CN102352366B - Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof - Google Patents
Macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and application thereof Download PDFInfo
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Abstract
The invention relates to a macrobrachium rosenbergii bicistronic messenger ribonucleic acid (mRNA) viral genome complete sequence and an application thereof, which belong to the technical field of virus genomics. The invention provides the macrobrachium rosenbergii bicistronic mRNA viral genome complete sequence shown as SEQIDNO: 1. In addition, the invention provides a macrobrachium rosenbergii bicistronic mRNA viral specific primer, a probe or a reagent kit. The macrobrachium rosenbergii bicistronic mRNA viral genome complete sequence and the application lay the reliable foundation to the purposes of macrobrachium rosenbergii dicistrovirus (MRDV) detection, MRDV infection path study and the like.
Description
Technical field
The invention belongs to the viral genome field that learns a skill, be specifically related to genome sequence and the application thereof of Macrobrachium rosenbergii bicistronic mRNA virus.
Background technology
Macrobrachium rosenbergii (Macrobrachium rosenbergii DeMan, 1879) claims fresh water long-armed prawn again, belongs to pond crayfish and belongs to (Macrobrachium), and be the main breed variety of China fresh water Crustacean.In statistical information in 2009,207749 tons that ultimate production reached in 2008 were cultured in the whole world of Macrobrachium rosenbergii according to Food and Argriculture OrganizationFAO (FAO), and wherein Asia output is 127627 tons then, accounts for global output 98%, and China is the maximum breed state in the whole world.
Virus is the main pathogen in Macrobrachium rosenbergii breeding and the breeding process, break out Macrobrachium rosenbergii young syndromes (the Macrobrachium rosenbergii larval Syndrome in 2009 and 2010, MRLS), cause Macrobrachium rosenbergii mass mortality to occur at larval stage, particularly particularly serious in the death of young 6-9 age in days, mortality ratio is generally 80-90%, serious reaches 100%, relate to China main Macrobrachium rosenbergii breeding zone, the financial loss of causing for the Macrobrachium rosenbergii breeding industry can't be estimated.Find that after deliberation its main pathogen is a kind of new bicistronic mRNA virus, temporary called after Macrobrachium rosenbergii bicistronic mRNA virus (Macrobrachium rosenbergii dicistrovirus, MRDV), MRDV is single positive chain RNA virus, be classified as the bicistronic mRNA Viraceae, since being found, because there not being this viral genome relevant information, more lack detection method, seriously hindered diagnosis and prevention work that this virus causes disease, so the parsing of this virus genome complete sequence and related application thereof seem particularly important.
Summary of the invention
At the problem that prior art exists, the objective of the invention is to design genome sequence that Macrobrachium rosenbergii bicistronic mRNA virus is provided and the technical scheme of application thereof, the present invention establishes reliable basis for detecting purposes such as MRDV, research MRDV route of infection.
Described a kind of isolated nucleic acid molecule is characterized in that described nucleic acid molecule contains nucleotide sequence or its antisense sequences shown in the SEQ ID NO:1.
Described a kind of isolated nucleic acid molecule is characterized in that described nucleic acid molecule is DNA or RNA.
Described a kind of isolated nucleic acid molecule is for the preparation of the primer, probe or the test kit that detect Macrobrachium rosenbergii bicistronic mRNA virus.
Described a kind of isolated DNA molecule is characterized in that containing sequence or its antisense sequences shown in the SEQ ID NO:1.
Described a kind of isolated DNA molecule is characterized in that containing 150-10300 continuous in sequence shown in the SEQ ID NO:1 or its antisense sequences Nucleotide.
Described a kind of isolated DNA molecule is characterized in that containing 300-10300 continuous in sequence shown in the SEQ ID NO:1 or its antisense sequences Nucleotide.
Described a kind of isolated DNA molecule is characterized in that containing 1000-10300 continuous in sequence shown in the SEQ ID NO:1 or its antisense sequences Nucleotide.
Described a kind of primer is characterized in that containing 15-100 continuous nucleotide in sequence shown in the SEQ ID NO:1 or its antisense sequences.
