CN108060265A - For detecting the primer sets of the oyster herpetovirus of infection blood clam and probe and its application - Google Patents
For detecting the primer sets of the oyster herpetovirus of infection blood clam and probe and its application Download PDFInfo
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- CN108060265A CN108060265A CN201711229920.0A CN201711229920A CN108060265A CN 108060265 A CN108060265 A CN 108060265A CN 201711229920 A CN201711229920 A CN 201711229920A CN 108060265 A CN108060265 A CN 108060265A
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- probe
- primer
- primer sets
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- C—CHEMISTRY; METALLURGY
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
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- C—CHEMISTRY; METALLURGY
- 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
- 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
Abstract
The invention discloses a kind of for detecting the primer sets and probe of the oyster herpetovirus of infection blood clam, the primer sets are made of forward primer and reverse primer, the base sequence of the forward primer is as shown in sequence table SEQ ID NO.1, and the base sequence of the reverse primer is as shown in sequence table SEQ ID NO.2；The base sequence of the probe is as shown in sequence table SEQ ID NO.3.The invention also discloses above-mentioned primer sets and probe to prepare for the application in the oyster herpetovirus reagent of RPA detection infection blood clams.Take that short, yield is high during reagent detection virus, specificity is high, and yin and yang attribute result difference is notable, and verification rate is high, more obvious reliable.
Description
Technical field
The invention belongs to oyster herpetovirus technical fields, and in particular to a kind of oyster bleb for detection infection blood clam
The primer sets and probe of virus and its application.
Background technology
Oyster herpetovirus (Ostreid herpesvirus, OsHV) belongs to Herpesvirus, is a kind of to shellfish culture
Industry endangers more serious cause of disease.The virus can infect and lethal a variety of bivalve shellfish, young and stalwart including oyster, scallop, clam
Blood clam etc..Since the 1990s, the long oyster (Crassostrea gigas) of the multiple countries and regions in whole world cultivation and I
In summer high temperature season massive mortality often occurs for the bivalve shellfish such as state's cultivation Chlamys farreri (Chlamys farreri), gives
Shellfishery has brought tremendous economic losses.At present, effective therapy, viral early stage be there is no for the virosis
Accurate detection prevention of disease and control are particularly important, therefore establish a kind of easy, quick, sensitive, accurate viral diagnosis
Method is of great significance to the prevention and control of virosis.At present, the most commonly used is the cause of diseases with PCR (PCR) for representative
Detection technique.But PCR reaction to accurate temperature control instrument it is more demanding, take it is longer, sensitivity is relatively low, application above have one
It settles finally sex-limited.
Recombinase polymeric enzymatic amplification (RPA) is that a kind of nucleic acid that can be carried out at room temperature occurred in recent years quickly detects skill
Art, without temperature control device, specificity is high, have than PCR also than good sensitivity, can complete to react in 15 minutes, well
Compensate for the limitation of PCR detection method.
In previous research work, present inventor is internal in cultivation blood clam (Scapharca subcrenata) for the first time
It was found that the OsHV cause of diseases that carrying rate is very high.The genome of other species such as the in vivo virus genom DNA of blood clam and oyster, scallop
DNA has certain similitude, but also has apparent difference, is detected not yet specifically for the nucleic acid amplification of blood clam OsHV both at home and abroad
Method.RPA detections primer sets used and probe are its core components, they decide the detection result of RPA.But RPA primers
Of high cost with probe, design difficulty is larger, and without the special designing software of similar PCR primer, also no guide may be referred to,
The sensitivity and specificity of detection place one's entire reliance upon design of primers person experience and substantial amounts of verification experimental verification.Sometimes waited from tens of sets
1 set of available detection sequence can not all be screened by selecting in primer combination of probe, therefore design of primers is the another of the exploitation present invention
It is crucial.
The content of the invention
It is an object of the invention to provide a kind of for detecting the primer sets and probe of the oyster herpetovirus of infection blood clam.
The present invention also aims to provide above-mentioned primer sets and probe preparing for the male of RPA detection infection blood clams
Application in oyster herpesviral reagent.
