CN1448518A - Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof - Google Patents
Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof Download PDFInfo
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- 208000015181 infectious disease Diseases 0.000 title 1
- 230000002458 infectious effect Effects 0.000 title 1
- 230000017074 necrotic cell death Effects 0.000 title 1
- 241001051238 Infectious spleen and kidney necrosis virus Species 0.000 claims abstract description 40
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
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- 238000003745 diagnosis Methods 0.000 claims description 9
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- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 5
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Abstract
The present invention is mandarin fish infectious spleen and kidney necrosis virus gene diagnosing kit and its usage. The kit and its usage is designed based on two pairs of primers for the conservation sequence of mandarin fish infectious spleen and kidney necrosis virus gene. By means of PCR technology, the present invention detects qualitatively the specific DNA nucleic acid segment of mandarin fish infectious spleen and kidney necrosis virus fast, simply, specifically and sensitively. The present invention may be used in the tracing detection of different stages of mandarin fish cultivation and environment monitoring.
Description
Technical field
The present invention relates to the diagnostic kit and the detection method of aquatic economic animal disease, mainly be test kit and detection method at mandarin fish infectious spleen and kidney necrosis virus (ISKNV, Infectious Spleen and Kidney Necrosis Virus) gene diagnosis.
Background technology
Mandarin fish is the famous-brand and high-quality freshwater aquiculture kind of China.Along with the success of mandarin fish artificial propagation and seeding raising technology, the mandarin fish aquaculture is comparatively fast developed, and meanwhile the disease problem is also serious day by day.The mandarin fish disease mainly contains virus disease, bacteriosis and parasitosis.Wherein, virus disease is popular soon, sickness rate is high, and does not still have effective measure of control at present, has a strong impact on the development of mandarin fish aquaculture.In recent years, culture the fulminant prevailing disease of mandarin fish occurrence of large-area, cause the mandarin fish mass mortality, cause serious loss.At present for the also not specific methods of treatment of mandarin fish virus disease, the healthy aquaculture of carrying out mandarin fish is proper prophylactic methods, and this mainly depends on early stage rapid detection.Detection method for mandarin fish virus comprises that mainly traditional Histological method, electron microscope technique, biological finger-length measurement, biochemistry detection method, immunological method (mainly contain fluorescent-antibody technique, enzyme-linked immunosorbent assay (ELISA), immunoelectronmicroscopy, cell culture processes, molecular biology method (mainly containing molecular hybridization method, polymerase chain reaction (PCR)) etc. at present.These detection methods some require high to technical qualification, detection time is long, some needed material preparation trouble, the expense height, some method sensitivity is not high, and specificity is not strong, therefore numerous raisers to grasp crucial technical difficulty very big, be difficult to promote, limited these The Application of Technology and development.
Early stage quick diagnosis mandarin fish infectious spleen and kidney necrosis virus is present mandarin fish main preventive measures of culturing and the effective way that reduces the mandarin fish loss, therefore, easy fast, well highly sensitive again diagnostic kit and the detection method thereof of specificity be that numerous aquaculturists are anxious for.
Polymerase chain reaction (polymerase chain reaction, be called for short round pcr), be the technology of a kind of amplification in vitro specific DNA fragment of getting up of development in recent years because have fast, characteristics such as sensitivity, high specificity, so after this method is set up, be applied to very soon in the practice.
Summary of the invention
The objective of the invention is to utilize two pairs of primers of ISKNV gene conserved regions sequences Design, set up ISKNV sleeve type PCR reaction system, optimize on this basis and design, the gene diagnosis kit and the detection method of mandarin fish infectious spleen and kidney necrosis virus is provided.This test kit can be produced in batches, and is simple to operate, and easy to be quick, specificity is good, and is highly sensitive.Can apply to virus in the mandarin fish breeding process in each in period and follow the tracks of and detect, also can be used for environmental monitoring, avoid virus disseminating popular, improve scientific management efficient, have very high practical value.
