CN102634532A - Preparation method for standard sample of infectious hematopoietic organ necrosis virus molecule - Google Patents
Preparation method for standard sample of infectious hematopoietic organ necrosis virus molecule Download PDFInfo
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Abstract
The invention discloses a preparation method for a standard sample of an infectious hematopoietic organ necrosis virus molecule. The preparation method is characterized by including following steps: extracting an infectious hematopoietic organ necrosis virus RNA (ribonucleic acid); performing RT-PCR (reverse transcription-polymerase chain reaction); purifying target genes; performing connection transformation of target segments; recycling plasmid; performing in vitro transcription; performing RNA purification after transcription; and performing phenol chloroform extraction. The preparation method is time-saving and labor-saving in a preparation process, the standard sample of prepared infectious hematopoietic organ necrosis virus ribonucleic acid is high in stability and fine in uniformity, standard samples can be provided for infectious hematopoietic organ necrosis virus detection researches, medicine researches, application researches and the like, so that comparison of different laboratory results is realized, and quality control of laboratories is guaranteed.
Description
Technical field
The present invention relates to a kind of preparation method of viruses molecule standard model, infectious hematopoietic necrosis's poison molecular criteria sample of especially a kind of time saving and energy saving, stable height, good uniformity.
Background technology
Infectious hematopoietic necrosis's poison belongs to rhabdovirus (Infectious haematopoietic necrosis virus; IHNV) the grain Rhabdovirus (Novirhabdovirus) of section; Generic hydrocoles virus also has flounder rhabdovirus (Hirame rhabdovirus; HRV), the murrel rhabdovirus (Snakehead rhabdovirUS, SRV) and viral haemorrhagic septicaemia virus (Viral haemorrhagic septicaemia, VHSV).IHNV is the representative species of this genus, and the representative strains of IHNV is WRAC strain (ATCC is encoded to VR 1), and viral particle morphology is the bullet shape, diameter 80 ~ 90 nm, and length is 160 ~ 180 nm, and cyst membrane is arranged; The buoyant density of viral nucleic acid in cesium sulfate is 1.59 g/ml, to heat, acid, ether is unstable.
(Infectious haematopoietic necrosis IHN) is a kind of serious communicable disease in the fish such as salmon, trout to the infectious hematopoietic necrosis, because that this virus causes is very harmful, OIE classifies it as important transmissible disease.China is a fishery big country; Salmon trout class is as a kind of large-scale economic fish; In China's culture fishery, occupy considerable status, the salmon trout amount of import simultaneously increases year by year, and the infectious hematopoietic necrosis also has been brought into China; Nineteen ninety, Benxi, Liaoning reported first the infectious hematopoietic necrosis break out in China.In order to protect China's culture fishery to develop in a healthy way, need carry out the strictness check to import salmon trout class and avoid virus to flow into, and domestic this sick plant that found is carried out control epidemic situation, prevents that the propagation of epidemic situation from worsening.In more than ten years in the past; Some are used to detect and infect and the method for the lHNV of non-Infection Status mainly comprises immuno-gold labeling method, ElISA and the RT-PCR that carries out in situ hybridization, immunohistochemistry, detects in vivo with the specific probe of nucleoprotein gene; In these methods, PCR is proved to be the most rapidly and sensitive method.But because this virus belongs to RNA viruses, its RNA is degraded extremely easily, and RNA viruses is extracted in the laboratory separately, not only wastes time and energy, and is difficult to keep its stability and homogeneity, is difficult to guarantee breadboard quality control.
Summary of the invention
The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, the preparation method of infectious hematopoietic necrosis's poison molecular criteria sample of a kind of time saving and energy saving, stable height, good uniformity to be provided.
