CN113278737A - Specific primer, probe and rapid detection kit for detecting small RNA virus-1 of pelteobagrus fulvidraco - Google Patents

Abstract

The invention discloses a specific primer, a probe and a rapid detection kit for detecting a small RNA virus-1 of pelteobagrus fulvidraco, wherein the fluorescent quantitative detection kit for the small RNA virus-1 of the pelteobagrus fulvidraco comprises a specific primer group A and a Taqman fluorescent probe B; the invention adopts a fluorescence quantitative technology to detect the Pelteobagrus fulvidraco small RNA virus-1 by detecting the capsid protein gene of the Pelteobagrus fulvidraco small RNA virus-1, which is the primer group and the kit for detecting the Pelteobagrus fulvidraco small RNA virus-1 which are firstly developed at home and abroad at present. The development of the kit lays a foundation for monitoring and preventing the small RNA virus-1 disease of the pelteobagrus fulvidraco.

Description

Specific primer, probe and rapid detection kit for detecting small RNA virus-1 of pelteobagrus fulvidraco

Technical Field

The invention belongs to the field of rapid detection of target RNA fragments, and particularly relates to a specific primer, a probe and a rapid detection kit for detecting a yellow catfish small RNA virus-1.

Background

Pelteobagrus fulvidraco small RNA virus-1 (YCPRV-1) is a potential pathogenic pathogen of Pelteobagrus fulvidraco. The compound has certain pathogenicity on the pelteobagrus fulvidraco, often causes body surface bleeding and enteritis of the pelteobagrus fulvidraco, and has bleeding spots in the livers of a small number of diseased fishes. The disease caused by the compound has great harm to the yellow catfish breeding industry. YCPRV-1 is a single-stranded RNA virus, belongs to the family of picornaviridae, is not classified in a certain genus, and genome comparison shows that the virus with the highest homology with the virus genome is the Japanese sea eel picornavirus (Conger japonica Calicivirus), and the homology is 38 percent, which indicates that the pelteobagrus fulvidraco picornavirus-1 is a brand-new virus and is different from any other virus in the known family of picornaviridae. The virus is separated from the yellow catfish in 2018 month 6 by the inventor, the genome of the virus is firstly obtained by the inventor, the genome comprises two coding frames, and the total length is 7149 bp. The disease incidence of the yellow catfish affects the economic income of the majority of fish people, and in recent years, the disease of the yellow catfish causes huge economic loss to the majority of farmers. The yellow catfish small RNA virus-1 is not reported at home and abroad, and the virus has no detection kit and detection method report so far, which seriously hinders the early warning and prevention work of diseases caused by the virus, so the research on the development of the virus detection kit and the detection technology is urgent.

Disclosure of Invention

In view of the above, the invention provides a specific primer, a probe and a rapid detection kit for detecting the yellow catfish small RNA virus-1, aiming at the problems, the primer, the probe and the kit have strong specificity and high sensitivity, and can realize rapid, effective and accurate detection of the yellow catfish small RNA virus-1; the kit is a fluorescence quantitative detection kit, the result is judged under the closed condition, the amplification product does not pollute the detection environment, and the kit can be used for monitoring and early warning and preventing the yellow catfish small RNA virus-1 disease. The invention adopts a fluorescence quantitative technology to detect the small RNA virus-1 of the pelteobagrus fulvidraco by detecting the capsid protein gene of the small RNA virus-1 of the pelteobagrus fulvidraco, which is a kit for detecting the small RNA virus-1 of the pelteobagrus fulvidraco, which is firstly developed at home and abroad at present; the development of the kit lays a foundation for monitoring and preventing the small RNA virus-1 disease of the pelteobagrus fulvidraco.

In order to solve the technical problems, the invention also discloses a primer probe mixture for detecting the Pelteobagrus fulvidraco small RNA virus-1, which consists of a primer group A and a Taqman fluorescent probe B, wherein the 5 'end of the probe B is marked with a fluorescent reporter group, and the 3' end of the probe B is marked with a fluorescent quenching group.

