CN103215388B - RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof - Google Patents
RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a RT-PCR (reverse transcription-polymerase chain reaction) detection kit for viral haemorrhagic septicaemia virus and a preparation method thereof, and relates to an RT-PCR detection kit. The RT-PCR detection kit for viral haemorrhagic septicaemia virus is provided with a kit body, an operation instruction and a detection reagent, wherein the operation instruction and the detection reagent are arranged in the kit body; and the detection reagent comprises a lysis solution, an adsorption solution, a washing solution, DEPC treatment water, an inverse transcription reaction solution, a VHSV-PCR reaction solution, a positive control and a negative control. The preparation method comprises the following steps of: preparing the detection reagent; and putting the operation instruction, the lysis solution, the adsorption solution, the washing solution, the DEPC treatment water, the inverse transcription reaction solution, the VHSV-PCR reaction solution, the positive control and the negative control into the kit body to obtain the RT-PCR detection kit for viral haemorrhagic septicaemia virus. The reagent is complete, and the operation is simple.
Description
Technical field
The present invention relates to a kind of RT-PCR detection kit, especially relate to RT-PCR detection kit of viral haemorrhagic septicaemia virus (Viral hemorrhagic septicemia virus, VHSV) and preparation method thereof.
Technical background
Viral hemorrhagic septicemia (Viral hemorrhagic septicemia, VHS) is a kind of lethality communicable disease that infects the multiple fish such as salmonid, trout, turbot, perch, Paralichthys lethostigma, brown trout, grayling.This disease causes fish-skin skin, kidney and liver hemorrhage, and mortality ratio is about 90%.The cause of disease of VHS is viral haemorrhagic septicaemia virus (VHSV), belongs to Rhabdoviridae Novirhabdovirus and belongs to.VHSV genome is one section of minus-strand RNA, the about 12kp of its length, 5 albumen of glm gene group coding, comprise: nucleocapsid protein (N albumen), two structural protein (P and M albumen), glycoprotein (G albumen), RNA polymerase (L albumen) and a Nonstructural Protein (NV albumen), order is 3 '-Leader-N-P-M-G-NV-L-Trailer-5 '.VHSV virus particle is about 180nm, wide about 70nm.In inward animal one, the two class transmissible disease registers of International Animal Health tissue and China, be all listed in and must report epidemic disease.International organization adopts some restrictions conventionally, thereby the fish that avoid catching an illness flow to other areas and national, and past VHS Major Epidemic is in North America and some countries of Europe.In recent years, along with the sharply increase of hydrocoles and products thereof foreign trade, VHSV has imported China into, and Bing China partial area are popular.
Reverse transcription chain polymerization enzyme reaction (RT-PCR) is a kind of for detection of virus and the most frequently used method of viroid, be applied in the detection of a series of fish disease virus, comprised: nona irido virus (Sleeping disease virus), Oncorhynchi infectious anemia syndrome virus (Infectious salmon anaemia virus), Aquareovirus (Fish aquareovirus), septicemia virus (Hemopoietic necrosis virus).