First purpose of the present invention is achieved by the following technical solution：A kind of oyster for detection infection blood clam
The primer sets and probe of herpesviral, the primer sets are made of forward primer and reverse primer, the forward primer
Base sequence is as shown in sequence table SEQ ID NO.1, the base sequence such as sequence table SEQ ID NO.2 of the reverse primer
Shown in；The base sequence of the probe is as shown in sequence table SEQ ID NO.3.
The primer sets and probe sequence of the present invention are according to (the GenBank accession of oyster herpetovirus ORF 95
No.AY509253.2) nucleotide sequence is designed and obtained.
Specifically, the primer sets are made of forward primer FP and reverse primer RP, sequence is as follows：
Forward primer FP：catgtttacg tggaaatgtt ggattggcta
Reverse primer RP：atgtcaaata ggttgttggc agtgatggtc.
The base sequence of probe is：
tacagcatcgcccgatgcacttcgtgat(dT-FAM)ca(THF)tt(dT-BHQ1)
atcggctcaatatata(C3spacer)。
Second object of the present invention is achieved by the following technical solution：Above-mentioned primer sets and probe are preparing use
Application in the oyster herpetovirus reagent for detecting infection blood clam in RPA.
Compared with prior art, the invention has the advantages that：
(1) present invention devises 1 set and draws for infecting the fluorescent quantitation RPA (qRPA) of the oyster herpetovirus of blood clam detections
Object group and probe sequence, the fluorescent quantitation RPA detection methods developed based on them have high sensitivity, high specificity, anti-
Rapid, instrument and equipment is answered to require low advantage, is highly suitable for aquatic livestock Pathogen test；
(2) detection sensitivity of the OsHV-1-qRPA detection architectures in the present invention is similar to qPCR, but detection time is only
20 ± 0.50 minutes, the time greatly shortened compared to qPCR；Reaction temperature is 37 DEG C of constant temperature, without heating and cooling system, suitable for field
It operates on the spot.
Description of the drawings
Fig. 1 be in embodiment 1 primer specificity detection fluorescence curve figure, 107The base containing OsHV-1 mesh is represented with 10
The recombinant plasmid of cause, NC are blank control, other genomic controls be RGNNV, WSSV, AbHV, Vibrio harveyi genome
DNA, healthy Penaeus Vannmei genomic DNA, Babylonia genomic DNA, abalone culture water body DNA；
Fig. 2 is OsHV-1-qRPA detection architecture amplification curve diagrams, and different curves represents 106~5 various concentration plasmid
Copy number, every amplification curve are the average value of duplicate measurements three times；
Fig. 3 is that various concentration standard items reach the corresponding period of fluorescence threshold in OsHV-1-qRPA detection architectures；
Fig. 4 is the scatter diagram of OsHV-1-qRPA and OsHV-1-qPCR testing results.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and examples, but the invention is not limited in any way.
The design and specific detection of 1 primer sets of embodiment and probe
Primer sets and spy are designed according to virus O RF 95 (GenBank accession no.AY509253.2) nucleotide sequence
Pin group, wherein target gene sequence are as follows：
Primer sets are：
Forward primer FP:catgtttacg tggaaatgtt ggattggcta 30
React primer RP:atgtcaaata ggttgttggc agtgatggtc 30
Probe sequence is：
tacagcatcg cccgatgcac ttcgtgat(dT-FAM)ca(THF)tt(dT-BHQ1)atcggctc
aatatata(C3spacer)
Specific detection：Establish the positive control containing OsHV-1 plasmids, ddH2The negative control of O, prawn disease are malicious (WSSV)
Genomic DNA control, Bao herpeslike virus (AbHV) control, grouper viral nervous necrosis malicious (RGNNV) control, Ha Wei
Vibrios genomic DNA control, Babylonia genomic DNA control, water body DNA controls and healthy prawn genomic DNA control, with this
A little genomic DNAs are detected as template.
