CN102108416B - Detection method and kit for pike juvenile rhabdovirus RT-PCR - Google Patents

Abstract

The invention discloses a detection method and a kit for pike juvenile rhabdovirus RT-PCR. The detection method and the kit of the invention detects PFRV by a specific primer which has a good initiation property and a wide range of Mg2+ concentration, primer concentration, dNTP concentration and annealing temperature; the RT-PCR detection method established by the invention has good specificity and high sensitivity, and the sensitivity for detecting nucleic acids extracted from a virus suspension can reach 102.5TCID50.

Description

Pike juvenile rhabdovirus RT-PCR detection method and test kit

Technical field

The present invention relates to Detecting method and detection kit, particularly relate to pike juvenile rhabdovirus RT-PCR detection method and test kit.

Background technology

According to international virusology classification (the International comittee on taxonomy ofviruses of the council, ICTV) the 8th report, Rhabdoviridae (Rhabdoviridae) is divided into 6 genus, comprise infection plant Cytorhabdovirus ( Cytorhabdovirus) and Nucleorhabdovirus ( Nucleorhabdovirus), and infect vertebrate vesicular stomatitis virus belong to ( Vesiculovirus), Lyssavirus ( Lyssavirus), Ephemerovirus (Ephemerovirus) and short outer Rhabdovirus (also claiming the non-virion protein Rhabdovirus) ( Novirhabdovirus), also have the part rhabdovirus not yet to classify, such as Sigma virus, Flanders virus etc.The rhabdovirus that hitherto reported infects fish has tens kinds, comprise infectious hematopoietic necrosis's poison (Infetioushematopoietic necrosis virus, IHNV), viral haemorrhagic septicaemia virus (Viralhaemorrhagic septicemia virus, VHSV), flounder rhabdovirus (Hirame rhabdovirus, HRV), snakehead fish rhabdovirus (Snakehead rhabdovirus, SHRV), SVCV (Spring viraemia of carp virus, SVCV), pike juvenile rhabdovirus (Pike fryrhabdovirus, PFRV), (Rhabdovirus 903/87 for rhabdovirus 903/87,903/87), mandarin fish rhabdovirus (Siniperca chuatsi rhabdovirus, SCRV), mullet rhabdovirus (Chinesesucker rhabdovirus) and bull trout rhabdovirus 28/97 (Sea trout rhabdovirus 28/97, (Granoff and Webster, 1999 such as STRV); Zhang Qiya and Li Zhengqiu, 1999; Zhang et al., 2000; Johansson et al., 2001,2002).In the 6th report of ICTV, SVCV, PFRV are listed in vesicular stomatitis virus and belong to tentative species (Wunner et al., 1995).In the 7th report of ICTV, formally IHNV, VHSV, HRV, SHRV are classified as the outer Rhabdovirus of grain, SVCV, PFRV etc. still is classified as the tentative species (Walker et al., 2000) that vesicular stomatitis virus belongs to.The classification position of other kinds of fish Rhabdovirus is not yet definite.

Along with the fast development of fish farming industry, Virus disease of fish has become the maximum disease of cultured fishes.Owing to lacking effective methods for the treatment of, the control of fishes virus disease is putting prevention first, thereby the diagnoois and test technical study of virus has become very active field in the fishes virus research.Kinds of fish Rhabdovirus has very strong pathogenic, and particularly VHSV, IHNV, HRV, SHRV and SVCV are widely current all over the world, causes great financial loss for the aquatic products aquaculture.Thereby these viral epidemiology enjoy the concern of researcher.

Pike juvenile rhabdovirus (PFRV) is a kind of virus very high with SVCV (SVCV) homology, does not cause also that at present people pay much attention to.PFRV is popular in European pike juvenile (Wolf, 1988) mainly.Should virus infect pike juvenile in Holland in 1972, and cause extensive death, from then on the first strain PFRV criticizes in the ill pike juvenile and separates (De Kinkelin, 1973).PFRV and SVCV sibship are nearest, the homology of G albumen＞70%.PFRV, SVCV and the kinds of fish Rhabdovirus G protein part nucleotide sequence relevant with their serology are carried out phylogenetic analysis, the result shows that they form 4 genotype, PFRV and SVCV form respectively a genotype, be respectively genotype III and I, other viruses form genotype II and IV (Stone et al., 2003).SVC main infection cyprinid fish, being classified as by OIE must notifiable disease, and the Ministry of Agriculture classifies SVC as the secondary animal epidemic.Along with the pay attention to day by day to SVC, to the increasing of SVCV control and monitoring, the virus that these and SVCV sibship are high also will more and more cause people's attention.Thereby seek fast detection method of special, sensitivity, very crucial for generation and the development of prevention and control disease.

