CN113637797A - Micro-drop digital PCR method for detecting fish nervous necrosis virus and corresponding kit - Google Patents
Micro-drop digital PCR method for detecting fish nervous necrosis virus and corresponding kit Download PDFInfo
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Abstract
The invention belongs to the technical field of detection of fish nervous necrosis viruses, and particularly relates to a droplet type digital PCR method and a corresponding kit for detecting the fish nervous necrosis viruses, in order to develop a nervous necrosis virus detection method with higher sensitivity, the invention establishes a droplet type digital PCR method for detecting the fish nervous necrosis viruses, namely, after a blood sample of fish to be detected is collected, total RNA of the blood sample is extracted, and cDNA is synthesized; and then, cDNA is taken as a template, and the droplet type digital PCR is adopted to accurately detect whether the fish to be detected is infected with or carries NNV.
Description
Technical Field
The invention belongs to the technical field of detection of fish nervous necrosis viruses, and particularly relates to a droplet type digital PCR method for detecting fish nervous necrosis viruses and a corresponding kit.
Background
The fish Nervous Necrosis Virus (NNV) is a viral pathogen capable of infecting more than 40 important economic fishes such as sea bass, turbot, grouper and the like. NNV infection can cause epidemic viral encephalopathy and retinopathy of fish, the mortality rate of larval fish and juvenile fish is up to 100%, and the disease has been classified as an important fish disease by the International animal and epidemic Organization (OIE).
NNV viruses are classified as belonging to the Nodaviridae (Nodaviridae) β nodavirus (Betanodaviruses) as non-enveloped single-stranded positive-stranded icosahedral RNA viruses with diameters of 23-25 nm. The genome comprises two RNA molecules, RNA1 and RNA 2. Wherein RNA1 encodes an RNA-dependent RNA polymerase responsible for viral replication; RNA2 encodes the viral capsid protein CP, which assembles to form the viral coat. NNVs can be divided into four genotypes according to their regions of variation in the variable sequence of RNA 2: takifugu rubripes nervous necrosis virus (TPNNV), decapterus japonicus nervous necrosis virus (SJNNV), verasper moseri nervous necrosis virus (BFNNV), and red-spotted grouper nervous necrosis virus (RGNNV).
Despite the different genotypes of NNV, diseased fish after NNV infection typically exhibit signs of dark skin color, poor appetite, lethargy, inversion on the water surface or lying on the bottom, bradykinesia, altered swimming, horizontal rotation, floating on the water surface or spiral swimming, etc., with histological diagnosis showing extensive necrosis of the central nervous system, the presence of large numbers of viral particles in the cytoplasm of diseased nerve cells, extensive vacuolization of the midbrain, hindbrain and retina, and neuronal degeneration. NNV causes fish infectious diseases with strong pathogenicity, and causes huge economic loss for domestic and foreign aquaculture.
Currently, the detection of NNV mainly focuses on fluorescent quantitative PCR, but the method has limited sensitivity, is difficult to detect under the condition of low NNV copy number, and is easy to generate false negative. Therefore, it is necessary to develop a highly sensitive NNV detection method.
The research separates NNV from the diseased sea bass juvenile fish in Guangdong province, carries out whole gene sequencing on the NNV to obtain a complete NNV nucleic acid sequence, and establishes a method for detecting the NNV based on micro-drop digital PCR by designing a probe for NNV specific amplification. The method has small wound on cultured fishes, particularly parents, and high sensitivity, and the method is not reported in NNV detection.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the droplet type digital PCR method for detecting the fish nervous necrosis virus, whether the fish nervous necrosis virus is infected or carried by the fish nervous necrosis virus can be accurately detected by adopting a fish venous blood sample, the sensitivity is high, and the method has a better application prospect in breeding or selecting cultured fish parents.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a droplet type digital PCR method for detecting fish nervous necrosis virus, which comprises the following steps:
s1, collecting a blood sample of the fish to be detected;
s2, extracting total RNA by taking the blood sample of the step S1 as a template, and synthesizing cDNA;
s3, using the cDNA of the step S2 as a template to detect whether the NNV is infected or carried by the cDNA by adopting a droplet digital PCR method.