The universal RNA Mini Kits of grouper viral nervous necrosis poison RNA extraction uses (Magen, extensively
East, China), cDNA synthesis uses TaKaRa (Dalian, China) reverse transcription reagent box.Oyster herpetovirus genomic DNA, Bao class
Hsv gene group DNA, prawn disease poison (WSSV) genomic DNA, Babylonia extracting genome DNA are carried using marine animal tissue
Kit (Dongsheng, Guangdong, China) is taken, upper instruction carries out to specifications for concrete operations.
Water body DNA and Vibrio harveyi extracting genome DNA use DNA of bacteria extracts kit (Magen, Guangdong, China),
Upper instruction carries out to specifications for concrete operations.
Reaction system：
Rehydration buffer (Twistdx kits, TAEXO02KIT)：29.5μL
RPA FP(10μM)：2.1μL
RPA RP(10μM)：2.1μL
Probe(10μM)：0.6μL
dd H2O：11.2μL
DNA (50~500ng)：2μL
MgAC(280nM)：2.5μL
Reaction condition：37 DEG C are incubated 30 minutes.
Result is judged by the amplification curve for observing each template.The results show that these genomic DNAs and feminine gender
Control is without amplified signal, and positive control has apparent amplified signal (Fig. 1).As it can be seen that the primer sets established of the present invention and
Probe specificity is strong.
2 sensitivity of embodiment is verified
Utilize RPA product construction recombination plasmids.The plasmid is by target gene segment and PMD18-T vector carriers company
It connects.
Carrier sequence is disclosed：http://wenku.baidu.com/view/
385564bdf121dd36a32d8271.html。
It is detected using absolute quantitation method：By the plasmid standard ddH of structure2O makees 10 times of gradient dilutions, and structure is not
With concentration gradient 106、105、104、103、102, 10 and 5 make standard curve, use ddH2O is as negative control, each concentration weight
Again three times.RPA amplified reactions are carried out with real-time fluorescence quantitative PCR instrument (Eppendorf).
Reaction system：
Rehydration buffer (Twistdx kits, TAEXO02KIT)：14.7μL
RPA FP(10μM)：1.1μL
RPA RP(10μM)：1.1μL
Probe(10μM)：0.3μL
dd H2O：4.6μL
DNA (50~500ng)：2μL
MgAC(280nM)：1.2μL
Reaction condition：37 DEG C are incubated 30 minutes.
The amplification curve and amplification efficiency of each concentration are established according to each concentration amplification, statistical result is established
Standard curve, as a result referring to Fig. 2 and Fig. 3.As it is clear from fig. 2 that the detection method is limited to 5 copies to the detection of OsHV-1.
Different curves represents 10 in Fig. 26~5 various concentration plasmid copy number, every amplification curve are to repeat three times
The average value of measurement.
Various concentration standard items in OsHV-1-qRPA detection architectures are embodied in Fig. 3 and reach corresponding the cycling of fluorescence threshold
Number.
3 feasibility of embodiment is verified
In order to further determine the reliability of qRPA detection architectures of the present invention, we extract 50 blood clams out at random, while right
Its in vivo OsHV-1 content carries out qRPA and qPCR and detects respectively.Blood clam DNA extractions use mollusk animal extracts reagent
Box (Magen, Guangdong, China), concrete operations indicate to carry out to specifications.
QRPA detection methods, primer sets and probe are the same as embodiment 2.
QPCR detection methods using it has been reported that SYBR Green detection methods (Pepin, Riou et al.
(2008).“Rapid and sensitive detection of ostreid herpesvirus 1in oyster
samples by real-time PCR”.Journal of Virological Methods).The construction method of qPCR mark songs
It is similar with the construction method of qRPA mark songs in embodiment 2.
The primer sequence is：
C9：GAGGGAAATTTGCGAGAGAA
C10：ATCACCGGCAGACGTAGG
QPCR reaction systems：
SYBR Green PCR Master Mix(QIAGEN,Germany)：10μL
C9：1.4μL:
C10:1.4μL
ddH2O:5.2μL
DNA:2μL
Reaction condition：95 DEG C of pre-degeneration 2min；95 DEG C of denaturation 2s, 60 DEG C of annealing/extension 10s, 40cycles.
