CN103215388A - 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 viral haemorrhagic septicaemia virus (Viral hemorrhagic septicemia virus, RT-PCR detection kit VHSV) and preparation method thereof.
Technical background
(Viral hemorrhagic septicemia VHS) is a kind of lethality communicable disease that infects multiple fish such as salmonid, trout, turbot, perch, Paralichthys lethostigma, brown trout, grayling to viral hemorrhagic septicemia.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.The VHSV genome is one section 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 '.The 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, all be listed in and report eqpidemic disease.International organization adopts some restrictions usually, thereby the fish that avoid catching an illness flow to other areas and national, and past VHS mainly is popular in North America and more European countries.In recent years, along with the rapid increase of hydrocoles and products thereof foreign trade, VHSV has imported China into, and popular in China partial area.
Reverse transcription chain polymerization enzyme reaction (RT-PCR) is a kind of be used to detect 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).
The viral haemorrhagic septicaemia virus lethality rate is very high, causes enormous economic loss to cage culture.The efficient manner of control virus infection is to prevent contact virus, carries out routine examination regularly, before and after the fish transportation, carries out stricter detection.Avoid the plant of contact infection in principle, use the bait feeding of virus-free infection, uses virus-free aquaculture water, using after testing fish-egg is the effective measure of controlling VHSV.Also do not have a kind of effective methods of treatment at present, therefore detect virus and just seem extremely important in initial infection.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 at first to isolate virus by cell cultures, re-use special method such as immunofluorescence monoclonal antibody (IFAT) or antibody neutralization test and detect ([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).Among China 2008 GB GB/T15805.3-2008, also adopted this method by State Administration for Quality Supervision and Inspection and Quarantine's issue.Enzyme-linked immunosorbent assay ([12] Encinas P in addition commonly used 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 the requirement that has.The RT-PCR technology be a kind of fast, specificity is good and sensitive high method for detecting virus, be widely used in bait, aquaculture water and the environmental monitoring.Also relevant for the report of VHSVRT-PCR detection method, still only reported not form test kit by Auele Specific Primer that need buy a large amount of matched reagents during operation, process was loaded down with trivial details in the past.
Summary of the invention
But the object of the present invention is to provide the primer of specific detection viral haemorrhagic septicaemia virus.
RT-PCR detection kit that provides a kind of viral haemorrhagic septicaemia virus and preparation method thereof is provided another object of the present invention.
According to disclosed viral haemorrhagic septicaemia virus genome sequence, choose the nucleoprotein gene sequence of viral haemorrhagic septicaemia virus, analyze design, but the primer of described specific detection viral haemorrhagic septicaemia virus is:
VHSN-F: its nucleotide sequence is shown in SEQ ID NO.1;
VHSN-F: its nucleotide sequence is 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 the box body, and described detection reagent comprises lysate, adsorption liquid, washings, DEPC treating 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) preparation box body;
2) preparation detection reagent:
Lysate is: the 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 suspends, and then add isopyknic nitric acid reaction, add aqueous suspension at last again and get final product, 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, adds isopyknic dehydrated alcohol again;
DEPC (diethylpyrocarbonate) treating water: adding DEPC in the ultrapure water is 0.1% to final concentration, behind the vibration 2h, and autoclave sterilization 2 times;
Inverse transcription reaction liquid: per 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 fluid; 0.25 μ L RNase inhibitor; 0.25 μ L reversed transcriptive enzyme is available from the precious biotech firm of DaLian, China; 0.3 μ L glycerine; 1.2 μ L DEPC treating water;
The VHSV-PCR reaction solution: per 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 fluid; 1.5 μ L glycerine; 11.25 μ L distilled water;
Negative control: DEPC treating 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 treating water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control are inserted in the box body, promptly get 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 primer VHSV-long-F and VHSV-long-R that are used to make up in-vitro transcription VHSV nucleoprotein gene RNA fragment recombinant plasmid pSP-VHSV, carry Hind III and BamH I restriction enzyme site respectively:
VHSV-long-F: its nucleotide sequence is shown in SEQ ID NO.3;
VHSV-long-R: its nucleotide sequence is shown in SEQ ID NO.4;
(2) contain the structure of testing goal fragment recombinant plasmid pSP-VHSV and as the segmental preparation of the RNA of positive control, concrete steps are:
A) pSP-VHSV construction of recombinant plasmid
Use VHSV-long-R as the reverse transcription primer, with the viral RNA sample as template, through the reverse transcription PCR reaction, obtained the purpose fragment, then the reverse transcription product that obtains is carried out pcr amplification as template, the PCR product is reclaimed the back do double digestion, re-use E.Z.N.A. with Hind III and BamH I restriction endonuclease
TM(OMEGA USA) reclaims enzyme and cuts product Cycle Pure test kit, then enzyme is cut product and is connected to pSP64poly (A) (Promega USA) on the carrier, is converted among the competence E.coliDH5 α screening positive clone and sequence verification;
B) in-vitro transcription obtains the RNA fragment as positive control
The pSP-VHSV plasmid that extraction builds reclaims plasmid after the EcoRI linearizing, obtains small RNA fragments with Promega in-vitro transcription test kit in-vitro transcription again, detects RNA quality and concentration with 1.2% agarose gel electrophoresis and spectrophotometer; Determine unit volume RNA molecule number according to the RNA molecular weight, 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 been used for the RT-PCR detection of VHSV, and has detected the 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, and is simple to operate, for popular detection and the prevention and control to this disease later on provide testing tool.
