CN110592269A - RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) - Google Patents

Abstract

The invention discloses a grass carp hemorrhage disease type 2 virus (GCRV-2) RAA constant-temperature fluorescence detection method and a detection kit. The detection kit comprises a forward primer SEQ ID NO.1, a reverse primer SEQ ID NO.2, a specific fluorescent probe SEQ ID NO.3, reaction liquid, reverse transcriptase, recombinant polymerase and a reference substance. The kit has strong specificity; the detection sensitivity is high and can reach 1 fg/. mu.L; the accuracy is high and reliable; the method is simple, convenient and quick to operate, is suitable for field detection, and has wide application scenes.

Description

RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2)

Technical Field

The invention belongs to the technical field of molecular biology, relates to a detection method of marine aquaculture industry, and particularly relates to an RAA constant-temperature fluorescence detection method and a kit for grass carp hemorrhagic disease type 2 virus.

Background

Grass carp hemorrhage type 2 virus (GCRV) belongs to the aquatic reovirus genus, is a new member of reoviridae, and is the first fish virus isolated from mainland China. The virus mainly causes the grass carp species in fresh water culture in Asian countries such as China, Vietnam, Myanmar and the like to generate leukemia at the fingerling stage, and the death rate can reach more than 60 percent. The virus has wide prevalence, great harm, high mortality, long morbidity season and serious threat to fishery production. The grass carp reovirus has a double-layer capsid, the average diameter of virus particles is 60 nm-70 nm, the icosahedron is symmetrical, and has no envelope, and the genome consists of 11 segmented double-stranded RNAs. More than 20 isolates have been reported at present, including GCRV854, GCRV861, GCRV873, GCRV875, GCRV876, GCRV991, GCRV H962, ZV-8802, GCRV-854, GCRVHZ 08, GCRV JX09-01, GCRV JX09-02, GCRV GD10, GCRV GCRV104 and the like, and different isolates have large differences in genome sequence, genome banding, cytopathic effect, pathogenicity to grass carp and the like. The strains which have been subjected to whole genome sequence analysis so far include strain GCRV873, strain GCRV HZ08, GCRV GD10, GCRV104 and the like, and many strains only complete sequencing work of partial segments or partial sequences. The grass carp reovirus is relatively complex, and the reassortment and antigen drift phenomena exist in each gene segment of different isolates, so that the subtype classification of the grass carp reovirus on serology or genotype is not carried out at present. However, there should be at least three types of isolates from the existing isolate sequence information, and the representative strains of each type are: the first type is 873 strains and JX09-01 strains, the second type is HZ08 strains and GD10 strains, and the third type is 104 strains. In the related research reports, the research on genotyping is mentioned, namely, the first, second and third types are classified into I, II and III according to the difference of gene sequences.

Currently, three subtypes of epidemic strains separated from all over the country are reported, and some subtypes are infected singly and also are infected mixedly. According to the grass carp hemorrhage monitoring and epidemiological investigation and analysis in recent years, the grass carp hemorrhage epidemics and outbreaks are mainly GCRV type II and are secondarily GCRV type I at present.

The method for detecting the grass carp bleeding type 2 virus has various advantages and defects, virus separation, electron microscope observation and nucleic acid banding pattern analysis are still generally adopted methods for detecting the grass carp bleeding type 2 virus, but the methods are time-consuming, labor-consuming, complex to operate, incapable of rapidly diagnosing the virus and not beneficial to epidemiological investigation work of grass carp bleeding disease, but the existing methods have certain defects, such as: the histopathology-based method is not only tedious and time-consuming to operate, but also low in sensitivity; although the method based on immunology has the characteristics of high sensitivity, quick detection and the like, and can be used for detecting a large number of samples, the false positive problem generated when a fresh sample is detected also limits the wide application of the method to a certain extent; the PCR method is sensitive, accurate and rapid, can replace the etiology detection, but is not suitable for the field rapid detection and the basic popularization and application due to the need of expensive instruments and equipment, higher detection cost and higher technical requirements on detection personnel. Compared with the common PCR technology, the fluorescent PCR detection technology simplifies the operation steps, can eliminate the cross contamination caused by the amplification product and reduce the occurrence of false positive. However, the real-time fluorescent PCR is long in time consumption and high in cost, and is not applied to conventional pathogen detection of aquaculture animals at present. The LAMP isothermal amplification technology is also relatively limited in application in aquatic pathogen detection due to higher false positive and low accuracy. The invention establishes a method for detecting grass carp hemorrhagic disease type 2 virus by RAA constant temperature fluorescence, is quick, convenient, accurate and reliable, is suitable for the time requirements of port quick detection and general customs, and has important effect on promoting the marine culture of China and the trade of products thereof.

