality control product is deactivation and II type GCRV-HZ08 strain.
5. the double fluorescent quantitative PCR detection method of a GCRV I type and II type is characterized in that: comprise the following steps:
1) extract the viral RNA in testing sample;
2) to take the RNA extracted be template, uses GCRV I type
Upstream primer: GCRV-I-F:5 '-CTCTCTGGCAGAAACACTTAGAC-3 ';
Downstream primer: GCRV-I-R:5 '-CGTTGTAGAACTCATTTGGGTAGG-3 ';
Fluorescent probe: GCRV-I-P:5 '-FAM-CCGCCATGACCATGCTAACACCTGACA-BHQ1-3 ' and GCRV II type
Upstream primer: GCRV-II-F:5 '-TGATGAGTTCAGTGCCTACG-3 ';
Downstream primer: GCRV-II-R:5 '-GAGCCATGAGGTGTGTCTAC-3 ';
Fluorescent probe: GCRV-II-P:5 '-CY5-ATCATAGCAGCCGCCGTCCGTAT-BHQ1-3 '
Carry out the double fluorescent quantitative PCR detection, wherein, the fluorescent probe reporter group of GCRV I type is FAM, and the fluorescent probe reporter group of GCRV II type is CY5, and quenching group is BHQ1;
3) interpretation of result: reaction judges according to the fluorescence Ct value of testing sample whether testing sample is GCRV I type, the GCRV II type positive after finishing.
6. the double fluorescent quantitative PCR detection method of a kind of GCRV I type according to claim 5 and II type is characterized in that: the reaction system of described double fluorescent quantitative PCR comprises viral RNA template, double fluorescent quantitative reaction solution, enzyme mixation, RT-PCR reaction solution and without RNA enzyme deionized water.
7. the double fluorescent quantitative PCR detection method of a kind of GCRV I type according to claim 5 and II type, it is characterized in that: the reaction conditions of described double fluorescent quantitative PCR is 42 ℃ of 30min, 94～96 ℃ of 3～8min, then gather 40 circulations of fluorescent signal through 94～96 ℃ of 7～10s and 58～62 ℃ of 32 ~ 38s.
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| CN110257385A (en) * | 2019-07-04 | 2019-09-20 | 广西科学院 | Anti- grass carp exhales aptamer and its construction method and the application of the lonely I type virus of intestines |
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| CN110894551A (en) * | 2018-09-13 | 2020-03-20 | 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 I virus (GCRV-I) |
| 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 |
| CN110257385A (en) * | 2019-07-04 | 2019-09-20 | 广西科学院 | Anti- grass carp exhales aptamer and its construction method and the application of the lonely I type virus of intestines |
| CN110257385B (en) * | 2019-07-04 | 2022-06-17 | 广西科学院 | Aptamer for resisting grass carp reovirus I, and construction method and application thereof |
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