CN108070678A - Constant temperature detects the RPA kits of II type carp herpesvirals and its primer special and probe in real time - Google Patents
Constant temperature detects the RPA kits of II type carp herpesvirals and its primer special and probe in real time Download PDFInfo
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- CN108070678A CN108070678A CN201810102971.5A CN201810102971A CN108070678A CN 108070678 A CN108070678 A CN 108070678A CN 201810102971 A CN201810102971 A CN 201810102971A CN 108070678 A CN108070678 A CN 108070678A
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Abstract
Detect the RPA kits of II type carp herpesvirals and its primer special and probe in real time the invention discloses constant temperature, the forward primer sequence of the RPA primer specials is CCCAGGAGACCAGCAGACTGTTGAACCCGTAC, reverse primer sequences GTATCCGCCTCGTCCATCATAGAGCCGAAACC；Probe sequence is cactctggcgacgcgtttgtggttgaaccgcca (BHQ1 dt) c (THF) g (FAM dT) ggaggcttcaaaggc (C3spacer), the 34th bp position, there are one (BHQ1 dt) groups, in 36bp positions, there are one (THF) groups, in 38bp positions there are one (FAM dT) group, afterbody is containing there are one (C3spacer) modifications.Carp herpesviral CyHV II can quickly and successfully be detected using the RPA kits including above-mentioned primer and probe.
Description
Technical field
The invention belongs to biological technical field, it is related to the detection technique of II type carp herpesvirals, and in particular to constant temperature is real-time
The RPA kits and its primer special and probe of detection II type carp herpesvirals and the application in detection II type carp herpesvirals.
Background technology
Simplex keratitis Hematopoietic Necrosis disease (herpesviral haematopoietic necrosis, HVHN) be
A kind of highly pathogenic virosis of golden (crucian carp) fish, cause of disease for II type carps herpesviral (cyprinid herpesvirus 2,
CyHV-II), above-mentioned disease takes place frequently causes damaging influence to crucian cultivation industry.Carp Herpesvirus (Cyprinivirus)
Belong to have cyst membrane containing double stranded big DNA genomic virals, nucleocapsid is into hexagon, a diameter of 175~200nm[1-5],
CyHV-II infection crucian can cause its viscera tissue, the gill and body surface extensive bleeding, cause crucian carp the death rate be up to 90% with
On.
In CyHV-II conventional detection technologies, LAMP technology reaction principle is complicated, and design of primers is cumbersome, and is set in primer
Timing is more stringent for the required conservative section of target sequence, and reaction product is inhomogenous, is unfavorable for the sequencing structure in later stage
Clone, therefore be not widely used；Round pcr takes longer, and cost is higher, and precision temperature is needed to cycle instrument,
The serious application limited during round pcr detects at the scene.In recent years, the appearance of constant temperature nucleic acid amplification technology solves round pcr
Limitation, and it is more convenient, rapid, it is a kind of practicable techniques means of quick detection CyHV-II.
Recombinase polymeric enzymatic amplification technology (recombinase poly-merase amplification, RPA) is constant temperature
One kind of nucleic acid amplification technologies, RPA utilize restructuring zymoprotein uvs X and uvs Y, the single strand binding protein of T4 phage encodeds
Three kinds of enzymes of gp32 and Bsu archaeal dna polymerases carry out isothermal duplication at 37~42 DEG C to target fragment, can be completed in 20~40min
Billions of DNA copies.RPA is combined with fluorescent quantitative PCR technique (Real-Time PCR), and RPA technologies are applied to diagnosis and examine
During survey, the specific type probe of specificity is added in into reaction system, real-time fluorescence detection can be reached.It is applied when being detected
Twist Amp exo probes, this probe carry a fluorophor and a fluorescence quenching, respectively with a thymidine knot
It closes, centre is separated by tetrahydrofuran (THF) base, and when this structural integrity, fluorescence intensity is low.The 3' ends of probe are sealed
It closes, blocks and primer is served as with this oligonucleotide sequence expanded.When probe is combined with target sequence, connect fluorophor and quench
The THF base positions of group of going out will be identified and digested that the quenching group in downstream is released by endonuclease, and fluorescence intensity increases
By force；The trip 3'-OH generated after digestion as archaeal dna polymerase target spot and expand this probe, fluorescence intensity also can with its amplification
And enhance, detection time also greatly shortens.