The gene diagnosis kit of mandarin fish infectious spleen and kidney necrosis virus of the present invention is made of following parts: 1). sample diluting liquid A liquid, and 2 pipes, interior dress phosphoric acid buffer (1 * PBS), pH7.4; 2). template extract B liquid, 1 pipe, interior dress phenol/chloroform/primary isoamyl alcohol, ratio is 25: 24: 1; 3) .C liquid, 1 pipe, interior dress 5M NaCL; 4) .D liquid, 1 pipe, interior dress dehydrated alcohol; 5) .E liquid, 1 the pipe, in adorn 70% ethanol; 6) .F liquid, 1 pipe, interior dress sterilization distilled water; 7) .PCR reaction solution G liquid, 1 pipe, interior dress PCR one expands reaction solution, comprises ddH
2O, 10 * Buffer (contain mg
2+), dNTP, outer
Primers F 1, outer primer R1 and TaqE; 8) .PCR reaction solution H liquid, 1 pipe, interior dress PCR two expands reaction solution, comprises ddH
2O, 10 * Buffer (contain mg
2+), dNTP, in
Primers F 2, inner primer R2 and TaqE; 9). positive control I liquid, 1 pipe, the positive DNA of interior dress ISKNV; 10). box; 11). a cystose, its size is identical with the bottom surface of above-mentioned box, is loaded in the box; Have on the cystose and be no less than the little of above-mentioned tubule quantity
The hole, above-mentioned each tubule correspondence respectively is positioned in these apertures.
Inside and outside two pairs of primers described in the above-mentioned ISKNV gene diagnosis kit are according to ISKNV gene conserved regions sequences Design, and its dna sequence dna is as follows respectively:
F1：5’-AGA?CCC?ACT?TGT?ACG?GCG
R1：5’-CCC?ATG?TCC?AAC?GTA?TAG?C
F2：5’-CGT?GAG?ACC?GTG?CGT?AGT
R2：5’-AGG?GTG?ACG?GTC?GAT?ATG
Method with mentioned reagent box detection mandarin fish infectious spleen and kidney necrosis virus of the present invention follows these steps to carry out: 1). and sample thief adds 10～20 times of sample diluting liquid A liquid dilutions, ice bath homogenate in homogenizer; 2) the centrifugal 10～15min of .4000～6000r/min; 3). get supernatant 600 μ l template extract B liquid extracting, mixing several times turns upside down; 4) the centrifugal 10～15min of .10000～12000r/min; 5). get the 500ul supernatant and add 20 μ lC liquid, add 1mlD liquid again, precipitate 1 hour in-20 ℃; 6) the centrifugal 10～15min of .10000～12000r/min; 7). abandon supernatant, add the flushing of E liquid, the centrifugal 5～10min of 10000～12000r/min washes twice; 8). abandon supernatant, seasoning or on Bechtop, dry up; 9). add the resuspended dissolving of 20～30 μ lF liquid as template; 10). difference delivery plate and I liquid 2 μ l, join in the PCR reaction solution (G liquid), the centrifugal several seconds behind the mixing, place on the PCR; 11). increase by following condition: 95 ℃ of pre-sex change of 2min, → 72 ℃ of 10min → 4 ℃ preservations of 30 circulations of → 94 ℃ of 30sec
55℃30sec
72 ℃ of 1min12). get one respectively and expand reaction solution, join in the PCR reaction solution H liquid, the centrifugal several seconds places on the PCR behind the mixing.By above-mentioned condition amplification; 13). get 5～10 μ l after an expansion reaction finishes and add 4 μ l tetrabromophenol sulfonphthalein mixings, through 0.8% agarose gel electrophoresis 20～30 minutes (5V/cm)
The back is observed on ultraviolet device.If the reaction band that occur to become clear at 1080bp place (with positive control at same position), then be ISKNV
The positive illustrates that testing sample carries infectious spleen and kidney necrosis virus, if reactionless band shows that then carrying out PCR two expands reaction.Two expand instead
Get 5～10 μ l after should finishing and add 4 μ l tetrabromophenol sulfonphthalein mixings, behind 0.8% agarose gel electrophoresis 20～30 minutes (5V/cm) in ultraviolet
Observe on the instrument.If the reaction band that occur to become clear at 562bp place (with positive control at same position), be the ISKNV positive then, say
Bright testing sample carries infectious spleen and kidney necrosis virus, otherwise negative.