Described a kind of probe is characterized in that containing 25-7000 continuous nucleotide in sequence shown in the SEQ ID NO:1 or its antisense sequences.
Described a kind of test kit is characterized in that containing 50-500 continuous nucleotide in sequence shown in the SEQ ID NO:1 or its antisense sequences.
The present invention checks order to the genome of Macrobrachium rosenbergii bicistronic mRNA virus, obtained the genom sequence of Macrobrachium rosenbergii bicistronic mRNA virus, and the present invention utilizes this complete sequence for the preparation of the primer, probe or the test kit that detect Macrobrachium rosenbergii bicistronic mRNA virus, establishes reliable basis for detecting purposes such as MRDV, research MRDV route of infection.
Embodiment
Nucleic acid among the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.
Genom sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
First aspect the invention provides a nucleic acid, and this nucleic acid contains the genome nucleotide sequence in the Macrobrachium rosenbergii bicistronic mRNA virus shown in the SEQ ID NO:l.Also provide and contained the nucleic acid that the sequence of sequence homogeny is arranged with nucleotide sequence disclosed herein.According to concrete sequence, the degree of sequence homogeny should be greater than 50% (for example 60%, 70%, 80%, 90%, 95%, 99% or higher).These sequences comprise for example mutant and allele variant.
The present invention also provides the nucleic acid that comprises one or more nucleotide sequence fragments disclosed herein.These fragments should comprise in all sequences n continuous amino acid at least, and according to concrete sequence, and n is 10 or higher (for example, 11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45,50,60,75,100 or higher).Preferably, this fragment is that Macrobrachium rosenbergii bicistronic mRNA viral genome is distinctive, does not in other words exist in the genome of other biological body.More preferably, this fragment is that the genome of Macrobrachium rosenbergii bicistronic mRNA virus stain is distinctive.The present invention also provides the nucleic acid that provides with this paper that the nucleic acid of hybridization takes place.Hybridization conditions as described herein.
The present invention also provides a nucleic acid, and this nucleic acid comprises and the sequence of above-mentioned sequence complementation (for example, be used for antisense, be used for probe or be used for amplimer).
Certainly, nucleic acid of the present invention can prepare (for example, chemosynthesis, make etc. from the DNA library or from organism itself) with several different methods, and can adopt various forms (for example strand, two strands, carrier, probe, primer etc.).Term " nucleic acid " comprises DNA and RNA, and their analogue, as contains those analogues of modifying skeleton, also comprises peptide nucleic acid(PNA) (PNA) etc.
Should be understood that because SEQ ID NO:l has represented the complete genome group of Macrobrachium rosenbergii bicistronic mRNA virus.
The present invention also provides the carrier (as expression vector, sequencing vector, cloning vector etc.) that contains nucleotide sequence of the present invention and with these carrier transformed host cells.
Second aspect, the present invention also provides a kind of protein, and this protein contains coded aminoacid sequence in the Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences shown in this article.Also provide and contained the albumen that the sequence of sequence homogeny is arranged with these protein.
According to concrete sequence, sequence homogeny degree should be greater than 50% (for example 60%, 70%, 80%, 90%, 95%, 99% or higher).The sequence homogeny is determined with aforesaid method.These homologous proteins comprise coded mutant and allele variant in the Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences shown in this article.
The present invention also provides a kind of protein, and this protein contains in the ordered list coded aminoacid sequence fragment in the disclosed Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences.These fragments should comprise in all sequences n continuous amino acid at least, and according to concrete sequence, and n is 7 or higher (for example, 8,10,12,14,16,18,20 or higher).These fragments should comprise the epi-position of this sequence.
The present invention also provides the nucleic acid of code book invention albumen.
The third aspect, the present invention also provides computer, computer memory, computer-readable storage medium (as floppy disk, hard disk, CD-ROM etc.) and/or the Computer Database of the nucleotide sequence that contains nucleic acid of the present invention.Preferably, it contains one or more Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences that this paper lists.
This can be used for analyzing the listed Macrobrachium rosenbergii bicistronic mRNA of this paper viral nucleotide sequences.For example, can be used for retrieving to differentiate open reading frame (ORF) or encoding sequence in the sequence.
