CN108504783A - A kind of detection method of grass carp reovirus - Google Patents

A kind of detection method of grass carp reovirus Download PDF

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CN108504783A
CN108504783A CN201810580424.8A CN201810580424A CN108504783A CN 108504783 A CN108504783 A CN 108504783A CN 201810580424 A CN201810580424 A CN 201810580424A CN 108504783 A CN108504783 A CN 108504783A
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grass carp
carp reovirus
reovirus
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吴明林
崔凯
李海洋
李文娟
蒋阳阳
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Fisheries Research Institute of Anhui AAS
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Fisheries Research Institute of Anhui AAS
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Abstract

The present invention relates to a kind of detection methods of grass carp reovirus.The present invention uses atraumatic technique, and, to grass carp without any wound, the welfare of this aquatic livestock of grass carp is effectively increased without slaughtering grass carp from grass carp tail vein acquisition blood as grass carp reovirus detection sample；The present invention chooses I type virus S8 segment portion nucleotide sequences, II types, type III virus S10 segment portion nucleotide sequences are as amplification target, design has synthesized the nest-type PRC primer of grass carp reovirus, respectively 3 pairs of grass carp reovirus Outside primers and 3 pairs of grass carp reovirus inner primers, totally 6 pairs of primers.I type virus S8 segments, II types, type III virus S10 segments are non-homogeneous segment, can effectively avoid false positive caused by non-specific amplification.The present invention can be detected low-abundance virus or a small amount of sample, and compared with single PCR, specificity is stronger, and required sample is less.

Description

A kind of detection method of grass carp reovirus
Technical field
The invention belongs to viral molecular biology detection fields, and in particular to a kind of detection side of grass carp reovirus Method.
Background technology
Grass carp (Ctenopharyngodon idellus) is one of the principal item of CHINESE FRESHWATER cultivation, and cultured output is First of all cultured fishes.Grass carp cultivation is made that weight for the guarantee agricultural products in China market supply and the raising of living standards of the people It contributes.However, grass carp is also a kind of fish that disease is the most multiple in aquiculture animal, by grass carp reovirus Hemorrhagic disease of grass carp caused by (Grass carp reovirus, GCRV) infection is disease most commonly seen in grass carp cultivation, is become One of distinct issues the most in grass carp cultivation.Hemorrhagic disease of grass carp period of disease is long, and infectiousness is strong, and prevention is difficult, does not prevent in advance such as Control, once breaking out, death rate of the onset is up to 80% or more, thus economic loss caused by disease, per every year at 1,000,000,000 yuan or more.Grass Fish reovirus is in addition to it can infect grass carp, moreover it is possible to infect pseudorasbora parva, black carp, silver carp etc., harm is wide, has seriously affected China's water Produce the sound development of aquaculture.
Grass carp reovirus different plant type is in virulence level, cell culture characteristic, challeng and to the cause of grass carp Sick power etc. all has larger otherness, and huge difficulty is brought to the prevention of the virus.According to existing separation strains sequence Column information carries out nucleotide sequence and generally divides with amino acid alignment and structure Phylogenetic analysis, institute's toxic strain For 3 genotype (I type, II type, III type).Grass carp reovirus different genotype, the independent infection having, some mixing senses Dye.When carrying out hemorrhagic disease of grass carp prevention, due to lacking the tracking and monitoring of cause of disease, blindness prevention and treatment is extremely difficult to be immunized The purpose of prevention and control.All hemorrhagic disease of grass carp can be caused to break out because of not immune or immuning failure every year, economic loss is huge.Therefore, really Current Major Epidemic plant type is examined, the purpose of control hemorrhagic disease of grass carp prevalence can be just really achieved, utmostly reduce the disease to fishing Industry produces the grave danger for causing economic loss.
It when traditional grass carp reovirus detects, is both needed to slaughter grass carp, the tissues such as acquisition liver, kidney, intestines, then Virus purification culture, viral purification, Electronic Speculum observation, nucleic acid band analysis are carried out to the tissue of acquisition, take longer, work Work amount is big, more demanding to equipment and technology, can not be used for quickly detecting to virus, is unfavorable for the timely anti-of hemorrhagic disease of grass carp Control.In the grass carp reovirus latent infection phase, since viral load is few, separation is more difficult, and false negative easily occurs in testing result, Carry out drug therapy again until virus is broken out, it is late, it suffers heavy losses.