Further, the primer set A comprises a primer YCPRV-1-q85F and a primer YCPRV-1-q 85R;

the nucleotide sequence of the primer YCPRV-1-q85F is shown as SEQ ID NO: 1 is shown in the specification;

the nucleotide sequence of the primer YCPRV-1-q85R is shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the probe B is shown as SEQ ID NO: 3, respectively.

Further, the fluorescence reporter group is selected from one or more of 6-carboxyfluorescein, hexachloro-6-methyl fluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethylrhodamine, sulforhodamine, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxy fluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and cyanine 5.5; the fluorescence quenching group is selected from one or more of 6-carboxytetramethyl rhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.

The invention also discloses a yellow catfish small RNA virus-1 fluorescence quantitative detection kit, which comprises a specific primer group A and a Taqman fluorescent probe B.

Furthermore, HEX is marked at the 5 'end of the nucleotide sequence of the Taqman fluorescent probe B, and BHQ1 is marked at the 3' end.

Further, the kit also comprises a virus nucleic acid extracting solution, a one-step RT-qPCR reaction solution, a positive quality control product and a negative quality control product which are independently packaged, wherein the virus nucleic acid extracting solution is a buffer solution containing 4mol/L guanidine isothiocyanate, 0.5% sodium dodecyl sarcosinate, 40mmol/L DTT, 25mmol/L sodium citrate, 20 mu g glycogen and pH8.0, the one-step RT-qPCR reaction solution is a probe method fluorescent quantitative one-step RT-qPCR reaction solution, the negative quality control product is sterilized physiological saline, and the positive quality control product is a carrier containing a coat protein gene of the pseudobagrus fulvidraco small RNA virus-1.

The invention also discloses an application of the fluorescence quantitative detection kit for detecting the yellow catfish small RNA virus-1 in the detection of the yellow catfish small RNA virus-1, which comprises the following steps:

extracting virus RNA from a sample to be detected by using a virus nucleic acid extracting solution, respectively adding the virus RNA into a primer probe mixed solution, then adding a one-step method RT-qPCR reaction solution, uniformly mixing, and then carrying out fluorescent quantitative PCR under the conditions of 42 ℃ for 20 minutes and 95 ℃ for 5-10 minutes; then reacting at 95 ℃ for 5-15 seconds and at 60 ℃ for 20-40 seconds for 38-42 cycles; setting HEX during the collection of the fluorescence signal, and setting the collection of the fluorescence signal at 60 ℃;

and (5) judging a result: if the S-type amplification curve does not appear in the detection channel, the yellow catfish small RNA virus-1 is judged to be negative; if the S-type amplification curve appears in the detection channel and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco small RNA virus-1 is judged to be positive.

Compared with the prior art, the invention can obtain the following technical effects:

1) the primer group provided by the invention is used for detecting the small RNA virus-1 of the pelteobagrus fulvidraco, and the existence of the target gene can be judged according to whether amplification is carried out or not because of high specificity;

2) the rapid diagnosis kit of the invention utilizes the fluorescence quantitative technology to rapidly detect the Pelteobagrus fulvidraco small RNA virus-1, the detection sensitivity is high, and the amplification template only needs 15.6 virus copies;

3) the rapid diagnostic kit disclosed by the invention is rapid and efficient in amplification, can complete amplification in less than 1 hour, and is high in efficiency;

4) the rapid diagnosis kit is simple to operate, has low requirements on the technical quality of detection personnel, can establish a rapid screening system with low cost, and realizes on-site high-flux rapid detection;

5) the rapid diagnosis kit not only enables the qualitative detection of the pelteobagrus fulvidraco small RNA virus-1 to be simpler and faster, has high specificity and high sensitivity, but also is the first kit for detecting the pelteobagrus fulvidraco small RNA virus-1, fills the gap that the pelteobagrus fulvidraco small RNA virus-1 has no detection method, and has high scientific research and economic values.

Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a specific test chart of the yellow catfish small RNA virus-1 fluorescent quantitative detection kit of the invention; wherein, 1: aeromonas hydrophila; 2: crucian herpes virus; 3: carp edema virus; 4: yellow catfish arterivirus 5: grass carp hemorrhagic disease virus; 6: koi herpesvirus; 7: pelteobagrus fulvidraco small RNA virus-1; NTC: normal pelteobagrus fulvidraco RNA negative control;

FIG. 2 is a sensitivity detection diagram of the yellow catfish small RNA virus-1 fluorescence quantitative detection kit of the invention; wherein 1 to 7 each represent 1.56X 10⁶copies、1.56×10⁵copies、1.56×10⁴copies、1.56×10³copies、1.56×10²copies、1.56×10¹copies、1.56×10⁰copies。

Detailed Description

The following detailed description of the embodiments of the present invention will be provided with reference to the accompanying drawings and examples, so that how to implement the embodiments of the present invention by using technical means to solve the technical problems and achieve the technical effects can be fully understood and implemented. The reagents and materials used in the following are well known to those skilled in the art unless otherwise specified.

Example 1

The primer probe mixture for detecting the yellow catfish small RNA virus-1 consists of a primer group A and a Taqman fluorescent probe B, wherein the 5 'end of the probe B is marked with a fluorescent reporter group, and the 3' end of the probe B is marked with a fluorescent quenching group.

The primer group A comprises a primer YCPRV-1-q85F and a primer YCPRV-1-q 85R;

the primer YCPRV-1-q85F has a sequence shown in SEQ ID NO: 1 is shown in the specification;

the primer YCPRV-1-q85R has a sequence shown in SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the probe B is shown as SEQ ID NO: 3, respectively.

Wherein, the fluorescence reporter group is selected from one or more of 6-carboxyl fluorescein, hexachloro-6-methyl fluorescein, VIC fluorescent dye, tetrachloro-6-carboxyl fluorescein, carboxyl-X-rhodamine, 6-carboxyl tetramethyl rhodamine, sulforhodamine, 6-carboxyl-4 ', 5' -dichloro-2 ', 7' -dimethoxy fluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and cyanine 5.5; the fluorescence quenching group is selected from one or more of 6-carboxytetramethyl rhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.

More preferably, the fluorescent reporter and the fluorescent quencher are selected as shown in Table 1 below.

TABLE 1

Fluorescence quenching group	Fluorescent reporter groups
		DABCYL	At least one of 6-FAM, TET, JOE, HEX and Cy3
TAMRA	6-FAt least one of AM, TET, JOE and HEX
		BHQ1	At least one of 6-FAM, TET, JOE, HEX and Cy3
BHQ2	At least one of TAMRA, Cy3, ROX and Texas Red
		BHQ3	Cy5 or Cy5.5

Most preferably, the fluorescent reporter is HEX; the fluorescence quenching group is BHQ 1.

Example 2

The yellow catfish small RNA virus-1 fluorescent quantitative detection kit comprises a virus nucleic acid extracting solution, a one-step RT-qPCR reaction solution, a positive quality control product, a negative quality control product and a primer probe mixed solution which are independently packaged.

The primer probe mixed solution consists of a primer group A and a Taqman fluorescent probe B, wherein the final concentrations of the upstream primer and the downstream primer of the primer group A are both 0.15 mu M, and the final concentration of the probe is 0.1 mu M.

The 5 'end of the nucleotide sequence of the Taqman fluorescent probe B is marked with HEX, and the 3' end of the nucleotide sequence of the Taqman fluorescent probe B is marked with BHQ 1;

the negative quality control product is sterilized normal saline; the positive quality control product is pseudovirus which is obtained by taking a purified pelteobagrus fulvidraco small RNA virus-1 genome as a template, carrying out PCR amplification by using a primer group A, connecting an amplification product with a vector, confirming the correctness through gene sequencing and then packaging;

the virus nucleic acid extracting solution is a buffer solution containing 4mol/L guanidine isothiocyanate, 0.5 percent sodium dodecyl sarcosine, 40mmol/L DTT, 25mmol/L sodium citrate, 20 mu g glycogen and pH8.0;

the one-step RT-qPCR reaction solution is a probe method fluorescent quantitative RT-PCR reaction solution and contains AMV reverse transcriptase, PCR enzyme, dNTP and Mg²⁺。

