CN111485035A - Fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis viruses of mandarin fish and corresponding kit - Google Patents
Fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis viruses of mandarin fish and corresponding kit Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and a corresponding kit. The invention skillfully applies specific gene detection to distinguish the mandarin fish infectious spleen and kidney necrosis virus from other species of strains or viruses, and obtains accurate genus information through comprehensive judgment. Compared with the existing mainstream detection kit, the kit for detecting the infectious spleen and kidney necrosis virus of the mandarin fish has the advantages of high sensitivity, rapidness, convenience, good specificity, rigorous and accurate judgment and the like, and has good application prospect and market value.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a method for carrying out fluorescent quantitative PCR (polymerase chain reaction) detection on infectious spleen and kidney necrosis viruses of mandarin fish through specific genes and a corresponding detection kit.
Background
Infectious Spleen and Kidney Necrosis Virus (ISKNV) belongs to the genus of cytomegaviruses in the iridoviridae family, mature virions are icosahedral, have a diameter of 100nm-150nm, have a double-stranded DNA genome, and generally consist of an envelope, an electron-dense region and a core. The mandarin fish invades the body of the mandarin fish by endocytosis and adheres to spleen tissue cells, so that a large number of alkalophilic and cytoplasm-homogenized swelling cells with the diameter of about 15-20 mu m appear in organs.
Siniperca chuatsi mandarin fish has tender meat and delicious taste, is one of high-quality freshwater fish varieties in China, the mandarin fish breeding industry gradually develops and grows since the artificial mandarin fish breeding technology is successful, however, as the mandarin fish breeding scale and density are increased year by year, diseases of the mandarin fish are increased, particularly infectious spleen and kidney necrosis viruses are prevalent, the diseases are main diseases in mandarin fish breeding, the incidence rate is over 60 percent, the death rate is over 50 percent, and the healthy development of the mandarin fish industry is seriously influenced.
The head, the lower jaw, the gill cover, the abdomen, the tail fin and other parts of the mandarin fish infected with infectious spleen and kidney necrosis virus have bleeding points, mucus on the gill cover is increased, spleen is enlarged, congestion and necrosis occur, cavities appear, a large amount of white blood cells infiltrate into the cavities, and vascular bulbs of renal tubules and renal sacs shrink, so that anemia and multiple organs of diseased fish die due to exhaustion.
At present, the detection method of ISKNV comprises a pathological section method, an electron microscope observation method, PCR detection and a nucleic acid probe in-situ hybridization method, but the method has complex operation and long time consumption, can only confirm diagnosis in a molecular laboratory generally, and cannot meet the requirements of quick, simple and sensitive detection. The fluorescent quantitative PCR detection method has strong specificity and high sensitivity, can detect diseases qualitatively or quantitatively, is the most accurate and rapid disease detection means which is generally accepted at present, does not establish a systematic QPCR (quantitative polymerase chain reaction) diagnosis technology of ISKNV aiming at the conserved sequence of ISKNV at present, finds three conserved sequences ATPase, MCP and IRB6 by comparing and analyzing the ISKNV whole genome sequence, designs a primer probe with high specificity, detects ISKNV qualitatively or quantitatively, and has important significance for diagnosis and prevention and control of mandarin fish pathogens.
Disclosure of Invention
One of the purposes of the invention is to provide a fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish.
Specifically, the method comprises the following steps:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting specific genes ATPase, MCP and IRB6 genes of the infectious spleen and kidney necrosis viruses of the mandarin fish by utilizing fluorescent quantitative PCR;
s4, reading the Ct value of the amplification; when the Ct value of at least 1 gene in the specific genes of the infectious spleen and kidney necrosis viruses of the mandarin fish is less than 35, the detection result of the infectious spleen and kidney necrosis viruses of the mandarin fish is positive; and when the Ct values of the specific genes ATPase, MCP and IRB6 of the infectious spleen and kidney necrosis viruses of the mandarin fish are all larger than 35, the detection result of the infectious spleen and kidney necrosis viruses of the mandarin fish is negative.
