CN106929604A - Primer sets and probe sequence for detecting Bao herpeslike virus - Google Patents

Primer sets and probe sequence for detecting Bao herpeslike virus Download PDF

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CN106929604A
CN106929604A CN201710173957.XA CN201710173957A CN106929604A CN 106929604 A CN106929604 A CN 106929604A CN 201710173957 A CN201710173957 A CN 201710173957A CN 106929604 A CN106929604 A CN 106929604A
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primer sets
probe sequence
bao
herpeslike
virus
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CN106929604B (en
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姜敬哲
高芳
王江勇
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South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

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Abstract

The invention discloses primer sets and probe sequence for detecting Bao herpeslike virus, described primer sets are：FP:CTTTCTTACCGCTTTCAATCTGATCCGTGG, RP:GAACAGGGGTAATTGTATAGCAACTGCGTA；The probe sequence：GCGTACAGTAAAACGAAAACCATGGCACA（dT‑FAM）GC（THF）CA（dT‑BHQ1）TGAAAACATCCAAGC（C3spacer）.The invention discloses above-mentioned primer sets and probe sequence in the application for preparing the reagent for RPA detection Bao herpeslike viruses.Based on above-mentioned primer sets and probe sequence develop fluorescent quantitation RPA detection methods have sensitivity high, high specificity, be swift in response, the low advantage of instrument and equipment requirement, be highly suitable for aquatic livestock Pathogen test.