Technical solution of the present invention is: a kind of preparation method of infectious hematopoietic necrosis's poison molecular criteria sample is characterized in that carrying out as follows:
A. the extraction of infectious hematopoietic necrosis's poison RNA
In centrifuge tube, add Trizol lysate 600 μ l, infectious hematopoietic necrosis's poison cell culture 200 μ l add chloroform 200 μ l concussion mixing, the centrifugal 15min of 10 000 g; Draw supernatant 500 μ l and join in the 500 μ l Virahols, put upside down mixing repeatedly, the centrifugal 15min of 10 000 g; Supernatant discarded is inverted on the thieving paper, is stained with dry liquids, adds 75% alcohol 600 μ l, puts upside down washing, the centrifugal 10min of 10 000 g; Supernatant discarded is inverted on the thieving paper, is stained with dry liquids; The centrifugal 10sec of 4 000g blots the pipe liquid at the end, drying at room temperature 3 min; The DEPC water that adds 11.5 μ l, mixing dissolves the RNA in the tube wall gently, the centrifugal 5sec of 2 000g, 4 ℃ of preservations, subsequent use as template;
B. RT-PCR reaction
B-1. reverse transcription system
5 * M-MLV Buffer, 4 μ l, dNTPs (each 2.5mmol/L) 2 μ l, M-MLV (5U/ μ l) 1 μ l, RNA enzyme inhibitors 0.5 μ l, reverse transcription primer (20pmol/ μ L) 1 μ l, a step gained RNA template 11.5 μ l; Said reverse transcription trip primer is 5 '-TCAAGGGGGGAGTCCTCGA-3 ', and the total reaction system is 20 μ l;
B-2. reverse transcription condition
42 ℃ of water-bath 1h ~ 2h;
The b-3.PCR amplification reaction system
10 * PCR Buffer (contains Mg
2+) 2.5 μ l, dNTPs (each 2.5mmol/L) 0.5 μ l,
TaqArchaeal dna polymerase (5U/ μ l) 1 μ l, upstream primer, each 1 μ L of downstream primer (20pmol/ μ L), the reverse transcription product 0.5 μ l of b-2 is supplemented to 25 μ l with sterilized water;
Said upstream primer is 5 '-TCAAGGGGGGAGTCCTCGA-3 ';
Said downstream primer is 5 '-CACCGTACTTTGCTGCTAC-3 ';
B-4. pcr amplification condition: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 1min, 53 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 30 circulations; 72 ℃ of 5min;
C. the purifying of target gene
Adopt TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 test kit, reclaim the target molecule dna fragmentation;
D. the segmental connection of purpose transforms
The d-1 linked system
The purpose fragment 4 μ l of the resulting purifying of c step, 2 * Rapid ligation buffer, 5 μ l, PGEM-T carrier 0.5 μ l, T4 DNA Ligase 0.5 μ l, room temperature connects 1h;
D-2 transforms
The connection product of a last step is joined in the competent cell mixing, ice bath 20min, 42 ℃ of heat stress 1min; Ice bath 2min adds 800 μ l SOC substratum, and 150g shakes bacterium 1.5h; The centrifugal 10min of 1 000g discards nutrient solution, adds fresh SOC substratum 200 μ l; Be coated in behind the mixing on the LB flat board that contains Amp+, air-dry after, be inverted to cultivate 12h ~ 16h for 37 ℃;
The screening of d-3 positive colony
Picking mono-clonal bacterium is cultivated, and carries out the screening of positive colony with PCR method, PCR system and reaction conditions such as b-3, b-4;
E. reclaim plasmid;
F. in-vitro transcription
E step gained plasmid is carried out single endonuclease digestion with SalI, and reaction system is: 10 * H Buffer, 5 μ l, SalI 1 μ l, plasmid 10 μ l, distilled water 34 μ l; 37 ℃ of 2h reclaim test kit with Omega and reclaim linearization plasmid; The responsive transcription system is: 10 * T7 RNA polymerase buffer, 5 μ l, DTT 5 μ l, NTP 10 μ l, Rnasin 1 μ l, linearization plasmid 5 μ l, T7 RNApolymerase 1 μ l, DEPC treating water 28 μ l; 37 ℃ of 2h, and then add 10U DnaseI 2 μ l, 37 ℃, 30min;
G. transcribe back RNA purification process
The RNA that transcribes is carried out DNase handle, reaction system and condition are following: the responsive transcription liquid 20ul of said f step, RNase free DNase I (5u/ul) 4 ul, Total 24ul, 37 ℃ of 1h;
H. phenol chloroform extracting
Said g step is carried out the extracting of phenol chloroform through the reaction solution that DNase handles, finally complement to 100 ul with RNase-free dH2O.