As a preferred technical scheme, a detection primer of the mandarin fish infectious spleen and kidney necrosis virus specific gene ATPase is shown as SEQ ID NO 1-2;
SEQ ID NO:1(5’-GCGGCAAGTCGGTGCTA-3’)；
SEQ ID NO:2(5’-CGGCCGCGGGAATTA-3’)。
the sequence of the TaqMan probe is shown in SEQ ID NO. 3;
SEQ ID NO:3(5’-FAM-ATCATTGCGGCCAAGCGGCAC-BHQ1-3’)；
the detection primer of the mandarin fish infectious spleen and kidney necrosis virus specific gene MCP is shown as SEQ ID NO of 4-5;
SEQ ID NO:4(5’-GCAATCTCAGGTGCAAACGTAA-3’)；
SEQ ID NO:5(5’-GGGTCTCCATCGCATCAAA-3’)。
the sequence of the TaqMan probe is shown as SEQ ID NO. 6;
SEQ ID NO:6：5’-FAM-CAGCGGGTTCATCGACATCTCCG-BHQ1-3’。
the detection primer of the mandarin fish infectious spleen and kidney necrosis virus specific gene IRB6 is shown as SEQ ID NO. 7-8;
SEQ ID NO:7(5’-TGTCATCGTAGTCGTCCATTCC-3’)；
SEQ ID NO:8(5’-CGACAGACACCGATGACGTT-3’)。
the sequence of the TaqMan probe is shown as SEQ ID NO. 9;
SEQ ID NO:9(5’-FAM-CTGCCCCCATCGTCAAGCAGTGT-BHQ1-3’)。
as a preferred technical scheme, the reaction system for the fluorescent quantitative PCR detection is 25 mu l, and comprises 12.5 mu l of 2 × TaqPCR Mix, 1 mu l of total10uM Primers Mix, 0.5 mu l of TaqMan probe, 2 mu l of DNA input, H2O 9μl。
As a preferred technical scheme, the reaction conditions of the fluorescent quantitative PCR detection are pre-denaturation at 95 ℃ for 2min30s, denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 30s, fluorescent signal collection and 40 cycles.
The invention also aims to provide a fluorescent quantitative PCR kit for detecting the infectious spleen and kidney necrosis viruses of the mandarin fish, which comprises detection primers of the specific genes ATPase, MCP and IRB6 of the infectious spleen and kidney necrosis viruses of the mandarin fish and a TaqMan probe.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the method, an orphan gene capable of distinguishing the infectious spleen and kidney necrosis viruses of the mandarin fish and the strains and viruses of other species is excavated from a large amount of early research data on the infectious spleen and kidney necrosis viruses of the mandarin fish and the strains and viruses of other species, whether the infectious spleen and kidney necrosis viruses of the mandarin fish are contained in other strains or virus positive samples and environmental samples or not is accurately judged by detecting the specific gene, the early large data mining and the comparison between different species are based, and the selected specific gene has the specificity of species and genus.
(2) In the invention, by optimizing the design conditions and the experimental conditions, the primers which are optimized for each gene detection amplification condition are selected as the detection primers, so that the amplification efficiency of the primers is improved, the detection primers with stability, high efficiency, high sensitivity and reproducibility can be achieved, and the correct detection of a trace sample is realized.
(3) Compared with other detection methods in the market, the detection method provided by the invention has the advantages that the infection of the siniperca chuatsi infectious spleen and kidney necrosis virus and the infection of other viruses or strains can be intuitively judged, and the result is more rigorous and accurate.
(4) The invention can realize that the positive sample is from 101Diluting to 10 times5The detection range of the dilution is doubled, the sensitivity is high, the application range is wide, a sample with higher concentration can be detected through dilution conversion, the detection result is stable, and the reproducibility is good.
(5) The detection method can be applied to screening and detecting samples from various aspects such as culture water body samples or mandarin fish samples and the like, accurately judges whether the culture water body samples contain the infectious spleen and kidney necrosis viruses of the mandarin fish, and has convenient, rapid and sensitive operation. The detection kit can be applied to the rapid detection of various culture samples and culture environment samples, does not need additional microbial culture, enables gene detection to be applied to sample detection and culture production, and has the advantages of high sensitivity, strong operability, rapidness, high efficiency and the like.