Viral haemorrhagic septicaemia virus lethality rate is very high, to cage culture, causes huge financial loss.The efficient manner of control virus infection is to prevent contact virus, carries out routine examination regularly, before and after fish transportation, carries out stricter detection.Avoid in principle the plant of contact infection, use the bait feeding of virus-free infection, use virus-free aquaculture water, using fish-egg is after testing the effective measure of controlling VHSV.Also there is no at present a kind of effective methods for the treatment of, therefore in initial infection, detect virus and just seem extremely important.This just needs that a kind of cost is low, quick, specificity and highly sensitive detection method.In the past few decades, set up the method for several detection viral haemorrhagic septicaemia virus.The detection method of traditional standard is first by cell cultures, to isolate virus, re-use special method as immunofluorescence monoclonal antibody (IFAT) or antibody neutralization test detection ([11] Fregeneda-GrandesJM, Olesen NJ.Detection of rainbow trout antibodies against viral haemorrhagicsepticaemia virus (VHSV) by neutralisation test is highly dependent on the virus isolate used.Dis Aquat Organ, 2007,74 (2): 151-158).In the GB GB/T15805.3-2008 of China 2008 Nian You State Administration for Quality Supervision and Inspection and Quarantine issue, also adopted the method.Enzyme-linked immunosorbent assay ([12] the Encinas P in addition commonly using at present, Gomez-Casado E, Estepa A, Coll JM.An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein.J Virol Methods, 176 (1-2): 14-23) and real-time quantitative RT-PCR ([13] Chico V, Gomez N, Estepa A, Perez L.Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR.J Virol Methods, 2006, 132 (1-2): 154-159).But the length consuming time that these methods have, expensive equipment and reagent are used in some requirements.RT-PCR technology is a kind of quick, good and sensitive high method for detecting virus of specificity, is widely used in bait, aquaculture water and environmental monitoring.Also relevant for the report of VHSVRT-PCR detection method, still only reported and do not formed test kit by Auele Specific Primer in the past, during operation, need to buy a large amount of matched reagents, process is loaded down with trivial details.
Summary of the invention
The object of the present invention is to provide can specific detection viral haemorrhagic septicaemia virus primer.
RT-PCR detection kit providing a kind of viral haemorrhagic septicaemia virus and preparation method thereof is provided another object of the present invention.
According to published viral haemorrhagic septicaemia virus genome sequence, choose the nucleoprotein gene sequence of viral haemorrhagic septicaemia virus, analyze design, described primer that can specific detection viral haemorrhagic septicaemia virus is:
VHSN-F: its nucleotide sequence is as shown in SEQ ID NO.1;
VHSN-F: its nucleotide sequence is as shown in SEQ ID NO.2.
The RT-PCR detection kit of described viral haemorrhagic septicaemia virus is provided with box body, process specifications and detection reagent; Process specifications and detection reagent are located in box body, and described detection reagent comprises that lysate, adsorption liquid, washings, DEPC process water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control.
The preparation method of the RT-PCR detection kit of described viral haemorrhagic septicaemia virus is as follows:
1) prepare box body;
2) preparation detection reagent:
Lysate is: 5mol/L Guanidinium hydrochloride, and 0.1mol/L EDTA, deionized water is settled to 1L, and pH is 8.0;
Adsorption liquid is: 5g glass powder is placed in 25～30mL water and is suspended, and then add isopyknic nitric acid reaction, finally again add aqueous suspension, the concentration of described nitric acid can be 68%;
Washings is: 0.1mol/L sodium-chlor, and 1mmol/L EDTA, 10mmol/L Tris-HCl, deionized water is settled to 1L, and pH is 7.5, then adds isopyknic dehydrated alcohol;
DEPC (diethylpyrocarbonate) processes water: toward adding DEPC in ultrapure water, to final concentration, be 0.1%, and after vibration 2h, autoclave sterilization 2 times;
Inverse transcription reaction liquid: every 6 μ L inverse transcription reaction liquids contain 1 μ L50mM VHSN-R primer; 1 μ L dNTP mixture, every kind of dNTP concentration is 10mM; 2 μ L5 * Primescript damping fluids; 0.25 μ L RNase inhibitor; 0.25 μ L reversed transcriptive enzyme, purchased from the precious biotech firm of DaLian, China; 0.3 μ L glycerine; 1.2 μ L DEPC process water;
VHSV-PCR reaction solution: every 18 μ L VHSV-PCR reaction solutions contain 1 μ L10mM VHSN-F primer; 1 μ L10mM VHSN-F primer; 1 μ L dNTP mixture, every kind of dNTP concentration is 2.5mM; 0.25 μ L Taq archaeal dna polymerase; 2 μ L10 * PCR damping fluids; 1.5 μ L glycerine; 11.25 μ L distilled water;
Negative control: DEPC processes water;
Positive control: the VHSV nucleoprotein gene RNA fragment that in-vitro transcription is obtained is diluted to 10
4copy/μ L gained solution;
3) process specifications, lysate, adsorption liquid, washings, DEPC are processed to water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control and insert in box body, obtain the RT-PCR detection kit of viral haemorrhagic septicaemia virus.