Fourth aspect the invention provides the method for differentiating aminoacid sequence, and it comprises step: retrieve open reading frame or the albumen coded sequence of inferring in the listed Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences of this paper.Similarly, the invention provides herein open reading frame that listed Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences infers in retrieval or the purposes in the protein coding sequence.
Open reading frame or albumen coded sequence analysis are carried out with the biology information technology of standard usually on computers.The typical algorithm or the program that are used for analyzing comprise: ORFFINDER (NCBI), GENMARK[Borodovsky; Mcininth (1993) Computers Chem 17:122-133) and GLIMMER[Salzberg et al. (1998) Nucl Acids Res 26:544-548].
Retrieval to open reading frame or albumen coded sequence can comprise step: retrieve initiator codon and the terminator codon of retrieving in the upstream sequence in the frame that is in the listed Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences herein.Middle codon has just represented the albumen coded sequence of inferring.Usually, should retrieve 6 kinds of possible frames in the sequence.
The aminoacid sequence that identifies in this way can be expressed with any suitable system, to obtain albumen.This albumen can be used for producing antibody, and the epi-position of this antibody recognition in the aminoacid sequence that identifies.These antibody can be used for screening Macrobrachium rosenbergii bicistronic mRNA virus, whether have the protein that contains the aminoacid sequence of being differentiated to detect.
In addition, in case identify ORF or albumen coded sequence, so just this sequence and sequence library can be compared.The sequential analysis instrument can be located to find at NCB worker (http://www. ncbi. nlm. nih. gov), BLAST for example, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx and tBLASTx algorithm [also can be referring to people such as Altschul. (1997) Gapped BLAST and PSI-BLAST:new generation of protein database search programs. Nucleic Acids Research 25:2289-3402].Be used for appropriate database relatively and comprise nonredundancy GenBank, EMBL, DDBJ and PDB sequence, and nonredundancy GenBank CDS translation thing, PDB, SwissProt, Spupdate and PIR sequence.This comparison can provide the indication of protein function.
Hydrophobic region in the aminoacid sequence can be predicted with above algorithm, for example based on the algorithm [" hydrophilic crucial evaluation of membranin " (Critical cvaluation of the hydropathy of membrane proteins) (1990) Eur J Biochem 190:207-219] of people's such as Esposti statistical research.Hydrophobic region has represented potential film district or the hydrophobicity leader sequence of striding, and the surface can be secreted or be positioned to this hint protein.The normally good immunogenic sign of these characteristics.
Similarly, available psort algorithm (http://www. psort. nibb. ac. jp) comes predicted transmembrane district or leader sequence, and comes forecast function district (GCG Wisconsin﹠amp with the MOTIFS program; PROSITE).
The present invention also provides a kind of nucleic acid, and this nucleic acid contains open reading frame or the albumen coded sequence in the listed Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences of this paper.In addition, based on genome sequence provided by the present invention, also provide some kinds of protein, this protein contains described open reading frame or the coded aminoacid sequence of albumen coded sequence.For example, the ORF of the 392-6589 position length of just encoding is 2065 amino acid whose virus proteases and RNA polymerase precursor protein (pre-proprotein) (SEQ ID NO:2) among the SEQ ID NO:1, and the ORF of the 6961-10053 position length of just encoding is 1030 amino acid whose viral capsid precursor proteins (pre-proprotein) (SEQ ID NO:3) among the SEQ ID NO:1.
The 5th aspect the invention provides the antibody in conjunction with these albumen.They may be polyclonal or monoclonal, and available any appropriate method well known by persons skilled in the art makes.
Antibody of the present invention can use with various different modes, for example is used for confirming protein expression, or is used for confirming the place of protein expression.For example, the antibody of mark (as fluorescent mark to be used for FACS (flow cell sorter)) can be hatched with complete virus, and exists mark just to confirm the position of protein at virus surface.
The 6th aspect the invention provides the whole bag of tricks.
The invention provides a kind of method of producing albumen of the present invention, the method comprising the steps of: under the condition that inducible protein is expressed, cultivate host cell of the present invention.A kind of method also can comprise chemosynthesis albumen or chemosynthesis (at least part of synthetic) Nucleotide.
