CN105400908A - Primer and kit for detecting channel catfish viruses through pyrosequencing technology and detecting method - Google Patents

Abstract

The invention discloses a primer, a kit and a detection method for detecting channel catfish virus by using pyrosequencing technology. Specifically, the present invention relates to a set of primers for detecting channel catfish virus using pyrosequencing technology, including SEQ? ID? No.2 and SEQ? ID? The amplification primer pair shown in No.3, and SEQ? ID? The sequencing primer shown in No.4. The invention also discloses a detection kit containing these primers. The present invention uses pyrosequencing technology to detect channel catfish virus, which has the advantages of high throughput, rapidity and accuracy, can accurately analyze short DNA sequences, and the sequencing process is time-consuming, which is convenient for constructing a standardized operation process.

Description

A primer, kit and detection method for detecting channel catfish virus using pyrosequencing technology

technical field

The invention relates to a primer, a kit and a detection method for detecting channel catfish virus by using pyrosequencing technology, belonging to the field of molecular biology.

Background technique

Channelcatfish virus (Channelcatfishvirus, CCV) is an enveloped double-stranded DNA virus, also known as Ictaluridherpesvirus type I (Ictaluridherpesvirus1), which is classified as herpes in the latest virus classification system of the eighth report of the International Committee on Taxonomy of Viruses Viridae, Rhinovirus genus. The channel catfish virus disease (CCVD) caused by this virus is an acute fatal infectious disease, which can cause serious losses to the channel catfish breeding industry. , List of Parasitic Diseases, Class II Infectious Diseases. Channel catfish and other catfish are susceptible to CCV. In natural water, the virus only infects catfish juveniles and fry, and the mortality rate of hatchlings reaches 100%. The disease is mainly prevalent in North America, and channel catfish has been cultured on a large scale in my country in recent years, but there is no definite report of CCV yet. After the CCVD epidemic, the remaining fish are often invisible carriers, generally without clinical symptoms. Establishing CCV rapid detection and early warning technology, strengthening the quarantine of channel catfish seedlings and adult fish, and the monitoring of aquaculture water environment are of great significance to prevent virus infection, effectively cut off virus transmission, and ensure the healthy and sustainable development of channel catfish aquaculture.

So far, the standard method of CCV detection is mainly to separate and culture the virus with cells, and then use neutralization test, immunofluorescence, ELISA or PCR and other means to identify it. The judgment of CCV infection in fish without clinical symptoms is based on serological methods. By measuring fish CCV antibodies. The sensitivity of serological methods is low; although the PCR method is sensitive, there is still the possibility of false positives; other methods cannot achieve the purpose of rapid confirmation at the molecular level.

Pyrosequencing technology is an enzyme cascade chemiluminescence reaction in the same reaction system catalyzed by four enzymes, which is suitable for sequencing and analysis of known short sequences, without fluorescently labeled primers or nucleic acid probes, and without electrophoresis, and has the advantages of rapid analysis, Accurate, high sensitivity, economical, automatic and real-time detection features. On the basis of ordinary PCR, the method sequenced PCR products, which improved the specificity of detection, and gave the gold standard of molecular diagnosis—gene sequence, which reduced false positive results due to high sensitivity of PCR, and improved the detection accuracy. Pyrosequencing technology can quickly and accurately analyze short DNA sequences, has high throughput, is easy to operate, and facilitates the establishment of standardized operating procedures. Therefore, it is very popular with researchers at home and abroad, and has been widely used in the rapid identification of pathogenic microorganisms.

Contents of the invention

The technical problem to be solved by the present invention is to provide a set of primers for detecting channel catfish virus by pyrosequencing technology.

Another technical problem to be solved by the present invention is to provide a kit for detecting channel catfish virus using pyrosequencing technology.

The third technical problem to be solved by the present invention is to provide a detection method for detecting channel catfish virus using pyrosequencing technology.