The content of the invention
To overcome the drawbacks described above of the prior art, II type carp blister sores are detected in real time it is an object of the invention to provide constant temperature
The RPA kits and its primer special and probe of poison, based on recombinase polymeric enzymatic amplification technology (recombinase poly-
Merase amplification, RPA) it is combined with Real-time quantitative PCR (Real-Time PCR), realize II type carp blebs
The quick detection of viral (CyHV-II).
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
It detects the RPA primer specials of II type carp herpesvirals in real time for constant temperature, is according to II type carp herpesviral purposes
Gene design, and the II types carp herpesviral objective gene sequence is：
cccaggagaccagcagactgttgaacccgtaccttgccaccaagggacagcgggtcgatccgtctaacttgtacatt
cccgacggtatctttgtgacttacacgcctactggaccagaccccggagtggcgcacggtggcggttactattacag
accttcgctgcagggtttcggctctatgatggacgaggcggatacgttggacgatctgcaggccaacgttctgtata
gcaatcagggtcagtggacgagactggcgttgt。
Preferably, the forward primer sequence of the RPA primer specials is
CCCAGGAGACCAGCAGACTGTTGAACCCGTAC；The reverse primer sequences of the RPA primer specials are
GTATCCGCCTCGTCCATCATAGAGCCGAAACC。
The second aspect of the present invention detects the probe of II type carp herpesvirals for constant temperature in real time, is according to II type carp blisters
The design of exanthema virus target gene, and the II types carp herpesviral objective gene sequence is：
cccaggagaccagcagactgttgaacccgtaccttgccaccaagggacagcgggtcgatccgtctaacttgtacatt
cccgacggtatctttgtgacttacacgcctactggaccagaccccggagtggcgcacggtggcggttactattacag
accttcgctgcagggtttcggctctatgatggacgaggcggatacgttggacgatctgcaggccaacgttctgtata
gcaatcagggtcagtggacgagactggcgttgt。
Preferably, the probe sequence is：
cactctggcgacgcgtttgtggttgaaccgcca(BHQ1-dt)c(THF)g(FAM-dT)
ggaggcttcaaaggc(C3spacer)。
Preferably, the probe sequence has one the 34th bp position there are one (BHQ1-dt) group in 36bp positions
A (THF) group, in 38bp positions there are one (FAM-dT) group, afterbody is containing there are one (C3spacer) modifications.
It should be further noted that according to RPA design of primers principles, requirements of the RPA to primer length is 30~35bp,
When amplified production is within 500bp, amplification efficiency is higher；Since primer Individual base difference can have an impact expanding effect,
It is quick, Sensitive Detection II type carp herpesvirals, it is necessary to carry out multiple primer screening.In a preferred embodiment of the invention, entrust
Shanghai bio-engineering corporation design is ask then to pass through for the specific primer sequence and probe sequence of CyHV-II target gene
A series of gradient candidate drugs are designed in conservative gene region, and optimal primer is therefrom selected according to RPA gel detections result.
The third aspect of the present invention, constant temperature detects the RPA kits of II type carp herpesvirals in real time, special including above-mentioned RPA
With primer and above-mentioned probe.
Preferably, constant temperature detects the RPA kits of II type carp herpesvirals in real time, further includes the sun containing ORF72 plasmids
Property control.
Preferably, constant temperature detects the RPA kits of II type carp herpesvirals in real time, further includes containing sterile ddH2The moon of O
Property control.
Compared with prior art, positive effect of the invention is：
(1) present invention can obtain the CyHV-II of needs for the RPA primer specials and probe of CyHV-II viral designs
Target gene is the key that detect II type carp herpesvirals with RPA technologies, currently without for II type carp herpesvirals
RPA primer specials and probe have specificity.