The invention provides a kind of method that detects polynucleotide of the present invention, this method comprises the following steps: that (a) makes nucleic acid probe of the present invention contact with biological sample under hybridization conditions, forms duplex; (b) detect described duplex.
The invention provides a kind of detection method of protein of the present invention, this method comprises the following steps: that (a) makes antibody of the present invention contact with biological sample under the condition that is fit to formation antibody one antigen mixture; (b) detect described mixture.
The 7th aspect, the invention provides and detect selective binding in the method for the antibody of antigen or polypeptide or protein, these antigens or polypeptide or protein are to any seed culture of viruses of Macrobrachium rosenbergii bicistronic mRNA virus or strain specific, be preferably specific to Macrobrachium rosenbergii bicistronic mRNA virus stain, but more preferably special to Macrobrachium rosenbergii bicistronic mRNA virus.The method comprising the steps of: (a) under the condition that is fit to formation antibody-antigenic compound, antigen of the present invention or polypeptide or protein are contacted with biological sample; (b) detect described mixture.
The method summary
The invention provides Macrobrachium rosenbergii bicistronic mRNA virus MRDV nucleotide sequence and its coded aminoacid sequence.Utilize these disclosed sequences, can produce nucleic acid probe test and expression cassette and carrier.But protein is chemosynthesis also.Expression vector can change host cell over to produce albumen.Many skins of purifying or separation can be used for producing antibody to detect MRDV albumen.And host cell or extract can be used for biological test to separate agonist and antagonist.In addition, utilize these sequences, people can retrieve to identify open reading frame (ORF) and identify aminoacid sequence.Protein also can be used for immunogenic composition and is used as vaccine component.
Unless description is arranged in addition, enforcement of the present invention will be adopted molecular biology, microbiology, recombinant DNA and immunologic routine techniques, and these all are known to those skilled in the art.
Nucleotide and amino acid whose standardized abbreviations have been adopted in this manual.
The immunodiagnosis test
The recombinant antigen of Macrobrachium rosenbergii bicistronic mRNA virus antigen of the present invention or exploitation can be used to prepare corresponding laboratory animal antibody, and this antibody-like can be used for the virus antigen level that immunity test detects clinical Macrobrachium rosenbergii sample.The design of immunity test can be made wide variation, and its various schemes all are as known in the art.
With suitable material (comprising composition of the present invention) and test other required reagent and material (for example suitable damping fluid, salts solution etc.) and suitable test specification sheets are packaged in the suitable containers, constitute the test kit that is applicable to immunodiagnosis and contains the reagent of suitable mark.
Nucleic acid hybridization
" hybridization " refers to the combination by hydrogen bond each other of two nucleotide sequences.Usually, a sequence is fixed in solid carrier, and another will be free in the solution.Then, two sequences are in contact with one another being conducive to form under the condition of hydrogen bond.The factor that influences this combination comprises: the type of solvent and volume; Temperature of reaction; Hybridization time; Stir; The reagent of confining liquid facies-suite and solid carrier non-specific binding (Denhardt's reagent or bovine lacto transfer technique optimizer); The concentration of each sequence; Whether use compound to increase the speed (T 500 or polyoxyethylene glycol) of sequence combination; And the rigorous degree of post-hybridization washing condition.
The nucleic acid probe test
Adopt the method (as PCR, branched DNA probe test or engram technology) of nucleic acid probe of the present invention can determine the existence of cDNA or mRNA.Stably be enough to the duplex or the double-stranded mixture that are detected if probe and sequence of the present invention can form, then claim probe and sequence of the present invention " hybridization ".
Nucleic acid probe will be hybridized with Macrobrachium rosenbergii bicistronic mRNA viral nucleotide sequences of the present invention (including justice and antisense strand).Although many different nucleotide sequence coded these aminoacid sequences are arranged, natural Macrobrachium rosenbergii bicistronic mRNA virus sequence is preferable, because it is the sequence that actually exists in the cell.MRNA represents a kind of encoding sequence, thus probe should with this encoding sequence complementation; Strand cDNA and mRNA complementation, thus the cDNA probe should with the non-coding sequence complementation.