In order to achieve the above object, the main principle of the present invention is to utilize pyrosequencing technology to catalyze four kinds of enzymes through DNA polymerase (DNAploymerase), adenosine triphosphate sulfurylase (ATPsulfurylase), luciferase (luciferase) and diphosphatase (apyrase). The enzymatic cascade chemiluminescence reaction between the DNA single strand to be sequenced and the sequencing primer, the reaction substrates are 5'-o-acylsulfate (adenosine5'phosphossulfate, APS) and fluorescein. In each round of sequencing reaction, one dNTP is added. If the dNTP is paired with the template, the polymerase can incorporate it into the primer strand and release an equimolar number of pyrophosphate groups (PPi). Sulfylase catalyzes APS and PPi to form ATP, which drives luciferase-mediated conversion of luciferin to oxyluciferin, and emits a visible light signal proportional to the amount of ATP, which is detected by a CCD camera and reacted by ^PyrogramTM as Peak, the peak height of each light signal is proportional to the number of nucleotides incorporated in the reaction. ATP and unincorporated dNTPs are degraded by bisphosphatases, which quench the light signal and regenerate the reaction before adding the next dNTP. The sequence of the final sequence to be tested can be read from the signal peak of the reaction light intensity.

The present invention provides a set of primers for detecting channel catfish virus using pyrosequencing technology, including amplification primer pairs and sequencing primers:

Amplification primer pair: upstream primer: 5'-GGCTTGGGCTTGATGGAC-3' (SEQ ID No.2)

Downstream primer: 5'-Biotin-GAGGAGGACAACGCGACTG-3' (SEQ ID No.3)

Sequencing primer: 5'-CTTGGGCTTGATGGA-3' (SEQ ID No.4)

Wherein, the 5' end of the downstream primer is labeled with biotin.

The channel catfish virus-specific primers and pyrosequencing primers of the present invention are designed according to the conserved region sequence of the channel catfish virus protein kinase (proteinkinase, PK) gene by AssayDesignSW software to amplify a specific single band, Then, pyrosequencing primers were designed according to the specific nucleotide sequence contained in the amplified region (shown in the specific target sequence SEQ ID No. 1) to determine the channel catfish virus. The target sequence of the channel catfish virus PK gene: CCGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No. 1); the PCR amplification fragment length is 144bp.

The present invention also provides a kit for detecting channel catfish virus by pyrosequencing technology, which kit includes the above-mentioned primer set for detecting channel catfish virus, that is, a pair of amplification primers and a sequencing primer. Further, the kit also includes the materials and reagents needed to complete the PCR reaction and pyrosequencing, such as DNA extraction reagents (can be self-prepared according to the methods disclosed in the prior art, or commercially purchased kits can be used); PCR Reaction reagents (can be self-prepared according to the methods disclosed in the prior art, or commercially purchased kits can be used); single-stranded template preparation reagents; pyrosequencing reagents (can be self-prepared according to the methods disclosed in the prior art, or Commercially purchased kits) and the like can be used, and the above-mentioned materials and reagents can be mixed and packaged, or packaged separately, preferably, packaged separately.

The present invention also provides a method for detecting channel catfish virus using pyrosequencing technology, the method comprising:

(1) Extract the DNA of the sample to be tested as a template, which can be extracted using known extraction methods or commercial kits in the prior art;

(2) Use the above amplification primers to carry out PCR amplification reaction;

(3) If the channel catfish virus-specific PCR reaction is positive, that is, the amplified fragment is 144bp, recover the PCR amplification product, prepare a pyrosequencing single-stranded template, and then use the above sequencing primers to perform pyrosequencing. (SEQ ID No.1) specific target sequence is consistent, then the sample to be tested contains channel catfish virus; if the specific PCR reaction of channel catfish virus is negative, then the sample is determined to be non-channel catfish virus.

The advantages of the present invention are: the present invention uses pyrosequencing technology to detect channel catfish virus. Compared with the prior art, this technology has the advantages of high throughput, fast and accurate, and can accurately perform short DNA sequence analysis and sequencing The process takes a short time and facilitates the establishment of standardized operating procedures. The PCR product can be directly used for sequencing without secondary treatment such as product purification. The operation is simple and the amount of sample required is small.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. Any equivalent replacements in the field made according to the disclosure of the present invention shall fall within the scope of protection of the present invention.