Description

Primer sets and probe sequence for detecting Bao herpeslike virus
Technical field
The present invention relates to primer and probe, and in particular to primer sets and probe sequence for detecting Bao herpeslike virus.
Background technology
Bao herpesviral（Abalone herpes-like virus, AbHV）As the main pathogen of Bao low temperature virosis, The pernicious epidemic disease that Haliotis diversicolor is cultivated since the southern china Bao Zhuyang areas spring in 1999 is caused, to China Haliotis diversicolor Aquaculture causes destructive strike.The sick infectiousness is strong, can infect each specification Haliotis diversicolor, but seldom cause disk Bao Dengfa Disease, with species specificity；The disease breaks with tremendous force, as long as occurring to the full pond dead generally 3 day time from symptom.At present, for The virosis there is no effective treatment method, and the early stage of virus, accurately detection prevention of disease and control were particularly important, therefore Set up a kind of prevention and control of easy, quick, sensitive, accurate method for detecting virus to virosis significant.At present, commonly use Be with PCR（PCR）It is the Pathogen test technology for representing.But this process needs accurate instrument and specialty Test site, time-consuming, ageing low, it is impossible to meets the on-site diagnosis demand of aquaculture.Recombinase polymeric enzymatic amplification （RPA）It is a kind of nucleic acid Fast Detection Technique that can be carried out at room temperature for occurring in recent years, without temperature control device, reaction can be Quickly completed in 15 minutes, with the sensitivity and specificity as PCR, be particularly suitable for the quick inspection in scene of aquatic products cause of disease Survey.RPA detections primer sets used and probe are its core components, and they decide the Detection results of RPA.But RPA primers and The design difficulty of probe is larger, and the special designing software without similar PCR primer, the sensitivity and specificity of detection are completely dependent on In the experience and substantial amounts of verification experimental verification of design of primers person.Sometimes all it is difficult to screen from tens of set candidate drugs probe combinations 1 set of available detection sequence, therefore design of primers is the key of RPA detection method success or failure.
The content of the invention
An object of the present invention is to provide a kind of primer sets and probe sequence for detecting Bao herpeslike virus.
Primer sets of the invention and probe sequence are the ORF 38 according to virus（GenBank accession no.JX453331.1）What gene order was designed.
Primer sets and probe sequence provided by the present invention for detecting Bao herpeslike virus, wherein, described primer Group is：
FP : CTTTCTTACCGCTTTCAATCTGATCCGTGG
RP : GAACAGGGGTAATTGTATAGCAACTGCGTA
Probe sequence：
GCGTACAGTAAAACGAAAACCATGGCACA（dT-FAM）GC（THF）CA（dT-BHQ1）TGAAAACATCCAAGC （C3spacer）.
The second object of the present invention is to provide and is being prepared for RPA detection Bao class blisters using above-mentioned primer sets and probe sequence The application of the reagent of exanthema virus.
Beneficial effects of the present invention：
The present invention provides a set of fluorescent quantitation RPA for Bao herpeslike virus（qRPA）Detection primer and probe groups, with them Based on develop fluorescent quantitation RPA detection methods have sensitivity high, high specificity, be swift in response, instrument and equipment requirement it is low The advantages of, it is highly suitable for aquatic livestock Pathogen test.The detection sensitivity of AbHV-qRPA detection architectures is similar to qPCR, but Detection time is only 20 ± 0.50 minutes, and the time greatly shortens compared to qPCR；Reaction temperature is 37 DEG C of constant temperature, without heating and cooling system System, is suitable to field and operates on the spot.
Brief description of the drawings
Fig. 1 is the electrophoretogram of amplified production in embodiment one.
Fig. 2 is AbHV-qRPA detection architecture amplification curve diagrams；
Different curves represent 10710 various concentrations plasmid copy number, every amplification curve is the flat of three duplicate measurements Average.
Fig. 3 is the standard curve of AbHV-qRPA detection architectures；
Various concentrations standard items reach the corresponding period of fluorescence threshold in embodying AbHV-qRPA detection architectures.
Fig. 4 is the scatter diagram of AbHV-qRPA and AbHV-qPCR testing results；
Embody the correlation between AbHV-qRPA and AbHV-qPCR testing results.
Specific embodiment
The present invention is described in further detail with reference to specific embodiments and the drawings, but protection scope of the present invention is not It is limited to following embodiment.
The design and specific detection of the primer sets of embodiment 1 and probe
According to the ORF 38 of virus（GenBank accession no.JX453331.1）Gene order designs primer sets and spy Pin, wherein target gene sequence are：
TGAACAGGGGTAATTGTATAGCAACTGCGTACAGTAAAACGAAAACCATGGCACATGCCATTGAAAACATCCA AGCAAACACCCAAGGCAAGTTTGTTGTTCCTTTATGCAAACAGATTCTCAAAACGACTAAAGAAACCACGGATCAGA TTGAAAGCGGTAAGAAAGA。
FP : CTTTCTTACCGCTTTCAATCTGATCCGTGG
RP : GAACAGGGGTAATTGTATAGCAACTGCGTA
Probe sequence：
GCGTACAGTAAAACGAAAACCATGGCACA（dT-FAM）GC（THF）CA（dT-BHQ1）TGAAAACATCCAAGC （C3spacer）.
Specific detection：Set up the positive control containing AbHV, ddH2The negative control of O, prawn disease poison（WSSV）Genome DNA controls, egg-shaped pompano genomic DNA control, Babylonia genomic DNA control, oyster genomic DNA control, DNA pairs, water body According to Aeromonas hydrophila genomic DNA control, using these genomic DNAs template, detected.
Bao hsv gene group DNA, prawn disease poison（WSSV）Genomic DNA, egg-shaped pompano genomic DNA, east wind tap bolt Because group DNA and oyster extracting genome DNA use marine animal tissue extraction kit（Dongsheng, Guangdong, China）, concrete operations Going up instruction to specifications is carried out.
Water body DNA and Aeromonas hydrophila extracting genome DNA use DNA of bacteria extracts kit（Magen, Suzhou, in State）, concrete operations go up instruction and carry out to specifications.
Reaction system：
Rehydration buffer（Twistdx kits（TABAS03KIT））: 14.8ul
RPA FP（10 μM）: 1.2 μL
RPA RP（10 μM）:1.2 μL
dd H2O :4.6 μL
DNA（50~500ng）:2 μL
MgAC（280 nM）:1.2 μL
Reaction condition：37 DEG C are incubated 30 minutes.
After reaction terminates, reactant passes through agarose gel electrophoresis（1.5% agarose）Result judged, as a result show, These genomic DNAs and negative control are without amplified signal, and positive control has obvious amplified signal（Fig. 1）.It can be seen that, this The primer sets and probe specificity for inventing foundation are strong.
The sensitivity of embodiment 2 is verified
Standard curve template is synthesized by Shanghai Invitrogen Corp..The template is by target gene fragment and PMD18-T vector Carrier is formed by connecting.Carrier sequence is disclosed：http://wenku.baidu.com/view/ 385564bdf121dd36a32d8271.html。
Detected using absolute quantitation method：The plasmid standard that will be built makees 10 times of gradient dilutions, structure with TE Buffer Build various concentrations gradient 107、106、105、104、103、102Standard curve is made with 10, ddH is used2O as negative control, each Concentration is in triplicate.With real-time fluorescence quantitative PCR instrument（Eppendorf）Carry out RPA amplified reactions.
Reaction system：
Rehydration buffer（Twistdx kits, TAEXO02KIT）: 14.7 μL
RPA FP（10 μM）: 1.1 μL
RPA RP（10 μM）:1.1 μL
Probe（10 μM）:0.3 μL
dd H2O :4.6 μL
DNA（50~500 ng）:2 μL
MgAC（280 nM）:1.2 μL
Reaction condition：37 DEG C are incubated 30 minutes.
The amplification curve and amplification efficiency of each concentration are set up according to each concentration amplification, statistics is set up Standard curve, as a result referring to Fig. 2 and Fig. 3.As it is clear from fig. 2 that detection of the detection method to AbHV is limited to 100 copies.
The feasibility of embodiment 3 is verified
In order to further determine that the reliability of qRPA detection architectures of the present invention, we extract 50 Haliotis diversicolors out at random, while to it Internal AbHV contents carry out qRPA and qPCR and detect respectively.Result shows (referring to Fig. 4), two testing results of diagnostic method Show preferable correlation（R2>0.8）
Bao musculature DNA is extracted and is used marine animal tissue extraction kit, and concrete operations indicate to carry out to specifications.
QRPA detection methods, primer sets and probe are with embodiment 2.
QPCR detection methods use it has been reported that TaqMan®Round pcr（Corbeil S,Williams L M et al,.Development and validation of a TaqMan® PCR assay for the Australian Abalone herpes-like virus Serge.Diseases of Aquatic Organisms.2010), the primer It is respectively with probe sequence：
F：AACCCACACCCAATTTTTGA
R：CCCAAGGCAAGTTTGTTGTT
Probe：6 FAM-CCGCTTTCAATCTGATCCGTGG-TAMRA.
The present invention can be summarized with others without prejudice to the concrete form of spirit or essential characteristics of the invention.Every foundation Any trickle amendment, equivalent variations and modification that substantial technological of the invention is made to above example, belong to skill of the present invention In the range of art scheme.
Sequence table
<110>Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
<120>Primer sets and probe sequence for detecting Bao herpeslike virus
<160> 7
<210> 1
<211> 30
<212> DNA
<213>Primers F P
<400> 1
ctttcttacc gctttcaatc tgatccgtgg 30
<210> 2
<211> 30
<212> DNA
<213>Primer RP
<400> 2
gaacaggggt aattgtatag caactgcgta 30
<210> 3
<211> 48
<212> DNA
<213>Probe sequence
<400> 3
gcgtacagta aaacgaaaac catggcaca（dT-FAM）gc（THF）ca（dT-BHQ1）tgaaaacatc caagc（C3spacer） 48
<210> 4
<211> 169
<212> DNA
<213> ORF 38
<400> 4
tgaacagggg taattgtata gcaactgcgt acagtaaaac gaaaaccatg gcacatgcca 60
ttgaaaacat ccaagcaaac acccaaggca agtttgttgt tcctttatgc aaacagattc 120
tcaaaacgac taaagaaacc acggatcaga ttgaaagcgg taagaaaga 169
<210> 5
<211> 20
<212> DNA
<213>Primers F
<400> 5
aacccacacc caatttttga 20
<210> 6
<211> 20
<212> DNA
<213>Primer R
<400> 6
cccaaggcaa gtttgttgtt 20
<210> 7
<211> 22
<212> DNA
<213>Primer R
<400> 7
6 FAM- ccgctttcaatctgatccgtgg -TAMRA 22