Drawings
FIG. 1 is a ATPase gene detection primer standard curve.
The MCP gene detection primer standard curve of fig. 2.
FIG. 3 is a standard curve of primers for detecting IRB6 gene.
FIG. 4 is the amplification curve of the ATPase gene in example 1.
FIG. 5 is a curve of MCP gene amplification in example 1.
FIG. 6 is the IRB6 gene amplification curve in example 1.
FIG. 7 is the ATPase gene amplification curve in example 2.
FIG. 8 is the MCP gene amplification curve in example 2.
FIG. 9 is the IRB6 gene amplification curve in example 2.
FIG. 10 is an amplification curve of three genes specifically tested in example 3.
FIG. 11 is the amplification curve of the ATPase gene at different dilution ratios in example 4.
FIG. 12 is an amplification curve of MCP gene at different dilution ratios in example 4.
FIG. 13 is the amplification curve of IRB6 gene at different dilution ratios in example 4.
Detailed Description
In order to make the technical contents of the present invention more clearly understandable, the following examples are described in detail with reference to the accompanying drawings, it being understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.
The following reagents used in the present invention can be purchased from conventional sources.
TABLE 1
In some embodiments, positive standard plasmids are constructed using the amplification products, and a standard curve of amplification of the primers is detected and plotted to obtain the amplification efficiency of each pair of detection primers, and the amplification efficiency and confidence level of the primer sequences provided by the invention are optimized.
In some embodiments, by detecting specific genes of different positive strains, samples infected by the siniperca chuatsi infectious spleen and kidney necrosis virus and samples infected by other species of bacteria and viruses can be correctly distinguished, so that the siniperca chuatsi infectious spleen and kidney necrosis virus can be accurately detected and judged.
In some embodiments, by pair 10110 times of210 times of310 times of4Multiple and 105The method can accurately detect the infectious spleen and kidney necrosis viruses of the mandarin fish under different sample concentrations by extracting and detecting double samples, and can ensure that positive samples are rareChinese medicine powder 105The detection can still be accurately carried out, and the microorganism sample with higher titer can be also accurately detected by diluting the microorganism sample to a detection interval.
In some embodiments, pure water, healthy mandarin fish, mandarin fish samples in the pearl sea and mandarin fish culture water samples are collected, and detection of specific genes can quickly and accurately detect whether the samples contain the infectious spleen and kidney necrosis viruses of the mandarin fish, so that convenience and rapidness are realized.
It will be understood by those skilled in the art that the genes to be detected and the specific primers to be designed in the method can be designed to achieve the corresponding detection purpose through the design of other sections of the same detection object.
The fluorescent quantitative PCR method and the kit for detecting the infectious spleen and kidney necrosis virus of the mandarin fish, which are described by the invention, can be widely applied to a plurality of inspection and quarantine categories such as mandarin fish sample detection, culture water body detection and the like, and can provide a convenient, rapid, accurate and efficient monitoring product based on gene detection for mandarin fish culture, propagation and the like.
Example 1 Standard Curve for primer amplification detection and plasmid sensitivity test
1. Cultivation of microorganisms
The L B culture medium is used as a culture medium of mandarin fish infectious spleen and kidney necrosis virus plasmids, the mandarin fish infectious spleen and kidney necrosis virus plasmids grow well in a plate with the pH value of 7.0-7.4, the colony diameter on the plate is 2mm, and the plate is round, smooth and transparent.
2. Genomic DNA extraction
Extraction of genomic DNA was performed according to the QIAamp DNA Mini Kit instructions from Qiagen.
3. Standard quality particle preparation of amplification product
DNA extracted from mandarin fish infectious spleen and kidney necrosis virus is amplified with ATPase, MCP and IRB6 gene primer separately, the amplified product is treated with A, T4 ligase is used to ligate into T vector, and the plasmid is amplified in large scale after transduction of competent cell. The primer sequences and TaqMan probe sequences of the 3 genes are shown in SEQ ID NO 1-9, and are specifically shown in Table 2.