Beneficial effect of the present invention:
The gene diagnosis kit of mandarin fish infectious spleen and kidney necrosis virus of the present invention and detection method are to design based on two pairs of primers according to ISKNV gene conserved regions sequences Design.Utilize this two pairs of primers, the related gene fragment of the testing sample that carries ISKNV virus of can increasing specifically, the sleeve type PCR reaction system of the optimization of being set up guarantees rapidity, accuracy and the stability of detected result.Therefore use test kit of the present invention and detection method, can be easy, quick, sensitive and detect the mandarin fish that infects ISKNV specifically, be much higher than tradition and conventional biological method, can apply to virus in the mandarin fish breeding process in each follows the tracks of and detects in period, also can be used for environmental monitoring, avoid virus disseminating popular, improve scientific management efficient, have very high practical value.
Description of drawings
Fig. 1 is that the PCR one that infects the mandarin fish sample of mandarin fish infectious spleen and kidney necrosis virus expands and two expansion detected results.M:DNA molecular weight standard wherein; 1: one expansion reacting positive result; 2: one expansion reaction negative results; 3: two expansion reacting positive results; 4: two expansion reaction negative results.
Embodiment
Below the invention will be further described by specific embodiment.
Embodiment 1: the gene diagnosis kit of mandarin fish infectious spleen and kidney necrosis virus
This test kit constitutes (10 sample part) by following parts: 1. release liquid (A liquid), and 2 pipes, the 5ml/ pipe, interior dress phosphoric acid buffer (1 * PBS), pH7.4.2. template extract (B liquid) is provided for oneself or is prepared, phenol/chloroform/primary isoamyl alcohol, and ratio is 25: 24: 1.3.C liquid, 1 pipe, 200 μ l/ pipe, interior dress 5M NaCL.4.D liquid is provided for oneself, 1ml/ part * 10 part, and 10ml is mainly dehydrated alcohol.5.E liquid is provided for oneself, is mainly 70% ethanol.6.F liquid, 1 pipe, 30 μ l/ parts * 10 parts, 300 μ l/ pipe, interior dress sterilization distilled water.7.PCR reaction solution (G liquid), 1 pipe, 25 μ l/ parts * 10 parts, 250 μ l/ pipe, interior dress PCR one expands reaction solution (25 μ l system), comprises ddH
2O, 10 * Buffer (contain mg
2+), dNTP, outer primer F1, outer primer R1 and TaqE.8.PCR reaction solution (H liquid), 1 pipe, 25 μ l/ parts * 10 parts, 250 μ l/ pipe, interior dress PCR two expands reaction solution (25 μ l system), comprises ddH
2O, 10 * Buffer (contain mg
2+), dNTP, inner primer F2, inner primer R2 and TaqE.9. positive control (I liquid), 1 pipe, 20 μ l/ pipe, interior dress ISKNV positive DNA.10. rectangular parallelepiped box, 8.5 * 5.8 * 6.2cm
311. a cystose, its size is identical with the bottom surface of rectangular parallelepiped box, and high 2.2cm has four rounds, four holes of first row, and aperture 1.3cm,
Five holes of second row, aperture 1.0cm, third and fourth row is six holes respectively, aperture 0.6cm.Above-mentioned each tubule correspondence respectively is positioned over
In the hole of cystose, be loaded in the rectangular parallelepiped box.