In China, fishes virus SVCV has caused the attention of people's height, and in annual spring and autumn national carp is carried out the SVCV monitoring.But the fishes virus PFRV very high with the SVCV sibship also do not cause people's attention at present, do not include the scope of detection in.In succession separate (Ahne, 1975 along with learning relevant virus with these 2 kinds of serum virus; Ahne et al., 1982; Fijan et al., 1984; Ahne and Thomsen, 1986; Haenen and Davidse, 1989; Jorgensen et al., 1989; Rowley et al., 2001; Stone et al., 2003; Way et al., 2003), people can get more and more to the concern of these viruses.Thereby need to set up practicable detection method.

At present, identify that PFRV reaches the virus relevant with its serology and generally takes to separate with sensitive cells first mode (De Kinkelin, 1973 of then identifying with serological method; Ahne, 1975; Ahne etal., 1982; Fijan et al., 1984; Ahne and Thomsen, 1986; Haenen and Davidse, 1989; Jorgensen et al., 1989; Rowley et al., 2001; Stone et al., 2003; Way et al., 2003).Adopt this detection mode not only the cycle long, and use immunological method, such as indirect fluorescent antibody test or enzyme linked immunosorbent assay, exist between PFRV and the SVCV cross reaction (

Et al., 1989; Way, 1991), detected result is inaccurate.Some rhabdoviruses and the PFRV serology homology that are separated in other fingerling classes except pike juvenile are very high, begin to be considered to PFRV, but measure its part G gene nucleotide series and carry out the phylogenetic analysis discovery, to such an extent as to these sequences enough can range different genotype (Rowley et al., 2001 greatly from the difference of PFRV corresponding sequence; Stone et al., 2003).Therefore, in the urgent need to setting up molecular biology method PFRV is detected.

Summary of the invention

The objective of the invention is for the deficiencies in the prior art, the RT-PCR detection method of a kind of pike juvenile rhabdovirus is provided.

Another object of the present invention is to provide the detection kit of a kind of pike juvenile rhabdovirus.

For achieving the above object, the present invention has adopted following technical scheme:

The invention discloses pike juvenile rhabdovirus (Pike fry rhabdovirus, PFRV) RT-PCR detection method, described method comprises utilizes Auele Specific Primer pair, carry out the RT-PCR reaction take virus genome RNA as template, described primer pair be for amplifying the primer pair of gene fragment of pike juvenile rhabdovirus G gene, and described primer pair is selected from lower at least one group:

A: the primer pair that contains respectively sequence shown in Seq ID No.1 and the Seq ID No.2;

Seq ID No.1：5’--AGTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.2：5’--ATGTTATCTCTTCCCTTCGATTGAT--3’

B: contain respectively the primer pair of sequence shown in Seq ID No.3 and the Seq ID No.4,

Seq ID No.3：5’--GTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.4。5’--GTGCAAGCTCTCCTTTTTTCTC--3’。

In the preferred embodiment of the present invention, described primer pair is selected from lower at least one group:

C: the primer pair with sequence shown in Seq ID No.1 and the Seq ID No.2;

D: the primer pair with sequence shown in Seq ID No.3 and the Seq ID No.4.

The reverse transcription of RT-PCR of the present invention and PCR preferably carry out in same reaction system, describedly refer in same reaction system: make traditional two-step approach RT-PCR into disposable adding reagent, then carry out the single stage method of reverse transcription and PCR reaction.

In the reaction system of described RT-PCR, Mg ²⁺Final concentration is preferably 1.6mM/L～3.6mM/L, more preferably 2.1mM/L～2.6mM/L; The dNTP final concentration is preferably 0.2mM/L～0.4mM/L, more preferably 0.4mM/L; The primer final concentration is preferably 0.05mM/L～1.0mM/L, more preferably 0.25mM/L; Annealing temperature is preferably 50 ℃～65 ℃, more preferably 55 ℃.

In the concrete embodiment of the present invention, the response procedures of described RT-PCR is: 42 ℃ of reverse transcription 30min, 94 ℃ of denaturation 4min, 35 circulations: 94 ℃ of sex change 50s, the 50s that under described annealing temperature, anneals, 72 ℃ are extended 1min, and 10min are extended in rear 72 ℃ of 35 circulations.

The present invention opens and discloses pike juvenile rhabdovirus (Pike fry rhabdovirus, PFRV) detection kit, described test kit contains the primer pair of the gene fragment that can amplify pike juvenile rhabdovirus G gene, and described primer pair is selected from lower at least one group:

A: the primer pair that contains respectively sequence shown in Seq ID No.1 and the Seq ID No.2;

Seq ID No.1：5’--AGTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.2：5’--ATGTTATCTCTTCCCTTCGATTGAT--3’

B: contain respectively the primer pair of sequence shown in Seq ID No.3 and the Seq ID No.4,

Seq ID No.3：5’--GTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.4。5’--GTGCAAGCTCTCCTTTTTTCTC--3’。

Preferably, described primer pair is selected from lower at least one group:

C: the primer pair with sequence shown in Seq ID No.1 and the Seq ID No.2;

D: the primer pair with sequence shown in Seq ID No.3 and the Seq ID No.4.