Definite length and the sequence of probe will depend on hybridization conditions, as temperature, salt concn etc.For example, for diagnostic use, according to the complexity of analyte sequence, nucleic acid probe contains 10-20 Nucleotide at least usually, and preferable 15-25, at least 30 better Nucleotide, but also can be shorter than this length.Short primer needs lesser temps usually, in order to form sufficiently stable hybridization complex with template.
Probe can produce with synthetic method, people's such as people's such as Matteucci [J. Am. Chem. Soc. (1981) 103: 3185] method or Urdea [Proc. Natl. Acad. Sci. USA (1983) 80: 7461] method for example, or synthetic with commercially available automatic oligonucleotide synthesizer.
Can select the chemical feature of probe according to preference.Use for some, DNA or RNA are suitable.For other application, can add modification, for example backbone modification, as thiophosphatephosphorothioate or methyl phosphorodithioate, can be used to increase the transformation period in the body, change RNA avidity, increase nuclease resistance etc. " for example referring to Agrawal and worker yer (1995) CurrOpin Biotechnol 6:12-19; Agrawal (1996) T worker BTECH14:376-387]; Also can adopt analogue such as skin nucleic acid [for example referring to Corey (1997) T worker BTECH15:224-229; People such as Buchardt (1993) TIBTECH 11:384-386].
In addition, polymerase chain reaction (PCR) is the means of another a small amount of target nucleic acid of knowing of detection.This test is in people such as Mullis [Meth. Enzymol. (1987) 155: 335-350]; United States Patent (USP) 4,683 is described in 195 and 4,683,202 to some extent.With two " primer " Nucleotide and target nucleic acid hybridization, and be used for guiding reaction.Primer can comprise not the sequence with amplified target sequence (or its complementary sequence) hybridization, helping the stability of duplex, or for example can insert an easy restriction site.The Macrobrachium rosenbergii bicistronic mRNA virus sequence that the common side joint of these sequences is required.
Utilize initial target nucleic acid as template, heat-staple polysaccharase can produce the copy of target nucleic acid from primer.Produce the target nucleic acid of critical amount at polysaccharase after, their available more traditional methods (as the Southern trace) detect.When adopting Southern trace method, the probe of mark will be hybridized with Macrobrachium rosenbergii bicistronic mRNA virus sequence (as its complementary sequence).
In addition, mRNA or cDNA also traditional engram technology of describing of people's (the same) such as available Sambrook detect.But separate mRNA or the cDNA that utilizes polysaccharase to produce from mRNA with gel electrophoresis purifying well.Then, with the nucleic acid blot on the gel on solid carrier such as nitrocellulose.Solid carrier is contacted with the probe of mark, and the probe that all are not hybridized is removed in washing then.Then, detect the duplex that contains label probe.This probe is marked with the radioactivity material usually.
The application of genome sequence
Based on the mensuration to Macrobrachium rosenbergii bicistronic mRNA virus genome complete sequence, can understand gene structure and the function relevant with this virus multiplication and important regulating and controlling function, the key point that finds its Molecular Physiological Mechanism and how to infect the host is sought virus and is caused a disease even lethal gene.
A kind of application method is with the Macrobrachium rosenbergii bicistronic mRNA virus infection Macrobrachium rosenbergii young, to set up the cDNA library.Measure the cDNA clone with two-way terminal method, or directly sequencing is carried out with the method for pcr amplification in the library of MRDV cDNA.Based on Macrobrachium rosenbergii bicistronic mRNA virus genome sequence provided by the present invention, can make things convenient for primer design and clone's process greatly.
Another kind of directly the application is to use primer of the present invention or probe, comes whether to exist in the test sample Macrobrachium rosenbergii bicistronic mRNA virus or its genetic material.
For probe, this detection method comprises step:
The DNA sample is contacted with probe of the present invention,
Observe whether form the DNA-probe complex, formed mixture and just represented to exist in the sample Macrobrachium rosenbergii bicistronic mRNA virus or genetic material.