The dna sequence dna of the inside and outside two pairs of primers in this test kit is as follows:
F1：5’-AGA?CCC?ACT?TGT?ACG?GCG
R1.?5’-CCC?ATG?TCC?AAC?GTA?TAG?C
F2：5’-CGT?GAG?ACC?GTG?CGT?AGT
R2：5’-AGG?GTG?ACG?GTC?GAT?ATG
The positive contrast liquid of I liquid in this test kit comprises the DNA plasmid control of ISKNV virus.Concrete operations are after the PCR reaction, reclaim associated clip with test kit, and enzyme is cut then, is connected on the plasmid by carrier, change intestinal bacteria E.coli over to, through ammonia benzyl culture medium flat plate enlarged culturing, picking positive colony, further enzyme is cut and is checked and verified, and measures the concentration of plasmid DNA, is diluted to 10
6Ratio is the positive control standard.
PCR one expansion reaction solution system (25 μ l system) is as follows:
ddH
2O 20μl
10 * Buffer (contains mg
2+) 2.5 μ l
dNTP 0.4μl
Outer primer F1 0.4 μ l
Outer primer R1 0.4 μ l
TaqE 0.3μl
PCR two expansion reaction solution systems (25 μ l system) are as follows:
ddH
2O 20μl
10 * Buffer (contains mg
2+) 2.5 μ l
dNTP 0.4μl
Inner primer F2 0.4 μ l
Inner primer R2 0.4 μ l
TaqE 0.3μl
Embodiment 2: the detection method of mandarin fish infectious spleen and kidney necrosis virus
Use the test kit of embodiment 1, follow these steps to carry out: 1. sample 0.1g adds 10 times of sample diluting liquid (A liquid) 1ml dilutions, ice bath homogenate in homogenizer.2.4000r/min centrifugal 10min.3. get supernatant 600 μ l/ template extract (B liquid) extracting, mixing several times turns upside down.4.12000r/min centrifugal 10min.5. get the 500ul supernatant and add 20 μ l/C liquid, add 1mlD liquid again, precipitate 1 hour in-20 ℃.6.12000r/min centrifugal 10min.7. abandon supernatant, add the flushing of E liquid, the centrifugal 5min of 12000r/min washes twice.8. abandon supernatant, seasoning or on Bechtop, dry up.9. add the resuspended dissolving of 20～30 μ lF liquid as template.10. difference delivery plate and I liquid 2 μ l join in the PCR reaction solution (G liquid), and the centrifugal several seconds places on the PCR behind the mixing.11. increase by following condition:
95 ℃ of pre-sex change of 2min, → 72 ℃ of 10min → 4 ℃ preservations of 30 circulations of → 94 ℃ of 30sec
55℃?30sec
72 ℃ of 1min12. get one respectively and expand reaction solution 2 μ l, join in the PCR reaction solution (H liquid), and the centrifugal several seconds places on the PCR behind the mixing.By above-mentioned condition
Amplification.13. one expands and to get 5～10 μ l after reaction finishes and add 4 μ l tetrabromophenol sulfonphthalein mixings, behind 0.8% agarose gel electrophoresis 20～30 minutes (5V/cm)
On ultraviolet device, observe.If the reaction band that occur to become clear at 1080bp place (with positive control at same position), then be the ISKNV sun
The property, illustrate that testing sample carries infectious spleen and kidney necrosis virus, if reactionless band shows that then carrying out PCR two expands reaction.Two expand reaction
Get 5～10 μ l after the end and add 4 μ l tetrabromophenol sulfonphthalein mixings, behind 0.8% agarose gel electrophoresis 20～30 minutes (5V/cm) in ultraviolet device
Last observation.If the reaction band that occur to become clear at 562bp place (with positive control at same position), then be the ISKNV positive, explanation
Testing sample carries infectious spleen and kidney necrosis virus, otherwise negative (see figure 1).