Owing to having adopted above technical scheme, the beneficial effect that the present invention is possessed is:

Detection method of the present invention and test kit use Auele Specific Primer that PFRV is detected, and two pairs of used primers all have good initiation characteristic, and it is at wider Mg ²⁺All can well cause template amplification in concentration, primer concentration, dNTP concentration and the annealing region, and when PFRV nucleic acid-templated be 10 ^3.5TCID ₅₀/ 0.1mL, 25 the circulation products concentration that increase have reached testing requirement.The RT-PCR detection method that the present invention sets up has good specificity, and the result who detects kinds of fish Rhabdovirus HRV, IHNV, VHSV, SVCV and other Common Fishs virus IPNV, VNNV is all negative; Have higher sensitivity, the sensitivity that detects viral suspension extracting nucleic acid all reaches 10 ^2.5TCID ₅₀

Description of drawings

Fig. 1 is Mg ²⁺Concentration optimization result's electrophoretic image,

1：Marker DL2000；2：1.6mM/L；3：2.1mM/L；4：2.6mM/L；5：3.1mM/L；6：3.6mM/L；7：Negative control。

Fig. 2 is dNTP concentration optimization result's electrophoretic image,

1：Marker DL2000；2：0.2mM/L；3：0.25mM/L；4：0.3mM/L；5：0.35mM/L；6：0.4mM/L；7：Negative control。

Fig. 3 is the electrophoretic image of primer concentration optimum result,

1：Marker DL2000；2：1.0mM/L；3：0.75mM/L；4：0.5mM/L；5：0.25mM/L；6：0.15mM/L；7：0.1mM/L；8：0.05mM/L；9：Negative control。

Fig. 4 is the electrophoretic image of annealing temperature optimum result,

2-5：F1/R1；7-10：F2/R2；1，6，11：Marker DL2000；2，7：50℃；3，8：55℃；4，9：60℃；5，10：65℃。

Fig. 5 is the electrophoretic image that reaction cycle is counted optimum result,

2-6：F1/R1；8-12：F2/R2；1，7，13：Marker DL2000；2，8：25；3，9：30；4，10：35；5，11：40；6，12：45。

Fig. 6 is the electrophoretic image of the RT-PCR amplification of primer specificity,

2-10：F2/R2；12-20：F1/R1；1，11：DL2000；2，12：IHNV；3，13：IPNV；4，14：VHS V；5，15：HRV；6，16：VNNV；7，17：SSS0604；8，18：9701；9，19：PFRV；10，20：Negative control。

Fig. 7 is primer sensitivity result's electrophoretic image,

1：Marker DL2000；2：10 ⁰；3：10 ^-1；4：10 ^-2；5：10 ^-3；6：10 ^-4；7：Negativecontrol。

Embodiment

Embodiment 1

1 materials and methods

1.1 virus and clone

PFRV is the F4 strain, is given by Inst. of Hydrobiology, Chinese Academy of Sciences, and SVCV and IHNV Reference Strains are so kind as to give by OIE reference laboratory professor B.Hill of Britain CEFAS; The VHSV Reference Strains by the veterinary institute OIE of Norway country reference laboratory Roar doctor Gudding be so kind as to give, IPNV, HRV, VNNV separate by this chamber.

IHNV, SVCV, VHSV, VNNV, IPNV, HRV and PFRV use respectively EPC, CO, RTG-2, SSN-1, CHSE-214, FHM and BF-2 cell amplification.The SSN-1 cell is available from hydrocoles health research institute of Thailand Kasetsart university, the RTG-2 cell is given by hydrobiont institute, the BF-2 cell is given by quarantine institute's (Brescia) cell preservation center, CHSE-214 cell, EPC cell by Britain's environment, fishery and aquaculture research establishment give, the FHM cell given by Univ Munich Germany and Inst. of Hydrobiology, Chinese Academy of Sciences, the CO cell is given by Inst. of Hydrobiology, Chinese Academy of Sciences.

EPC, CO, CHSE-214, FHM, BF-2 and RTG-2 cell are all cultivated with 199 nutrient solutions that contain 10% foetal calf serum, and the SSN-1 cell is cultivated with the L-15 nutrient solution that contains 10%FBS.Behind the virus inoculation, with added with antibiotic (100IU/mL penicillin; 100 μ g/mL Streptomycin sulphates) corresponding nutrient solution is cultivated.