It is time saving and energy saving that the present invention not only prepares process; And high, the good uniformity of prepared infectious hematopoietic necrosis poison molecular criteria sample stability; Can standard model be provided for infectious hematopoietic necrosis's poison detects research, medical research, applied research etc.; With realization different experiments chamber result's comparison, thereby guarantee breadboard quality control.
Description of drawings
Fig. 1 is an embodiment of the invention infectious hematopoietic necrosis poison purpose fragment electrophorogram.
Fig. 2 is an embodiment of the invention infectious hematopoietic necrosis toadstool liquid PCR electrophorogram.
Fig. 3 is an embodiment of the invention infectious hematopoietic necrosis poison linearization plasmid electrophorogram.
Fig. 4 is an embodiment of the invention infectious hematopoietic necrosis poison linearization plasmid in-vitro transcription product electrophorogram.
Fig. 5 is embodiment of the invention infectious hematopoietic necrosis poison linearization plasmid in-vitro transcription product distortion rear electrophoresis figure.
Embodiment
Fragment selected target fragment according in OIE and " the GB/T 15805.2-2008 fish quarantine method part 2: infectious hematopoietic necrosis's poison (IHNV) " is used for the laboratory quality control.
A. the extraction of infectious hematopoietic necrosis's poison RNA
In centrifuge tube, add Trizol lysate 600 μ l, infectious hematopoietic necrosis's poison cell culture 200 μ l, this culture is purchased in China typical culture collection center (GDV012), establishes positive control, negative control simultaneously; Add chloroform 200 μ l concussion mixing, the centrifugal 15min of 10 000 g; Draw supernatant 500 μ l and join in the 500 μ l Virahols, put upside down mixing repeatedly, the centrifugal 15min of 10 000 g; Supernatant discarded is inverted on the thieving paper gently, is stained with dry liquids, adds 75% alcohol 600 μ l, puts upside down washing, the centrifugal 10min of 10 000 g; Supernatant discarded is inverted on the thieving paper, is stained with dry liquids; The centrifugal 10sec of 4 000g blots the pipe liquid at the end, drying at room temperature 3 min with micro sample adding appliance as far as possible; The DEPC water that adds 11.5 μ l, mixing dissolves the RNA in the tube wall gently, the centrifugal 5sec of 2 000g, 4 ℃ of preservations, subsequent use as template;
B. RT-PCR reaction
B-1. reverse transcription system
5 * M-MLV Buffer, 4 μ l, dNTPs (each 2.5mmol/L) 2 μ l, M-MLV (5U/ μ l) 1 μ l, RNA enzyme inhibitors 0.5 μ l, reverse transcription primer (20pmol/ μ L) 1 μ l, a step gained RNA template 11.5 μ l; Said reverse transcription trip primer is 5 '-TCAAGGGGGGAGTCCTCGA-3 ', and the total reaction system is 20 μ l;
B-2. reverse transcription condition
42 ℃ of water-bath 1h ~ 2h;
The b-3.PCR amplification reaction system
10 * PCR Buffer (contains Mg
2+) 2.5 μ l, dNTPs (each 2.5mmol/L) 0.5 μ l,
TaqArchaeal dna polymerase (5U/ μ l) 1 μ l, upstream primer, each 1 μ L of downstream primer (20pmol/ μ L), the reverse transcription product 0.5 μ l of b-2 is supplemented to 25 μ l with sterilized water;
Said upstream primer is 5 '-TCAAGGGGGGAGTCCTCGA-3 ';
Said downstream primer is 5 '-CACCGTACTTTGCTGCTAC-3 ';
Above-mentioned primer is synthetic;
B-4. pcr amplification condition: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 1min, 53 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 30 circulations; 72 ℃ of 5min; The segmental electrophorogram of purpose is as shown in Figure 1, and the purpose band is arranged, wherein M:DL2000 Marker; 1: glue reclaims product;
C. the purifying of target gene