Claims (2)
1. the gene diagnosis kit of a mandarin fish infectious spleen and kidney necrosis virus is characterized in that this test kit comprises following parts: 1). sample diluting liquid A liquid, and 2 pipes, interior dress phosphoric acid buffer (1 * PBS), pH7.4; 2). template extract B liquid, 1 pipe, interior dress phenol/chloroform/primary isoamyl alcohol, ratio is 25: 24: 1; 3) .C liquid, 1 pipe, interior dress 5M NaCL; 4) .D liquid, 1 pipe, interior dress dehydrated alcohol; 5) .E liquid, 1 the pipe, in adorn 70% ethanol; 6) .F liquid, 1 pipe, interior dress sterilization distilled water; 7) .PCR reaction solution G liquid, 1 pipe, interior dress PCR one expands reaction solution, comprises ddH
2O, contain mg
2+10 * Buffer, dNTP, outer
Primers F 1, outer primer R1 and TaqE F1 are: 5 '-AGA CCC ACT TGT ACG GCG, R1 is: 5 '-CCC ATG
TCC AAC GTA TAG C; 8) .PCR reaction solution H liquid, 1 pipe, interior dress PCR two expands reaction solution, comprises ddH
2O, contain mg
2+10 * Buffer, dNTP, in
Primers F 2, inner primer R2 and TaqE; F2 is: 5 '-CGT GAG ACC GTG CGT AGT, R2:5 '-AGG GTG
ACG GTC GAT ATG; 9). positive control I liquid, 1 pipe, the positive DNA of interior dress ISKNV.10). box; 11). a cystose, its size is identical with the bottom surface of above-mentioned box, is loaded in the box; Have on the cystose and be no less than the little of above-mentioned tubule quantity
The hole, above-mentioned each tubule correspondence respectively is positioned in these apertures.
2. a method that detects the mandarin fish infectious spleen and kidney necrosis virus is characterized in that using the described test kit of claim 1, follows these steps to carry out: 1). and sample thief adds 10～20 times of sample diluting liquid A liquid dilutions, ice bath homogenate in homogenizer; 2) the centrifugal 10～15min of .4000～6000r/min; 3). get supernatant 600 μ l template extract B liquid extracting, mixing several times turns upside down; 4) the centrifugal 10～15min of .10000～12000r/min; 5). get the 500ul supernatant and add 20 μ lC liquid, add 1mlD liquid again, precipitate 1 hour in-20 ℃; 6) the centrifugal 10～15min of .10000～12000r/min; 7). abandon supernatant, add the flushing of E liquid, the centrifugal 5～10min of 10000～12000r/min washes twice; 8). abandon supernatant, seasoning or on Bechtop, dry up; 9). add the resuspended dissolving of 20～30 μ lF liquid as template; 10). difference delivery plate and I liquid, join in the PCR reaction solution G liquid, the centrifugal several seconds behind the mixing, place on the PCR; 11). increase by following condition:
95 ℃ of pre-sex change of 2min, → 72 ℃ of 10min → 4 ℃ preservations of 30 circulations of → 94 ℃ of 30sec
55℃30sec
72 ℃ of 1min12). get one respectively and expand reaction solution, join in the PCR reaction solution H liquid, the centrifugal several seconds places on the PCR behind the mixing.By above-mentioned condition amplification; 13). one expands and to get 5～10 μ l after reaction finishes and add 4 μ l tetrabromophenol sulfonphthalein mixings, through 0.8% agarose gel electrophoresis after 20～30 minutes in ultraviolet device
Last observation; If bright reaction band occurs at the 1080bp place, be the ISKNV positive then, illustrate that testing sample carries infectious spleen and kidney
Necrosis virus is if reactionless band shows that then carrying out PCR two expands reaction; Two expand and to get 5～10 μ l after reaction finishes and add 4 μ l tetrabromophenol sulfonphthaleins and mix
Even, on ultraviolet device, observe after 20～30 minutes through 0.8% agarose gel electrophoresis; If bright reaction band occurs at the 562bp place,
Be the ISKNV positive then, illustrate that testing sample carries infectious spleen and kidney necrosis virus, otherwise negative.
