CN108103246A - For detecting the RPA kits of II type carp herpesvirals and its primer special and probe - Google Patents
For detecting the RPA kits of II type carp herpesvirals and its primer special and probe Download PDFInfo
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- CN108103246A CN108103246A CN201810101977.0A CN201810101977A CN108103246A CN 108103246 A CN108103246 A CN 108103246A CN 201810101977 A CN201810101977 A CN 201810101977A CN 108103246 A CN108103246 A CN 108103246A
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Abstract
The invention discloses for detecting the RPA kits of II type carp herpesvirals and its primer special and probe, the forward primer sequence of RPA primer specials is CAATCAGGGTCAGTGGACGAGACTGGCGTTGT, reverse primer sequences CCTCCCAGAGCCATGTTACCCGGTCTGAAGGA；Probe sequence is (FAM) cactctggcgacgcgtttgtggttgaaccgccaTc (THF) gTggaggcttcaaaggc (C3spacer)；RPA kits including above-mentioned RPA primer specials and probe can be in 15min under the conditions of 37 DEG C of constant temperature, Visual retrieval II type carp herpesvirals.
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 for detecting
The RPA kits and its primer special and probe of II type carp herpesvirals and the application in detection II type carp herpesvirals.
Background technology
2009-2010 crucians " viral hemorrhagic disease " sporadicly occur in Jiangsu Province northern Suzhou Sheyang County crucian cultivation pond,
The crucian for causing morbidity pond is dead, and 8-9 months in 2011 occur breaking out for larger area in Yancheng Region, and cause large area
Death starts to find a small number of crucian pond morbidities, break out on a large scale June, pond occurs for the disease high in Sheyang County in March, 2012
Up to 80%, the incidence in other crucian cultivation areas of northern Suzhou is equally severe, this popular crucian " viral hemorrhagic disease " quilt
It is determined as crucian carp Hematopoietic Necrosis disease (Crucian Carp Hematopoietic Necrosis), cause of disease is carp blister sore
Malicious II type (CyprinidherpesvirusII, CyHV-II).
Herpetoviridae (Herpesviridae) belongs to double-stranded DNA virus, tool cyst membrane, spherical shape, various shapes, diameter
For 120-200nm, there is apparent cyst membrane on surface, has spike.20 face body of nucleocapsid is symmetrical, and diameter 100-110nm, surface has one
The overlay film that layer is made of sphere, the distribution to being in, quantity is not different yet for sphere.The big double-stranded DNA of package in viral capsid.
The coe virus has 3 subfamilies, is α types herpesviral (Alphaherpesvirinae), β type herpesvirals respectively
(Betaherpesvirinae) and γ types herpesviral (Gammaherpesvirinae).The herpesviral reported in fish
Section temporarily turns to herpesviral to be sorted by ICTV：Fish herpes like virus category (Ictalurid herpes-like viruses) disease
Disease, sick fish body is rubescent, and below lateral line scales and chest is particularly evident, gill cover swelling, during gill cover opening and closing (or fish body is jumped
During jump), watery blood can be flowed out from gill portion；After sick fish death, the gill cover has apparent bleeding, cuts off gill cover observation, the gill
Silk swelling simultaneously has a large amount of mucus.
The conventional detection technology such as LAMP technology of CyHV-II, reaction principle is complicated, and design of primers is cumbersome, and is drawing
More stringent for the required conservative section of target sequence when object designs, reaction product is inhomogenous, is unfavorable for the sequencing in later stage
Structure clone, therefore be not widely used；Round pcr takes longer, and cost is higher, and needs accurate temperature cycles
Instrument, the serious application limited during round pcr detects at the scene.In recent years, the appearance of constant temperature nucleic acid amplification technology can just solve
Round pcr limits to, more convenient, is a kind of possible ways of quick detection CyHV-II rapidly.Recombinase polymeric enzymatic amplification
Technology (recombinase poly-merase amplification, RPA) is one kind of constant temperature nucleic acid amplification technology, and RPA is sharp
With three kinds of restructuring zymoprotein uvs X and uvs Y, single strand binding protein gp32 and Bsu archaeal dna polymerase enzymes of T4 phage encodeds
Isothermal duplication is carried out to target fragment at 37-42 DEG C, billions of DNA copies can be completed in 20-40min, based on the technology
With gel electrophoresis, lateral flow immunochromatography technique (LFD) can realize the quick detection of CyHV-II.