Adopt TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 test kit (article No. DV805A), reclaim the target molecule dna fragmentation;
D. the segmental connection of purpose transforms
The d-1 linked system
The purpose fragment 4 μ l of the resulting purifying of c step, 2 * Rapid ligation buffer, 5 μ l, PGEM-T carrier 0.5 μ l, T4 DNA Ligase 0.5 μ l, room temperature connects 1h;
D-2 transforms
The connection product of a last step joined in the competent cell (purchase precious biotech firm), use the pipettor mixing gently, ice bath 20min, 42 ℃ of heat stress 1min in Dalian; Ice bath 2min adds 800 μ l SOC substratum, and 150g shakes bacterium 1.5h; The centrifugal 10min of 1 000g discards nutrient solution, adds fresh SOC substratum 200 μ l; Be coated in behind the mixing on the LB flat board that contains Amp+, air-dry after, be inverted to cultivate 12h ~ 16h for 37 ℃;
The screening of d-3 positive colony
Picking mono-clonal bacterium is cultivated, and carries out the screening of positive colony with PCR method, PCR system and reaction conditions such as b-3, b-4; Its bacterium liquid PCR electrophorogram is as shown in Figure 2: M:DL2000 Marker wherein; 1,2,3,4: bacterium liquid PCR product;
E. reclaim plasmid;
Use Omega company plasmid and reclaim kit method, reclaim plasmid.
Bacterium liquid PCR is identified that being the male recombinant plasmid send precious biological (Dalian) ltd to check order, and sequencing result is (dash area is a primer before and after the fragment) as follows:
CACCGTACTTTGCTGCTACCCCTGGGGCCTCTGCTACGATCCTCATCAGTCTTACAATGCGTCTAGCGGACTCCACCACTGACTTCATGCACAGATCTTCTAGAAGGTCTGCTGGAAGGGCGCCCAGGTTCTTTGCGGCTTGGTTGAACAGTCCCACCATTGTCATCCCTGTCCCGCTCAGTCTCATGGCGCACAGTGCCTTGGCTCTAGCGGCGGTGGCTGGGTCAGACAGGTTGATGAGAATGATCCCATAAACAGCCTTGGTGAGCTTCTGTCCAAGTCTAAGTACCGCCCCGATCTTGGCCAGCACTCCCCTGTTGGAGTTGAAGTTGACCAACTCGTCAATCCCTTGGCTGGTTGCAAGACGCTCGAGCTTGTTTTGGCAGTATATGGCCATCTTGTCCACGTCATACTTCGTCAGTAGAGCGCAGGTGAAGAGGAGGCCGGTCACAACCTCAAGGACATTTTCCTTTCCGATCGTTTCTGCAAGCTTGTTGTTGGGATCTGCGAAAGTGGCGTCCAGTGGTGCCCCAGTGTCCAAAGACTCCAGCAAGAAGCCCAGGGCTTCCAGAGTTTCGGCGACCGTGCCTGTCCCCGATGGGACCGTCTCCGCAATGACGAACGCGCACAAGAGGCCCAATGCCCTTCTCGTTCCCTCTCCTCCGACGTGACTCACCGCCCGCTTAATCATCTCTAGTTCAAAGTCTGCCTTGGAGAAGAAGAGAGGGAGGGTTATCGTACCGGGACGATACTCCGTCTCTGAGTCCTCGAGGACTCCCCCCTTGA
Sequential analysis:
The online nucleotide blast of NCBI analyzes, and search obtains 57 of high similar sequences, 2 complete virus gene sequences wherein, and 26 N gene complete sequences, other are N Gene Partial sequence.Utilize DNAStar software that the above-mentioned sequence of measuring (IHNV-DL) and 15 different sorts sequences of reporting are carried out the comparison of nucleotide sequence comparison and translated amino acid both at home and abroad; The result is as shown in table 1; The nucleic acid similarity is 93.3% ~ 99.6%; With IHNV be 93.6% with reference to strain WRAC similarity, with type strain RB-1 similarity be 96.3%; With the amino acid similarity that translates be 93% ~ 99.6%, with the strain zyx (NCBI id:HM099906.1) of Harbin City, Heilongjiang Province city report maximum similarity 99.6% is arranged.(sequence number of each strain in GeneBank is RB-76:AY442516.1; LWS-87:AY442515.1; Carson-89:AY442508.1; LB91KI:AY438975.1; LR-73:AY442513.1; SRCV:AY442517.1; LR-80:AY442514.1; Col-85:AY442510.1; Col-80:AY442509.1; WRAC:AY442518.1; 193-110:AY442507.1; CST-82:AY442511.1; HO-7:AY442512.1; Zyx:HM099906.1; RB-1:U50402.1)
The comparative result of the protein sequence of process nucleotide sequence and translation shows; Infectious hematopoietic necrosis's poison standard model (IHNV-DL) of the embodiment of the invention is higher with other IHNV all similar property; The highest with the similarity of zyx strain; Reach 99.6%, the two possibility sibship is nearer, for same strain development comes.