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| CNB031143679A CN1186459C (en) | 2003-05-06 | 2003-05-06 | Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof |
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|---|---|---|---|
| CNB031143679A CN1186459C (en) | 2003-05-06 | 2003-05-06 | Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof |
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| CN1186459C CN1186459C (en) | 2005-01-26 |
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|---|---|---|---|
| CNB031143679A Expired - Fee Related CN1186459C (en) | 2003-05-06 | 2003-05-06 | Mandarin fish infectious splenorenal necrosis virogene diagnostic kit and detecting method thereof |
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| CN100365131C (en) * | 2005-04-12 | 2008-01-30 | 中华人民共和国连云港出入境检验检疫局 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
| CN101956022A (en) * | 2010-08-17 | 2011-01-26 | 中国检验检疫科学研究院 | Detection method of infectious spleen and kidney necrosis virus Nest-PCR and kit thereof |
| CN102240399A (en) * | 2011-07-09 | 2011-11-16 | 中国水产科学研究院珠江水产研究所 | Application of siniperca chuatsi ISKNV (Infectious Spleen and Kidney Necrosis Virus) ORF093 protein |
| CN101530613B (en) * | 2009-04-17 | 2012-02-01 | 中山大学 | Infectious spleen and kidney necrosis virus vaccine and preparation method and application thereof |
| CN101624636B (en) * | 2009-06-12 | 2012-06-20 | 宁波大学 | LAMP-LFD detection method of infectious spleen and kidney necrosis virus (ISKNV) |
| CN104694483A (en) * | 2015-02-11 | 2015-06-10 | 中国水产科学研究院珠江水产研究所 | Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) |
| CN109762940A (en) * | 2019-02-02 | 2019-05-17 | 中国水产科学研究院珠江水产研究所 | For detecting the primer sets and kit of infectious spleen and kidney necrosis virus Yu mandarin fish rhabdovirus |
| CN111518957A (en) * | 2020-05-27 | 2020-08-11 | 电子科技大学中山学院 | PCR (polymerase chain reaction) rapid detection kit and method for infectious spleen and kidney necrosis of mandarin fish |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100365131C (en) * | 2005-04-12 | 2008-01-30 | 中华人民共和国连云港出入境检验检疫局 | Kit for testing three kinds of viruses of fish diseases synchronistically and test method thereof |
| CN101530613B (en) * | 2009-04-17 | 2012-02-01 | 中山大学 | Infectious spleen and kidney necrosis virus vaccine and preparation method and application thereof |
| CN101624636B (en) * | 2009-06-12 | 2012-06-20 | 宁波大学 | LAMP-LFD detection method of infectious spleen and kidney necrosis virus (ISKNV) |
| CN101956022A (en) * | 2010-08-17 | 2011-01-26 | 中国检验检疫科学研究院 | Detection method of infectious spleen and kidney necrosis virus Nest-PCR and kit thereof |
| CN101956022B (en) * | 2010-08-17 | 2011-12-28 | 中国检验检疫科学研究院 | Detection method of infectious spleen and kidney necrosis virus Nest-PCR and kit thereof |
| CN102240399A (en) * | 2011-07-09 | 2011-11-16 | 中国水产科学研究院珠江水产研究所 | Application of siniperca chuatsi ISKNV (Infectious Spleen and Kidney Necrosis Virus) ORF093 protein |
| CN104694483A (en) * | 2015-02-11 | 2015-06-10 | 中国水产科学研究院珠江水产研究所 | Proliferation method of siniperca chuatsi infectious spleen and kidney necrosis virus (ISKNV) |
| CN104694483B (en) * | 2015-02-11 | 2018-05-04 | 中国水产科学研究院珠江水产研究所 | A kind of enrichment procedure of mandarin fish infectious spleen and kidney necrosis virus ISKNV |
| CN109762940A (en) * | 2019-02-02 | 2019-05-17 | 中国水产科学研究院珠江水产研究所 | For detecting the primer sets and kit of infectious spleen and kidney necrosis virus Yu mandarin fish rhabdovirus |
| CN111518957A (en) * | 2020-05-27 | 2020-08-11 | 电子科技大学中山学院 | PCR (polymerase chain reaction) rapid detection kit and method for infectious spleen and kidney necrosis of mandarin fish |
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