Viral level is low in virus lays dormant infection period or early stage, grass carp body, traditional first extraction RNA, then anti- Change into cDNA, then carry out the technology of Standard PCR, can further increase virus quantity loss, often result in false negative as a result, prolonging The timely prevention of hemorrhage is missed.
Animal welfare can be defined as the impression that environmental condition brings animal, these impressions are dependent on animal needs and its completely Meaning degree.British agriculture animal welfare association, which proposes animal welfare, must make animal not by oneself of pain injury and threats By damage and disease risks should be minimized limit by feeding management system, and from pain and fear, should be used to animal pre- Anti- or quick diagnosis and treatment measure.Currently, the diagnosis to aquatic animal disease, typically using the tradition of anatomic observation Mode, which are relatively applicable in parasitic disease and organ disease.But in the latent infection phase of virus, even if nothing if dissection Method observes disease states, therefore it is frequently not necessary to dissect, and using atraumatic technique acquisition testing sample, can be effectively improved dynamic The welfare of object.
Invention content
In order to improve the detection efficiency of grass carp reovirus, realize that noninvasive acquisition testing sample, the present invention provide a kind of The detection method of grass carp reovirus.
A kind of detection method of grass carp reovirus to acquire Blood of Ctenopharyngodon as clinical detection sample, and extracts grass The RNA of fish blood；Using the reovirus genes of grass carps in grass carp reovirus nest-type PRC primer pair Blood of Ctenopharyngodon RNA Type is detected, and the nest-type PRC primer of the grass carp reovirus is made of 2 groups of primers, respectively grass carp reovirus Outside primer and grass carp reovirus inner primer；
The grass carp reovirus Outside primer is：
I type grass carp reovirus Outside primers
GCRV I-out-F:ACGCAACATCGTTGTCAATACT
GCRV I-out-R:TGCATCGAATGTAAGATGATTG
II type grass carp reovirus Outside primers
GCRV II-out-F:ACTGGAATTCAGTAATGGTCGT
GCRV II-out-R:CACGCCATATGAGATGTTCAGA
Type III grass carp reovirus Outside primer
GCRV III-out-F:CACGGTCCATCTGACAACACT
GCRV III-out-R:GTCTGGGTCAATCGTGGACAAC
The grass carp reovirus inner primer is：
I type grass carp reovirus inner primers
GCRV I-inner-F:TCACTAGCCATCAAGGTATCA
GCRV I-inner-R:CACTTAAGCACGGCTTGATCT
II type grass carp reovirus inner primers
GCRV II-inner-F:AGCGGGCATGGCTGTTCTTCG
GCRV II-inner-R:TCGAGCTCAACATTCCACGGC
Type III grass carp reovirus inner primer
GCRV III-inner-F:TGGCTTTCAATAAATACATAC
GCRV III-inner-R:CACTACCTGTATATAGTCTAC
Specific detection operating procedure is as follows：
(1) grass carp reovirus Outside primer is added in reverse transcription system to obtain inverse using a step reverse transcription PCR Transcribe PCR product；
(2) 1% agarose gel electrophoresis detection is carried out to the reverse transcription PCR product that step (1) obtains, it is preliminary to judge to be examined Whether sample infects grass carp reovirus and judges the genotype of virus, specific criterion such as the following table 1：
1 reverse transcription PCR testing result of table and genotype criterion
Stripe size and combination Genotype judges
620bp I
355bp II
451bp III
620bp+355bp I+II
620bp+451bp I+III
355bp+451bp II+III
620bp+355bp+451bp I+II+III
From table 1：
If only there is the band of 620bp, judge to infect I type grass carp reovirus；
If only there is the band of 355bp, judge to infect II type grass carp reovirus；
If only there is the band of 451bp, judge to infect type III grass carp reovirus；
If there are two bands of 620bp, 355bp, mixed infection I, II type grass carp reovirus is judged；
If there are two bands of 620bp, 451bp, mixed infection I, type III grass carp reovirus are judged；
If there are two bands of 355bp, 451bp, mixed infection II, type III grass carp reovirus are judged；
If there are tri- bands of 620bp, 355bp, 451bp, judge that mixed infection I, II, type III grass carp exhale the lonely disease of intestines Poison；
(3) it is template by 50-100 times of the reverse transcription PCR product dilution that step (1) obtains, in grass carp reovirus Side primer carries out nested PCR amplification, obtains nested PCR amplified fragments；
(4) 1% agarose gel electrophoresis detection is carried out to the nested PCR amplified fragments that step (3) obtains, according to electrophoresis knot Fruit judges whether institute's sample product infect grass carp reovirus and judge the genotype of virus, specific criterion such as the following table 2：
2 nest-type PRC testing result of table and genotype criterion
Stripe size and combination Genotype judges
499bp I
252bp II
351bp III
499bp+252bp I+II
499bp+351bp I+III
252bp+351bp II+III
499bp+252bp+351bp I+II+III
From table 2：
If only there is the band of 499bp, judge to infect I type grass carp reovirus；
If only there is the band of 252bp, judge to infect II type grass carp reovirus；
If only there is the band of 351bp, judge to infect type III grass carp reovirus；
If there are two bands of 499bp, 252bp, mixed infection I, II type grass carp reovirus is judged；
If there are two bands of 499bp, 351bp, mixed infection I, type III grass carp reovirus are judged；
If there are two bands of 252bp, 351bp, mixed infection II, type III grass carp reovirus are judged；
If there are tri- bands of 499bp, 252bp, 351bp, judge that mixed infection I, II, type III grass carp exhale the lonely disease of intestines Poison.