Table 1 IHNV-DL compares with the strain homology of reporting both at home and abroad
(upper right nucleic acid a, left side is amino acid down)
1:RB-76；?2:LWS-87；?3:Carson-89；?4:LB91KI；?5:LR-73；?6:SRCV；?7:LR-80；?8:Col-85；9:Col-80；?10:WRAC；?11:193-110；?12:CST-82；?13:HO-7；?14:zyx；?15:RB-1；?16:IHNV-DL。
F. in-vitro transcription
E step gained plasmid is carried out single endonuclease digestion with SalI, and reaction system is: 10 * H Buffer, 5 μ l, SalI 1 μ l, plasmid 10 μ l, distilled water 34 μ l; 37 ℃ of 2h reclaim test kit with Omega and reclaim linearization plasmid; The responsive transcription system is: 10 * T7 RNA polymerase buffer, 5 μ l, DTT 5 μ l, NTP 10 μ l, Rnasin 1 μ l, linearization plasmid 5 μ l, T7 RNApolymerase 1 μ l, DEPC treating water 28 μ l; 37 ℃ of 2h, and then add 10U DnaseI 2 μ l, 37 ℃, 30min; To reclaim the linearization plasmid electrophorogram as shown in Figure 3: M: λ-Nind III digeet DNA Marker wherein; 1: linearization plasmid; M
1: quantitative Marker200mg;
Plasmid in-vitro transcription product electrophorogram is as shown in Figure 4; M:100bp DNA ladder marker wherein; 1: transcription product;
G. transcribe back RNA purification process
The RNA that transcribes is carried out DNase handle, reaction system and condition are following: the responsive transcription liquid 20ul of said f step, RNase free DNase I (5u/ul) 4 ul, Total 24ul, 37 ℃ of 1h;
H. phenol chloroform extracting
Said g step is carried out the extracting of phenol chloroform through the reaction solution that DNase handles, finally complement to 100 ul with RNase-free dH2O.Get 10 times of 1ul dilutions carry out sex change (65 ℃, 10min) after, carry out 3% agarose gel electrophoresis, the result is as shown in Figure 5: M:RNA Marker RL1000bp wherein; 1: the transcription product of sex change.
Homogeneity and stability test:
Homogeneity:
The uniformity result of the embodiment of the invention sees table 2, and its Evaluation for Uniformity result sees table 3.Can know from table 3, not have significant difference between two sample sets, can think that sample is uniform.