The Recombinase-aid Amplification (RAA) technique is also a method by which nucleic acids can be rapidly amplified at a constant temperature. Unlike RPA, RAA amplification uses a recombinase obtained from bacteria or fungi, which binds tightly to the primer DNA at a constant temperature of 37 ℃ to form an aggregate of the enzyme and the primer, when the primer searches for a sequence on the template DNA that is completely complementary to the primer, the template DNA is melted with the help of single-strand DNA binding protein (SSB), and a new complementary strand of DNA is formed under the action of DNA polymerase, and the reaction product is exponentially increased, and usually an amplified fragment that can be detected by agarose gel electrophoresis can be obtained within 1 hour. The fluorescent group is added into the RAA reaction system, the whole RAA amplification process is monitored in real time by utilizing the accumulation of fluorescent signals, and the quantitative and qualitative analysis of the initial template can be realized within 20 minutes. The whole reaction is simple and quick, and high-temperature circulation is not needed, so the method is particularly suitable for being used in non-laboratory detection places with a large number of samples, and is suitable for the field of quick detection of foods.

Disclosure of Invention

In view of the above, the present invention provides a RAA constant temperature fluorescent nucleic acid detection kit and a detection method for grass carp hemorrhagic disease type 2 virus (GCRV-2).

In order to achieve the purpose, the invention adopts the following technical scheme:

a detection kit for grass carp hemorrhagic disease type 2 virus (GCRV-2) nucleic acid comprises: the kit comprises a grass carp bleeding disease type 2 virus forward primer, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the grass carp bleeding disease type 2 virus forward primer is shown as SEQ ID No.1, the nucleotide sequence of the grass carp bleeding disease type 2 virus reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, a fluorescent reporter group is marked at the 5 'end of the specific fluorescent probe, and a fluorescent quenching group is marked at the 3' end of the specific fluorescent probe.

In some embodiments, the fluorescent reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC Red460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.

In some embodiments, the nucleic acid detection kit further comprises at least one of a primer mixture, a specific fluorescent probe, an a Buffer, a B Buffer, a RAA dry powder reagent, a grass carp hemorrhagic disease type 2 virus standard, and DEPC treated water.

In some embodiments, the kit, the reverse transcription system consists of a RTE reverse transcriptase, an rnase inhibitor.

In some embodiments, the kit of (a), wherein the a Buffer is 20% PEG; b Buffer is 280mM MgAc.

In some embodiments, the kit, wherein the composition of the RAA dry powder reagent is as follows: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ LRad51, 30ng/μ L Bsu DNA polymerase, 30ng/mL RTE reverse transcriptase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.

In some embodiments, the nucleic acid detection kit and the grass carp hemorrhagic disease type 2 virus standard are positive plasmids containing partial sequences of conserved genes of grass carp hemorrhagic disease type 2 viruses.

In some embodiments, the kit comprises a positive plasmid containing a partial sequence of a grass carp hemorrhagic disease type 2 virus conserved region gene as shown in SEQ ID No. 4.