The technical solution further limited is as follows：
In step (1), reverse transcription reaction system 20-50 μ L, the final concentration of every grass carp reovirus Outside primer is equal It is 0.4-0.8 μM, Blood of Ctenopharyngodon rna content<1μg；Reverse transcription reaction condition is：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, totally 30 recycle.
In step (3), nest-type PRC reaction system 20-50 μ L, the final concentration of every grass carp reovirus inner primer is equal It is 0.2-0.4 μM, 50 μM of dNTP Mixture final concentrations, MgCl2500 μM of final concentration, reverse transcription PCR product dilution 2-10 μ L；Nested PCR amplification condition is：94℃3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 cycles；72℃8min.
The acquisition Blood of Ctenopharyngodon sample is noninvasive acquisition, and concrete operations are as follows：Fish for 10-20 item grass at random from pond Fish, after anesthetic impregnates anesthesia, 75% alcohol disinfecting is taken using the 1-5mL syringes of anti-coagulants rinse from grass carp tail vein Blood.
The anesthetic is MS-222 or caryophyllus oil；The final concentration of 25-35mg/L of MS-222, caryophyllus oil are final concentration of 10-20mg/L。
The anti-coagulants is EDTA or sodium citrate；A concentration of 2.5-3mg/mL of EDTA, sodium citrate concentration are 1.5-2mg/mL。
The TRIzol of 5-10 times of volume is added in Blood of Ctenopharyngodon, extracts RNA.
The advantageous effects of the present invention embody in the following areas：
1. the present invention is comparing and is analyzing 3 genotype reovirus genes of grass carps group features and sieved to multipair primer On the basis of choosing, I type virus S8 segment portion nucleotide sequences, II types, type III virus S10 segment portion nucleotides sequences are chosen Row have synthesized 6 pairs of primers as amplification target, design.I type virus S8 segments, II types, type III virus S10 segments are non-homogeneous Segment can effectively avoid false positive caused by non-specific amplification.
2. present invention acquisition Blood of Ctenopharyngodon as detection sample, has the advantages that noninvasive, grass carp this water is effectively increased Produce the welfare of animal.
3. the present invention uses a step reverse transcription PCR, 3 genes of grass carp reovirus are added simultaneously in reverse transcription system 3 pairs of Outside primers of type can simultaneously be detected the grass carp reovirus of 3 genotype, and there is only purposes in product Gene order segment, interference is small, and amplification efficiency is higher.For this method in primary first-order equation, RNA → cDNA → PCR operations are equal It is carried out continuously in same reaction system, compared with the conventional two-step method for first inverting again PCR, avoids multi-step operation and cause Target fragment loss, time-saving and efficiency, it is ensured that the timeliness and accuracy of detection.
4. in the grass carp reovirus latent infection phase, the characteristics of virion content is low, difficult detection, the present invention adopts With triple nide PCR technology, specificity and the sensitivity of PCR amplification are increased, it can be to low-abundance virus or a small amount of sample It is detected, compared with single PCR, specificity is stronger, and required sample is less.