Description of drawings

Fig. 1 is the agarose gel electrophoresis result of the channel catfish virus PCR amplification product of the present invention; wherein M is DNAMarker, 1 is channel catfish virus, and 2 is a negative control.

Fig. 2 is the result of pyrosequencing of the channel catfish virus PCR amplification product in the present invention.

Fig. 3 is the result that the present invention carries out agarose gel electrophoresis to the PCR amplification product of sample tissue DNA; Wherein M is DNAMarker, 1 is the catfish tissue sample of channel catfish virus infection, 2 is normal catfish sample, 3 is negative control.

Fig. 4 is the sequencing result of DNA PCR amplification product of channel catfish virus-infected catfish tissue according to the present invention.

Fig. 5 is the sensitivity detection result of pyrosequencing of the channel catfish virus PCR amplification product of the present invention. Among them, AG in turn is the pyrosequencing detection result of the channel catfish virus template diluted from 10 ^-1 to 10 ^-7 PCR amplification products.

Fig. 6 is the agarose gel electrophoresis result of PCR amplification specific detection of channel catfish virus in the present invention. Among them, M is DNA Marker, 1 is channel catfish virus, 2 is goldfish hematopoietic organ necrosis virus, 3 is koi herpes virus, 4 is epidemic hematopoietic organ necrosis virus, 5 is soft-shelled turtle iridescent virus, and 6 is a negative control.

detailed description

Embodiment 1 detects the design of the primer of channel catfish virus

Through the comparative analysis of the published channel catfish virus gene sequences in the NCBI gene bank (GenBank) of the National Center for Bioinformatics in the United States, specific PCR primer pairs and pyrosequencing primers were designed according to the specific sequence of the CCVPK gene, and a set of optimal primers was screened out. Primers for subsequent PCR reactions and sequencing.

The channel catfish virus-specific primer designed according to the specific region sequence of the channel catfish virus PK gene can amplify a specific single band for the channel catfish virus DNA, and the length of the PCR amplification fragment is 144 bp. Then, pyrosequencing primers were designed according to the specific nucleotide sequence contained in the amplified region (shown in the specific target sequence SEQ ID No. 1) to determine the channel catfish virus. The target sequence of the channel catfish virus PK gene: CCGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No. 1).

The channel catfish virus-specific PCR amplification primer pair and sequencing primers designed by the present invention after screening are respectively:

Amplification primer pair: upstream primer (CCVF): 5'-GGCTTGGGCTTGATGGAC-3' (SEQ ID No.2);

Downstream primer (CCVR): 5'-Biotin-GAGGAGGACAACGCGACTG-3' (5' labeled with biotin) (SEQ ID No.3);

Sequencing primer (CCVS): 5'-CTTGGGCTTGATGGA-3' (SEQ ID No.4).

Embodiment 2 kit composition

The kit includes the primer set designed in Example 1 for detecting channel catfish virus, that is, a pair of amplification primers and a sequencing primer. The kit also includes the materials and reagents needed to complete the PCR reaction and pyrosequencing, such as DNA extraction reagents (DNeasyBlood&TissueKit, QIAGEN Company), PCR reaction reagents (PyroMarkPCRKit, QIAGEN Company), single-stranded template preparation reagents (streptavidin Coated magnetic beads, GE company), pyrosequencing reagents (PyroMarkGoldQ96SQARagents, denaturing buffer, washing buffer, QIAGEN company), etc., the above-mentioned materials and reagents can be mixed and packaged, and can also be packaged separately, preferably separately.