Experiments show that the microdroplet type digital PCR has higher sensitivity in NNV detection than RT-PCR, and the digital PCR and the RT-PCR show that in low-concentration NNV detection, when NNV copy number is low, the RT-PCR cannot be effectively detectedNNV in the sample was measured. Meanwhile, the micro-drop digital PCR can effectively detect the content of NNV in blood of the sea bass after being attacked by toxin, and NNV (2X 10) with different concentration titers can be detected3And 2X102 TCID50) Healthy sea bass species are injected into the abdominal cavity, tail vein blood collection is carried out after three days of toxicity attack, NNV can be detected in sea bass blood, and the copy number of the NNV in different concentration titer groups does not change in a linear trend. In addition, the micro-drop digital PCR can effectively detect whether the wild cultured sea bass parents carry viruses and the copy number of the viruses, blood is collected through the tail veins of the wild cultured sea bass parents, RNA is extracted and reverse transcription is carried out on the blood, and the micro-drop digital PCR detects that different parents carry NNV with different copy numbers.
Preferably, the primers used in the digital micro-droplet PCR are shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the probes used are shown in SEQ ID NO. 3.
Preferably, the reaction system of the droplet digital PCR is: mu.L Mix (2X), 2.4. mu.L forward primer, 2.4. mu.L reverse primer, 0.75. mu.L probe, 1. mu.L cDNA template, and 8.45. mu.L water to make up to 30. mu.L.
Furthermore, the concentration of the upstream primer and the concentration of the downstream primer are both 10 μ M, the concentration of the probe is 250nmol/L, and the concentration of the cDNA template is less than 50 ng/uL.
Preferably, the amplification reaction conditions of the droplet digital PCR are: pre-denaturation at 95 ℃ for 10 min; 39 cycles: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 15s, and ramp rate of 1.5 ℃/s.
Preferably, the fish species include, but are not limited to, farmed fish species and import-export frozen fish species.
The invention also provides a micro-drop digital PCR kit for detecting the fish nervous necrosis virus, which comprises specific primers and probes for detecting the fish nervous necrosis virus genome.
Preferably, the specific primers are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the probes are shown as SEQ ID NO. 3.
Preferably, the fish species include, but are not limited to, farmed fish species and import-export frozen fish species.
Preferably, the reaction system of the micro-drop digital PCR kit is as follows: mu.L Mix (2X), 2.4. mu.L forward primer, 2.4. mu.L reverse primer, 0.75. mu.L probe, 1. mu.L cDNA template, and 8.45. mu.L water to make up to 30. mu.L.
Furthermore, the concentration of the upstream primer and the concentration of the downstream primer are both 10 μ M, the concentration of the probe is 250nmol/L, and the concentration of the cDNA template is less than 50 ng/uL.
Preferably, the amplification reaction conditions of the digital micro-drop PCR kit are as follows: pre-denaturation at 95 ℃ for 10 min; 39 cycles: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 15s, and ramp rate of 1.5 ℃/s.
Preferably, the digital PCR kit further comprises a total RNA extraction reagent or kit, a reverse transcription reagent or kit.
Compared with the prior art, the invention has the beneficial effects that:
the method is used for developing a nervous necrosis virus detection method with higher sensitivity, is used for detecting whether fish parents carry nervous necrosis viruses, and provides reference for breeding or selecting high-quality fish parents. The invention establishes a droplet type digital PCR method for detecting the fish nervous necrosis virus, namely, after collecting a blood sample of fish to be detected, extracting total RNA of the blood sample and synthesizing cDNA; and then, cDNA is taken as a template, and the droplet type digital PCR is adopted to accurately detect whether the fish to be detected is infected with or carries NNV.
Drawings
FIG. 1 is a comparison of the accuracy and sensitivity of the droplet digital PCR and the fluorescent quantitative PCR;
in FIG. 1, A, B and C are copy numbers of NNV template after different fold-ratio dilutions (10 fold dilutions) for digital quantitative PCR detection; D. e and F are C after NNV template is diluted by different times (10 times) in fluorescent quantitative PCR detectiontThe value is obtained.
FIG. 2 is a graph of the detection of blood samples from Lateolabrax japonicus after NNV challenge by droplet digital PCR.