For primer, this detection method comprises step:
Use Auele Specific Primer of the present invention, sample carried out the PCR reaction,
Whether observing increases has produced specific amplified production, has produced specific amplification products and has just represented to exist in the sample Macrobrachium rosenbergii bicistronic mRNA virus or genetic material.
In an example of the present invention, the genome sequence of Macrobrachium rosenbergii bicistronic mRNA virus obtains by the following method, and the method comprising the steps of:
1. separation and purification MRDV virus is obtained high purity RNA sample.
2. be template with this RNA sample, carry out reverse transcription reaction with the random primer that has anchor series, with synthesizing single-stranded cDNA.Continuation is template with this cDNA, utilizes the anchor series primer, carries out pcr amplification under the effect of Taq and Deep Vent DNA polymerase, and extension amplification outcome is gone into the T carrier, carries out sequencing, obtains most of virus sequence
3. with the sequence input computer that obtains, utilize software (as Innerpeace software) to splice, postmenstruation, work obtained complete virus genome complete sequence again.
Through repeatedly order-checking repeatedly, obtained the NO as SEQ ID on average each base about 6 times basis of checking order: the Macrobrachium rosenbergii bicistronic mRNA virus genome complete sequence shown in the l.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York: Cold Spring Harbor Laboratory Press, l989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The extraction of viral RNA
Get the Macrobrachium rosenbergii young that Macrobrachium rosenbergii bicistronic mRNA virus strain CN-NTH infects, organize grinding, add the PBS damping fluid with volume ratio 1:10, ultrasonication 3-5 second is with cytoclasis.Get supernatant liquor behind 4 ℃ of centrifugal 30min of 6000rpm, with the degerming of 0.22um membrane filtration, carry out the sucrose discontinuous density gradient centrifugation again, obtain high purity MRDV virus, extract test kit with viral RNA and extract viral RNA.
Reverse transcription PCR (RT-PCR)
Design and synthesize on the primer: 5'-GCCGGAGCTCTGCAGAATTCNNNNNN-3'(SEQ ID NO: 4).The RNA viruses 5uL that gets aforementioned preparation places the special-purpose tubule of pcr amplification of 200 u L, the primer 1 that adds 100pmol, after placing 65 ℃ of sex change 10min, place immediately on ice, add 10 * reverse transcription damping fluid, 2 uL, l mmol/L 4 * dNTP, 20U RNasin, with 50U Expand Reverse Transcriptase, add water to final volume 20 u L.42 ℃ of incubation 1h behind the mixing.
Pcr amplification
Design and synthesize following primer 2.
Primer 2: 5 '-GCCGGAGCTCTGCAGAATTC-3'(SEQ ID NO: 5).
Get in the special-purpose tubule of pcr amplification that above-mentioned reverse transcription product 5uL places 200 u L, add 10 * PCR damping fluid, 2 u L, 350 nmol, 4 * dNTP, 700nmol primer 2,8U contain the Taq archaeal dna polymerase of Deep Vent, and the rearmounted PCR instrument of mixing increases, loop parameter: 1 circulation of 95 ℃ of 3min, 94 ℃ of 30s, 54 ℃ of 30s, 40 circulations of 68 ℃ of 3min.Reaction product is got 50uL and is detected in 1% agarose gel electrophoresis, and is that the fragment of 1-2Kb is cut off the recovery purifying with size.
Ligation
Say that by following condition and pMD20-T plasmid row is connected:
| 10 * ligase enzyme damping fluid | 1μL |
| DNA | 7.5μL |
| pMD20-T | 0.5μL |
| The T4 ligase enzyme | 1μL |
Transform
Get the bacillus coli DH 5 alpha competent cell of 50u1, add the ligation product, placed 30 minutes on ice.Placed 1 minute under 42 ℃ of conditions, carried out ice bath again 2 minutes.Add the SOC nutritive medium of 37 ℃ of 500u1,37 ℃ were shaken bacterium one hour, coated plate.
Order-checking
Then, picking intestinal bacteria positive colony carries out PCR.The PCR product is carried out terminal dideoxy method order-checking, the sequence that obtains is carried out sequence assembly and comparison, and adopt PCR method to fill a vacancy, obtain virus terminal sequence by the RACE method.
