CN104032038A - Detection kit and detection method for siniperca chuatsi rhabdoviruses - Google Patents

Abstract

The invention belongs to the technical field of detection and diagnosis of aquatic animal pathogeny, and discloses a detection kit and detection method for siniperca chuatsi rhabdoviruses. The reverse transcription-polymerase chain reaction type rapid detection kit for the siniperca chuatsi rhabdoviruses is filled with a 5* reverse transcription buffer solution, reverse transcriptase, a random primer, 1.0 mL of 2*reaction mixed buffer solution, a preferably-designed sense primer, a preferably-designed reverse primer, Taq DNA polymerase, a positive control solution, a negative control solution and ddH2O. The novel reverse transcription-polymerase chain reaction type rapid detection kit and detection method for the siniperca chuatsi rhabdoviruses overcome shortcomings of an existing detection method for the siniperca chuatsi rhabdoviruses, are practicable when applied to clinical diagnosis of the siniperca chuatsi rhabdoviruses, have the advantages of rapidity, accuracy and specificity, meet requirements of the clinical diagnosis, and provide convenience for detection of the siniperca chuatsi rhabdoviruses.

Description

The detection kit of mandarin fish rhabdovirus and detection method

Technical field

The invention belongs to detection and the diagnostic techniques field of aquatic animal cause of disease, relate to the detection method of a kind of mandarin fish rhabdovirus, particularly a kind of method that adopts reverse transcription-polymerase chain reaction (being called for short RT-PCR) technology for detection mandarin fish rhabdovirus.

Background technology

In recent years, the Foshan in Guangdong Province, Zhong Shan, large-scale epidemic disease is broken out in the snakehead on the ground such as Jining and Hubei in Shandong, mandarin fish, Micropterus salmonoides plant, causes huge financial loss.Laboratory is detected analysis from bacteriology and virusology two aspects to ill fish, confirm to cause that the disease of these cultured fishes fulminant death is rhabdovirus disease, its cause of disease is mandarin fish rhabdovirus (Siniperca chuatsi rhabdovirus, SCRV).

Rhabdovirus (Rhabdovirus) is a kind of minus-stranded rna virus of sub-thread non-segmented negative, and virus particle size is (100-430) nm × (45-100) nm, and form is like bar-shaped or bullet shaped.Rhabdovirus kind is more, comprises 175 kinds of above members, can infect vertebrates, invertebrates and plant.Kinds of fish Rhabdovirus is because infection host is wide, strain kind is many, virulence is strong, the various fresh water of serious harm and sea water fish.Hitherto reported infects the existing kind more than 20 of rhabdovirus of fish, and what wherein harm was larger has SVCV (SVCV).Viral haemorrhagic septicaemia virus (VHSV), infectious hematopoietic necrosis's poison (IHNV), flounder rhabdovirus (HRV) and mandarin fish rhabdovirus (SCRV) etc.Understand and find by inquiry, the snakehead of natural infection mandarin fish rhabdovirus, mandarin fish, Micropterus salmonoides mortality ratio are up to 90%.In order to find early whether cultured fishes are infected by SCRV, be necessary to set up a kind of quick, accurate, sensitive detection method.

The diagnosis of tradition virus disease need to be carried out virus and separate, cell cultures, and the methods such as electron microscopic observation and immunodetection, lack virus are carried out to quick, accurate, responsive detection method.The advantages such as it is simple to operate, special, quick, responsive that PCR method has, are just progressively applied in the detection and research of pathogenic micro-organism.

Summary of the invention

Technical problem to be solved by this invention is, overcomes the deficiencies in the prior art, and reverse transcription-polymerase chain-reaction test method of a kind of mandarin fish rhabdovirus is provided, can be fast and efficiently for the detection of mandarin fish rhabdovirus.

Reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus, described test kit comprises:

(1) 5 × reverse transcription damping fluid, 100 μ L;

(2) reversed transcriptive enzyme 25 μ L;

(3) random primer 25 μ L;

(4) 2 × polymerase chain reaction cocktail buffer 1.0mL, comprise following composition:

(5) design that detects primer is synthesized: according to the gene order of the mandarin fish rhabdovirus of issuing in GenBank, design a pair of specific detection primer at its conserved sequence, upstream primer P1:5 '-ATAAGGGTAGTTGAGAAGAAG-3 ', downstream primer P2:5 '-CTTCTTGTTGCTCTTCTTAAA-3 ', concentration is 10 μ M;

(6) Taq enzyme 5U/ μ L;

(7) ddH of sterilizing ₂o 1.0mL;

(8) positive control solution: extract the total RNA of mandarin fish rhabdovirus, reverse transcription obtains cDNA, then taking cDNA as template, taking Pl, P2 as primer, carry out pcr amplification, be connected to pMD18-T carrier by reclaiming the amplified production of purifying, using the recombinant plasmid of positive colony as positive control;

(9) negative controls: with ddH ₂o is as negative control.