The establishment of embodiment 3 detection method

1. Extraction of channel catfish virus DNA

Using CCV (preserved in our laboratory) as raw material, DNeasyBlood&TissueKit (QIAGEN, Cat. No. 69506) was used to extract DNA. The kit operation steps are as follows:

(1) Pipette 20 μl proteinase K to the bottom of a 1.5ml centrifuge tube. (2) Add 200 μl of the virus liquid (the virus titer is 10 ⁷ TCID ₅₀ /0.1 ml) to be extracted into the centrifuge tube. (3) Add 200 μl BufferAL to the sample, and vortex for 15 seconds to mix well. (4) Incubate at 56°C for 10 minutes. (5) Centrifuge quickly to remove the liquid droplets remaining in the lid of the 1.5ml centrifuge tube. (6) Add 200 μl (equal volume of the sample) of ethanol (96-100%), and vortex for 15 seconds to mix. After shaking, quickly centrifuge to remove the liquid droplets remaining in the cap of the 1.5ml centrifuge tube. (7) Transfer the mixture obtained in step 6 to a QIAampMini spin column. Close the lid and centrifuge at 6000g for 1min. (8) Put the QIAampMin spin column into a new clean 2ml receiving tube, and discard the filtrate together with the used collection tube. (9) Open the QIAampMini spin column carefully and add 500μl BufferAW1. Close the lid tightly and centrifuge at 6000g for 1min. Transfer the QIAampMini spin column to a new 2ml collection tube and discard the filtrate along with the used collection tube. Discard the filtrate, carefully open the QIAampMini spin column, and add 500μl BufferAW2. Close the lid tightly and centrifuge at maximum speed for 3 minutes. (10) Transfer the QIAampMini spin column to a new 1.5ml collection tube. Carefully open the spin column and add 50 μl BufferAE or double distilled water. Incubate at room temperature (15-25° C.) for 1 min, then centrifuge at 6000 g for 1 min. Store in -20°C refrigerator for later use.

2. PCR reaction system and conditions

Using the extracted CCVDNA as a template, use PyroMarkPCRKit (QIAGEN company, product number 978703) to carry out PCR to amplify the target fragment. The upstream and downstream primers are the CCVF and CCVR primers designed in Example 1. The PCR reaction system is as follows:

The reaction program was: pre-denaturation at 95°C for 15 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, annealing at 72°C for 30 s, and a total of 45 cycles; and extension at 72°C for 10 min.

3. Agarose gel electrophoresis

Perform agarose gel electrophoresis on the PCR product, take 0.6g of agarose, heat it in 30ml of 1×TAE electrophoresis buffer, fully melt, add 2μl of GoldView ^TM DNA dye 10000 times diluted solution, make gel, add electrophoresis to the electrophoresis tank Buffer solution, so that the liquid level is just above the gel; take 5 μl of PCR amplification products and mix them with 1 μl 6×LoadingBuffer respectively, and apply the sample; 10V/cm constant voltage electrophoresis, until the bromophenol blue indicator migrates to the middle of the gel, stop the electrophoresis, The gel was placed under an ultraviolet gel imaging system to observe the electrophoresis results, and the electrophoresis results are shown in Figure 1.

4. Preparation of single-stranded template for pyrosequencing

Use 20 μl of biotin-labeled PCR products to mix with 30 μl ddH ₂ O and then mix with 200 μg streptavidin-coated magnetic beads, and incubate on a shaker at 1400 rpm for 15 minutes at room temperature, and use a vacuum adsorption pump to combine with magnetic beads The final PCR product was sucked up, and then passed through 70% ethanol for 5s; denaturing buffer for 5s; washing buffer for 5s, and finally moved to the top of the PSQ96-well plate where 45 μl of binding buffer containing 0.3mol/L sequencing primers was added to each well. Release the magnetic beads, heat the 96-well plate in an oven at 80°C for 2 minutes, then take it out and cool it to room temperature.

5. Pyrosequencing

The reaction was carried out on a pyrosequencer (PYROMARKQ96). At 20-28°C, the base addition sequence used was 15 (ATGC), and the running time of each round of sequencing detection was 65 minutes. The primer chains changed with the addition of different dNTPs. Extension, along with the combination of nucleic acid, the CCD camera detects the emitted light signal, the channel catfish virus-specific pyrosequencing results are shown in Figure 2, the read sequence is CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No.1).

6. Result judgment

The electrophoresis results of the PCR product showed that a 144bp target fragment was amplified, which was consistent with the standard; after pyrosequencing, the target sequence determined was consistent with the specific target sequence of the channel catfish virus PK gene, which was CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQIDNo.1), so it can be determined For channel catfish virus.