Through repeatedly order-checking repeatedly, on average each base checks order about 6 times basis, obtained the NO as SEQ ID: the Macrobrachium rosenbergii bicistronic mRNA virus genome complete sequence shown in the l.
Embodiment 2
Checking to Macrobrachium rosenbergii bicistronic mRNA virus genome sequence
According to the nucleotide sequence shown in the SEQ ID NO:l, by every 800bp left and right sides Design of length primer, carry out the PCR reaction, the PCR product is carried out sequence verification.The result shows that the nucleotide sequence of SEQ ID NO:l is correct.
Embodiment 3
Detect the test kit of Macrobrachium rosenbergii bicistronic mRNA virus disease strain
Based on the genome sequence shown in the SEQ ID NO:l, synthetic following PCR primer and probe:
Sense primer 1: sequence is among the SEQ ID NO:l the 9625-9644.
Antisense primer 2: sequence is the 9755-9774 complementary sequence among the SEQ ID NO:l.
Probe 3: sequence is the complementary sequence of 9646-9667 position among the SEQ ID NO:l.
Prepare a test kit (detecting 100 times), it contains:
| Title | Concentration |
| Adopted primer 1 is arranged | 100pmol |
| Antisense primer 2 | 100pmol |
| Probe 3 | 100pmol |
| The PCR reaction solution | Contain Taq enzyme dNTP magnesium ion PCR reaction buffer |
Get Macrobrachium rosenbergii bicistronic mRNA virus strain CN-NTH and infect the Macrobrachium rosenbergii young, obtain Macrobrachium rosenbergii bicistronic mRNA virus strain and obtain the cDNA sample by RT-PCR as embodiment 1.This cDNA diluted sample is made sample A for 104,105,106 and 107 times, B, C, D carries out pcr amplification and hybridization detection with above-mentioned test kit, and positive control and negative control are set simultaneously.
The result shows that it is 10 that this test kit can detect extent of dilution
4, 10
5, 10
6With 10
7MRDV sample doubly.
Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (6)
1. an isolated nucleic acid molecule is characterized in that described nucleic acid molecule is nucleotide sequence or its antisense sequences shown in the SEQ ID NO:1.
2. an isolated DNA molecule is characterized in that being sequence or its antisense sequences shown in the SEQ ID NO:1.
3. a sense primer is characterized in that being sequence shown in the 9625-9644 among the SEQ ID NO:l.
4. antisense primer is characterized in that being the 9755-9774 complementary sequence among the SEQ ID NO:l.
5. a probe is characterized in that the complementary sequence for 9646-9667 position among the SEQ ID NO:l.
6. a test kit is characterized in that by sense primer 100 pmol, antisense primer 100pmol, probe 100pmol and contains Taq enzyme dNTP magnesium ion PCR reaction buffer constituting; Described sense primer sequence is sequence shown in the 9625-9644 among the SEQ ID NO:l; Described antisense primer sequence is the 9755-9774 complementary sequence among the SEQ ID NO:l; Described probe sequence is the complementary sequence of 9646-9667 position among the SEQ ID NO:l.
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| CN1438330A (en) * | 2003-03-06 | 2003-08-27 | 中国科学院武汉病毒研究所 | Method for detecting Nuoda Virus mucleic-acid point cross-breeding of luoshi shrimp in natural pond |
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| CN1451764A (en) * | 2003-05-19 | 2003-10-29 | 中国科学院武汉病毒研究所 | Method for compound rverse transcription polymerase chain reaction detection of Ro's pond shrimp viral |
| CN101710125A (en) * | 2009-12-08 | 2010-05-19 | 中山大学 | Methods for detecting bicistronic mRNA virus of scylla serrata and kits thereof |
| CN101717434A (en) * | 2009-12-08 | 2010-06-02 | 中山大学 | Mud crab bicistronic mRNA virus structural protein 1, monoclonal antibody thereof and application |
| CN101875980A (en) * | 2010-07-16 | 2010-11-03 | 中国水产科学研究院黄海水产研究所 | Kit and method for detecting Macrobrachium rosenbergii Nodavirus |
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