The method that adopts test kit to detect mandarin fish rhabdovirus is:

(1) extraction of testing sample RNA: dissect fish sample to be detected, get the tissues such as liver,spleen,kidney and carry out homogenate, adopt traditional Trizol method or business-like RNA to extract test kit and extract RNA, use ddH ₂o dissolves, and is test RNA template;

(2) the synthetic cDNA of reverse transcription: the RNA template 7 μ L that get testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) pcr amplification: the PCR reaction system that adopts 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, check the expection size of PCR product;

(5) result and judgement: observe under ultraviolet lamp and contrast with standard molecular weight, occur bright band at 372bp place if detect sample, and negative control not having object band, is the mandarin fish rhabdovirus positive; If the visible object band of positive control occur without object band and detect sample, negative, do not have or be not the object mandarin fish rhabdovirus that will detect.

Compared with prior art, mandarin fish rhabdovirus reverse transcription-polymerase chain reaction detection kit of the present invention and detection method thereof have following characteristics and advantage:

(1) the present invention adopts reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction, be called for short RT-PCR) method of technology for detection mandarin fish rhabdovirus, be applicable to mandarin fish rhabdovirus to carry out rapid detection, can be widely used in the particularly supervision of epidemic disease of the culturing area such as mandarin fish, Micropterus salmonoides, snakehead of fish.

(2) compared with prior art, beneficial effect of the present invention comprises: 1. detection is quick, efficiency is high: judge and only need 4 hours from nucleic acid extraction, reverse transcription (RT) and polymerase chain reaction (PCR) to result; Once can carry out the detection of multiple samples, there is high efficiency.2. detect accurately: detect primer and be only combined specifically with mandarin fish rhabdovirus, all there is no cross reaction with other fishes virus.3. detection sensitivity is high, is suitable for the early stage monitoring of mandarin fish rhabdovirus.

Brief description of the drawings

Fig. 1 is the RT-PCR amplification that infects mandarin fish rhabdovirus snakehead viscera tissue.Wherein, in X-coordinate: the negative contrast of label 1 (using negative controls as template); The positive contrast of label 2 (using positive control solution as template); Label 3 is rhabdovirus infection snakehead viscera tissue sample; Label M is molecular weight standard DL2000 (purchased from precious biotechnology company limited); In ordinate zou: bp is base pair, the quantity of 2000,1000,750,500,250,100 base pairs that are homologous segment.

Fig. 2 is the RT-PCR amplification to Different Kinds of Pathogens.Wherein, in X-coordinate: label 1 is mandarin fish rhabdovirus; Label 2 is infectious hematopoietic necrosis's poison; Label 3, viral haemorrhagic septicaemia virus; Label 4 is, flounder rhabdovirus; Label 5 is SVCV; Label 6 is shuttle fry rhabdovirus, and label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, the quantity of 2000,1000,750,500,250,100 base pairs that are homologous segment.

Fig. 3 is the RT-PCR amplification of 8 tail snakehead tissue samples.Wherein, in X-coordinate: the positive contrast of label 1 (using positive control solution as template); The negative contrast of label 2 (using negative controls as template); Label 3-10 is the different snakehead tissue samples of 8 tails; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, the quantity of 2000,1000,750,500,250,100 base pairs that are homologous segment.

Fig. 4 is the RT-PCR amplification of 2 parts of mandarin fish tissue samples.Wherein, in X-coordinate: the positive contrast of label 1 (using positive control solution as template); The negative contrast of label 2 (using negative controls as template); Label 3-4 is 2 parts of different mandarin fish tissue samples; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, the quantity of 2000,1000,750,500,250,100 base pairs that are homologous segment.

Fig. 5 is the RT-PCR amplification of 7 tail Micropterus salmonoides tissue samples.Wherein, in X-coordinate: the positive contrast of label 1 (using positive control solution as template); The negative contrast of label 2 (using negative controls as template); Label 3-9 is the different Micropterus salmonoides tissue samples of 7 tails; Label M is molecular weight standard DL2000; In ordinate zou: bp is base pair, the quantity of 2000,1000,750,500,250,100 base pairs that are homologous segment.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail:

Embodiment mono-: reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus

All chemical reagent of reverse transcription-polymerase chain reaction detection kit of the mandarin fish rhabdovirus described in the present embodiment and primer are all bought from professional reagent company.But mentioned reagent and primer source do not form any limitation of the invention, the present invention can prepare relevant reagent and synthetic relevant primer voluntarily.