In step 2) in, the preparation method of described positive control is as follows:
(1) design of primers, concrete grammar is:
According to acquired VHSV nucleoprotein gene sequence, design following two for building primer VHSV-long-F and the VHSV-long-R of in-vitro transcription VHSV nucleoprotein gene RNA fragment recombinant plasmid pSP-VHSV, carry respectively Hind III and BamH I restriction enzyme site:
VHSV-long-F: its nucleotide sequence is as shown in SEQ ID NO.3;
VHSV-long-R: its nucleotide sequence is as shown in SEQ ID NO.4;
(2) structure that contains testing goal fragment recombinant plasmid pSP-VHSV and as the preparation of the RNA fragment of positive control, concrete steps are:
A) structure of pSP-VHSV recombinant plasmid
Use VHSV-long-R as reverse transcription primer, using viral RNA sample as template, through reverse transcription PCR reaction, obtained object fragment, then the reverse transcription product of acquisition is carried out to pcr amplification as template, after PCR product is reclaimed, with Hind III and BamH I restriction endonuclease, do double digestion, re-use E.Z.N.A.
tMcycle Pure test kit (OMEGA, USA) reclaims enzyme and cuts product, then enzyme is cut to product and is connected on pSP64poly (A) (Promega, USA) carrier, is converted in competence E.coliDH5 α screening positive clone sequence verification;
B) in-vitro transcription obtains the RNA fragment as positive control
The pSP-VHSV plasmid that extraction builds, reclaims plasmid after EcoRI linearizing, then obtains small RNA fragments by Promega in-vitro transcription test kit in-vitro transcription, with 1.2% agarose gel electrophoresis and spectrophotometer, detects RNA quality and concentration; According to RNA molecular weight, determine unit volume RNA molecule number, by 10 times of gradient dilutions, dilution is 1 * 10 respectively
6to 1 * 10
1copy/μ L, the positive RNA fragment of acquisition concentration gradient.
The present invention has designed two pairs of new primers according to VHSV nucleoprotein (N) gene order and has detected for the RT-PCR of VHSV, and has detected specificity and the sensitivity of this primer; In-vitro transcription a bit of viral RNA as positive control, thereby set up the VHSV detection kit of rapid sensitive.
The invention provides a kind of special, easy and viral haemorrhagic septicaemia virus detection kit fast, reagent is complete, simple to operate, for providing testing tool to popular detection and the prevention and control of this disease later.
Accompanying drawing explanation
Fig. 1 is the RT-PCR detection kit cross section view (being composition and the position view arranging in box) of viral haemorrhagic septicaemia virus.In Fig. 1, be respectively labeled as: 1: lysate, 2: washings, 3: box body, 4: process specifications, 5: adsorption liquid, 6:DEPC process water, 7: inverse transcription reaction liquid, 8:VHSV-PCR reaction solution, 9: positive control:, 10: negative control.
Fig. 2 is embodiment 1 primer specificity experiment electrophorogram.In Fig. 2, M is DL Marker2000(Takara, China); 1 positive contrast (VHSV transcribes gained RNA fragment), 2 is VHSV, and 3 is IHNV, and 4 is IPNV, and 5 is SVCV, 6 negative contrasts.
Fig. 3 is embodiment 2 sensitivity test experience electrophorograms.In Fig. 3, M is DL Marker2000(Takara, China); 1～6 is 2 * 10
6, 2 * 10
5, 2 * 10
4, 2 * 10
3, 2 * 10
2, 2 * 10
1copy/μ L in-vitro transcription gained positive control RNA; 7 negative contrasts.
Fig. 4 is embodiment 3 rate of recovery experiment electrophorograms.In Fig. 4, M is DL Marker2000(Takara, China); 1～3 is 2 * 10
6, 2 * 10
5, 2 * 10
4copy/μ L in-vitro transcription gained positive control RNA; 4～6 is 2 * 10
6, 2 * 10
5, 2 * 10
4the RNA reclaiming after copy/μ L positive control RNA biased sample; 7 negative contrasts.