In FIG. 2, A and B are 2X10, respectively3 TCID50And 2x102 TCID50Toxic pathogen attacking the bloodThe number of copies of NNV detected in the sample; c and D are positive and negative controls, respectively.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 comparison of the sensitivity and accuracy of the droplet digital PCR with RT-PCR in NNV detection
(1) Design of micro-drop type digital PCR primer and probe and specificity verification thereof
The primers used in the experimental process are designed and optimized through Primer 5.0, and the 5 'end and the 3' end of the probe respectively carry a luminescent group and a quenching group. Firstly, downloading the gene group data of the sea bass (NCBI accession number: PRJNA408177), establishing a gene group database of the sea bass, and analyzing the NNV primer sequence designed by local BLAST without a complementary sequence in the genome of the sea bass. The method comprises the steps that a laboratory separates NNV virus from diseased sea bass juvenile fish in a Zhujiang mouth culture area in Guangdong province, RNA is extracted after the NNV virus infects brain cells of the sea bass, NNV template is obtained after reverse transcription, and genome segment of the NNV is inserted into a pcDNA3.1(Invitrogen) vector after PCR amplification to obtain pc-RDRP; sea bass fry is purchased from a farmer in the infanta area of Zhuhai city, Guangdong province, temporarily cultured in a laboratory for two weeks, intraperitoneally injected with NNV virus (MOI 100) after the sea bass fry is completely stabilized, three days after infection, different tissues are taken to extract RNA, and the RNA is used as a detection template after reverse transcription. Taking a pc-RDRP plasmid and an NNV infected sea bass tissue RNA reverse transcription sample as templates, firstly, carrying out amplification by using an upstream primer F (NNV-ddPCR-F) and a downstream primer R (NNV-ddPCR-R) (a reaction system is 1 mu L of NNV-ddPCR-F/R, 0.25 mu L of HS Taq enzyme (Takara), 2.5 mu L of Buffer, 2 mu L of dNTP, 1 mu L of template and 17.25 mu L of L H2O); then, the probe and the downstream primer R are used for amplification (a reaction system is 1 muL of probe and 1 muL of NNV-ddPCR-R, 0.25 muL of HS Taq enzyme, 2.5 muL of Buffer, 2 muL of dNTP, 1 muL of template and 17.25 muL of L H2O), and the two amplifications are single bands, thereby proving that the upstream primer F, the downstream primer R and the probe obtained by screening have good specificity. The specific primer and probe sequences used for detection are as follows:
upstream primer F- - -NNV-ddPCR-F: TGTGGCTACGAGCCATATGTC (SEQ ID NO: 1);
downstream primer R- - -NNV-ddPCR-R: ATCAACTCATGCATGTCCACGT (SEQ ID NO: 2);
and (3) probe: 5'-FAM-TTGGATACCACGGGTCACGTCAGT-3' -TAMRA (SEQ ID NO:3), wherein FAM (carboxyfluorescein) is a luminescent group and TAMRA (6-carboxytetramethylrhodamine) is a quenching group.
(2) Optimization of droplet type digital PCR detection conditions
Digital PCR system optimization was optimized from temperature, primer and probe concentrations. By optimizing conditions such as annealing temperature (62 ℃,60 ℃,58 ℃,56 ℃) and primer concentration (1000nmol/L,800nmol/L,600nmol/L), with a probe concentration of 250nmol/L (kit recommendation), we found that annealing temperature of 60 ℃ and primer concentration of 800nmol/L is the best experimental condition. The reaction system is as follows: mu.l Mix (2X), 2.4. mu.l forward primer F (10. mu.M, see example 1 for sequence), 2.4. mu.l reverse primer R (10. mu.M, see example 1 for sequence), 0.75. mu.l probe (see example 1 for sequence), 1. mu.l cDNA template (template <50ng/uL), make up to 30. mu.l with 8.45. mu.l water. The amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; 39 cycles: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 15s, and ramp rate of 1.5 ℃/s. The universal kit for micro-droplet detection is purchased from manufacturing technologies (Beijing) Co., Ltd., product catalog number (10002).
(3) And (3) detecting NNV genome plasmids with different dilution times by using micro-droplet digital PCR and RT-PCR.
Plasmids of NNV genomic fragments were diluted 10, 100, 1000 and 10000 times and their copy number or amplification curves were examined by digital PCR and RT-PCR. When diluted 10 times, the digital PCR detection result is found to be full positive, which indicates that the plasmid concentration is too high and exceeds the maximum threshold value of the detection; when the plasmid is diluted 100 times, both the droplet digital PCR and the fluorescent quantitative PCR can accurately detect the NNV template (shown in FIGS. 1A and D); when the plasmid is diluted to be more than 1000 times or 10000, the amplification cycle number of the RT-PCR is over 35, the RT-PCR cannot effectively detect the NNV in the sample, and the copy number of the NNV in the sample can still be accurately detected by the digital PCR (shown in FIG. 1B, C, E and F).