Example 4 Utilizes Pyrosequencing Technology to Detect Samples

Take 1 channel catfish virus-infected catfish tissue sample and 1 normal catfish tissue sample, and use the method in Example 3 to detect the samples.

1. Extraction of DNA from channel catfish tissue samples

Genomic DNA was extracted from channel catfish tissue samples using DNeasy Blood & Tissue Kit (QIAGEN, Cat. No. 69506), and stored in a -20°C refrigerator for later use. See step 1 of Example 3.

2. PCR reaction system and conditions

Refer to the corresponding steps in Example 3 for the PCR reaction system and conditions. Using the extracted channel catfish tissue sample DNA as a template, PCR was performed with PyroMark PCR Kit (QIAGEN, Cat. No. 978703) to amplify the target fragment.

3. Agarose gel electrophoresis

For the electrophoresis method, refer to Step 3 of Example 3. The electrophoresis results of this embodiment are shown in FIG. 3 .

4. Preparation of single-stranded template for pyrosequencing

Refer to Step 4 of Example 3 for the method.

5. Pyrosequencing

The reaction was carried out on a pyrosequencer (PYROMARKQ96). At 20-28°C, the base addition sequence used was 15 (ATGC), and the running time of each round of sequencing detection was 65 minutes. The primer chains changed with the addition of different dNTPs. Extension, along with the combination of nucleic acid, the CCD camera detects the emitted light signal, the result of pyrosequencing of the DNA PCR amplification product of the catfish tissue infected with CCV is shown in Figure 4, and the read sequence is CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No.1).

6. Result judgment

The results of electrophoresis of PCR products showed that the channel catfish virus-infected catfish tissue amplified a 144bp target fragment, which was consistent with the detection result of CCV; after pyrosequencing, the determined target sequence was consistent with the specific target sequence of the channel catfish virus PK gene, It is CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No. 1), so it can be determined that sample No. 1 is infected with channel catfish virus. The target fragment of 144bp was not amplified in normal catfish tissue, so it can be determined that there is no channel catfish virus in sample No. 2.

Example 5 Sensitivity experiment using pyrosequencing technology to detect channel catfish virus

Using the CCVDNA extracted in Example 3 as a template, sequentially perform 10-fold dilution to 10 ^-7 , and set a negative control at the same time. The steps in Example 3 were used for sensitivity testing.

1. PCR reaction system and conditions

Refer to the corresponding steps in Example 3 for the PCR reaction system and conditions. Using the diluted viral DNA as a template, PCR amplification was carried out with PyroMarkPCRKit (QIAGEN, Cat. No. 978703). For the reaction system and reaction procedure, refer to Example 3.

3. Agarose gel electrophoresis

For the electrophoresis method, refer to Step 3 of Example 3.

4. Preparation of single-stranded template for pyrosequencing

Refer to Step 4 of Example 3 for the method.

5. Pyrosequencing

The reaction was carried out on a pyrosequencer (PYROMARKQ96). At 20-28°C, the base addition sequence used was 15 (ATGC), and the running time of each round of sequencing detection was 65 minutes. The primer chains changed with the addition of different dNTPs. Extension, along with the binding of nucleic acid, the CCD camera detects the emitted light signal, and the pyrosequencing result of the CCVDNA PCR amplification product is shown in Figure 5, and the read sequence is CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No.1).

6. Result judgment

The results of electrophoresis showed that the 144bp target fragment was still amplified after the CCV DNA template was diluted from 10 ^-1 to 10 ^-7 , and the band brightness gradually weakened; after pyrosequencing, the CCV DNA template was diluted to 10 ^-6 , and the measured CCV target sequence and PK The specific target sequence of the gene was consistent, which was CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No.1); when diluted to 10 ^-7 , no specific target sequence was detected by pyrosequencing. This example shows that the method established by the present invention for detecting channel catfish virus by using pyrosequencing technology is relatively sensitive and can be diluted to 10 ^-6 , ie 20TCID ₅₀ .