Description of drawings
Fig. 1 is the RT-PCR detection kit cross section view (being composition and the position view that is provided with in the box) of viral haemorrhagic septicaemia virus.In Fig. 1, respectively be labeled as: 1: lysate, 2: washings, 3: box body, 4: process specifications, 5: adsorption liquid, 6:DEPC treating water, 7: inverse transcription reaction liquid, 8:VHSV-PCR reaction solution, 9: positive control:, 10: negative control.
Fig. 2 is embodiment 1 a 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 electrophorogram.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 that reclaims behind the copy/μ L positive control RNA biased sample; 7 negative contrasts.
Embodiment
One, Auele Specific Primer of the present invention, according to disclosed 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 shown in SEQ ID NO.1;
VHSV-R: its nucleotide sequence is 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 the box body 3, and described detection reagent comprises that lysate 1, adsorption liquid 5, washings 2, DEPC treating water 6, inverse transcription reaction liquid 7, VHSV-PCR reaction solution 8, positive control 9, negative control 10(are referring to Fig. 1);
The preparation method of the RT-PCR detection kit of described viral haemorrhagic septicaemia virus is as follows:
1. preparation box body;
2. preparation detection reagent:
Lysate is: the 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 the 25-30mL water suspends, and then add the nitric acid reaction of equal-volume concentration 68%, add aqueous suspension at last again and get final product.
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, adds isopyknic dehydrated alcohol again;
DEPC (diethylpyrocarbonate) treating water: adding DEPC in the ultrapure water is 0.1% to final concentration, behind the vibration 2h, and autoclave sterilization 2 times.
Inverse transcription reaction liquid: per 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 fluid; 0.25 μ LRNase inhibitor; 0.25 μ L reversed transcriptive enzyme is available from the precious biotech firm of DaLian, China; 0.3 μ L glycerine; 1.2 μ L DEPC treating water.
The VHSV-PCR reaction solution: per 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 fluid; 1.5 μ L glycerine; 11.25 μ L distilled water.
Negative control: DEPC treating 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:
According to acquired VHSV nucleoprotein gene sequence, design following two primer VHSV-long-F and VHSV-long-R that are used to make up in-vitro transcription VHSV nucleoprotein gene RNA fragment recombinant plasmid pSP-VHSV, carry Hind III and BamH I restriction enzyme site respectively:
VHSV-long-F: its nucleotide sequence is shown in SEQ ID NO.3;
VHSV-long-R: its nucleotide sequence is shown in SEQ ID NO.4.