Example 3

The application of the kit for the quantitative fluorescence detection of the Pelteobagrus fulvidraco small RNA virus-1 comprises the steps of adding 0.05g of a sample to be detected into 1mL of a virus nucleic acid extracting solution for full grinding, centrifuging at 12000r/min for 10min, taking 800uL of supernatant, adding 400uL of ethanol, centrifuging at 12000r/min for 5min after room temperature 5min, washing precipitates once with 75% ethanol, drying at room temperature for 5min, adding 50uL of DEPC (diethylpyrocarbonate) to dissolve RNA, taking 5uL to add into a reaction tube, adding 2uL of a primer probe mixed solution, adding 10uL of a one-step RT-qPCR reaction solution, adding 3uL of DEPC water, uniformly mixing, and carrying out quantitative fluorescence RT-PCR under the conditions of 42 ℃ for 20 min and 95 ℃ for 5-10 min; then reacting for 5-15 seconds at 95 ℃ and 20-40 seconds at 60 ℃ for 45 cycles; setting HEX during the collection of the fluorescence signal, and setting the collection of the fluorescence signal at 60 ℃;

and (5) judging a result:

if the S-type amplification curve does not appear in the detection channel, the yellow catfish small RNA virus-1 is judged to be negative; if the S-type amplification curve appears in the detection channel and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco small RNA virus-1 is judged to be positive.

The primer group A and the probe B are designed aiming at the capsid protein gene of the Pelteobagrus fulvidraco small RNA virus-1, the result detection is carried out by adopting the fluorescent group capture of the specific probe, and the kit has the characteristics of high sensitivity and convenient use and is also the universal standard of the international detection kit.

Example 4 application example of yellow catfish small RNA virus-1 fluorescent quantitative detection kit

(1) Extracting total viral RNA:

taking 10-20mg of pelteobagrus fulvidraco liver or spleen tissue into a 1.5mL centrifuge tube, adding 1mL of virus nucleic acid extracting solution for full grinding, centrifuging at 12000r/min for 10min, taking 800uL of supernatant into a new 1.5mL centrifuge tube, adding 400uL of ethanol, centrifuging at 12000r/min for 5min after room temperature is 5min, washing precipitates with 75% ethanol once, drying at room temperature for 5min, adding 50uL of DEPC water to dissolve RNA, and taking the RNA as a template to be detected.

(2)RT-qPCR：

10 mu L of RT-PCR reaction liquid, 2 mu L of primer-probe mixed liquid and 3uL of DEPC water are respectively added into 8 reaction tubes to prepare a reaction system, and then 5.0 mu L of template to be detected is respectively added.

Fluorescent quantitative RT-PCR reaction conditions: 20 minutes at 42 ℃; 10 minutes at 95 ℃; 95 ℃, 10 seconds, 60 ℃, 32 seconds, 40 cycles.

A LightCycler 96System fluorescent quantitative PCR instrument is adopted, HEX fluorescein is set during fluorescent signal collection, and the fluorescent signal collection is set at 60 ℃.

Negative quality control products (sterilized normal saline) and positive quality control products (pseudoviruses of the yellow catfish small RNA virus-1) are set in each batch of reaction.

(3) And (5) judging a result: the detection channel has no S-type amplification curve and is judged to be negative to the yellow catfish small RNA virus-1; if the S-type amplification curve appears in the detection channel and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco small RNA virus-1 is judged to be positive.

And (5) result verification: and (3) carrying out gene sequencing on the amplification product with positive detection, wherein the sequencing result is consistent with the gene sequence of the coat protein of the yellow catfish small RNA virus-1.

The specificity and sensitivity test results of the yellow catfish small RNA virus-1 are shown in the attached drawing 1 and the attached drawing 2 respectively by adopting the yellow catfish small RNA virus-1 fluorescence quantitative detection kit and the method for rapidly detecting the yellow catfish small RNA virus-1.

FIG. 1 is a specific detection diagram of the Pelteobagrus fulvidraco picornavirus-1, as shown in the figure, after Real-time RT-PCR quantitative detection, only the Pelteobagrus fulvidraco picornavirus-1 has a specific amplification curve, and no amplification curve appears in other viruses and bacteria, which indicates that the method has good specificity.