TABLE 2
4. Plasmid extraction and Standard Curve gradient configuration
Respectively plating standard clone bacteria of primer amplification products corresponding to ATPase, MCP and IRB6 genes, collecting monoclonal bacterial plaques, culturing and shaking bacteria through L B for overnight amplification, finally obtaining 5ml of Plasmid positive strains in exponential growth phase, extracting Plasmid DNA according to instructions of QIAGEN Plasmid Midi Kit, detecting the concentration of the Plasmid DNA after extraction, and configuring 7 standard products with concentration gradients of 1 fg/mu L and 1 × 10 by a 10-fold dilution method, wherein the concentration gradients are respectively 1 fg/mu L and 1 × 101fg/μL，1×102fg/μL，1×103fg/μL，1×104fg/μL，1×105fg/μL，1×106fg/μL。
5. Drawing a standard curve of the detection primer
And (3) performing qPCR amplification of a concentration gradient standard curve according to the instruction of the TaKaRa Taq PCR Mix instruction, and drawing a corresponding amplification curve to obtain the amplification efficiency of each pair of primers. 3 biological repetitions and 3 technical repetitions are set for each qPCR reaction, the qPCR reaction system and amplification reaction conditions are shown in Table 3, and the drawn standard curves are shown in FIGS. 1-3.
TABLE 3
6. Analysis of results
The result of the detection primer amplification standard curve shows that primers corresponding to ATPase, MCP and IRB6 genes are optimized primer design and a reaction system. Linear standard curve confidence R2>0.990, the primer design and the reaction system were confirmed to be optimal. Analysis of 3 genesThe amplification curve of (1), wherein 1 × 101fg/μL～1×106CT values measured under the 6 concentrations of fg/mu L are all less than 35 cycles and all meet the requirements of an optimal state, 3 pairs of primers can meet the detection requirements under the reaction conditions, which shows that the plasmid sensitivity of the method is high, and the variation coefficient among the CT values measured by 3 technical repeated experiments is less than 5.0 percent, which proves that the method has high repeatability, the detection results are shown in tables 4-6 and figures 4-6, figures 4-6 are amplification curves of three genes under different plasmid concentrations, and the concentrations of the amplification curves from left to right are 1 × 10 in sequence6fg/μL，1×105fg/μL，1×104fg/μL，1×103fg/μL，1×102fg/μL，1×101fg/μL，1×100fg/μL。
TABLE 4
TABLE 5
TABLE 6
Example 2 plasmid reproducibility test
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
qPCR detection of 3.3 target genes
The qPCR amplification of the samples was performed according to the instructions of TaKaRa Taq PCR Mix, and Ct value readings corresponding to each pair of primers were obtained, the reaction set for qPCR of this experiment was 2 concentrations of 1 × 10 respectively6fg/. mu. L and 1 × 105fg/μ L, 4 biological replicates and 5 technical replicates were set at each concentration, respectively, and the qPCR reaction system and amplification reaction conditions are shown in table 7.
TABLE 7
4. Analysis of results
In the embodiment, correlation coefficients and credibility of amplification curves of 3 genes and amplification efficiency under 2 concentrations are analyzed, CT values of measurement are less than 35 cycles and all meet the requirement of an optimal state, 3 pairs of primers can meet the detection requirement under the reaction condition, 4 groups are set for each concentration condition, each group is repeatedly measured for 5 times, the measurement result shows that the variation coefficients are less than 5.0 percent, the plasmid repeatability of the method is good, the stability is high, the detection results are shown in tables 8-10 and in figures 7-9, figures 7-9 are amplification curves of repeated experiments under different plasmid concentrations of three genes, the concentration of the amplification curves from left to right is 1 × 10 in sequence6fg/. mu. L and 1 × 105fg/μL。
TABLE 8
TABLE 9
EXAMPLE 3 Positive and negative sample detection
1. Cultivation of microorganisms
The same as in example 1.