Description of the drawings
Fig. 1 be the embodiment of the present invention 1 RNA agarose gel electrophoresis characteristic pattern (1-6 be RNA samples, M Marker trans 2k plus)。
Fig. 2 is the detection of 2 single virus of the embodiment of the present invention, reverse transcription PCR amplification agarose gel electrophoresis characteristic pattern (1-7 is Virus Sample, and N is negative control, and M is Marker 2000).
Fig. 3 is the detection of 2 single virus of the embodiment of the present invention, nested PCR amplification result agarose gel electrophoresis characteristic pattern (1- 7 be Virus Sample, and N is negative control, and M is Marker 2000).
Fig. 4 is 2 grass carp reovirus combine detection of the embodiment of the present invention, reverse transcription PCR amplification Ago-Gel Electrophoretic Characterization figure (1-4 is Virus Sample, and N is negative control, and M is Marker 2000).
Fig. 5 is 2 grass carp reovirus combine detection of the embodiment of the present invention, nested PCR amplification result Ago-Gel electricity Swimming characteristic pattern (1-4 is Virus Sample, and N is negative control, and M is Marker 2000).
Fig. 6 is the morbidity grass carp reovirus detection of the embodiment of the present invention 3, reverse transcription PCR amplification Ago-Gel Electrophoretic Characterization figure (1-10 is grass carp sample, and P positive controls, N is negative control, and M is Marker 2000).
Fig. 7 is the morbidity grass carp reovirus detection of the embodiment of the present invention 3, nested PCR amplification result Ago-Gel electricity Swimming characteristic pattern (1-10 is grass carp sample, and P positive controls, N is negative control, and M is Marker 2000).
Fig. 8 is that the embodiment of the present invention 3 is not fallen ill grass carp reovirus detection, and reverse transcription PCR amplification agarose is solidifying Gel electrophoresis characteristic pattern (1-10 is grass carp sample, and P positive controls, N is negative control, and M is Marker 2000).
Fig. 9 be the embodiment of the present invention 3 do not fall ill grass carp reovirus detection, nested PCR amplification result Ago-Gel Electrophoretic Characterization figure (1-10 is grass carp sample, and P positive controls, N is negative control, and M is Marker 2000).
Specific implementation mode
With reference to embodiment, the present invention is further illustrated, and however, it is not limited to this.
Embodiment 1
The noninvasive acquisition technique detection of Blood of Ctenopharyngodon sample
1. the noninvasive acquisition of Blood of Ctenopharyngodon sample
20 grass carps are selected at random, after the MS-222 anesthesia of final concentration 30mg/L, 75% alcohol disinfecting fish body is latter half of Point.Then, it is in from the side line of caudal peduncle front with the 5mL syringes of the sodium citrate anticoagulant rinse of final concentration 1.8mg/mL 45° angle is slowly inserted into tail vein, stops movement when blood point occurs in syringe needle, slowly pulls out syringe piston, and 1-2mL blood is sucked out The TRIzol of 10 times of volumes is added in liquid immediately.
It is as follows that TRIzol methods extract the step of blood total serum IgE：200 μ L are added in the mixed liquor 1mL of draw blood+TRIzol Chloroform stands 5min after vibrating mixing, and 12000rpm centrifuges 15min at 4 DEG C, and supernatant is taken to be added isometric isopropanol, mixing, It is placed at room temperature for 10min, 12000rpm centrifuges 10min at 4 DEG C, abandons supernatant, 75% ethyl alcohol cleaning precipitation, the 7500rpm at 4 DEG C 5min is centrifuged, being dissolved in 35 μ L after precipitation drying at room temperature dezymotizes water.
2. electrophoresis result
The RNA of above-mentioned 20 samples is detected into row agarose gel electrophoresis, testing result shows：The 5S of all samples RRNA, 18S rRNA, 28S rRNA bands are high-visible, and no degradation, part electrophoresis result is shown in Fig. 1；OD260/OD280 exists Within the scope of 1.8-2.1, meet RNA quality index, can be used for subsequent experiment.