1.2 equipment, reagent and substratum

Equipment: PCR instrument (T3, Biometra), constant incubator (Binder, Germany produces), the gel imaging instrument (BDA TI1 type, Biometra).

Reagent: Taq archaeal dna polymerase, dNTP, EB and DEPC are purchased from Shanghai biotechnology company limited; Reversed transcriptive enzyme, RNA enzyme inhibitors and DNA Marker are purchased from Dalian precious biotechnology company limited; 199 and L-15 be Gibco company product; Other reagent are analytical pure.Experiment institute water is DEPC and processes water, and experimental ware all passes through high-temperature sterilization 2 times.

199 nutrient solutions: get one bag of 199 substratum pulvis (GIBCO) and add the 900mL deionized water, fully stir, until without till the insoluble substance, add the foetal calf serum of 100mL deactivation, cumulative volume is transferred to 1000mL, stir evenly, add NaHCO ₃Regulate pH to 7.2-7.8, sterile filtration, packing ,-20 ℃ save backup.

199+HEPES: adding final concentration in 199 nutrient solutions is 10%FCS, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, 0.02M HEPES, regulates pH value to 7.6;

Cell dissociation buffer: with the 2.5g pancreatin, 40g NaCl, 2.0g KCl, 5.0g glucose, 2.9g NaHCO ₃, 1.0g EDTA is dissolved in the super steaming of the 500mL water successively, until completely dissolved, filtration sterilization, packing ,-20 ℃ save backup.

The L-15 nutrient solution: the L-15 substratum pulvis at configuration 1L dosage adds the 900mL deionized water, fully stirs, until without till the insoluble substance, add the foetal calf serum of 100mL deactivation, cumulative volume is transferred to 1000mL, stirs evenly, and adds NaHCO ₃Regulate pH to 7.2-7.8, sterile filtration, packing ,-20 ℃ save backup.

1.3 design of primers

According to PFRV G gene (AJ538069) sequence among the Genbank, use 2 pairs of primers of Primer Premier5.0 software design.And after carrying out BLAST checking primer sequence specificity by ncbi database, synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Primer sequence sees Table 1.

Table 1PFRV RT-PCR primer

^aSequence location is with reference to the sequence among the Genbank No.AJ538069.

1.4 the mensuration of virus titer

Virus titer is measured and is adopted Endpoint Dilution Method, with reference to the method for Reed and Muench (1938).

Concrete grammar is as follows:

1) pass the BF-2 cell and enter 96 orifice plates, place 25 ℃ of incubators cultivate (＜24h);

2) 10 times of serial dilution viruses to be measured;

3) each dilution viral suspension is inoculated into cell and grows up in single 96 orifice plates that become, every extent of dilution adds 3 holes, and every hole adds 100 μ L, and control group adds 100 μ L nutrient solutions;

4) place the optimal temperature incubator to cultivate 96 orifice plates, until CPE is complete;

5) observe under the inverted microscope, calculate virus titer (TCID ₅₀/ 0.1mL).

1.5 the extraction of viral nucleic acid

Viral nucleic acid extracts and operates with reference to TaKaRa MiniBEST Viral RNA/DNA Extraction Kit specification sheets.

1.6 One Tube RT-PCR reaction condition optimization

The present invention makes traditional two-step approach RT-PCR into disposable adding reagent, then carries out the single stage method of reverse transcription and PCR reaction, and add fashionable reagent with reference to two-step approach at reagent and add dosage, and to Mg ²⁺, dNTP and primer concentration be optimized.When carrying out the individual event reaction condition optimization, the add-on of other reagent is carried out with reference to following prescription:

*5×AMV RT Buffer	4μL
			10×PCR buffer	8μL
MgCl 2(25mM)	8μL
		dNTPs Mixture(10mM/L each)	4μL
RNasin Inhibitor(40U/μL)	1μL
		AMV RTase XL(5U/μL)	1μL
Taq Polymerase(5U/μL)	1μL
		F Primer(50pmol)	1μL
R Primer(50pmol)	1μL
		RNA	10μL
RNase Free dH 2O	61μL
		Total	100μL

^*5×AMV RT Buffer(250mM Tris-HCl pH 8.3，375mM KCl，40mMdithiothreitol，40mM MgCl ₂).

The RT-PCR response procedures is as follows: 42 ℃ of reverse transcription 30min, and 94 ℃ of sex change 4min then carry out 35 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 50s, optimal temperature annealing 50s), and next 72 ℃ are extended 10min.Reaction with amplified production point sample electrophoresis in containing the sepharose of 1.5%EB, is taken pictures under the ultraviolet after finishing.

Mg ²⁺Concentration optimization: add Mg in the reaction system ²⁺, make its final concentration be respectively 1.6mM/L, 2.1mM/L, 2.6mM/L, 3.1mM/L and 3.6mM/L.