Embodiment
One, Auele Specific Primer of the present invention, according to published viral haemorrhagic septicaemia virus genome sequence, choose the nucleoprotein gene sequence of viral haemorrhagic septicaemia virus, analyze design, can specificity differentiate viral haemorrhagic septicaemia virus, this comprises two to primer:
VHSV-F: its nucleotide sequence is as shown in SEQ ID NO.1;
VHSV-R: its nucleotide sequence is as shown in SEQ ID NO.2.
Two, the RT-PCR detection kit of viral haemorrhagic septicaemia virus of the present invention is provided with box body 3, process specifications 4 and detection reagent; Process specifications 4 and detection reagent are located in box body 3, and described detection reagent comprises that lysate 1, adsorption liquid 5, washings 2, DEPC process water 6, inverse transcription reaction liquid 7, VHSV-PCR reaction solution 8, positive control 9, negative control 10(referring to Fig. 1);
The preparation method of the RT-PCR detection kit of described viral haemorrhagic septicaemia virus is as follows:
1. prepare box body;
2. prepare detection reagent:
Lysate is: 5mol/L Guanidinium hydrochloride, and 0.1mol/L EDTA, deionized water is settled to 1L, and pH is 8.0;
Adsorption liquid is: 5g glass powder is placed in 25-30mL water and is suspended, and then add the nitric acid reaction of equal-volume concentration 68%, finally again add aqueous suspension.
Washings is: 0.1mol/L sodium-chlor, and 1mmol/L EDTA, 10mmol/L Tris-HCl, deionized water is settled to 1L, and pH is 7.5, then adds isopyknic dehydrated alcohol;
DEPC (diethylpyrocarbonate) processes water: toward adding DEPC in ultrapure water, to final concentration, be 0.1%, and after vibration 2h, autoclave sterilization 2 times.
Inverse transcription reaction liquid: every 6 μ L inverse transcription reaction liquids contain 1 μ L50mM VHSV-R primer; 1 μ L dNTP mixture, every kind of dNTP concentration is 10mM; 2 μ L5 * Primescript damping fluids; 0.25 μ LRNase inhibitor; 0.25 μ L reversed transcriptive enzyme, purchased from the precious biotech firm of DaLian, China; 0.3 μ L glycerine; 1.2 μ L DEPC process water.
VHSV-PCR reaction solution: every 18 μ L VHSV-PCR reaction solutions contain 1 μ L10mMVHSV-F primer; 1 μ L10mM VHSV-R primer; 1 μ L dNTP mixture, every kind of dNTP concentration is 2.5mM; 0.25 μ L Taq archaeal dna polymerase; 2 μ L10 * PCR damping fluids; 1.5 μ L glycerine; 11.25 μ L distilled water.
Negative control: DEPC processes water.
Positive control: the VHSV nucleoprotein gene RNA fragment that in-vitro transcription is obtained is diluted to 10
4copy/μ L gained solution.
In step 2, the preparation method of described positive control is as follows:
Step 1, design of primers, concrete grammar is:
According to acquired VHSV nucleoprotein gene sequence, design following two for building primer VHSV-long-F and the VHSV-long-R of in-vitro transcription VHSV nucleoprotein gene RNA fragment recombinant plasmid pSP-VHSV, carry respectively Hind III and BamH I restriction enzyme site:
VHSV-long-F: its nucleotide sequence is as shown in SEQ ID NO.3;
VHSV-long-R: its nucleotide sequence is as shown in SEQ ID NO.4.