Embodiment 2
Design of primers and specific detection
1. the design and synthesis of primer
Its genome of grass carp reovirus difference separation strains is all made of 11 segments, by molecular size range, 11 pieces Section can be divided into 3 groups, i.e., larger segment (L1, L2, L3), medium segment (M4, M5, M6) and smaller fragment (S7, S8, S9, S10, S11).3 genotype reovirus genes of grass carps group features are analyzed, I type virus S8 segment portion nucleotide sequences are chosen, As amplification target, design has synthesized 6 pairs of primers for II types, type III virus S10 segment portion nucleotide sequences：
Grass carp reovirus 3 is as follows to Outside primer：
I type grass carp reovirus Outside primers
GCRV I-out-F:ACGCAACATCGTTGTCAATACT
GCRV I-out-R:TGCATCGAATGTAAGATGATTG
II type grass carp reovirus Outside primers
GCRV II-out-F:ACTGGAATTCAGTAATGGTCGT
GCRV II-out-R:CACGCCATATGAGATGTTCAGA
Type III grass carp reovirus Outside primer
GCRV III-out-F:CACGGTCCATCTGACAACACT
GCRV III-out-R:GTCTGGGTCAATCGTGGACAAC
The grass carp reovirus 3 is as follows to inner primer：
I type grass carp reovirus inner primers
GCRV I-inner-F:TCACTAGCCATCAAGGTATCA
GCRV I-inner-R:CACTTAAGCACGGCTTGATCT
II type grass carp reovirus inner primers
GCRV II-inner-F:AGCGGGCATGGCTGTTCTTCG
GCRV II-inner-R:TCGAGCTCAACATTCCACGGC
Type III grass carp reovirus inner primer
GCRV III-inner-F:TGGCTTTCAATAAATACATAC
GCRV III-inner-R:CACTACCTGTATATAGTCTAC
2. experiment strain
873 plants of GCRV, HZ08 plants of GCRV, 104 plants of GCRV, Koi herpesvirus (KHV), infectious hematopoietic organ are bad Dead virus (IHNV), Micropterus salmoides irido virus (LMBV), infectious spleen and kidney necrosis virus (ISKNV) are preserved simultaneously by this laboratory Proliferation.
The extraction of 3.RNA and a step reverse transcription PCR
It is as follows that TRIzol methods extract the step of virus total RNA：The mixed liquor 1mL of virus+TRIzol is drawn, 200 μ L are added Chloroform stands 5min after vibrating mixing, and 12000rpm centrifuges 15min at 4 DEG C, and supernatant is taken to be added isometric isopropanol, mixing, It is placed at room temperature for 10min, 12000rpm centrifuges 10min at 4 DEG C, abandons supernatant, 75% ethyl alcohol cleaning precipitation, the 7500rpm at 4 DEG C 5min is centrifuged, being dissolved in 30 μ L after precipitation drying at room temperature dezymotizes water.
Select a step reverse transcription PCR kit PrimeScriptTMOne Step RT-PCR Kit Ver.2 (Takara, RR055A), reverse transcription PCR is completed, PCR product is obtained.20 μ L of reverse transcription reaction system, each group are divided into： 0.8 μ L, 2 × 1Step buffer of PrimeScript1Step Enzyme Mix, 10 μ L, 6 grass carp reovirus outsides Primer (20 μM) respectively takes 0.4 μ L, 0.5 μ L, RNase Free dH of experiment strain total serum IgE2O 6.3μL.Reaction condition is specially： 50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, totally 30 recycle.
4. nest-type PRC
After diluting 50 times to the PCR product that step 3 obtains, the second wheel is carried out with 3 pairs of grass carp reovirus inner primers Nested PCR amplification.20 μ L of nest-type PRC reaction system, each group are divided into：0.1 μ L, 10 × PCRBuffer (Mg of Taq enzyme (5U/ μ L)2+ Free) 2 μ L, MgCl21.6 μ L of (25mM) 1.2 μ L, dNTP Mixture (2.5mM), 6 grass carp reovirus inner primers (10 μM) respectively take 0.4 μ L, 2 μ L, the RNase Free dH of reverse transcription PCR product after dilution2O10.7μL.Amplification condition is specific For：94℃3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 cycles；72℃8min.
5. electrophoresis result
(1) single virus detects
(2) grass carp reovirus combine detection
6. Analysis of test results
(1) it in first round reverse transcription PCR, can expand to obtain the target fragment of 620bp for template with GCRV 873；With GCRV HZ08, which are template, can expand to obtain the target fragment of 355bp；It can expand to obtain 451bp for template with GCRV 104 Target fragment (see Fig. 2)；In the second wheel nest-type PRC, can expand to obtain the purpose piece of 499bp for template with GCRV 873 Section；It can expand to obtain the target fragment of 252bp using GCRV HZ08 as template；It can expand to obtain for template with GCRV 104 The target fragment of 351bp (see Fig. 3)；Using other Aquatic animals virus as template, 2 PCR cannot expand to obtain any segment.