DNTP concentration optimization: add dNTP in the reaction system, make its final concentration be respectively 0.2mM/L, 0.25mM/L, 0.3mM/L, 0.35mM/L and 0.4mM/L.

Primer concentration is optimized: add primer in the reaction system, make the final concentration of every primer be 0.05mM/L, 0.1mM/L, 0.15mM/L, 0.25mM/L, 0.5mM/L, 0.75mM/L and 1.0mM/L.

Annealing temperature is optimized: the RT-PCR amplification is carried out in the component configuration after selecting to optimize, and with 50 ℃, 55 ℃, 60 ℃ and 65 ℃ carry out RT-PCR for annealing temperature, choose Optimal Temperature.

The suitableeest cycle number optimization: be diluted to 10 with PFRV is nucleic acid-templated ^3.5TCID ₅₀/ 0.1mL.Annealing temperature behind the component disposition and distribution optimization after selecting to optimize is carried out 25 of RT-PCR amplifications, and 30,35,40 and 45 circulations are to determine its suitableeest cycle number.

1.7RT-PCR specific test

Extraction fishes virus PFRV, HRV, IHNV, VHSV, SVCV, IPNV, VNNV nucleic acid carry out PFRV RT-PCR as template and detect, and observations is determined its specificity.

1.8RT-PCR sensitivity test

Get PFRV virus liquid 250 μ L, extract viral RNA, RNA is made 10 times of serial dilutions, carry out RT-PCR with each dilution nucleic acid as template.

2 results

2.1 virus titer measurement result

10 times of serial dilution PFRV viral suspensions are inoculated 96 orifice plate BF-2 monolayer cells, and 20 ℃ to be cultured to CPE complete, adopt the Reed-MuenchShi method to calculate, and the titre of PFRV is 10 ^5.5TCID ₅₀/ 0.1mL.

2.2 the component of optimizing configuration and reaction conditions

Mg ²⁺Concentration optimization: electrophoresis result is seen Fig. 1, Mg ²⁺Final concentration is 1.6mM/L-3.6mM/L, and the product of two couples of primer RT-PCR all forms brighter feature band.When using F1/R1, Mg ²⁺Final concentration is 2.6mM/L, and the brightness of feature band is the highest.When using F2/R2, Mg ²⁺Final concentration is 2.1mM/L, and the feature band is the brightest.Therefore, primers F 1/R1 and F2/R2 Mg ²⁺Optimal final concentration be respectively 2.6mM/L, 2.1mM/L.

The dNTP concentration optimization: electrophoresis result is seen Fig. 2, the dNTP final concentration is 0.2mM/L-0.4mM/L in the reaction system, the product of 2 couples of primer RT-PCR all forms brighter feature band, and the increase with the dNTP add-on, brightness has the trend of increase, is that 0.4mM/L is the suitableeest final concentration therefore 2 pairs of primers are all selected the dNTP final concentration.

Primer concentration is optimized: electrophoresis result is seen Fig. 3, and the primer final concentration is 0.05mM/L-1.0mM/L, and the product of 2 couples of primer RT-PCR all forms brighter feature band.When 2 pairs of primer final concentrations are 0.25mM/L, primer dimer is minimum, and the brightness of feature band is very high, therefore 0.25mM/L is the suitableeest final concentration of 2 pairs of primers.

Annealing temperature is optimized: carry out RT-PCR according to the component configuration reaction system after optimizing and optimize annealing temperature, electrophoresis result is seen Fig. 4, annealing temperature is from 50 ℃-65 ℃, template all can increase, when annealing temperature was 55 ℃, 2 pairs of primer initiation amplified production electrophoretic bands were brighter, increase under the higher or lower annealing temperature, electrophoretic band brightness has reducing tendency, therefore select 55 ℃ to be the suitableeest annealing temperature.

The suitableeest cycle number optimization: with the component configuration reaction system of optimizing, select 55 ℃ to carry out RT-PCR for annealing temperature cycle number is optimized.Electrophoresis result is seen Fig. 5, and PFRV is nucleic acid-templated to be 10 ^3.5TCID ₅₀During/0.1mL, carry out 25-45 circulations, electrophoresis all has the feature band to produce, and 30 the amplified production electrophoretic band is all very bright more than the circulation, in order to save time, and makes amplified production reach maximum, and selecting 35 circulations is the RT-PCR amplification cycles.