Table 2 infectious hematopoietic necrosis poison molecular criteria sample homogeneity is measured the result
| Sample number | The OD value of |
The OD value of |
| 1 | 1.42 | 1.45 |
| 2 | 1.52 | 1.39 |
| 3 | 1.49 | 1.50 |
| 4 | 1.46 | 1.49 |
| 5 | 1.47 | 1.44 |
| 6 | 1.45 | 1.49 |
| 7 | 1.41 | 1.38 |
| 8 | 1.40 | 1.40 |
| 9 | 1.44 | 1.48 |
| 10 | 1.34 | 1.44 |
| 11 | 1.37 | 1.40 |
| 12 | 1.51 | 1.46 |
| 13 | 1.34 | 1.43 |
| 14 | 1.42 | 1.46 |
| 15 | 1.39 | 1.38 |
Table 3 infectious hematopoietic necrosis poison molecular criteria sample homogeneity evaluation result
| Soruces of variation | Sum of squares | Degree of freedom | All square MS | The F ratio |
| Between group (between bottle) | 0.001 | 1 | 0.01 | 0.351 |
| In the group | 0.068 | 28 | 0.02 | ? |
| Summation | 0.069 | 29 | ? | ? |
Stability:
Adopt two types stability test: a kind of is stability test under-20 ℃, gets each sample (sample A and B) at random, detects 2 parts every month; Another kind is the stability test under higher temperature (4 ℃), and sample is got 14 parts, detects 2 parts in per 1 day and amounts to that 7 days test results see the following form 4, table 5.
Table 4 stability test experiment (20 ℃)
F=0.002, F
Table 0.05=3.18, F＜F
Cr [0.05,5,5], there was no significant difference
Table 5 stability test experiment (4 ℃) result
F=1.274, F
Table 0.05=3.18, F＜F
Cr [0.05,5,5], there was no significant difference
Valued methods
Select infectious hematopoietic necrosis's poison molecular criteria sample of 10 embodiment of the invention at random; With carrying out the definite value test shown in " infectious hematopoietic necrosis's poison RT-PCR detection method " among the GB/T15805.2-2008; The result is in full accord, and it is positive to be infectious hematopoietic necrosis's poison nucleic acid.
Infectious hematopoietic necrosis's poison molecular criteria sample coordinated trials such as inspection and quarantine bureau, Zhuhai inspection and quarantine bureau, Xinjiang Entry-Exit Inspection and Quarantine Bureau, Shanxi inspection and quarantine bureau, disease prevention and control center, Dalian through Beijing of the embodiment of the invention, definite value result's statistics is seen table 6.
Table 6 coordinated trials definite value is table as a result
| Sequence number | Coordinated trials laboratory title (sample number) | Test result (qualitative) |
| 1 | Xinjiang Entry-Exit Inspection and Quarantine Bureau | Infectious hematopoietic necrosis's poison nucleic acid is positive |
| 2 | Beijing Administration for Entry-Exit Inspection and Quarantine | Infectious hematopoietic necrosis's poison nucleic acid is positive |
| 3 | Zhuhai Entry-Exit Inspection and Quarantine Bureau | Infectious hematopoietic necrosis's poison nucleic acid is positive |
| 4 | Shanxi Entry-Exit Inspection and Quarantine Bureau | Infectious hematopoietic necrosis's poison nucleic acid is positive |
| 5 | The disease prevention and control center, Daliang City | Infectious hematopoietic necrosis's poison nucleic acid is positive |
| 6 | Fujian Entry-Exit Inspection and Quarantine Bureau | Infectious hematopoietic necrosis's poison nucleic acid is positive |
| 7 | Shenyang Entry-Exit Inspection and Quarantine Bureau | Infectious hematopoietic necrosis's poison nucleic acid is positive |
Use the method for GB/T15805.2-2008 regulation, to the definite value experiment of embodiment of the invention infectious hematopoietic necrosis poison molecular criteria sample, it is positive that the result is infectious hematopoietic necrosis's poison nucleic acid.