1) pSP-VHSV construction of recombinant plasmid
Use VHSV-long-R as the reverse transcription primer, the viral RNA sample as template, through the reverse transcription PCR reaction, has been obtained the purpose fragment.Its reverse transcription reaction system and response procedures are as follows:
Mix back 42 ℃ of reaction 1h, place rapidly on ice after 70 ℃ of reaction 15min termination reactions, then the reverse transcription product that obtains is carried out pcr amplification as template, reaction system and response procedures are as follows:
The PCR program is: 95 ℃ of 3min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ of 5min;
The PCR product is reclaimed the back do double digestion, re-use E.Z.N.A. with Hind III and BamH I restriction endonuclease
TM(OMEGA USA) reclaims enzyme and cuts product Cycle Pure test kit, then enzyme is cut product and is connected to pSP64poly (A) (Promega USA) on the carrier, is converted among the competence E.coliDH5 α screening positive clone and sequence verification;
2) in-vitro transcription obtains the RNA fragment as positive control
The pSP-VHSV plasmid that extraction builds reclaims plasmid after the EcoRI linearizing, obtains small RNA fragments with Promega in-vitro transcription test kit in-vitro transcription again.In-vitro transcription system and condition are as follows:
Adding DEPC treating water to final volume is 20 μ L.
Centrifugal be placed on 37 ℃ the reaction 1h, react the back and added RQ1RNase-Free DNase2u, hatch 30min digestion for 37 ℃ and remove the pSP-VHSV plasmid, phenol/chloroform/primary isoamyl alcohol (25: 24: 1) and chloroform/primary isoamyl alcohol (24: 1) extracting RNA detect RNA quality and concentration with 1.2% agarose gel electrophoresis and spectrophotometer again; Determine unit volume RNA molecule number according to the RNA molecular weight, 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. with detection reagent: process specifications, lysate, adsorption liquid, washings, DEPC treating water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control are inserted in the box body, promptly get 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 the 0.5mL centrifuge tube, adds then and manage 8 of interior lysates for No. 1, fully smash (about 1～2min) to pieces with toothpick.
2.12000r/min centrifugal 3min is transferred to supernatant in the new 0.5mL centrifuge tube, adds the adsorption liquid (include white mass, fully shake up with preceding) in the 30 μ L5 pipes, mixing.Room temperature is placed 5min, during vibration 3 times.
3.12000r/min centrifugal 30s abandons supernatant, centrifugal again 3s blots residual liquid with pipettor, adds No. 2 and manages 10 of interior washingss, stirs gently to make fully suspension of precipitation.
4.12000r/min centrifugal 30s abandons supernatant, centrifugal again 3s blots residual liquid with pipettor, and room temperature is placed 3min.
5. add 20 μ LDEPC treating 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 the 0.5mL centrifuge tube, hatch 0.5h for 42 ℃, VHSV-PCR reaction solution with reverse transcription product 2 μ L and 18 μ L8 pipes mixes again, carries out the PCR reaction.The PCR reaction parameter is set to: 95 ℃ of pre-treatment 1min of elder generation, carry out 35 circulating reactions, and each circulation comprises 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s, after circulation was finished, 72 ℃ were extended 10min.The negative control of the positive control of No. 9 pipes and No. 10 pipes does not need to handle, 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. the result judges: the PCR reaction finishes, and gets 10 μ L reaction product electrophoresis detection on 1.5% sepharose, time 15～25min.Ultraviolet lamp is observed down, and positive control should have the clear band of a 471bp; Negative control does not have band at this parallel position; If sample has band at this parallel position, show that this sample may contain viral haemorrhagic septicaemia virus, if no band does not then contain viral haemorrhagic septicaemia virus in the sample.
Remarks: when test kit does not use, 7,8,9 and No. 10 reagent-20 ℃ preservations, 4 ℃ of preservations of all the other reagent behind the Kaifeng, should use up in 6 months.
Below in conjunction with specific embodiment the present invention is further elaborated, but specific embodiment is not done any qualification to the present invention.
The specificity experiment of embodiment 1 primer
In order to determine the specificity of this RT-PCR detection kit, use other viral RNAs or DNA as template respectively, 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 detect the purpose fragment (see figure 2) less than 471bp behind other viral agarose gel electrophoresis.Illustrate that the used primer specificity of this detection kit is good.
Get 2 μ L different concns gradients (1 * 10
6-1 * 10
1Copy/μ L) positive control RNA fragment detects as the using method of template according to test kit, uses 1.5% agarose gel electrophoresis detected result then, and the result shows (see figure 3), and this method can detect and be approximately 10
2Individual positive RNA both had been equivalent to 10
2Individual virus particle.