To Mg ²⁺, after dNTP and primer concentration be optimized, the add-on of reagent is as follows in the RT-PCR reaction system:

*5×AMV RT Buffer	4μL
			10×PCR buffer	8μL
MgCl 2(25mM)	4μL(F1/R1)，2μL(F2/R2)
		DNTPs Mixture (each 10mM)	4μL
RNasin Inhibitor(40U/μL)	1μL
		AMV RTase XL(5U/μL)	1μL
Taq Polymerase(5U/μL)	1μL
		Primer(25pmol/μL)	1μL
Primer(25pmol/μL)	1μL
		RNA	10μL
RNase Free dH 2O	61μL
		Total	100μL

^*5×AMV RT Buffer(250mM Tris-HCl pH 8.3，375mM KCl，40mMdithiothreitol，40mM MgCl ₂).

After annealing temperature and cycle number be optimized, the PCR response procedures was as follows: 42 ℃ of reverse transcription 30min, and 94 ℃ of sex change 4min then carry out 35 circulations (72 ℃ are extended 1min for 94 ℃ of sex change 50s, 55 ℃ of annealing 50s), and next 72 ℃ are extended 10min.Reaction with RT-PCR amplified production point sample electrophoresis in containing the sepharose of 1.5%EB, is taken pictures under the ultraviolet after finishing.

2.3RT-PCR specific test

Adopt and optimize afterreaction system and amplification condition, primer pair PFRV, HRV, IHNV, VHSV, SVCV, IPNV, VNNV with PFRV carry out RT-PCR, electrophoresis result is seen Fig. 6, when detecting PFRV with 2 pairs of primers, there is the feature band to produce, and when detecting other RNA viruses and negative control with 2 pairs of primers, do not have the feature band to produce, show that these 2 pairs of primers all can cause the nucleic acid-templated amplification of PFRV specifically.

2.4RT-PCR sensitivity test

Add 25 μ L DEPC water dissolution after getting the extracting of 250 μ L virus, get 10 μ L nucleic acid and carry out 10 times of serial dilutions, every extent of dilution is got 10 μ L and is adopted the reaction conditions after optimizing to carry out RT-PCR, and detected result is seen Fig. 7, and 2 pairs of positive greatest dilutions of primer detected result are 10 ^-3, corresponding sensitivity is 10 ^2.5TCID ₅₀

3 discuss

PFRV is popular in European pike juvenile (Wolf, 1988) mainly.Should virus infect pike juvenile in Holland in 1972, and cause extensive death, from then on the first strain PFRV criticizes in the ill pike juvenile and separates (De Kinkelin, 1973).PFRV and SVCV sibship are nearest, the homology of G albumen＞70%.PFRV, SVCV and the kinds of fish Rhabdovirus G protein part nucleotide sequence relevant with their serology are carried out phylogenetic analysis, the result shows that they form 4 genotype, PFRV and SVCV form respectively a genotype, be respectively genotype III and I, other viruses form genotype II and IV (Stone et al., 2003).SVC main infection cyprinid fish, being classified as by OIE must notifiable disease, and the Ministry of Agriculture classifies SVC as the secondary animal epidemic.Along with the pay attention to day by day to SVC, to the increasing of SVCV control and monitoring, the virus that these and SVCV sibship are high also will more and more cause people's attention.Thereby seek fast detection method of special, sensitivity, very crucial for generation and the development of prevention and control disease.

At present, cell culture technology is still " golden standard " of Aquatic animals virus diagnosis, but needs the professional to operate, and Diagnostic Time is long, generally needs 4d-7d.Although serum neutralization test, elisa technique high specificity because hydrocoles antibody generation level is on the low side, generally detect its antigen, and detection sensitivity are on the low side.Aforesaid method also has a shortcoming, and the virus that is difficult to detect identifies kind.Thereby in the quarantine of hydrocoles disease test, use seldom separately.Round pcr generally for the nucleotide sequence design primer of certain specific virus, can identify kind with virus, when determining whether some fish infects certain virus unique advantage is arranged, particularly in the situation that there is not sensitive cells to can be used for cultivating.Thereby in inspection and quarantine, round pcr becomes very important means.In fishes virus detected, round pcr and cell cultures were combined with, and sensitivity and viral recall rate are higher.

G albumen is a kind of glycosylated protein, and main code embrane-associated protein (McAllister andWagner, 1975) forms the membrane-associated protein projection on the virus particle surface of maturation, consists of antigenic determinant (Huang et al., 1996; Xu Yao waits first, and 2000).All rhabdovirus G albumen all belong to single I type transmembrane glycoprotein.Each virus particle comprises approximately 1200 glycoprotein molecules, is attached to virus particle surface (Gaudin etal., 1992) with the membrane spaning domain of 400 trimerical forms by its C-terminal.G albumen is comprised of about 500 amino acid, very low (the Walker and Kongsuwan of the rhabdovirus G albumen homology that does not belong to together, 1999), but rhabdovirus has common feature, comprise 2-6 potential glycosylation sites, 12-16 conservative cysteine residues, 7 hydrophobic amino acid of 2-3 section repeat, and the N end has 1 hydrophobic signal peptide that can be removed, the C end has 1 hydrophobic cross-film sequence and 1 hydrophobic tenuigenin tail (Coll, 1995).