The invention also provides an RAA constant temperature fluorescence detection method of the grass carp bleeding disease type 2 virus, which comprises the steps of extracting RNA of a sample to be detected, carrying out real-time fluorescence RAA reaction in the presence of a forward primer, a reverse primer, a specific fluorescence probe, an RAA dry powder reagent, A Buffer, B Buffer and DEPC (diethyl phthalate) treated water of the grass carp bleeding disease type 2 virus by taking the RNA of the sample to be detected as a template, and analyzing the sample to be detected according to a real-time fluorescence RAA amplification curve; the nucleotide sequence of the grass carp hemorrhage disease type 2 virus forward primer is shown as SEQ ID No.1, the nucleotide sequence of the grass carp hemorrhage disease type 2 virus reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.

In some embodiments, the RNA extraction of the grass carp hemorrhagic disease type 2 virus nucleic acid is performed by using a traditional Trizol-RNA reagent or an equivalent RNA extraction kit.

In some embodiments, the performing a fluorescent RAA reaction procedure is: at 39 ℃ for 40 s; at 39 ℃ for 20min, and 40 cycles in total;

according to the detection method, after the real-time fluorescence RAA reaction is required to be finished, the to-be-detected sample is analyzed according to the amplification curve of the real-time fluorescence RAA by using the analysis software of the real-time fluorescence RAA instrument. Preferably, the FAM channel fluorescence curve of the sample to be tested is S-shaped, the CT value is less than or equal to 35, and the sample to be tested is judged to be a positive result of the grass carp hemorrhagic disease type 2 virus; and when the curve of the sample to be detected is not S-shaped or the CT value is more than 35, judging that the grass carp has a virus negative result of type 2 hemorrhagic disease.

Advantageous effects

1. Fast and efficient: the whole amplification can be completed within 20-30min, and the amplification yield can reach 10⁹-10¹⁰A copy;

2. the operation is simple: no special reagent is needed, complicated steps such as deformation of double-stranded DNA and the like are not needed in advance, only a constant-temperature fluorometer is needed, and the conditions are mild;

3. high specificity: the invention is used for other diseases of fish, such as Viral Nervous Necrosis Virus (VNNV), Viral Hemorrhagic Septicemia Virus (VHSV), Infectious Hematopoietic Necrosis Virus (IHNV), carp spring viremia (SVCV), bastard Halibut Rhabdovirus (HRV), Infectious Salmon Anemia Virus (ISAV), and grass carp hemorrhagic disease type I virus (GCRV-I).

4. High sensitivity: the detection limit of the invention can reach 1 fg/reaction

5. The identification is simple: and the amplification result is directly judged according to the real-time fluorescence data, electrophoresis detection is not needed, and the method is suitable for field detection.

Drawings

FIG. 1 is a graph showing the amplification of 4 pairs of primers RAA involved in the present invention.

FIG. 2 is a graph showing the sensitivity test of the RAA detection method to GCRV-II, and the amplification results of positive standards of 100fg/μ L, 10fg/μ L, 1fg/μ L, 0.1fg/μ L and 0.01fg/μ L are shown from left to right.

FIG. 3 is a diagram showing the specificity experiment of RAA detection method for GCRV-II.

Detailed description of the invention

The present invention is further illustrated by the following specific examples, but is not limited thereto.

Example 1:

the invention searches gene sequences of grass carp hemorrhagic disease type 2 virus strains in a Genebank database for grass carp hemorrhagic disease type 2 virus, and compares multiple sequences by using DNAMAN 6.0 software to find out conserved segments. 4 sets of primers and probes were designed in the conserved regions and BLAST alignments were performed in the NCBI database, with the sequences of the primers and probes as shown in Table 1. The positive sample amplification curve is shown in FIG. 1.

Table 1 primer and probe sequences:

as can be seen from the results in FIG. 1, the amplification curves for the fourth set of primers and probes are most typical, with distinct exponential and plateau phases, with higher fluorescence intensity (ordinate values), and smaller CT values (abscissa corresponding to the intersection of the curve with the threshold line) and the results are analyzed in Table 2. The rise height of other primer probe curves is lower, the CT value is larger, and the plateau period is not obvious; or no amplification occurs and missed detection occurs. The fourth group of primers and the target products of the probes have higher replication speed, more quantity and higher amplification reaction efficiency.