Step 2, the structure that contains testing goal fragment recombinant plasmid pSP-VHSV and as the preparation of the RNA fragment of positive control, concrete steps are:
1) structure of pSP-VHSV recombinant plasmid
Use VHSV-long-R as reverse transcription primer, using viral RNA sample as template, through reverse transcription PCR reaction, obtained object fragment.Its reverse transcription reaction system and response procedures are as follows:
Mix rear 42 ℃ of reaction 1h, after 70 ℃ of reaction 15min termination reactions, be placed in rapidly on ice, then the reverse transcription product of acquisition is carried out to pcr amplification as template, reaction system and response procedures are as follows:
PCR program is: 95 ℃ of 3min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 5min;
After PCR product is reclaimed, with Hind III and BamH I restriction endonuclease, do double digestion, re-use E.Z.N.A.
tMcycle Pure test kit (OMEGA, USA) reclaims enzyme and cuts product, then enzyme is cut to product and is connected on pSP64poly (A) (Promega, USA) carrier, is converted in competence E.coliDH5 α screening positive clone sequence verification;
2) in-vitro transcription obtains the RNA fragment as positive control
The pSP-VHSV plasmid that extraction builds, reclaims plasmid after EcoRI linearizing, then obtains small RNA fragments by Promega in-vitro transcription test kit in-vitro transcription.In-vitro transcription system and condition are as follows:
Adding DEPC processing water to final volume is 20 μ L.
Centrifugal be placed on 37 ℃ reaction 1h, after having reacted, add RQ1RNase-Free DNase2u, hatch 30min digestion for 37 ℃ and remove pSP-VHSV plasmid, phenol/chloroform/primary isoamyl alcohol (25: 24: 1) and chloroform/primary isoamyl alcohol (24: 1) extracting RNA, then detect RNA quality and concentration with 1.2% agarose gel electrophoresis and spectrophotometer; According to RNA molecular weight, determine unit volume RNA molecule number, by 10 times of gradient dilutions, dilution is 1 * 10 respectively
6to 1 * 10
1copy/μ L, the positive RNA fragment of acquisition concentration gradient.
3. by detection reagent: process specifications, lysate, adsorption liquid, washings, DEPC process water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control and insert in box body, obtain the RT-PCR detection kit of viral haemorrhagic septicaemia virus.The quality of stochastic sampling detection kit again.
Three, the application of the RT-PCR detection kit of viral haemorrhagic septicaemia virus is as follows:
1. the sample of obtaining is added in 0.5mL centrifuge tube, then adds and manage 8 of interior lysates for No. 1, with toothpick, fully smash (approximately 1～2min) to pieces.
The centrifugal 3min of 2.12000r/min, is transferred to supernatant in new 0.5mL centrifuge tube, adds the adsorption liquid (include white mass, fully shake up with front) in 30 μ L5 pipes, mixes.Room temperature is placed 5min, during vibrate 3 times.
The centrifugal 30s of 3.12000r/min, abandons supernatant, more centrifugal 3s, with pipettor, blots residual liquid, adds 10 of No. 2 washingss in pipe, stirs and makes to precipitate abundant suspension gently.
The centrifugal 30s of 4.12000r/min, abandons supernatant, more centrifugal 3s, with pipettor, blots residual liquid, and room temperature is placed 3min.
5. add 20 μ LDEPC to process water, abundant dissolution precipitation, the centrifugal 30s of 12000r/min, the inverse transcription reaction liquid 6 μ L that draw in supernatant 4 μ L and No. 7 pipes are added in 0.5mL centrifuge tube, hatch 0.5h for 42 ℃, again the VHSV-PCR reaction solution of reverse transcription product 2 μ L and 18 μ L8 pipes is mixed, carry out PCR reaction.PCR reaction parameter is set to: first 95 ℃ of pre-treatment 1min, carry out 35 circulating reactions, and each circulation comprises 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, after having circulated, 72 ℃ are extended 10min.The negative control of the positive control of No. 9 pipes and No. 10 pipes does not need to process, and directly gets 4 μ L and carries out reverse transcription and follow-up PCR reaction, and reaction conditions detects identical with previous sample.
6. result is judged: PCR reaction is complete, gets 10 μ L reaction product electrophoresis detection on 1.5% sepharose, time 15～25min.Under ultraviolet lamp, observe, positive control should have the clear band of a 471bp; Negative control at this parallel position without band; If sample has band at this parallel position, show that this sample may contain viral haemorrhagic septicaemia virus, if do not contain viral haemorrhagic septicaemia virus in sample without band.