Example 6 The Specificity Experiment of Using Pyrosequencing Technology to Detect Channel Catfish Viruses

Extract the DNA of goldfish hematopoietic necrosis virus (GFHNV), koi herpes virus (KHV), epidemic hematopoietic necrosis virus (EHNV) and soft-shelled turtle iridescent virus (STIV) respectively (all are preserved in this laboratory), according to Example 3 The CCVDNA extracted from was used as a positive control, and a negative control was set at the same time. The specificity detection was performed using the steps in Example 3.

1. Extraction of virus sample DNA

Viral DNA was extracted by using DNeasyBlood & TissueKit (QIAGEN, 69506). For the operation steps of the kit, refer to step 1 of Example 3.

2. PCR reaction system and conditions

Using the extracted virus and control virus sample DNA as templates, PyroMarkPCRKit (QIAGEN, 978703) was used for PCR amplification. For the reaction system and reaction procedure, refer to step 2 of Example 3.

3. Agarose gel electrophoresis

For the electrophoresis method, refer to Step 3 of Example 3. The electrophoresis results of this embodiment are shown in FIG. 6 .

4. Preparation of single-stranded template for pyrosequencing

Refer to Step 4 of Example 3 for the method.

5. Pyrosequencing

The reaction was carried out on a pyrosequencer (PYROMARKQ96). At 20-28°C, the base addition sequence used was 15 (ATGC), and the running time of each round of sequencing detection was 65 minutes. The primer chains changed with the addition of different dNTPs. Extension, along with the combination of nucleic acid, the CCD camera detects the emitted light signal, and the pyrosequencing result of the CCVDNAPCR amplification product is shown in Figure 2, and the read sequence is CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No.1).

6. Result judgment

The results of electrophoresis showed that GFHNV, KHV, EHNV and STIV did not amplify the target fragment of 144bp; CCV amplified the target fragment of 144bp, which was consistent with the standard; after pyrosequencing, the target sequence of CCV and the specific target sequence of PK gene were determined Consistent, it is CGGTCGTGTCGGTGTCGGTGGCTGTGTCGGCTTCCGTTTCACCG (SEQ ID No. 1). This example shows that the method for detecting channel catfish virus established by the present invention using pyrosequencing technology has good specificity and accuracy. Those skilled in the art can construct a kit for detecting channel catfish virus according to the sequencing primers and target sequences provided by the present invention. The kit can be used for rapid detection of channel catfish virus. It has the characteristics of high throughput and low cost. The product can be directly used for sequencing without secondary treatment. The operation is very simple, the required sample volume is small, and it has good Application prospect.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the implementation of the present invention. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. All the implementation manners cannot be exhaustively listed here. All obvious changes or variations derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims (5)

1. A group of primers utilizing pyrosequencing technology to detect channel catfish virus, characterized in that: including the pair of amplification primers shown in SEQIDNo.2 and SEQIDNo.3 in the sequence listing, and as shown in SEQIDNo.4 in the sequence listing sequencing primers. 2. A group of primers utilizing pyrosequencing technology to detect channel catfish virus according to claim 1, characterized in that: the 5' end of the primer shown in SEQ ID No.3 is labeled with biotin. 3. A kit for detecting channel catfish virus using pyrosequencing technology, characterized in that the kit comprises a set of primers for detecting channel catfish virus using pyrosequencing technology according to claim 1 or 2. 4. The kit according to claim 3, characterized in that, the kit also includes DNA extraction reagents, PCR reaction reagents, single-stranded template preparation reagents and pyrosequencing reagents. 5. A detection method utilizing pyrosequencing technology to detect channel catfish virus, characterized in that the method comprises: (1) extract the sample DNA to be tested as a template; (2) use the amplification primer described in claim 1 or 2 to carry out PCR amplification reaction; (3) If the channel catfish virus-specific PCR reaction is positive, then recover the PCR amplification product, prepare the pyrosequencing single-stranded template, and then use the sequencing primers described in claim 1 to carry out pyrosequencing, if the target sequence is the same as that in the sequence list If the specific target sequence shown in SEQIDNo.1 is consistent, the sample to be tested contains channel catfish virus; if the channel catfish virus-specific PCR reaction is negative, the sample is determined to be non-channel catfish virus.