One section sequence on the viral G gene relevant with the PFRV serology has been indexed to ncbi database, and we select, and corresponding one section sequence makes things convenient for us to design the high primer of specificity as the template of design primer like this on the PFRV G gene.The reason that the G gene is suitable as design primer template also has 2 points: 1, the G gene is oligogene (Huang et al., 1996 of coding for antigens determinant; Xu Yao waits first, and 2000), this gene should be more conservative in virus of the same race; 2, according to the homology analysis result of each albumen of last chapter PFRV and other vesicular stomatitis virus corresponding proteins, the SVCV the highest with homology compares, G albumen is the 4th conservative protein, compare with other vesicular stomatitis virus, the G gene is the 3rd conservative protein, thereby easily designs the high primer of specificity.

The present invention has designed 2 pairs of RT-PCR primers that detect PFRV, and the reaction conditions of 2 couples of primer RT-PCR is optimized.Find in optimizing process, these 2 pairs of primers have good initiation characteristic, and it is at wider Mg ²⁺All can well cause template amplification in concentration, primer concentration, dNTP concentration and the annealing region, and when PFRV nucleic acid-templated be 10 ^3.5TCID ₅₀/ 0.1mL, 25 the circulation products concentration that increase have reached testing requirement.The RT-PCR method that this research is set up has good specificity, and the result who detects kinds of fish Rhabdovirus PFRV, HRV, IHNV, VHSV, SVCV and other Common Fishs virus IPNV, VNNV is all negative; Have higher sensitivity, the sensitivity that detects viral suspension extracting nucleic acid all reaches 10 ^2.5TCID ₅₀Thereby these 2 pairs of primers can be used for clinical detection.

4 brief summaries

According to PFRV G gene conserved sequence, design 2 pairs of Auele Specific Primers; Reactive component Mg to RT-PCR ²⁺, dNTP, primer concentration and reaction annealing temperature, cycle number be optimized; Carry out specific test and sensitivity test according to optimizing good RT-PCR condition, the result shows: when detecting IHNV, SVCV, VHSV, VNNV, IPNV, HRV and PFRV, only have PFRV amplified production electrophoresis the feature band to occur; Detection sensitivity to viral suspension extracting nucleic acid is 10 ^2.5TCID ₅₀

Embodiment 2

A kind of detection kit, the RT-PCR that is used for pike juvenile rhabdovirus (Pike fry rhabdovirus, PFRV) detects.Contain following primer sequence in the described test kit:

Seq ID No.1：5’--AGTTGTCGTACATCCTGTTCATTTAG--3’(F1)

Seq ID No.2：5’--ATGTTATCTCTTCCCTTCGATTGAT--3’(R1)

Above-mentioned primer can amplify the portion gene fragment of pike juvenile rhabdovirus G gene.

Described test kit also contains working instructions.

Embodiment 3

A kind of detection kit, the RT-PCR that is used for pike juvenile rhabdovirus (Pike fry rhabdovirus, PFRV) detects.Contain following primer sequence in the described test kit:

Seq ID No.3：5’--GTTGTCGTACATCCTGTTCATTTAG--3’(F2)

Seq ID No.4。5’--GTGCAAGCTCTCCTTTTTTCTC--3’(R2)。

Above-mentioned primer can amplify the portion gene fragment of pike juvenile rhabdovirus G gene.

Described test kit also contains working instructions.

Attached 1 viral list of abbreviations

Initialism	English name	Chinese name
			PFRV	Pike fry rhabdovirus	The pike juvenile rhabdovirus
IHNV	Infetious hematopoietic necrosis	Infectious hematopoietic necrosis's poison
			IPNV	Infectious pancreatic necrosis virus	Infectious pancreas necrosis virus
SVCV	Spring viraemia of carp virus	SVCV
			VHSV	Viral haemorrhagic septicemia virus	Viral hemorrhagic septicemia, VHS virus
VNNV	Viral nervous necrosis virus	The viral nervous necrosis poison
			HRV	Hirame rhabdovirus	Flounder rhabdovirus