(2) in first round reverse transcription PCR, using GCRV 873, GCRV HZ08 as template can expand to obtain 620bp, The target fragment of 355bp；It can expand to obtain the target fragment of 620bp, 451bp for template with GCRV 873, GCRV 104；With GCRV HZ08,104 GCRV, which are template, can expand to obtain the target fragment of 355bp, 451bp；With GCRV 873, GCRV HZ08, GCRV 104, which is template, can expand to obtain the target fragment of 620bp, 355bp, 451bp (see Fig. 4)；Nest is taken turns second When formula PCR, it can expand to obtain the target fragment of 499bp, 252bp using GCRV 873, GCRV HZ08 as template；With GCRV 873, GCRV 104, which is template, can expand to obtain the target fragment of 499bp, 351bp；It is mould with GCRV HZ08,104 GCRV Plate can expand to obtain the target fragment of 252bp, 351bp；It can be expanded for template with GCRV 873, GCRV HZ08,104 GCRV Increasing obtains the target fragment of 499bp, 252bp, 351bp (see Fig. 5).
Embodiment 3
Clinical application is tested
1. the acquisition of blood sample
20 parts of grass carp samples are detected, wherein having 10 tail of grass carp of apparent bleeding, without apparent bleeding 10 tail of grass carp, after the MS-222 anesthesia of 30mg/L, 75% alcohol disinfecting fish body latter half, the citric acid of final concentration 1.8mg/mL The 5mL syringes of sodium anti-coagulants rinse are slowly inserted into tail vein from the side line of caudal peduncle front in 45° angle, when blood point occurs in syringe needle When stop movement, slowly pull out syringe piston, 1-2mL blood be sucked out, the TRIzol of 10 times of volumes is added immediately.
2. extraction and the multiplex-nested PCR of blood sample RNA
According to the TRIzol methods in embodiment 1, the total serum IgE of Blood of Ctenopharyngodon is extracted.It is reversed according to the method for embodiment 2 Record PCR and nest-type PRC reaction.
3. pair reverse transcription PCR and nested PCR amplification product are identified with 1% agarose gel electrophoresis.
I representatives detect I type grass carp reovirus；II representatives detect II type grass carp reovirus；III represents inspection Measure type III grass carp reovirus；Grass carp reovirus is not detected in representative.
4. Analysis of test results
10 morbidity grass carps, wherein 8, II type grass carp reovirus is infected in detection in 2 PCR；Separately there are 2, Infection II type grass carp reovirus is not detected in 2 PCR, it may be possible to bacillary bleeding.10 grass carps that do not fall ill, When reverse transcription PCR, infection grass carp reovirus is not detected, but in nest-type PRC, wherein 3 detection latent infections II type grass carp reovirus illustrates that nest-type PRC has higher sensitivity, and the lonely disease of intestines is exhaled to the grass carp in the latent infection phase Malicious high specificity.

Claims (7)

1. a kind of detection method of grass carp reovirus, it is characterised in that：Blood of Ctenopharyngodon is acquired as clinical detection sample, and Extract the RNA of Blood of Ctenopharyngodon；The lonely disease of intestines is exhaled using the grass carp in grass carp reovirus nest-type PRC primer pair Blood of Ctenopharyngodon RNA Virus gene type is detected, and the nest-type PRC primer of the grass carp reovirus is made of 2 groups of primers, and respectively grass carp exhales intestines Lonely virus Outside primer and grass carp reovirus inner primer；
The grass carp reovirus Outside primer is：
I type grass carp reovirus Outside primers
GCRV I-out-F:ACGCAACATCGTTGTCAATACT
GCRV I-out-R:TGCATCGAATGTAAGATGATTG
II type grass carp reovirus Outside primers
GCRV II-out-F:ACTGGAATTCAGTAATGGTCGT
GCRV II-out-R:CACGCCATATGAGATGTTCAGA
Type III grass carp reovirus Outside primer
GCRV III-out-F:CACGGTCCATCTGACAACACT
GCRV III-out-R:GTCTGGGTCAATCGTGGACAAC
The grass carp reovirus inner primer is：
I type grass carp reovirus inner primers
GCRV I-inner-F:TCACTAGCCATCAAGGTATCA
GCRV I-inner-R:CACTTAAGCACGGCTTGATCT
II type grass carp reovirus inner primers
GCRV II-inner-F:AGCGGGCATGGCTGTTCTTCG
GCRV II-inner-R:TCGAGCTCAACATTCCACGGC
Type III grass carp reovirus inner primer
GCRV III-inner-F:TGGCTTTCAATAAATACATAC
GCRV III-inner-R:CACTACCTGTATATAGTCTAC
Specific detection operating procedure is as follows：