FIG. 2 is a diagram showing the sensitivity detection of Pelteobagrus fulvidraco picornavirus-1, which is an amplification diagram showing serial dilution of Pelteobagrus fulvidraco picornavirus-1 RNA after Real-time RT-PCR quantitative detection, and when 15.6 copies of the virus RNA are added into the reaction system, the amplification and color development result is positive.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> research institute for fresh water aquatic products in Zhejiang province

<120> specific primer, probe and rapid detection kit for detecting small RNA virus-1 of pelteobagrus fulvidraco

<130> 2021

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tgagtcgtct gagttgacct gttgggattg gtcgcctcta ctccttagga gtttggccga 5340

ttacattgaa gttcaggctc ttaaaaagaa aggcctggtt ttccacatcc tcaacaaatg 5400

cagggccatt ccgggatcta tttcttattc cctggtgaat cggtttaaaa cggtactcat 5460

gtacttcttc cctaaaatga agtgctttag ggggaaaaga actgatctgg tggagcccat 5520

cacagttgtg tcaatgggca cctcctttaa cggttttttg accccccaga atgagtttta 5580

ttgcccggac ttctatggag agtccataac actttctgat ccacctcgtc ccaggctttt 5640

ggagctacgt ggtgatacag gggggtgttc aataacatgc agagcttgcg gggtcaagat 5700

caagaagtct aacctgctca agcatctcca agtgtgcccc aagaggaccc agccacccag 5760

acccccttgt cctccaaaga tgacatacaa aaagacacca gcaggacaac ttaaggtgaa 5820

acaagctggc cccaagcaaa tagcccccat accaatgcgt gatgatccca agggaaaggg 5880

taggtggtgg atttcttcca acctcgctgc ctattcccca gcccatgctg accaacacaa 5940

ggggttggcc tgtggtgacc gtggtaagtc acttaaatgg actgatgtgg tggagaaatc 6000

ctacacattt aatgaggttg gagacctcct ggtcctggga actgggcttg tggagtcacc 6060

gtatgcagtg gtggcaacca aagatgggtc aacgtccact ggatctaccc ttccagacat 6120

tttccacccc gtgatgtgtg tgggagtgaa tgacagagat gaagacgacc tggatgtcac 6180

tggatcgaaa gacgactcca agctggtcat aacaaaaatg tttgacacac ctgaaagtca 6240

tgaaaaacac cgcatggttg gaagctccat cactatcaca gtaagtggag aagctggcag 6300

agtgaacatc gccgaactca cctctggacc tgagaaggac gatttgaaaa ccgtccacat 6360

gaccagatta caccaaactg acggtcatac agcacataat gggaaggagg gcctctacat 6420

catcagtaag ccagcacatg atcatgatcc aaccaactgg atggataatg gtgatgtgat 6480

cccagcactg acatccctgg tttctatgga agatggaggt gccgacaagg agcacaacat 6540

cacctggaag actgcagagg caatgcatct gagcggacgc gggctgcagt tcgcgaagct 6600

tgatggggtg cacgctggtg tgactgttac tgttaaggtt gtgctgcaca tggagtacaa 6660

ggttgaccca acccactccc atgccaggta cctcacccct acaaacgtag tcggaggagt 6720

tcctggggcg gcccatcgca acgtgtacca gcgcatttgg tctggctacc cagcccatta 6780

caacgacctc aagggcttct tagcaggtct tgcaagtgtg ctagcgtcaa tcggagctat 6840

cgttgctgtc ttcatccccc ctgcaggagc cgtaattgga gccgtgggcg caggggttgg 6900

tggcgtcgca gcaatcctct aaattgaacc aaaaaccaac caacatcaac cctctcataa 6960

ttcgaagttt gtaactactc tcttcttact catttgaggt ttgctagcat ttcaaccata 7020

atggaaagag agcacaggta gtgcctcaag tacaaaagcc ccatacaacc agtgtgttgc 7080

gggatcagat acctgaacta aacagaacgg gaacggtaac tgagttgatt gctgcaaatt 7140

ctttcgctga ggtagggaaa cataaaattt cctactttct ttcccatcct tcctcagcgg 7200

atacctttaa tgcgtgtgtc caccccagtg gtgtaccacg catctccttc agtatatctg 7260

tgggacgtct caaaggcgtt tcaacacgga tctcctgtgg ggggaaaccg tgttctcaag 7320

gcttgtcacc aaaggtggcg tgcaatgggt gcacgttact tttccccctc ggggggttca 7380

tggtgaatat gcttatttga gacagaaata cacttctttt ataaacatca aaaaaaaaaa 7440

aaaaaaaaa 7449

Claims (7)