(2) present invention can quickly and successfully detect carp blister sore for detecting the RPA kits of II type carp herpesvirals
Malicious CyHV-II, fastest response 10min can determine whether detection Sample Positive or negative at 37 DEG C.
Description of the drawings
Fig. 1 is the Evaluation on specificity effect of embodiment 2RPA detections；
Fig. 2 is the sensitivity assessment effect of embodiment 3RPA detections.
Specific embodiment
Present pre-ferred embodiments are provided below in conjunction with the accompanying drawings, with the technical solution that the present invention will be described in detail, but the present invention
Protection domain be not limited to following embodiments.
Embodiment 1 detects the RPA primer specials of II type carp herpesvirals in real time for constant temperature and probe designs and screening
The present embodiment commission Shanghai bio-engineering corporation design for CyHV-II target gene specific primer sequence and
Probe sequence, wherein：
Forward primer sequence is CCCAGGAGACCAGCAGACTGTTGAACCCGTAC；
Reverse primer sequences are GTATCCGCCTCGTCCATCATAGAGCCGAAACC.
Probe sequence is：
cactctggcgacgcgtttgtggttgaaccgcca(BHQ1-dt)c(THF)g(FAM-dT)
ggaggcttcaaaggc(C3spacer)。
The Evaluation on specificity of 2 RPA of embodiment detections
It is viral first, in accordance with following steps extraction CyHV-II, including：
(1) add in appropriate PBS buffer solution to be uniformly mixed, 5min is centrifuged with 8000rpm, abandons supernatant；
(2) 1ml buffer solutions are added in and 20 μ L protease keeps the temperature 3h in 56 DEG C of water-baths；
(3) 5min is centrifuged in 8000rpm, taken in 750 μ L supernatants in new centrifuge tube, it is 25 to add in volume ratio:24:1
Phenol：Chloroform：Isoamyl alcohol to centrifuge 5min after the speed mixing 10min of 50rpm with 8000rpm, then takes 650 μ L supernatants
In new centrifuge tube, repeat aforesaid operations and ensure not suck albumin layer；
(4) 600 μ L supernatants are taken in new centrifuge tube, it is 24 to add in volume ratio:1 chloroform：With 50rpm's after isoamyl alcohol
Speed mixes 10min, then 8000rpm centrifugations 5min；
(5) 480 μ L supernatants are taken in new centrifuge tube, add in the sodium acetate and 960 μ L4 DEG C ethyl alcohol of 40 μ L3mol/L, gently
Shake visible white floccule；
(6) 10min, abandoning supernatant are centrifuged with 12000rpm after -20 DEG C of placement 20-30min；
(7) washed with the ethyl alcohol of 200ul75%, 14000rpm centrifugation 5min abandon supernatant；After repeating the above steps once,
The milli-Q water of 50ul is used after drying, is saved backup in -20 DEG C.
RPA-real timePCR reactions are carried out according still further to the reaction system of consisting of and proportioning, wherein, sense primer
2.1 μ l, 2.1 μ l of anti-sense primer, fluorescence probe 0.6 μ l, Rehydration Buffer29.5 μ l, viral DNA 2 μ l, ddH2O
11.2 μ l, total amount are 47.5 μ l.
Then, the molecule for being detected above-mentioned RPA primer and probes with II type carp herpes virus DNAs at optimum conditions is special
The opposite sex chooses the DNA of CyHV-II, IHHNV, WSSV, and 106 plants of 102 plants of GCRV, GCRV carries out fluorescence as template analysis to be determined
Round pcr (Real-Time PCR) is measured to differentiate, experimental result as shown in Figure 1, wherein, No. 1 sample is sick for positive CyHV-II
Malicious sample, 2-6 are respectively IHHNV, WSSV, GCRV102 plant, GCRV106 plants and water.