Attached 2 cells and other list of abbreviations

Initialism	English name	Chinese name
			BF-2	Blue gill fry	Copper kiss phosphorus gill sunfish inoblast
CO	Overy of grass carp	The grass carp gonadal cell
			CHSE-214	Chinook salmon embryo	The king salmon embryonic cell
EPC	Epithelioma papulosum cyprini	Carp epithelioma cell
			FHM	Fathead minnow	Seawater cyprinid fish cell
GCF	Grass carp fin	Grass carp fin ray cell
			PG	Pike gonad	The pike gonadal cell
SSN-1	Stripped snakehead	Line sweet wine cell
			N	Nucleoprotein	Nucleoprotein
P	Phosphoprotein	Phosphorylated protein
			M	Matrix protein	Stromatin
G	Glycoprotein	Glycoprotein
			L	RNA-dependent RNA polymerase	The RNA polymerase that RNA relies on
NV	Non-virion	Non-virion protein
			ELISA	Enzyme-linked immunosorbent assay	Enzyme linked immunosorbent assay
RT-PCR	Reverse transcription polymerase chain	Inverse transcription polymerase chain reaction
			LAMP	Loop-mediated isothermal amplification	Ring matchmaker constant-temperature amplification
FIP	Forward inner primer	Front inner primer
			BIP	Backward inner primer	Rear inner primer
RACE	Rapid-amplification of cDNA ends	The terminal rapid amplifying of cDNA
			IUdR	5-iodo-2’-deoxyuridine	5 '-iodo-2 '-deoxidation uridylic

Sequence table

＜110〉Animal ﹠. Plant Inspection and Quarantine Techn Center, Shenzhen Bureau of Impor

＜120〉pike juvenile rhabdovirus RT-PCR detection method and test kit

<130>DHC0910506

<160>4

<170>PatentIn version 3.3

<210>1

<211>26

<212>DNA

＜213〉artificial sequence

<400>1

agttgtcgta catcctgttc atttag 26

<210>2

<211>25

<212>DNA

＜213〉artificial sequence

<400>2

atgttatctc ttcccttcga ttgat 25

<210>3

<211>25

<212>DNA

＜213〉artificial sequence

<400>3

gttgtcgtac atcctgttca tttag 25

<210>4

<211>22

<212>DNA

＜213〉artificial sequence

<400>4

gtgcaagctc tccttttttc tc 22

Claims (6)

1. pike juvenile rhabdovirus (Pike fry rhabdovirus, the detection method of the non-diagnostic purpose of RT-PCR PFRV), described method comprises utilizes Auele Specific Primer pair, carry out the RT-PCR reaction take virus genome RNA as template, it is characterized in that: described primer pair be for amplifying the primer pair of gene fragment of pike juvenile rhabdovirus G gene, and described primer pair is selected from lower at least one group:

A: the primer pair that is respectively sequence shown in Seq ID No.1 and the Seq ID No.2;

Seq ID No.1：5’--AGTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.2：5’--ATGTTATCTCTTCCCTTCGATTGAT--3’

B: be respectively the primer pair of sequence shown in Seq ID No.3 and the Seq ID No.4,

Seq ID No.3：5’--GTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.4：5’--GTGCAAGCTCTCCTTTTTTCTC--3’。

2. the detection method of non-diagnostic purpose according to claim 1, it is characterized in that: the reverse transcription of described RT-PCR and PCR carry out in same reaction system.

3. the detection method of non-diagnostic purpose according to claim 2 is characterized in that: in the reaction system of described RT-PCR, and Mg ²⁺Final concentration is 1.6mM～3.6mM, and the dNTP final concentration is 0.2mM～0.4mM, and the primer final concentration is 0.05mM～1.0mM, and annealing temperature is 50 ℃～65 ℃.

4. the detection method of non-diagnostic purpose according to claim 3 is characterized in that: in the reaction system of described RT-PCR, and Mg ²⁺Final concentration is 2.1mM～2.6mM, and the dNTP final concentration is 0.4mM, and the primer final concentration is 0.25mM, and annealing temperature is 55 ℃.

5. the detection method of non-diagnostic purpose according to claim 4, it is characterized in that: the response procedures of described RT-PCR is: 42 ℃ of reverse transcription 30min, 94 ℃ of denaturation 4min, 35 circulations: 94 ℃ of sex change 50s, 50s anneals under described annealing temperature, 72 ℃ are extended 1min, 35 rear 72 ℃ of extension 10min of circulation.

6. pike juvenile rhabdovirus (Pike fry rhabdovirus, PFRV) detection kit, it is characterized in that: described test kit contains the primer pair of the gene fragment that can amplify pike juvenile rhabdovirus G gene, and described primer pair is selected from lower at least one group:

A: the primer pair that is respectively sequence shown in Seq ID No.1 and the Seq ID No.2;

Seq ID No.1：5’--AGTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.2：5’--ATGTTATCTCTTCCCTTCGATTGAT--3’

B: be respectively the primer pair of sequence shown in Seq ID No.3 and the Seq ID No.4,

Seq ID No.3：5’--GTTGTCGTACATCCTGTTCATTTAG--3’

Seq ID No.4：5’--GTGCAAGCTCTCCTTTTTTCTC--3’。

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