TABLE 2 analysis of primer Probe screening results

Group \ result	CT value	Intensity of fluorescence
			First group	7.79	50,000
Second group	10.68	30,000
			Third group	5.73	73,000
Fourth group	2.46	62,000

Real-time example 2: the kit is used for treating grass carp hemorrhagic disease type 2 virus

The nucleic acid detection kit also comprises a primer mixed solution, a specific fluorescent probe, an ABuffer, a B Buffer, an RAA dry powder reagent, a grass carp hemorrhage disease type 2 virus standard substance and DEPC treated water.

The kit of the invention, wherein the ABuffer is 20% PEG; b Buffer is 280mM MgAc.

The kit of the invention, wherein the RAA dry powder reagent comprises the following components: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease, 30ng/mL RTE reverse transcriptase.

In the primer mixture, the base sequence of the forward primer is shown as SEQ ID NO.1, the base sequence of the reverse primer is shown as SEQ ID NO.2, and the molar ratio of the forward primer to the reverse primer is SEQ ID NO. 1: SEQ ID NO.2 is 1: 1.

The base sequence of the specific probe for the grass carp hemorrhagic disease type 2 virus is shown in SEQ ID NO.3, the 5 'end of the probe is marked with an FAM fluorescent reporter group, and the 3' end of the probe is marked with a BHQ1 fluorescent quenching group.

The grass carp hemorrhage type 2 virus standard provided by the invention comprises a positive plasmid of a gene sequence of a conserved region of grass carp hemorrhage type 2 virus, and the base sequence of the plasmid is shown as SEQ ID NO. 4.

Base sequence of plasmid (SEQ ID NO.4):

ATGGAGCGATCCACTTACAATATCTGCACCCCAGGCTTCTTCGGCGCGAATGTTCCGCCTTTTAAAACAATAGACATACAACGATCTACGACGGGTGGTAATACGCTTTGGAATGCGCGCGGTCACGATGCTTTCAGGACTTACCCTAAAGTGGTATCTCATGAGAAGGATTTCCCTCTAATTTATACAGAGCAGTTTACATTCAACTTGCTTATTGGCGCCTTCCTCCAGCAACCCCTACTGCAGAACTCGATTGACCGACAATGGAGGGGTATGATTTGGACATCTGATCGGCTTAGCTCGTTGCGTATAGCGCCACCGAATTCACGTGTTGCTGATCTGCCTCGCGCATATCGTACGTTGGACCTTGCGAATTACCCACTATGGGGAACTGCACCTGCTGCCCTACAGACACTATGGATGGACAGTTGTCTGATGACATTAGAGAGTTTATCTGCGAGAGGACCCTTTCTTTACCTCCGGCATCCTCAAGCTCGACCAGACGGGAATGTTTTACGAGCCTTACAGCAGCATATTTCCAAGCCAATGGAGGCCATAGTCTCGGAAGCCTACCAATCAATAGCTGTGGGACCTTTGACATTGCAAGACGGGTATTATCGCGCTCTGTCAGTGATCACCCTCATCTATCTAGCCTCTCTGACCGGTCGTCTGGGCCCTGACCGTACATATTATGGTTTCTACGTCCAATTCCCTAAGAAACGGAAATTTGAGGATCTCGGATACTTTGCGTACAATGCTGATGGACGTAACGTCGCTGTCCTGCAATCCATTAACGCCTACATCTACTGTGCTTCACCTGACTGGCAGTACAGCTGTGCCCTCTACTACTTGCACGTCCTTTCTGCCCTATCGCTCTCCTGGACTGATCCAGTTGGAATGATAGATGGTTTCTCGTGTGTCAATCAATTCACGGACGTTCCTGGTTGGTTTGCCACAAACCGTGCTTTGCACACGCATAGCTTCAACTGGTTCAACCTACTGGAGGACGCTATTGACACACTAGTTGCACGTAGATACTGGACCAATGCCGTGGGACAGGCCATACGGCAGGAATGGACGGCGGCGCGAGACAGGTGGCGAGTGATCATGGACGCAACCCGTGATGAGGATGACTTGGTGGTGTTCCGTACGCCAGACGATTGTCGTAGAAGGCTTAAACCTTATGGGGACAATAATTGGACGCGTGCTTACGATACTGCCGATGTGGTGCGGGTG

the DEPC treated water provided by the invention is purchased from Solambio company.