Test kit forms (12 sample part) by following part:

(1) 5 × reverse transcription damping fluid, 100 μ L;

(2) reversed transcriptive enzyme 25 μ L;

(3) random primer 25 μ L;

(4) 2 × polymerase chain reaction cocktail buffer 1.0mL, comprise following composition:

(5) design that detects primer is synthesized: according to the gene order of the mandarin fish rhabdovirus of issuing in GenBank, design a pair of specific detection primer at its conserved sequence, upstream primer P1:5 '-ATAAGGGTAGTTGAGAAGAAG-3 ', downstream primer P2:5 '-CTTCTTGTTGCTCTTCTTAAA-3 ', concentration is 10 μ M;

(6) Taq enzyme 5U/ μ L;

(7) ddH of sterilizing ₂o 1.0mL;

(8) positive control solution: the recombinant plasmid of positive colony;

(9) negative controls: ddH ₂o;

Above-mentioned positive control is to extract the total RNA of mandarin fish rhabdovirus, and reverse transcription obtains cDNA, then taking cDNA as template, taking Pl, P2 as primer, carry out pcr amplification, be connected to pMD18-T carrier by reclaiming the amplified production of purifying, using the recombinant plasmid of positive colony as positive control; With ddH ₂o is as negative control.

RT system (10 μ L) is:

RNA template 7 μ L

Random primer 0.5 μ L

Reversed transcriptive enzyme 0.5 μ L

RT reaction conditions is:

37℃ 15min

85℃ 5sec

Last 4 DEG C of preservations

Pcr amplification reaction system (25 μ L) is:

Pcr amplification reaction condition is:

4 DEG C of preservations of amplified production.

Embodiment bis-: reverse transcription-polymerase chain-reaction test method of mandarin fish rhabdovirus

Test kit described in use embodiment mono-, follows these steps to carry out:

(1) get 50mg sample to be checked, add the distilled water of 600uL sterilising treatment, after fully grinding with glass homogenizer, be placed in-20 DEG C of refrigerator multigelations 3 times, 6000rpm low-temperature centrifugation 5 minutes, get supernatant, adopt traditional Trizol method or business-like RNA to extract test kit and extract RNA, use ddH ₂o dissolves, and is test RNA template.

(2) get respectively the RNA template 7 μ L of testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) the PCR reaction system of employing 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, if there is bright reaction band at 372bp place, it is the mandarin fish rhabdovirus positive; If reactionless band occurs, negative;

(5) interpretation: as shown in Figure 1, there is bright reaction band in 372bp place in the electrophoresis band of label 3 (testing sample), in the electrophoresis band of label 2 (positive control), also there is same reaction band, and in the electrophoresis band of label 1 (negative control), do not occur showing that the detection effect of this PCR test kit meets expection by reaction band.

Embodiment tri-: reverse transcription-polymerase chain reaction fast test agent box specificity experiment of mandarin fish rhabdovirus

Test kit described in use embodiment mono-, follows these steps to carry out:

(1) respectively with the RNA of the infectious hematopoietic necrosis's poison, the viral haemorrhagic septicaemia virus that extract, flounder rhabdovirus, SVCV, shuttle fry rhabdovirus, carry out RT-PCR detection.

(2) get respectively the RNA template 7 μ L of testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) the PCR reaction system of employing 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of (4) pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, if there is bright reaction band at 372bp place, it is the mandarin fish rhabdovirus positive; If reactionless band occurs, negative;

(5) interpretation: as shown in Figure 2, bright reaction band appears in the electrophoresis band of label 1 at 372bp place, show the prawn baculovirus positive.At 372bp place, reactionless band occurs the electrophoresis band of label 2～6 (infectious hematopoietic necrosis's poison, viral haemorrhagic septicaemia virus, flounder rhabdovirus, SVCV, shuttle fry rhabdovirus), shows prawn baculovirus feminine gender.Show the high specificity of test kit of the present invention to prawn baculovirus.

Embodiment tetra-: the detection of reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus to morbidity snakehead

Test kit described in use embodiment mono-, follows these steps to carry out:

(1) respectively using the RNA of extraction in the 8 tails morbidity snakehead tissues of getting pond from morbidity as template, carry out RT-PCR detection.

(2) get respectively the RNA template 7 μ L of testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) the PCR reaction system of employing 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, if there is bright reaction band at 372bp place, it is the mandarin fish rhabdovirus positive; If reactionless band occurs, negative;

Detect the 8 tail snakeheads of getting, 6 tail snakehead mandarin fish rhabdoviruses are positive, and show by further order-checking, and its detected result is mandarin fish rhabdovirus, and nucleotide homology is all more than 99%, and RT-PCR detected result is shown in Fig. 3.

Embodiment five: the detection of reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus to morbidity mandarin fish

Test kit described in use embodiment mono-, follows these steps to carry out:

(1) respectively using the RNA of extraction in the 2 tails morbidity mandarin fish tissues of getting pond from morbidity as template, carry out RT-PCR detection.