1. The primer probe mixture for detecting the yellow catfish small RNA virus-1 is characterized by consisting of a primer group A and a Taqman fluorescent probe B, wherein the 5 'end of the Taqman fluorescent probe B is marked with a fluorescent reporter group, and the 3' end of the Taqman fluorescent probe B is marked with a fluorescent quenching group.

2. The primer-probe mixture of claim 1, wherein the primer set a comprises primers YCPRV-1-q85F and YCPRV-1-q 85R;

the nucleotide sequence of the primer YCPRV-1-q85F is shown as SEQ ID NO: 1 is shown in the specification;

the nucleotide sequence of the primer YCPRV-1-q85R is shown as SEQ ID NO: 2 is shown in the specification;

the nucleotide sequence of the Taqman fluorescent probe B is shown as SEQ ID NO: 3, respectively.

3. The Pelteobagrus fulvidraco picornavirus-1 Taqman fluorescent probe B according to claim 1, wherein the fluorescent reporter group is selected from one or more of 6-carboxyfluorescein, hexachloro-6-methylfluorescein, VIC fluorescent dye, tetrachloro-6-carboxyfluorescein, carboxy-X-rhodamine, 6-carboxytetramethylrhodamine, sulforhodamine, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein succinimidyl ester, cyanine 3, cyanine 3.5, cyanine 5 and cyanine 5.5; the fluorescence quenching group is selected from one or more of 6-carboxytetramethyl rhodamine, 4- (4-dimethylamino phenylazo) benzoic acid, a black hole quenching agent 1, a black hole quenching agent 2 or a black hole quenching agent 3.

4. A yellow catfish small RNA virus-1 fluorescence quantitative detection kit is characterized by comprising a specific primer group A and a Taqman fluorescent probe B.

5. The fluorescence quantitative detection kit for detecting the yellow catfish small RNA virus-1 according to claim 4, characterized in that HEX is marked at the 5 'end of the nucleotide sequence of the Taqman fluorescent probe B, and BHQ1 is marked at the 3' end.

6. The fluorescence quantitative detection kit for detecting the pseudobagrus fulvidraco small RNA virus-1 according to claim 4, characterized by further comprising an independently packaged viral nucleic acid extracting solution, a one-step RT-qPCR reaction solution, a positive quality control product and a negative quality control product, wherein the viral nucleic acid extracting solution is a buffer solution containing 4mol/L guanidinium isothiocyanate, 0.5% sodium dodecyl sarcosinate, 40mmol/L DTT, 25mmol/L sodium citrate, 20 μ g glycogen and pH8.0, the PCR reaction solution is a probe method fluorescence quantitative PCR reaction solution, the negative quality control product is sterilized physiological saline, and the positive quality control product is a carrier containing the pseudobagrus fulvidraco small RNA virus-1 capsid protein gene.

7. The application of the fluorescence quantitative detection kit for detecting the yellow catfish small RNA virus-1 in the detection of the yellow catfish small RNA virus-1 in the claim 4 is characterized by comprising the following steps:

extracting virus total RNA from a sample to be detected by using a virus nucleic acid extracting solution, and performing fluorescent quantitative RT-qPCR by using a one-step RT-qPCR kit under the reaction conditions of 20 minutes at 42 ℃ and 10 minutes at 95 ℃; then reacting at 95 ℃ for 10 seconds and at 60 ℃ for 32 seconds for 40 cycles; setting HEX during the collection of the fluorescence signal, and setting the collection of the fluorescence signal at 60 ℃;

and (5) judging a result: if the S-type amplification curve does not appear in the detection channel, the yellow catfish small RNA virus-1 is judged to be negative; if the S-type amplification curve appears in the detection channel and the Ct value is less than or equal to 35, the pelteobagrus fulvidraco small RNA virus-1 is judged to be positive.

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