2. Genomic DNA extraction
The same as in example 1.
qPCR detection of 3.3 target genes
qPCR amplification of the samples was performed as indicated by TaKaRa Taq PCR Mix instructions and Ct value readings were obtained for each pair of primers. 3 biological replicates and 3 technical replicates were set for each qPCR reaction, and the qPCR reaction system and amplification reaction conditions were the same as in example 2.
4. Analysis of results
As shown in table 11 and fig. 10, the qPCR amplification results with 3 pairs of primers showed that the mandarin fish infectious splenorenal necrosis virus showed all positive results for ATPase, MCP and IRB6 genes, while all other viruses or genera showed negative results. Therefore, the detection of ATPase, MCP and IRB6 genes is carried out on the infectious spleen and kidney necrosis viruses and other viruses or genera of the mandarin fish, so that the infectious spleen and kidney necrosis viruses and other viruses or genera of the mandarin fish can be better distinguished. In conclusion, the corresponding primers of ATPase, MCP and IRB6 genes can meet the detection and judgment of the infectious spleen and kidney necrosis virus of the mandarin fish. In FIG. 1, the amplification curves are, from left to right, the MCP gene, IRB6 gene and ATPase gene in this order.
TABLE 11
EXAMPLE 4 sensitivity of Positive samples
1. Sample collection and processing
Respectively collecting healthy mandarin fish samples and infectious spleen and kidney necrosis positive samples according to the ratio of 10110 times of210 times of310 times of4Multiple and 105The respective dilutions were performed in a gradient and the genomic DNA was extracted as indicated in the QIAampDNA Mini Kit instruction from Qiagen.
2. Genomic DNA extraction
The same as in example 1.
qPCR detection of 3.3 target genes
The same as in example 1.
4. Analysis of results
As shown in Table 12 and FIGS. 11-13, qPCR amplification with 3 pairs of primersThe results show that from 10110 times of210 times of310 times of4Multiple and 105In the range of the dilution sample, the mandarin fish infectious spleen and kidney necrosis virus shows positive results of ATPase, MCP and IRB6 genes. Therefore, the detection of the ATPase, MCP and IRB6 genes can be preferably performed to 10 degrees1Multiple to 105And detecting the mandarin fish infectious spleen and kidney necrosis virus in a dilution range. Simultaneously, diluting 10 from the sample1Multiple to 105In the double range, the detection of the infectious spleen and kidney necrosis viruses of the mandarin fish in the sample can be better completed through the detection of ATPase, MCP and IRB6 genes, and the detection range can be extended to 10 dilution of the sample1Multiple to 105And the higher strain concentration in the sample can be diluted to be within the optimal detection range by a dilution method, so that the expected detection requirement can be met.
In fig. 11 to 13, fig. 11 to 13 are amplification curves of three genes at different positive sample concentrations, and the dilution times of the amplification curves from left to right are as follows: 10110 times of210 times of310 times of4Multiple and 105And (4) doubling.
TABLE 12
EXAMPLE 5 detection of environmental samples
1. Environmental sample collection and extraction of genomic DNA
Respectively collecting pure water samples, healthy mandarin fish samples, a plurality of mandarin fish samples in the Zhuhai and a plurality of mandarin fish culture water samples, and extracting genome DNA according to the instruction of QIAamp DNA Mini Kit of Qiagen.
qPCR detection of 2.3 target genes
The same as in example 2.