(1) grass carp reovirus Outside primer is added in reverse transcription system and reverse transcription is obtained using a step reverse transcription PCR PCR product；
(2) 1% agarose gel electrophoresis detection is carried out to the reverse transcription PCR product that step (1) obtains, tentatively judges institute's sample product Whether infect grass carp reovirus and judges that the genotype of virus, specific criterion are as follows：
If only there is the band of 620bp, judge to infect I type grass carp reovirus；
If only there is the band of 355bp, judge to infect II type grass carp reovirus；
If only there is the band of 451bp, judge to infect type III grass carp reovirus；
If there are two bands of 620bp, 355bp, mixed infection I, II type grass carp reovirus is judged；
If there are two bands of 620bp, 451bp, mixed infection I, type III grass carp reovirus are judged；
If there are two bands of 355bp, 451bp, mixed infection II, type III grass carp reovirus are judged；
If there are tri- bands of 620bp, 355bp, 451bp, mixed infection I, II, type III grass carp reovirus are judged；
(3) it is template by 50-100 times of the reverse transcription PCR product dilution that step (1) obtains, is drawn with grass carp reovirus inside Object carries out nested PCR amplification, obtains nested PCR amplified fragments；
(4) 1% agarose gel electrophoresis detection is carried out to the nested PCR amplified fragments that step (3) obtains, is sentenced according to electrophoresis result Whether disconnected institute sample product infect grass carp reovirus and judge that the genotype of virus, specific criterion are as follows：
If only there is the band of 499bp, judge to infect I type grass carp reovirus；
If only there is the band of 252bp, judge to infect II type grass carp reovirus；
If only there is the band of 351bp, judge to infect type III grass carp reovirus；
If there are two bands of 499bp, 252bp, mixed infection I, II type grass carp reovirus is judged；
If there are two bands of 499bp, 351bp, mixed infection I, type III grass carp reovirus are judged；
If there are two bands of 252bp, 351bp, mixed infection II, type III grass carp reovirus are judged；
If there are tri- bands of 499bp, 252bp, 351bp, mixed infection I, II, type III grass carp reovirus are judged.
2. a kind of detection method of grass carp reovirus according to claim 1, it is characterised in that：It is inverse in step (1) Responsive transcription system 20-50 μ L, the final concentration of every grass carp reovirus Outside primer is 0.4-0.8 μM, Blood of Ctenopharyngodon Rna content<1μg；Reverse transcription reaction condition is：50 DEG C of 30min, 94 DEG C of 2min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, totally 30 A cycle.
3. a kind of detection method of grass carp reovirus according to claim 1, it is characterised in that：In step (3), nest Formula PCR reaction system 20-50 μ L, the final concentration of every grass carp reovirus inner primer is 0.2-0.4 μM, dNTP 50 μM of Mixture final concentrations, MgCl2500 μM of final concentration, reverse transcription PCR product dilution 2-10 μ L；Nested PCR amplification condition For：94℃3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 cycles；72℃8min.
4. a kind of detection method of grass carp reovirus according to claim 1, it is characterised in that：The acquisition grass carp Blood sample is noninvasive acquisition, and concrete operations are as follows：Fish for 10-20 grass carp at random from pond, after anesthetic impregnates anesthesia, 75% alcohol disinfecting takes blood using the 1-5mL syringes of anti-coagulants rinse from grass carp tail vein.
5. a kind of detection method of grass carp reovirus according to claim 4, it is characterised in that：The anesthetic is MS-222 or caryophyllus oil；The final concentration of 25-35mg/L of MS-222, the final concentration of 10-20mg/L of caryophyllus oil.
6. a kind of detection method of grass carp reovirus according to claim 4, it is characterised in that：The anti-coagulants is EDTA or sodium citrate；A concentration of 2.5-3mg/mL of EDTA, sodium citrate concentration 1.5-2mg/mL.
7. a kind of detection method of grass carp reovirus according to claim 4, it is characterised in that：In Blood of Ctenopharyngodon The TRIzol of 5-10 times of volume is added, extracts RNA.
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