Therefore, when the NNV with low concentration is detected by the digital PCR and the RT-PCR, the RT-PCR can not effectively detect the NNV in the sample when the copy number of the NNV is lower, which shows that the micro-drop digital PCR has higher sensitivity in NNV detection than the RT-PCR.
Example 2 detection of NNV-challenged Lateolabrax japonicus blood sample and wild cultured Lateolabrax japonicus parent blood sample by micro-droplet digital PCR
(1) Laboratory sea bass toxin counteracting
NNV is separated from diseased sea bass juvenile fish in a Zhujiang mouth culture area in Guangdong province, NNV is infected with brain cells of the sea bass, repeated freeze thawing is carried out for three times at minus 80 ℃, NNV suspension is obtained after centrifugation and filtration through a 0.22 mu m filter membrane, and the prepared NNV suspension is stored at minus 80 ℃ for standby. 100 sea bass species were purchased in the glottis of the pearl sea, weighing about 40g, and were first tested to be free of NNV virus. The feed is normally fed in a laboratory circulating water culture for two weeks, and is fed twice a day. Two weeks later, the sea bass was anesthetized with eugenol, and the prepared NNV suspension was injected into the chest of the sea bass at the pectoral fin. Two experimental groups, each injected with 2x10, and one control group were set up3 TCID50And 2x102 TCID50NNV virus suspension, control group injected with PBS, and 10 fish per group. Feeding and culturing normally after injection.
(2) Sampling for field culture of sea bass
The wild sea bass parents are cultured in a pond of a sluice river mouth fishery research institute base in the Zhuhai city of Guangdong province for 2-3 years, the wild sea bass parents are fed twice a day during the culture period, an oxygenator is arranged in the pond, and the culture conditions are good. Capturing 12 jewfish parents in a culture pond, placing the jewfish parents in a temporary culture pond, and anesthetizing the jewfish parents with eugenol.
(3) Blood sample collection and sample total RNA extraction
After the jewfish is anesthetized by eugenol, the tail part of the jewfish is wiped by an alcohol cotton ball for body surface disinfection, blood is collected through a tail vein of a 30-gauge needle inclined at 45 ℃, and the blood drawing position is pressed by a sterile cotton ball for about 20s after blood collection. Extracting 5-8 drops of blood, violently shaking and cracking in 800 mu L Trizol, adding 150 mu L chloroform, shaking and uniformly mixing, standing at room temperature for 3min, and centrifuging at 4 ℃ for 15min under 12000 rmp; taking 300 mu L of supernatant, adding isopropanol with the same amount into a centrifugal tube without RNase, gently mixing, standing at room temperature for 10min, and centrifuging at 4 ℃ for 15min under 12000 rmp; removing supernatant, adding 75% ethanol prepared from 700 μ L EDTA water, and centrifuging at 7500rmp and 4 deg.C for 5 min; removing supernatant, air drying RNA at room temperature and precipitating for 10 min; dissolving the RNA precipitate with 8 μ L of nucleic acid-free water to obtain total RNA of the cell, and measuring the concentration of the total RNA.
(4) cDNA Synthesis
Using GoScriptTMAnd carrying out RNA reverse transcription by using the reverse transcription kit. The reaction system is as follows: a premix containing 4. mu.L of Reaction Buffer and 2. mu.L of Enzyme Mix was prepared, and 1. mu.g of total RNA template was added to the mixture according to the RNA concentration of each blood sample, and the mixture was made up to 20. mu.L with non-nucleic acid water. The reverse transcription reaction procedure was as follows: reacting 20 mu L of reaction system at 42 ℃ for 50 min; then reacting at 85 ℃ for 5min, and storing at 4 ℃ to obtain the cDNA.
(5) Real-time fluorescent quantitative PCR
Promega GoTaq was usedTMAnd carrying out real-time fluorescent quantitative PCR by using the qPCR kit. The reaction system is as follows: mu.L of qPCR Mix (2X), 0.25. mu.L of NNV-ddPCR-F forward primer (10. mu.M), 0.25. mu.L of NNV-ddPCR-R reverse primer (10. mu.M), 1. mu.L of cDNA template, and 3.5. mu.L of water to make up to 10. mu.L. The amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; 42 cycles of: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 15 s.