3. Analysis of results
As shown in Table 13, the results of the 3-primer qPCR amplification showed that ATPase, MCP and IRB6 genes were detected in all samples except pure water samples, healthy mandarin fish samples, culture water samples and a mandarin fish sample from the place of the Zhuhai, and it was confirmed that the corresponding sample source contained the infectious spleen and kidney necrosis virus of mandarin fish. Finally, the criterion for judging the mandarin fish infectious spleen and kidney necrosis virus is that Ct values obtained by detecting 1 or more than 1 of ATPase, MCP and IRB6 genes are less than 35 cycles. If the Ct value obtained by detecting ATPase, MCP and IRB6 genes is more than 35 cycles, the mandarin fish infectious spleen and kidney necrosis virus is judged to be negative and not detected.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangdong Meige Gene science and technology Co., Ltd
<120> fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish and corresponding kit
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<211>23
<212>DNA
<213> MCP Probe (Artificial Sequence)
<400>6
cagcgggttc atcgacatct ccg 23
<210>7
<211>22
<212>DNA
<213> IRB6 Forward primer (Artificial Sequence)
<400>7
tgtcatcgta gtcgtccatt cc 22
<210>8
<211>20
<212>DNA
<213> IRB6 reverse primer (Artificial Sequence)
<400>8
<210>9
<211>23
<212>DNA
<213> IRB6 Probe (Artificial Sequence)
<400>9
ctgcccccat cgtcaagcag tgt 23
Claims (8)
1. The fluorescent quantitative PCR method for detecting the infectious spleen and kidney necrosis virus of the mandarin fish is characterized by comprising the following steps of:
s1, collecting a sample;
s2, extracting genome DNA;
s3, detecting specific genes ATPase, MCP and IRB6 genes of the infectious spleen and kidney necrosis viruses of the mandarin fish by utilizing fluorescent quantitative PCR;
s4, reading the Ct value of the amplification; when the Ct value of at least 1 gene in the specific genes of the infectious spleen and kidney necrosis viruses of the mandarin fish is less than 35, the detection result of the infectious spleen and kidney necrosis viruses of the mandarin fish is positive; and when the Ct values of the specific genes ATPase, MCP and IRB6 of the infectious spleen and kidney necrosis viruses of the mandarin fish are all larger than 35, the detection result of the infectious spleen and kidney necrosis viruses of the mandarin fish is negative.
2. The fluorogenic quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish of claim 1, wherein said step S2 further comprises the step of designing detection primers for ATPase, MCP and IRB6 genes and TaqMan probe.
3. The fluorescent quantitative PCR method for detecting the infectious spleen and kidney necrosis virus of mandarin fish of claim 2, wherein a detection primer of the specific gene ATPase of the infectious spleen and kidney necrosis virus of mandarin fish is shown as SEQ ID NO. 1-2, and a TaqMan probe sequence is shown as SEQ ID NO. 3.
4. The fluorescent quantitative PCR method for detecting the infectious spleen and kidney necrosis virus of mandarin fish of claim 1, wherein a detection primer of the specific gene MCP of the infectious spleen and kidney necrosis virus of mandarin fish is shown as SEQ ID NO. 4-5, and a TaqMan probe sequence is shown as SEQ ID NO. 6.
5. The fluorescent quantitative PCR method for detecting infectious spleen and kidney necrosis viruses of mandarin fish of claim 1, wherein a detection primer of the specific gene IRB6 of the infectious spleen and kidney necrosis viruses of mandarin fish is shown as SEQ ID NO. 7-8, and a TaqMan probe sequence is shown as SEQ ID NO. 9.
6. The fluorogenic quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish of claim 1, wherein the reaction system of the fluorogenic quantitative PCR detection is 25 μ l, comprising 2 × Taq PCR Mix 12.5 μ l, total10uM Primers Mix 1 μ l, TaqMan probe 0.5 μ l, DNA input 2 μ l, H2O 9μl。
7. The fluorogenic quantitative PCR method for detecting infectious spleen and kidney necrosis virus of mandarin fish of claim 1, wherein the reaction conditions of the fluorogenic quantitative PCR detection are pre-denaturation at 95 ℃ for 2min30s, denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 30s, and collection of fluorescent signals for 40 cycles.
8. A fluorescent quantitative PCR kit for detecting mandarin fish infectious spleen and kidney necrosis virus is characterized by comprising detection primers and TaqMan probes of specific genes ATPase, MCP and IRB6 of the mandarin fish infectious spleen and kidney necrosis virus.
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| CN117106980A (en) * | 2023-09-05 | 2023-11-24 | 长江大学 | Primer group, kit and method for detecting Infectious Spleen and Kidney Necrosis Virus (ISKNV) |
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