The sensitivity assessment of embodiment 3RPA detections
Then the extracting method of CyHV-II viruses takes the DNA of CyHV-II to carry out multiple dilution with embodiment 2, each dilute
Degree of releasing carries out RPA fluorescent quantitative PCRs, wherein, 1 and 2 be positive DNA sample, and 2 for 10 times of dilution positive DNA sample,
3 be negative control water.As shown in Figure 2, No. 1 positive can detect experimental result in the 5th Xun Huan (4min or so)
To apparent band, conspicuousness experimental result (8min or so) can be observed in the 10th Xun Huan；No. 2 samples are followed at the 10th
Ring (8min or so) can detect apparent band, in the 15th Xun Huan it is observed that a conspicuousness experimental result (16min left sides
It is right)；No. 3 negative samples are without non-specific amplification band.
4 constant temperature of embodiment detects the RPA kit effect assessments of II type carp herpesvirals in real time
Pass through CyHV-II conventional detections technology such as common fluorescent quantitative PCR, PCR, LAMP[2、6、7]It is real with constant temperature of the present invention
When detect the RPA kits of II type carp herpesvirals using RPA- fluorescence quantitative PCR detection CyHV-II, from reaction time, reaction
Temperature, instrument and reaction step are tested and illustrated, as shown in table 1.
Table 1 detects CyHV-II technology correction datas
RPA is can be seen that from the correction data in table 1 to be combined with fluorescent quantitative PCR technique (Real-Time PCR),
When detecting CyHV-II for diagnosing, the specific type probe of specificity is added in into reaction system, real-time fluorescence inspection can be reached
It surveys.Using the RPA kits containing primer special and probe in the present invention when being detected, wherein, probe is at the 34th bp
It puts there are one (BHQ1-dt) group, in 36bp positions there are one (THF) group, in 38bp positions, there are one (FAM-dT) bases
Group, afterbody is containing there are one (C3spacer) modifications.When probe is combined with target sequence, fluorophor and quenching group are connected
THF base positions will be identified and digested that the quenching group in downstream is released by endonuclease, fluorescence intensity enhancing；Digestion
The trip 3'-OH generated afterwards as archaeal dna polymerase target spot and expand this probe, fluorescence intensity also can with its amplification and enhance,
Detection time also greatly shortens, and testing result can be obtained within 10min；And following three kinds of enzymes of RPA technologies：It can combine single
Recombinase, single-stranded DNA binding protein (SSB) and the strand displacement archaeal dna polymerase of chain nucleic acid (Oligonucleolide primers) have at normal temperatures
Activity, can quickly and successfully detect carp herpesviral CyHV-II, and fastest response 10min can determine whether detection sample at 37 DEG C
It is positive or negative.
Bibliography：
Molecule diagnosis [J] Central China agricultural of the goldfish carp herpesvirusⅡtypes such as [1] Li Lijuan, Luo Yangzhi, Liu Xueqin is big
Learn journal:Natural science edition, 2013,32 (1):92-96.
[2] Zhou Yong, Zeng Lingbing, Zhang Hui wait building for carp herpesvirusⅡtype TaqMan real-time PCR detection methods
Stand and apply [J] aquatic product journals, 2013,37 (4):607-613.
[3] Ma Jie, Zhou Yong, Fan Yuding, the physics and chemistry and biological characteristics and ultra micro form for waiting carp herpesvirusⅡtypes occur
[J] aquatic product journals, 2016,40 (3):475-483.
[4] Lin Xiuxiu, leaf member soil, Wu Ping wait carp herpesvirusⅡtype in hybridized prussian carp Hematopoietic Necrosis's disease disease fish bodies
Electron microscopic observation and Ultrastructural Pathology feature [J] Fisheries Science magazines, 2016,29 (1):17-23.
[5] Xia Siyao, Wang Hao, Patorida Podok wait carps herpesvirusⅡtype to show hybridized prussian carp dorsal fin cell
Influence [J] aquatic product journals of titanium miniplate and immunogene expression, 2016,40 (12):1915-1922.)
[6] foundation of 4 kinds of detection methods of Luo Dan carps herpesvirusⅡtype and application [D] Gansu Agriculture Universities, 2014.
[7] research of summer Xiao Ming prawns WSSV and IHHNV virus recombinase polymeric enzymatic amplification (RPA) rapid detection method
[D] Shanghai Ocean Universities, 2015.
The above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment
Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within
It encloses.