Claims (2)

1. it is used to detect the primer sets and probe sequence of Bao herpeslike virus, it is characterized in that, described primer sets are：
FP : CTTTCTTACCGCTTTCAATCTGATCCGTGG
RP : GAACAGGGGTAATTGTATAGCAACTGCGTA
The probe sequence is：
GCGTACAGTAAAACGAAAACCATGGCACA（dT-FAM）GC（THF）CA（dT-BHQ1）TGAAAACATCCAAGC （C3spacer）.
2. the primer sets and probe sequence described in claim 1 are preparing answering for the reagent for RPA detection Bao herpeslike viruses With.
CN201710173957.XA 2017-03-22 2017-03-22 Primer group and probe sequence for detecting abalone herpes virus Active CN106929604B (en)

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Publication number Priority date Publication date Assignee Title
CN108060265A (en) * 2017-11-29 2018-05-22 中国水产科学研究院南海水产研究所 For detecting the primer sets of the oyster herpetovirus of infection blood clam and probe and its application
CN108103247A (en) * 2018-02-01 2018-06-01 上海海洋大学 Kit and its primer special and probe based on RPA technologies III type grass carp reovirus of detection
CN114085931A (en) * 2022-01-24 2022-02-25 中国水产科学研究院黄海水产研究所 Primer group for detecting abalone herpesvirus and application of primer group in construction of virus detection method and kit
CN114107573A (en) * 2022-01-27 2022-03-01 中国水产科学研究院黄海水产研究所 Abalone herpes virus HaHV-1 universal RPA nucleic acid isothermal amplification primer, kit and application thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108060265A (en) * 2017-11-29 2018-05-22 中国水产科学研究院南海水产研究所 For detecting the primer sets of the oyster herpetovirus of infection blood clam and probe and its application
CN108060265B (en) * 2017-11-29 2021-05-07 中国水产科学研究院南海水产研究所 Primer group and probe for detecting oyster herpesvirus infected with scapharca subcrenata and application of primer group and probe
CN108103247A (en) * 2018-02-01 2018-06-01 上海海洋大学 Kit and its primer special and probe based on RPA technologies III type grass carp reovirus of detection
CN114085931A (en) * 2022-01-24 2022-02-25 中国水产科学研究院黄海水产研究所 Primer group for detecting abalone herpesvirus and application of primer group in construction of virus detection method and kit
CN114107573A (en) * 2022-01-27 2022-03-01 中国水产科学研究院黄海水产研究所 Abalone herpes virus HaHV-1 universal RPA nucleic acid isothermal amplification primer, kit and application thereof

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