(6) Droplet digital PCR
The micro-drop digital PCR was performed using a sample preparation universal kit and a micro-drop detection universal kit (New chess manufacturing technologies, Inc.). The reaction system is as follows: mu.L Mix (2X), 2.4. mu.L forward primer (10. mu.M), 2.4. mu.L reverse primer (10. mu.M), 0.75. mu.L probe, 1. mu.L cDNA template (template <50ng/uL), make up to 30. mu.L with 8.45. mu.L water. The amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; 39 cycles: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 15s, and ramp rate of 1.5 ℃/s.
(7) Micro-drop digital PCR detection of toxic-attacking jewfish and field-cultured jewfish parent NNV
After NNV diluted by different times is adopted for toxicity attack, the NNV content in blood can be effectively detected in the toxicity-attacked jewfish through the micro-drop digital PCR, and the copy number of the NNV in the blood sample is not changed proportionally, and the NNV probably proliferates continuously after entering a jewfish body (as shown in figure 2).
In the field test of sea bass parents, only one of the 12 parents tested was found to carry no NNV virus, and the copy numbers of the other parents carrying NNV virus were also inconsistent (as shown in Table 1).
The results show that the micro-drop digital PCR can effectively detect the NNV content in the blood of the sea bass.
TABLE 1 micro-drop digital PCR detection of whether field cultured sea bass parents carry NNV
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
<110> university of Zhongshan; the modern agriculture development center of the Zhuhai city (management committee of the Industrial park of Taiwan farmers in the gold gulf region of the Zhuhai city, scientific research and promotion center of the agricultural fishery of the Zhuhai city)
<120> droplet type digital PCR method for detecting fish nervous necrosis virus and corresponding kit
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> upstream primer F (artificial sequence)
<400> 1
tgtggctacg agccatatgt c 21
<210> 2
<211> 22
<212> DNA
<213> downstream primer R (Artificial sequence)
<400> 2
atcaactcat gcatgtccac gt 22
<210> 3
<211> 24
<212> DNA
<213> Probe (Artificial Sequence)
<220>
<223> 5'- FAM- TTGGATACCACGGGTCACGTCAGT-3'- TAMRA
<400> 3
ttggatacca cgggtcacgt cagt 24
Claims (9)
1. The droplet type digital PCR method for detecting the fish nervous necrosis virus is characterized by comprising the following steps of:
s1, collecting a blood sample of the fish to be detected;
s2, extracting total RNA by taking the blood sample of the step S1 as a template, and synthesizing cDNA;
s3, using the cDNA of the step S2 as a template to detect whether the NNV is infected or carried by the cDNA by adopting a droplet digital PCR method.
2. The method for detecting the fish nervous necrosis virus (NENV) according to claim 1, wherein primers used in the micro-droplet digital PCR are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and a probe used in the micro-droplet digital PCR is shown as SEQ ID NO. 3.
3. A micro-drop digital PCR kit for detecting the fish nervous necrosis virus is characterized by comprising specific primers and probes for detecting the fish nervous necrosis virus genome.
4. The micro-drop digital PCR kit for detecting the fish nervous necrosis virus according to claim 3, wherein the specific primers are shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the probe is shown as SEQ ID NO. 3.
5. The digital PCR kit of claim 3, wherein the fish includes but is not limited to farmed fish and import-export frozen fish.
6. The micro-drop digital PCR kit for detecting the fish nervous necrosis virus according to claim 3, wherein the reaction system of the micro-drop digital PCR kit is as follows: mu.L Mix (2X), 2.4. mu.L forward primer, 2.4. mu.L reverse primer, 0.75. mu.L probe, 1. mu.L cDNA template, and 8.45. mu.L water to make up to 30. mu.L.
7. The micro-droplet digital PCR kit for detecting the fish nervous necrosis virus according to claim 6, wherein the concentration of the upstream primer and the concentration of the downstream primer are both 10 μ M, the concentration of the probe is 250nmol/L, and the concentration of the cDNA template is less than 50 ng/uL.
8. The digital PCR kit for detecting the fish nervous necrosis virus according to claim 3, wherein the amplification reaction conditions of the digital PCR kit are as follows: pre-denaturation at 95 ℃ for 10 min; 39 cycles: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 1min, extension at 72 ℃ for 15s, and ramp rate of 1.5 ℃/s.
9. The digital PCR kit for detecting the fish nervous necrosis virus according to claim 3, wherein the digital PCR kit further comprises a total RNA extraction reagent or kit, a reverse transcription reagent or kit.
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