The content of the invention
To overcome the drawbacks described above of the prior art, it is an object of the invention to provide for detecting II type carp herpesvirals
RPA kits and its primer special and probe, based on recombinase polymeric enzymatic amplification technology (recombinase poly-merase
Amplification, RPA) it is combined with lateral flow immunochromatography technique (LFD), it can be in 15min under the conditions of 37 DEG C of constant temperature
Visual retrieval II type carp herpesvirals.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
For detecting the RPA primer specials of II type carp herpesvirals, designed according to II type carp herpesvirals target gene
, and the II types carp herpesviral objective gene sequence is：
caatcagggtcagtggacgagactggcgttgtgcggtctggccaacacggccaactaccccatcgatctgtacgacc
agaaggacaccaagtacagaatcatcaacaccggggccgagcctctgcactctggcgacgcgtttgtggttgaaccg
ccatcggtggaggcttcaaaggcgcaggtggacagcatgaaaaacaacaccatcagagccgagggttccttcagacc
gggtaacatggctctggg；The forward primer sequence of the RPA primer specials is
CAATCAGGGTCAGTGGACGAGACTGGCGTTGT；The reverse primer sequences of the RPA primer specials are
CCTCCCAGAGCCATGTTACCCGGTCTGAAGGA。
The second aspect of the present invention is according to II type carp herpesviral mesh for detecting the probe of II type carp herpesvirals
Gene design, and the II types carp herpesviral objective gene sequence is：
caatcagggtcagtggacgagactggcgttgtgcggtctggccaacacggccaactaccccatcgatctgtacgacc
agaaggacaccaagtacagaatcatcaacaccggggccgagcctctgcactctggcgacgcgtttgtggttgaaccg
ccatcggtggaggcttcaaaggcgcaggtggacagcatgaaaaacaacaccatcagagccgagggttccttcagacc
gggtaacatggctctggg；The probe sequence is：
(FAM)cactctggcgacgcgtttgtggttgaaccgccaTc(THF)gTggaggcttcaaaggc
(C3spacer)。
It should be noted that according to RPA design of primers principles, requirements of the RPA to primer length is 30~35bp, amplification production
When object is within 500bp, amplification efficiency is higher；Since primer Individual base difference can have an impact expanding effect, for it is quick,
Sensitive Detection II type carp herpesvirals are, it is necessary to carry out multiple primer screening.In a preferred embodiment of the invention, Shanghai life is entrusted
The design of object engineering company is directed to the specific primer sequence and probe sequence of CyHV-II target gene, then by conservative base
Because a series of gradient candidate drugs are designed in region, and optimal primer is therefrom selected according to RPA gel detections result.
The third aspect of the present invention for detecting the RPA kits of II type carp herpesvirals, is drawn including above-mentioned RPA is special
Object and probe.
Preferably, for detecting the RPA kits of II type carp herpesvirals, it is right to further include the positive containing ORF72 plasmids
According to.
Preferably, for detecting the RPA kits of II type carp herpesvirals, further include containing sterile ddH2The feminine gender of O is right
According to.
It should be further noted that the side that the present invention is combined using RPA with lateral flow immunochromatography technique (LFD)
Method, reaction principle are the nucleic acid amplification products of the specific probe and biotin (Biotin) mark of Fluoresceincarboxylic acid (FAM) mark
Specific binding being added dropwise in will be combined in test strips with the antibody of the anti-FAM of colloid gold label, forming tri compound
Object, when being diffused into detection line, ternary complex is captured by biotin ligand, forms coloured detection line；It is tied with judgement
Pulp eye can interpretation, it is easy to detect, the reaction time in 10min, it is easy to carry the advantages that.
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, can in 15min Visual retrieval II type carp herpesvirals under the conditions of 37 DEG C of constant temperature.
Description of the drawings
Fig. 1 is the agarose gel analysis of reaction product as a result, wherein, 1, No. 2 sample is positive, and No. 3 samples are
Negative sample；
Fig. 2 is the test strips analysis result of reaction product, wherein, 1, No. 2 sample is positive test symbol, and NC is negative sample
Product；
Fig. 3 and Fig. 4 is the specific analysis result of reaction product, wherein No. 1-10 be respectively CyHV-II DNA,
IHHNV, WSSV, GCRV-jx01, GCRV-JX02, GCRV-104, KHV, SVC, YHV and water；
Fig. 5 is the sensitivity assessment result of agarose gel detection；
Fig. 6 is the sensitivity evaluation result of RPA ELISA test strips.