Remarks: when test kit is not used, 7,8,9 and No. 10 reagent-20 ℃ preservations, 4 ℃ of preservations of all the other reagent behind Kaifeng, should be finished in 6 months.
Below in conjunction with specific embodiment, the present invention is further elaborated, but specific embodiment is not done any restriction to the present invention.
The specificity experiment of embodiment 1 primer
In order to determine the specificity of this RT-PCR detection kit, use respectively other viral RNAs or DNA as template, using method according to test kit detects, described other viruses comprise infectious hematopoietic necrosis's virus (Infectious hematopoietic necrosis virus, IHNV), pancreas necrosis virus (Infectious pancreatic necrosis virus, IPNV) and carp pure blood disease necrosis virus (Spring viremia of carp virus, SVCV).Experimental result shows: except viral haemorrhagic septicaemia virus, all can't detect the object fragment (see figure 2) of 471bp after other viral agarose gel electrophoresis.Illustrate that this detection kit primer specificity used is good.
Embodiment 2 sensitivity test experience
Get 2 μ L different concns gradients (1 * 10
6-1 * 10
1copy/μ L) positive control RNA fragment detects according to the using method of test kit as template, then uses 1.5% agarose gel electrophoresis detected result, and result shows (see figure 3), and the method can detect and be approximately 10
2individual positive RNA, had both been equivalent to 10
2individual virus particle.
Embodiment 3 rate of recovery experiments
In order to analyze the efficiency of extracting viral RNA from sample, carried out rate of recovery experiment.By 20 μ L1 * 10
6, 1 * 10
5, 1 * 10
4the positive control RNA of copy/μ L and negative sample mix, and according to the using method of test kit, extract RNA, RNA is dissolved in to 20 μ LDEPC and processes in water.Then with each concentration gradient positive control RNA of 2 μ L and the new RNA extracting, as template, detect respectively.The demonstration of agarose gel electrophoresis detected result, the rate of recovery of this extracting method is greater than 50%(and sees Fig. 4).
Claims (5)
1. primer that can specific detection viral haemorrhagic septicaemia virus, is characterized in that described primer is:
VHSN-F: its nucleotide sequence is as shown in SEQ ID NO.1;
VHSN-F: its nucleotide sequence is as shown in SEQ ID NO.2.
2. the RT-PCR detection kit of viral haemorrhagic septicaemia virus, is characterized in that being provided with box body, process specifications and detection reagent; Process specifications and detection reagent are located in box body, and described detection reagent comprises that lysate, adsorption liquid, washings, DEPC process water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control.
3. the preparation method of the RT-PCR detection kit of viral haemorrhagic septicaemia virus as claimed in claim 2, is characterized in that comprising the following steps:
1) prepare box body;
2) preparation detection reagent:
Lysate is: 5mol/L Guanidinium hydrochloride, and 0.1mol/L EDTA, deionized water is settled to 1L, and pH is 8.0;
Adsorption liquid is: 5g glass powder is placed in 25～30mL water and is suspended, and then add isopyknic nitric acid reaction, finally again add aqueous suspension;
Washings is: 0.1mol/L sodium-chlor, and 1mmol/L EDTA, 10mmol/L Tris-HCl, deionized water is settled to 1L, and pH is 7.5, then adds isopyknic dehydrated alcohol;
DEPC processes water: toward adding DEPC in ultrapure water, to final concentration, be 0.1%, and after vibration 2h, autoclave sterilization 2 times;
Inverse transcription reaction liquid: every 6 μ L inverse transcription reaction liquids contain 1 μ L50mM VHSN-R primer; 1 μ L dNTP mixture, every kind of dNTP concentration is 10mM; 2 μ L5 * Primescript damping fluids; 0.25 μ L RNase inhibitor; 0.25 μ L reversed transcriptive enzyme, purchased from the precious biotech firm of DaLian, China; 0.3 μ L glycerine; 1.2 μ L DEPC process water;
VHSV-PCR reaction solution: every 18 μ L VHSV-PCR reaction solutions contain 1 μ L10mM VHSN-F primer; 1 μ L10mMVHSN-F primer; 1 μ L dNTP mixture, every kind of dNTP concentration is 2.5mM; 0.25 μ L Taq archaeal dna polymerase; 2 μ L10 * PCR damping fluids; 1.5 μ L glycerine; 11.25 μ L distilled water;
Negative control: DEPC processes water;
Positive control: the VHSV nucleoprotein gene RNA fragment that in-vitro transcription is obtained is diluted to 10
4copy/μ L gained solution;
3) process specifications, lysate, adsorption liquid, washings, DEPC are processed to water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control and insert in box body, obtain the RT-PCR detection kit of viral haemorrhagic septicaemia virus.