Example 3: the kit of the invention is used for grass carp hemorrhagic disease type 2 virus

1. Extraction of nucleic acids from Positive samples

1.1, nucleic acid extraction: conventional Trizol-RNA reagents or an equivalent RNA extraction kit were used.

2. Configuration of RAA reaction system: one RAA reaction dry powder tube was used for each test sample, and the reaction components and the added volume in each RAA reaction dry powder tube are shown in Table 3.

Table 3:

RAA reaction system component	Volume (μ L)
		A Buffer	12.5μL
B Buffer	2.5μL
		Primer mixture	4μL
Specific fluorescent probe	0.6μL
		DNA template	2μL
DEPC treated Water	28.4μL
		Total volume	50μL

A Buffer is 20% PEG; b Buffer is 280mM MgAc

3. Placing the RAA reaction tube with the prepared reaction system in an ABI7500 amplification instrument, and carrying out RAA amplification according to the following procedures: at 37 ℃ for 40 s; at 37 ℃ for 20min, for a total of 40 cycles. Fluorescence of FAM channels was collected for each cycle.

4. And after the amplification is finished, judging the positive or negative result of the grass carp hemorrhagic disease type 2 virus according to the fluorescence curve judgment and the CT value.

And (4) judging the result: the fluorescence curve of the FAM channel is S-shaped, the CT value is less than or equal to 35, and the virus is judged to be a positive result of grass carp hemorrhagic disease type 2 virus; and when the curve of the sample to be detected is not S-shaped or the CT value is more than 35, judging that the grass carp has a virus negative result of type 2 hemorrhagic disease.

Example 4: evaluation of RAA detection kit of the invention in clinical practical application

The kit is adopted to carry out clinical blind sample experiments, and 48 prawns are detected; experimental results show that the fourth primer pair can distinguish grass carp hemorrhagic disease type 2 viruses, and has high positive coincidence rate with reverse transcription PCR. In 50 parts, 26 parts of the reverse transcription PCR are positive results, 22 parts of the reverse transcription PCR are negative results, 27 parts of the results detected by the RAA method are positive, 21 parts of the results are negative results, and one part of the positive results are different, the sample is subjected to reverse transcription PCR amplification and sequencing, and the sequencing result shows that the sample is positive, so that the RAA detection reagent has higher accuracy.

Test example 5: sensitivity test of the kit of the invention

The grass carp hemorrhage type 2 virus standard plasmid provided by the kit in the embodiment 2 of the invention is extracted to obtain positive plasmids, the concentration of the positive plasmids is measured by using NanoDrop, and the positive plasmids are respectively diluted to 5 concentration gradients of 100 fg/muL, 10 fg/muL, 1 fg/muL, 0.1 fg/muL and 0.01 fg/muL to carry out sensitivity test.

The detection results are shown in figure 2, and are the amplification results of the positive standard substances of 100 fg/muL, 10 fg/muL, 1 fg/muL, 0.1 fg/muL and 0.01 fg/muL from left to right in sequence, so that the RAA fluorescence amplification reagent and the detection sensitivity can reach 1 fg/muL, the accuracy is superior to that of the common PCR detection method, and the RAA constant temperature fluorescence detection kit and the detection method have high sensitivity on GCRV-II diagnosis.

Test example 6: specificity test of the kit of the present invention

In order to detect the specificity of the kit, the detection method in example 3 is adopted to respectively detect the samples of viruses VNNV, IHNV, VHSV, SVCV, HRV, ISAV and GCRV-II, and the detection conditions of the kit on GCRV-II and other common viruses of prawns are analyzed.