The experiment of embodiment 3 rate of recovery
In order to analyze the efficient of from sample, extracting viral RNA, carried out rate of recovery experiment.With 20 μ L1 * 10
6, 1 * 10
5, 1 * 10
4The positive control RNA of copy/μ L and negative sample mix, and extract RNA according to the using method of test kit, and RNA is dissolved in the 20 μ LDEPC treating water.Detect as template with each concentration gradient positive control RNA of 2 μ L and the new RNA that extracts respectively then.The agarose gel electrophoresis detected result shows that the rate of recovery of this extracting method is seen Fig. 4 greater than 50%().
Claims (5)
1. but the primer of specific detection viral haemorrhagic septicaemia virus is characterized in that described primer is:
VHSN-F: its nucleotide sequence is shown in SEQ ID NO.1;
VHSN-F: its nucleotide sequence is 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 the box body, and described detection reagent comprises lysate, adsorption liquid, washings, DEPC treating water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control.
3. as the preparation method of the RT-PCR detection kit of viral haemorrhagic septicaemia virus as described in the claim 2, it is characterized in that may further comprise the steps:
1) preparation box body;
2) preparation detection reagent:
Lysate is: the 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 suspends, and then add isopyknic nitric acid reaction, add aqueous suspension at last again and get final product;
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, adds isopyknic dehydrated alcohol again;
The DEPC treating water: adding DEPC in the ultrapure water is 0.1% to final concentration, behind the vibration 2h, and autoclave sterilization 2 times;
Inverse transcription reaction liquid: per 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 fluid; 0.25 μ L RNase inhibitor; 0.25 μ L reversed transcriptive enzyme is available from the precious biotech firm of DaLian, China; 0.3 μ L glycerine; 1.2 μ L DEPC treating water;
The VHSV-PCR reaction solution: per 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 fluid; 1.5 μ L glycerine; 11.25 μ L distilled water;
Negative control: DEPC treating 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 treating water, inverse transcription reaction liquid, VHSV-PCR reaction solution, positive control and negative control are inserted in the box body, promptly get the RT-PCR detection kit of viral haemorrhagic septicaemia virus.
4. as the preparation method of the RT-PCR detection kit of viral haemorrhagic septicaemia virus as described in the claim 3, it 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 primer VHSV-long-F and VHSV-long-R that are used to make up in-vitro transcription VHSV nucleoprotein gene RNA fragment recombinant plasmid pSP-VHSV, carry Hind III and BamH I restriction enzyme site respectively:
VHSV-long-F: its nucleotide sequence is shown in SEQ ID NO.3;
VHSV-long-R: its nucleotide sequence is shown in SEQ ID NO.4;
(2) contain the structure of testing goal fragment recombinant plasmid pSP-VHSV and as the segmental preparation of the RNA of positive control, concrete steps are:
A) pSP-VHSV construction of recombinant plasmid
Use VHSV-long-R as the reverse transcription primer, with the viral RNA sample as template, through the reverse transcription PCR reaction, obtained the purpose fragment, then the reverse transcription product that obtains is carried out pcr amplification as template, the PCR product is reclaimed the back do double digestion, re-use E.Z.N.A. with Hind III and BamH I restriction endonuclease
TM(OMEGA USA) reclaims enzyme and cuts product Cycle Pure test kit, then enzyme is cut product and is connected to pSP64poly (A) (Promega USA) on the carrier, is converted among the competence E.coliDH5 α screening positive clone and sequence verification;
B) in-vitro transcription obtains the RNA fragment as positive control
The pSP-VHSV plasmid that extraction builds reclaims plasmid after the EcoRI linearizing, obtains small RNA fragments with Promega in-vitro transcription test kit in-vitro transcription again, detects RNA quality and concentration with 1.2% agarose gel electrophoresis and spectrophotometer; Determine unit volume RNA molecule number according to the RNA molecular weight, 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. as the preparation method of the RT-PCR detection kit of viral haemorrhagic septicaemia virus as described in the claim 3, it is characterized in that in step 2) in, the concentration of described nitric acid is 68%.
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| 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) |
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| 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 |
| WO2024058300A1 (en) * | 2022-09-16 | 2024-03-21 | 재단법인 대구경북첨단의료산업진흥재단 | Vhsv detection composition using gene scissors and loop-mediated isothermal amplification, kit, and vhsv detection method using same |
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