4. the preparation method of the RT-PCR detection kit of viral haemorrhagic septicaemia virus as claimed in claim 3, is characterized in that in step 2) in, the preparation method of described positive control is as follows:
(1) design of primers, concrete grammar is:
According to acquired VHSV nucleoprotein gene sequence, design following two for building primer VHSV-long-F and the VHSV-long-R of in-vitro transcription VHSV nucleoprotein gene RNA fragment recombinant plasmid pSP-VHSV, carry respectively Hind III and BamH I restriction enzyme site:
VHSV-long-F: its nucleotide sequence is as shown in SEQ ID NO.3;
VHSV-long-R: its nucleotide sequence is as shown in SEQ ID NO.4;
(2) structure that contains testing goal fragment recombinant plasmid pSP-VHSV and as the preparation of the RNA fragment of positive control, concrete steps are:
A) structure of pSP-VHSV recombinant plasmid
Use VHSV-long-R as reverse transcription primer, using viral RNA sample as template, through reverse transcription PCR reaction, obtained object fragment, then the reverse transcription product of acquisition is carried out to pcr amplification as template, after PCR product is reclaimed, with Hind III and BamH I restriction endonuclease, do double digestion, re-use E.Z.N.A.
tMcycle Pure test kit (OMEGA, USA) reclaims enzyme and cuts product, then enzyme is cut to product and is connected on pSP64poly (A) (Promega, USA) carrier, is converted in competence E.coliDH5 α screening positive clone sequence verification;
B) in-vitro transcription obtains the RNA fragment as positive control
The pSP-VHSV plasmid that extraction builds, reclaims plasmid after EcoRI linearizing, then obtains small RNA fragments by Promega in-vitro transcription test kit in-vitro transcription, with 1.2% agarose gel electrophoresis and spectrophotometer, detects RNA quality and concentration; According to RNA molecular weight, determine unit volume RNA molecule number, by 10 times of gradient dilutions, dilution is 1 * 10 respectively
6to 1 * 10
1copy/μ L, the positive RNA fragment of acquisition concentration gradient.
5. the preparation method of the RT-PCR detection kit of viral haemorrhagic septicaemia virus as claimed in claim 3, is characterized in that in step 2) in, the concentration of described nitric acid is 68%.
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| CN105543413B (en) * | 2016-01-25 | 2019-01-22 | 山东出入境检验检疫局检验检疫技术中心 | Viral haemorrhagic septicaemia virus visualizes nucleic acid test strip detection primer group and detection method |
| CN110592272A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Viral Hemorrhagic Septicemia (VHSV) |
| CN110387440B (en) * | 2019-09-06 | 2020-08-11 | 深圳海关动植物检验检疫技术中心 | Reagent for multiple detection of salmon and trout viruses and application thereof |
| KR20240038884A (en) * | 2022-09-16 | 2024-03-26 | 재단법인 대구경북첨단의료산업진흥재단 | Composition For Detecting Viral Hemorrhagic Septicemia Virus, Kit For Detecting the Same and Method of Detecting VHSV Using the Same based on loop mediated isothermal amplification combined with CRISPR/Cas diagnosis |
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