The detection result shows that: only GCRV-II samples showed normal amplification, and negative controls (DEPC treated water) and VNNV, IHNV, VHSV, SVCV, HRV, ISAV samples did not show amplification (as shown in fig. 3). The results show that the RAA constant temperature fluorescence detection kit can specifically amplify the target sequence in GCRV-II without cross reaction with other virus nucleic acids. The method and the kit have good specificity and do not generate false negative.

Meanwhile, the same specificity experiment is carried out on the primers 1-3 designed by the invention, and the primers can not distinguish different samples well and have poor specificity (the specific experimental data is slight).

The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

Sequence listing

<110> Hangzhou Zhongzhuang Biotech Co., Ltd

RAA constant-temperature fluorescence detection method and reagent for <120> grass carp hemorrhagic disease type 2 virus (GCRV-2)

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Claims (10)

1. A detection kit for grass carp hemorrhagic disease type 2 virus (GCRV-2) nucleic acid comprises: the kit comprises a grass carp bleeding disease type 2 virus forward primer, a reverse primer and a specific fluorescent probe, wherein the nucleotide sequence of the grass carp bleeding disease type 2 virus forward primer is shown as SEQ ID No.1, the nucleotide sequence of the grass carp bleeding disease type 2 virus reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, a fluorescent reporter group is marked at the 5 'end of the specific fluorescent probe, and a fluorescent quenching group is marked at the 3' end of the specific fluorescent probe.

2. The nucleic acid detection kit according to claim 1, wherein the fluorescence reporter group of the specific fluorescent probe is selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460, and the fluorescence quenching gene is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

3. The nucleic acid detection kit according to claims 1 and 2, further comprising at least one of a primer mixture, a specific fluorescent probe, an A Buffer, a B Buffer, a RAA dry powder reagent, a grass carp hemorrhagic disease type 2 virus standard, and DEPC treated water.

4. The kit of claim 3, wherein the reverse transcription system comprises RTE reverse transcriptase and RNase inhibitor.

5. The kit according to claim 3, wherein the A Buffer is 20% PEG; b Buffer is 280mM MgAc.

6. The kit of claim 5, wherein the RAA dry powder reagent is comprised of: 1mmol/L dNTP, 90ng/μ L SSB protein, 120ng/μ L recA recombinase protein (SC-recA/BS-recA) or 30ng/μ L Rad51, 30ng/μ L Bsu DNA polymerase, 30ng/mL RTE reverse transcriptase, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol, 100ng/μ L creatine kinase, Exo exonuclease.

7. The nucleic acid detection kit according to any one of claims 1 to 5, wherein the standard substance of the grass carp hemorrhagic disease type 2 virus is a positive plasmid containing a partial sequence of a gene of a conserved region of the grass carp hemorrhagic disease type 2 virus.

8. The kit according to claim 6, wherein the positive plasmid containing the partial sequence of the conserved region gene of grass carp hemorrhagic disease type 2 virus has the sequence shown in SEQ ID No. 4.

9. An RAA constant-temperature fluorescence detection method for grass carp bleeding disease type 2 virus comprises the steps of extracting RNA of a sample to be detected, carrying out real-time fluorescence RAA reaction in the presence of a forward primer, a reverse primer, a specific fluorescence probe, an RAA dry powder reagent, an A Buffer, a B Buffer and DEPC (diethylpyrocarbonate) treated water of the grass carp bleeding disease type 2 virus by taking the RNA of the sample to be detected as a template, and analyzing the sample to be detected according to a real-time fluorescence RAA amplification curve; the nucleotide sequence of the grass carp hemorrhage disease type 2 virus forward primer is shown as SEQ ID No.1, the nucleotide sequence of the grass carp hemorrhage disease type 2 virus reverse primer is shown as SEQ ID No.2, the nucleotide sequence of the specific fluorescent probe is shown as SEQ ID No.3, the 5 'end of the specific fluorescent probe is marked with a fluorescent reporter group, and the 3' end of the specific fluorescent probe is marked with a fluorescent quenching group.

10. The method as claimed in claim 9, wherein the RNA extraction of the grass carp hemorrhagic disease type 2 virus nucleic acid is performed by using a traditional Trizol-RNA reagent or an equivalent RNA extraction kit.