The template being processed as above is chosen, according to the shown qRPA reaction systems in embodiment 2 and reaction condition and embodiment 3
Middle qPCR reaction systems and condition are reacted, and are detected using real-time fluorescence quantitative PCR instrument (Eppendorf).According to glimmering
Fluorescent Quantitative PCR instrument software analyzes the CT values of sample to be tested, the standard curve and its amplification curve pair built according to embodiment 2 and 3
The criterion of virus carries out qualitative and quantitative analysis respectively to sample to be tested.The results show (Fig. 4), the inspection of two diagnostic methods
It surveys result and shows preferable correlation (R2>0.8).That is Fig. 4 embodies OsHV-1-qRPA and OsHV-1-qPCR testing results
Between correlation.
The present invention is not limited in the range of above-mentioned specific embodiment, and the embodiment above is used for the purpose of can be to this
The use process of invention is described in detail, and has the production method of equal function and technical detail to fall in the present invention
A part for appearance.In fact, those skilled in the art are according to description above, it becomes possible to according to each needing to find different tune
Perfect square case, these adjustment all should be in the scope of the claims by the appended claims herein.
Sequence table
<110>Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
Guangzhou Yi meter bio tech ltd
<120>For detecting the primer sets of the oyster herpetovirus of infection blood clam and probe and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Oyster herpetovirus (Ostreid herpesvirus)
<400> 1
catgtttacg tggaaatgtt ggattggcta 30
<210> 2
<211> 30
<212> DNA
<213>Oyster herpetovirus (Ostreid herpesvirus)
<400> 2
atgtcaaata ggttgttggc agtgatggtc 30
<210> 3
<211> 47
<212> DNA
<213>Oyster herpetovirus (Ostreid herpesvirus)
<400> 3
tacagcatcg cccgatgcac ttcgtgatct tatcggctca atatata 47
Claims (2)
1. it is a kind of for detecting the primer sets and probe of the oyster herpetovirus of infection blood clam, it is characterized in that：The primer sets
It is made of forward primer and reverse primer, the base sequence of the forward primer is as shown in sequence table SEQ ID NO.1, institute
The base sequence for the reverse primer stated is as shown in sequence table SEQ ID NO.2；Such as sequence table of the base sequence of the probe
Shown in SEQ ID NO.3.
2. primer sets described in claim 1 and probe are being prepared for the oyster herpetovirus reagent of RPA detection infection blood clams
In application.
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| CN201711229920.0A CN108060265B (en) | 2017-11-29 | 2017-11-29 | Primer group and probe for detecting oyster herpesvirus infected with scapharca subcrenata and application of primer group and probe |
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| CN201711229920.0A CN108060265B (en) | 2017-11-29 | 2017-11-29 | Primer group and probe for detecting oyster herpesvirus infected with scapharca subcrenata and application of primer group and probe |
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Cited By (3)
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| CN108103059A (en) * | 2017-12-12 | 2018-06-01 | 广州易米生物科技有限公司 | It is a kind of to be used to inhibit shrimp, the primer of oyster 18SrDNA sequence specific amplifications and its application |
| CN110760615A (en) * | 2019-07-05 | 2020-02-07 | 北京普生诺维生物科技有限责任公司 | II type herpes simplex virus detection kit and detection method |
| CN113293239A (en) * | 2021-07-21 | 2021-08-24 | 中国水产科学研究院黄海水产研究所 | Universal RPA nucleic acid isothermal amplification primer and kit for bivalve herpesvirus OsHV-1 infection and application of primer and kit |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103059A (en) * | 2017-12-12 | 2018-06-01 | 广州易米生物科技有限公司 | It is a kind of to be used to inhibit shrimp, the primer of oyster 18SrDNA sequence specific amplifications and its application |
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| CN110760615B (en) * | 2019-07-05 | 2022-04-22 | 北京普生诺维生物科技有限责任公司 | II type herpes simplex virus detection kit and detection method |
| CN113293239A (en) * | 2021-07-21 | 2021-08-24 | 中国水产科学研究院黄海水产研究所 | Universal RPA nucleic acid isothermal amplification primer and kit for bivalve herpesvirus OsHV-1 infection and application of primer and kit |
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| CN108060265B (en) | 2021-05-07 |
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