Sequence table
< 110>Dalian Nationality College
< 120>preparation method of infectious hematopoietic necrosis's poison molecular criteria sample
<160>?4
<210> 1
<211> 786
<212> DNA
< 213>infectious hematopoietic necrosis's poison N gene (Infectious heamatopoietic necrosis virus IHNV)
<400>?1
CACCGTACTTTGCTGCTACCCCTGGGGCCTCTGCTACGATCCTCATCAGTCTTACAATGCGTCTAGCGGACTCCACCACTGACTTCATGCACAGATCTTCTAGAAGGTCTGCTGGAAGGGCGCCCAGGTTCTTTGCGGCTTGGTTGAACAGTCCCACCATTGTCATCCCTGTCCCGCTCAGTCTCATGGCGCACAGTGCCTTGGCTCTAGCGGCGGTGGCTGGGTCAGACAGGTTGATGAGAATGATCCCATAAACAGCCTTGGTGAGCTTCTGTCCAAGTCTAAGTACCGCCCCGATCTTGGCCAGCACTCCCCTGTTGGAGTTGAAGTTGACCAACTCGTCAATCCCTTGGCTGGTTGCAAGACGCTCGAGCTTGTTTTGGCAGTATATGGCCATCTTGTCCACGTCATACTTCGTCAGTAGAGCGCAGGTGAAGAGGAGGCCGGTCACAACCTCAAGGACATTTTCCTTTCCGATCGTTTCTGCAAGCTTGTTGTTGGGATCTGCGAAAGTGGCGTCCAGTGGTGCCCCAGTGTCCAAAGACTCCAGCAAGAAGCCCAGGGCTTCCAGAGTTTCGGCGACCGTGCCTGTCCCCGATGGGACCGTCTCCGCAATGACGAACGCGCACAAGAGGCCCAATGCCCTTCTCGTTCCCTCTCCTCCGACGTGACTCACCGCCCGCTTAATCATCTCTAGTTCAAAGTCTGCCTTGGAGAAGAAGAGAGGGAGGGTTATCGTACCGGGACGATACTCCGTCTCTGAGTCCTCGAGGACTCCCCCCTTGA
<210>?2
<211>19
<212>?DNA
< 213>artificial sequence
<220>
<221>?misc?feature
< 223>primer
<400>2
TCAAGGGGGGAGTCCTCGA
<210>?3
<211>?19
<212>?DNA
< 213>artificial sequence
<220>
<221> misc?feature
< 223>primer
<400>?3
TCAAGGGGGGAGTCCTCGA
<210>?4
<211>?19
<212>?DNA
< 213>artificial sequence
<220>
<221>?misc?feature
< 223>primer
<400>?4
CACCGTACTTTGCTGCTAC
Claims (1)
1. the preparation method of infectious hematopoietic necrosis poison molecular criteria sample is characterized in that carrying out as follows:
A. the extraction of infectious hematopoietic necrosis's poison RNA
In centrifuge tube, add Trizol lysate 600 μ l, infectious hematopoietic necrosis's poison cell culture 200 μ l add chloroform 200 μ l concussion mixing, the centrifugal 15min of 10 000 g; Draw supernatant 500 μ l and join in the 500 μ l Virahols, put upside down mixing repeatedly, the centrifugal 15min of 10 000 g; Supernatant discarded is inverted on the thieving paper, is stained with dry liquids, adds 75% alcohol 600 μ l, puts upside down washing, the centrifugal 10min of 10 000 g; Supernatant discarded is inverted on the thieving paper, is stained with dry liquids; The centrifugal 10sec of 4 000g blots the pipe liquid at the end, drying at room temperature 3 min; The DEPC water that adds 11.5 μ l, mixing dissolves the RNA in the tube wall gently, the centrifugal 5sec of 2 000g, 4 ℃ of preservations, subsequent use as template;
B. RT-PCR reaction
B-1. reverse transcription system
5 * M-MLV Buffer, 4 μ l, dNTPs (each 2.5mmol/L) 2 μ l, M-MLV (5U/ μ l) 1 μ l, RNA enzyme inhibitors 0.5 μ l, reverse transcription primer (20pmol/ μ L) 1 μ l, a step gained RNA template 11.5 μ l; Said reverse transcription trip primer is 5 '-TCAAGGGGGGAGTCCTCGA-3 ', and the total reaction system is 20 μ l;
B-2. reverse transcription condition
42 ℃ of water-bath 1h ~ 2h;
The b-3.PCR amplification reaction system
10 * PCR Buffer (contains Mg
2+) 2.5 μ l, dNTPs (each 2.5mmol/L) 0.5 μ l,
TaqArchaeal dna polymerase (5U/ μ l) 1 μ l, upstream primer, each 1 μ L of downstream primer (20pmol/ μ L), the reverse transcription product 0.5 μ l of b-2 is supplemented to 25 μ l with sterilized water;
Said upstream primer is 5 '-TCAAGGGGGGAGTCCTCGA-3 ';
Said downstream primer is 5 '-CACCGTACTTTGCTGCTAC-3 ';
B-4. pcr amplification condition: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 1min, 53 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 30 circulations; 72 ℃ of 5min;
C. the purifying of target gene
Adopt TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 test kit, reclaim the target molecule dna fragmentation;