Sequence table
<110>Shanghai Ocean University
<120>Constant temperature detects the RPA kits of II type carp herpesvirals and its primer special and probe in real time
<130> 20180201
<141> 2018-02-01
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 264
<212> DNA
<213> Artificial Sequence
<220>
Claims (8)
- It is according to II type carp herpesviral purpose bases 1. detecting the RPA primer specials of II type carp herpesvirals in real time for constant temperature Because of design, and the II types carp herpesviral objective gene sequence is：cccaggagaccagcagactgttgaacccgtaccttgccaccaagggacagcgggtcgatccgtctaacttgta cattcccgacggtatctttgtgacttacacgcctactggaccagaccccggagtggcgcacggtggcggttactatt acagaccttcgctgcagggtttcggctctatgatggacgaggcggatacgttggacgatctgcaggccaacgttctg tatagcaatcagggtcagtggacgagactggcgttgt。
- 2. detect the RPA primer specials of II type carp herpesvirals in real time for constant temperature as described in claim 1, which is characterized in that The forward primer sequence of the RPA primer specials is CCCAGGAGACCAGCAGACTGTTGAACCCGTAC；The RPA is special to be drawn The reverse primer sequences of object are GTATCCGCCTCGTCCATCATAGAGCCGAAACC.
- 3. detecting the probe of II type carp herpesvirals in real time for constant temperature, designed according to II type carp herpesvirals target gene , and the II types carp herpesviral objective gene sequence is：cccaggagaccagcagactgttgaacccgtaccttgccaccaagggacagcgggtcgatccgtctaacttgta cattcccgacggtatctttgtgacttacacgcctactggaccagaccccggagtggcgcacggtggcggttactatt acagaccttcgctgcagggtttcggctctatgatggacgaggcggatacgttggacgatctgcaggccaacgttctg tatagcaatcagggtcagtggacgagactggcgttgt。
- 4. detect the probe of II type carp herpesvirals in real time for constant temperature as claimed in claim 3, which is characterized in that the probe Sequence is：cactctggcgacgcgtttgtggttgaaccgcca(BHQ1-dt)c(THF)g(FAM-dT)ggaggcttcaaaggc (C3spacer)。
- 5. detect the probe of II type carp herpesvirals in real time for constant temperature as described in claim 3 or 4, which is characterized in that described Probe sequence the 34th bp position there are one (BHQ1-dt) group, in 36bp positions there are one (THF) group, in 38bp Position is there are one (FAM-dT) group, and afterbody is containing there are one (C3spacer) modifications.
- 6. constant temperature detects the RPA kits of II type carp herpesvirals in real time, which is characterized in that including described in claim 1 or 2 Any one of RPA primer specials and claim the 3-5 probe.
- 7. constant temperature as claimed in claim 6 detects the RPA kits of II type carp herpesvirals in real time, which is characterized in that further includes Positive control containing ORF72 plasmids.
- 8. constant temperature as claimed in claim 6 detects the RPA kits of II type carp herpesvirals in real time, which is characterized in that further includes Contain sterile ddH2The negative control of O.
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| CN110592270A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for Koi Herpesvirus (KHV) |
| CN112126712A (en) * | 2019-12-10 | 2020-12-25 | 江苏省渔业技术推广中心 | Specific primer pair, probe and detection kit for detecting carp herpesvirus type II |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592269A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for grass carp hemorrhagic disease type 2 virus (GCRV-2) |
| CN110592270A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant-temperature fluorescence detection method and reagent for Koi Herpesvirus (KHV) |
| CN109913583A (en) * | 2018-12-30 | 2019-06-21 | 广州动佰生物科技有限公司 | A kind of primer and its method of quick detection Koi herpesvirus |
| CN112126712A (en) * | 2019-12-10 | 2020-12-25 | 江苏省渔业技术推广中心 | Specific primer pair, probe and detection kit for detecting carp herpesvirus type II |
| CN112126712B (en) * | 2019-12-10 | 2021-08-24 | 江苏省渔业技术推广中心 | Specific primer pair, probe and detection kit for detecting carp herpesvirus type II |
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