(2) get respectively the RNA template 7 μ L of testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) the PCR reaction system of employing 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, if there is bright reaction band at 372bp place, it is the mandarin fish rhabdovirus positive; If reactionless band occurs, negative;

Detect the 2 tail mandarin fishes of getting, 2 tail mandarin fish mandarin fish rhabdoviruses are all positive, and show by further order-checking, and its detected result is mandarin fish rhabdovirus, and nucleotide homology is all more than 99%, and RT-PCR detected result is shown in Fig. 4.

Embodiment six: the detection of reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus to morbidity Micropterus salmonoides

Test kit described in use embodiment mono-, follows these steps to carry out:

(1) respectively using the RNA of extraction in the 7 tails morbidity Micropterus salmonoides tissues of getting pond from morbidity as template, carry out RT-PCR detection.

(2) get respectively the RNA template 7 μ L of testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) the PCR reaction system of employing 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, if there is bright reaction band at 372bp place, it is the mandarin fish rhabdovirus positive; If reactionless band occurs, negative;

Detect the 7 tail Micropterus salmonoides of getting, 7 tail Micropterus salmonoides mandarin fish rhabdoviruses are all positive, and show by further order-checking, and its detected result is mandarin fish rhabdovirus, and nucleotide homology is all more than 99%, and RT-PCR detected result is shown in Fig. 5.

Claims (2)

1. reverse transcription-polymerase chain reaction detection kit of mandarin fish rhabdovirus, is characterized in that, described test kit comprises:

(1) 5 × reverse transcription damping fluid, 100 μ L;

(2) reversed transcriptive enzyme 25 μ L;

(3) random primer 25 μ L;

(4) 2 × polymerase chain reaction cocktail buffer 1.0mL, comprise following composition:

(5) design that detects primer is synthesized: according to the gene order of the mandarin fish rhabdovirus of issuing in GenBank, design a pair of specific detection primer at its conserved sequence, upstream primer P1:5 '-ATAAGGGTAGTTGAGAAGAAG-3 ', downstream primer P2:5 '-CTTCTTGTTGCTCTTCTTAAA-3 ', concentration is 10 μ M;

(6) Taq enzyme 5U/ μ L;

(7) ddH of sterilizing ₂o 1.0mL;

(8) positive control solution: extract the total RNA of mandarin fish rhabdovirus, reverse transcription obtains cDNA, then taking cDNA as template, taking Pl, P2 as primer, carry out pcr amplification, be connected to pMD18-T carrier by reclaiming the amplified production of purifying, using the recombinant plasmid of positive colony as positive control;

(9) negative controls: with ddH ₂o is as negative control.

2. test kit according to claim 1, is characterized in that, the method that adopts test kit to detect mandarin fish rhabdovirus is:

(1) extraction of testing sample RNA: dissect fish sample to be detected, get the tissues such as liver,spleen,kidney and carry out homogenate, adopt traditional Trizol method or business-like RNA to extract test kit and extract RNA, use ddH ₂o dissolves, and is test RNA template;

(2) the synthetic cDNA of reverse transcription: the RNA template 7 μ L that get testing sample, add in PCR reaction tubes, add again 5 × reverse transcription damping fluid, 2 μ L, reversed transcriptive enzyme 0.5 μ L, random primer 0.5 μ L, reaction cumulative volume 10 μ L, after fully mixing, be placed on PCR instrument 37 DEG C of 15min reverse transcription reactions, 85 DEG C of 5sec deactivation reverse transcriptions, obtain cDNA product;

(3) pcr amplification: the PCR reaction system that adopts 25 μ L, in 0.2mL PCR reaction tubes, add respectively 2 × reaction cocktail buffer, 12.5 μ L, the each 0.5 μ L of upstream primer P1, downstream primer P2,5U/ μ L Taq archaeal dna polymerase 0.5 μ L, cDNA template 3.0 μ L, add to cumulative volume with sterilizing ultrapure water, use respectively positive control solution and negative controls as template simultaneously, the positive and negative control group are set, mixing the rear centrifugal several seconds carries out PCR reaction, and response procedures is: 94 DEG C of denaturations 5 minutes; Then 94 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds, 72 DEG C are extended 30 seconds, circulate altogether 35 times; 72 DEG C are extended 10 minutes, last 4 DEG C of preservations;

(4) after the detection of pcr amplification product: PCR reaction finishes, get 10 μ L products on 1.5% sepharose (containing ethidium bromide 0.5 μ g/mL), use TAE damping fluid to carry out electrophoresis, under ultraviolet transilluminator, observe PCR product, check the expection size of PCR product;

(5) result and judgement: observe under ultraviolet lamp and contrast with standard molecular weight, occur bright band at 372bp place if detect sample, and negative control not having object band, is the mandarin fish rhabdovirus positive; If the visible object band of positive control occur without object band and detect sample, negative, do not have or be not the object mandarin fish rhabdovirus that will detect.