D. the segmental connection of purpose transforms
The d-1 linked system
The purpose fragment 4 μ l of the resulting purifying of c step, 2 * Rapid ligation buffer, 5 μ l, PGEM-T carrier 0.5 μ l, T4 DNA Ligase 0.5 μ l, room temperature connects 1h;
D-2 transforms
The connection product of a last step is joined in the competent cell mixing, ice bath 20min, 42 ℃ of heat stress 1min; Ice bath 2min adds 800 μ l SOC substratum, and 150g shakes bacterium 1.5h; The centrifugal 10min of 1 000g discards nutrient solution, adds fresh SOC substratum 200 μ l; Be coated in behind the mixing on the LB flat board that contains Amp+, air-dry after, be inverted to cultivate 12h ~ 16h for 37 ℃;
The screening of d-3 positive colony
Picking mono-clonal bacterium is cultivated, and carries out the screening of positive colony with PCR method, PCR system and reaction conditions such as b-3, b-4;
E. reclaim plasmid;
F. in-vitro transcription
E step gained plasmid is carried out single endonuclease digestion with SalI, and reaction system is: 10 * H Buffer, 5 μ l, SalI 1 μ l, plasmid 10 μ l, distilled water 34 μ l; 37 ℃ of 2h reclaim test kit with Omega and reclaim linearization plasmid; The responsive transcription system is: 10 * T7 RNA polymerase buffer, 5 μ l, DTT 5 μ l, NTP 10 μ l, Rnasin 1 μ l, linearization plasmid 5 μ l, T7 RNApolymerase 1 μ l, DEPC treating water 28 μ l; 37 ℃ of 2h, and then add 10U DnaseI 2 μ l, 37 ℃, 30min;
G. transcribe back RNA purification process
The RNA that transcribes is carried out DNase handle, reaction system and condition are following: the responsive transcription liquid 20ul of said f step, RNase free DNase I (5u/ul) 4 ul, Total 24ul, 37 ℃ of 1h;
H. phenol chloroform extracting
Said g step is carried out the extracting of phenol chloroform through the reaction solution that DNase handles, finally complement to 100 ul with RNase-free dH2O.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103173569A (en) * | 2013-04-03 | 2013-06-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for detecting infectious haematopoietic necrosis virus based on liquid-phase chip |
| CN103233005A (en) * | 2013-05-10 | 2013-08-07 | 国家海洋局第三海洋研究所 | Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit |
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| CN1860220A (en) * | 2003-07-02 | 2006-11-08 | 南卡罗来纳医科大学研究发展基金会 | Dsrna induced specific and non-specific immunity in crustaceans and other invertebrates and biodelivery vehicles for use therein |
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| CN1860220A (en) * | 2003-07-02 | 2006-11-08 | 南卡罗来纳医科大学研究发展基金会 | Dsrna induced specific and non-specific immunity in crustaceans and other invertebrates and biodelivery vehicles for use therein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103173569A (en) * | 2013-04-03 | 2013-06-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for detecting infectious haematopoietic necrosis virus based on liquid-phase chip |
| CN103173569B (en) * | 2013-04-03 | 2015-07-29 | 山东出入境检验检疫局检验检疫技术中心 | The method of infectious hematopoietic necrosis's poison is detected based on liquid-phase chip |
| CN103233005A (en) * | 2013-05-10 | 2013-08-07 | 国家海洋局第三海洋研究所 | Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit |
| CN103233005B (en) * | 2013-05-10 | 2015-04-08 | 国家海洋局第三海洋研究所 | Reverse transcription-polymerase chain reaction (RT-PCR) detection kit for infectious haematopoietic necrosis viruses (IHNV) and preparation method of kit |
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Application publication date: 20120815 |