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 is used to detect the RPA primer specials of II type carp herpesvirals 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 by designing a series of gradient candidate drugs in conservative gene region, and is therefrom selected according to RPA gel detections result
Select optimal primer.Wherein：
The forward primer sequence of RPA primer specials is
CAATCAGGGTCAGTGGACGAGACTGGCGTTGT；
The reverse primer sequences of RPA primer specials are
CCTCCCAGAGCCATGTTACCCGGTCTGAAGGA；
Probe sequence is：
(FAM)cactctggcgacgcgtttgtggttgaaccgccaTc(THF)gTggaggcttcaaaggc
(C3spacer)。
The agarose gel analysis of 2 reaction product of embodiment
First, in accordance with following steps extraction CyHV-II viruses：(1) add in and be uniformly mixed with adding in sample P BS buffer solutions, with
8000rpm centrifuges 5min, 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) exist
8000rpm centrifuges 5min, takes in 750 μ L supernatants in new centrifuge tube, and it is 25 to add in volume ratio:24:1 phenol：Chloroform：It is different
Amylalcohol to centrifuge 5min after the speed mixing 10min of 50rpm with 8000rpm, then takes 650 μ L supernatants in new centrifuge tube, weight
It answers aforesaid operations and ensures 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：10min, then 8000rpm centrifugations 5min are mixed with the speed of 50rpm after isoamyl alcohol；(5) take 480 μ L supernatants in newly from
In heart pipe, the sodium acetate and 960 μ L4 DEG C ethyl alcohol of 40 μ L3mol/L, jog visible white floccule are added in；(6) in -20 DEG C of placements
10min, abandoning supernatant are centrifuged with 12000rpm after 20-30min；(7) washed with the ethyl alcohol of 200ul75%, 14000rpm from
Heart 5min, abandons supernatant；It repeats the above steps once, the milli-Q water of 50ul is used after drying, is saved backup in -20 DEG C.
Pcr amplification reaction is carried out according still further to the reaction system of consisting of and proportioning, wherein, 2.1 μ l of sense primer, downstream
Primer 2 .1 μ l, Rehydration Buffer29.5 μ l, viral DNA 2 μ l, ddH211.8 μ l of O, total amount are 47.5 μ l, 37 DEG C
Lower reaction 15min carries out agarose gel analysis, as a result as shown in Figure 1, wherein 1, No. 2 sample is expanded to positive products,
Negative sample is without non-specific amplification.
The test strips analysis of 3 reaction product of embodiment
It is anti-to carry out PCR amplification with embodiment 2 according to the reaction system of consisting of and proportioning for the extraction of CyHV-II viruses
Should, wherein, 2.1 μ l of sense primer, 2.1 μ l of anti-sense primer, 0.6 μ l, RehydrationBuffer29.5 μ l of fluorescence probe, disease
Malicious DNA2 μ l, ddH2O11.2 μ l, total amount are 47.5 μ l.RPA ELISA test strip reaction steps are：1. after more than component is mixed
The several seconds is centrifuged after slight oscillatory；2. mixed liquor is added in the 0.2mLTwist Amp Basic reaction tubes containing lyophilized enzyme powder repeatedly
It inhales for several times；3. the magnesium acetate for adding in 2.5 μ L280mM is inhaled for several times repeatedly；4. being put into 4min in 38 DEG C of Metal constant temperature casees, mixing is taken out
It is put into 38 DEG C of Metal constant temperature case 36min again afterwards；5. it after reaction, is detected with nucleic acid test strip, as a result such as 2 institute of attached drawing
Show.When two band occur in test strips, one is located in quality control region, and one is located at detection zone, and result is the positive, is shown in sample
Contain CyHV-II viruses；When test strips only have quality control region a band occur, detection zone does not have band, the result is that it is negative, show
CyHV-II viruses are not contained in sample.
The molecular specificity analysis of 4 RPA primer and probes of embodiment
The extraction of CyHV-II viruses is with embodiment 2, and the molecular specificity of RPA primer and probes is in 38 DEG C of Metal constant temperature casees
Be detected after middle 15min with the DNA of CyHV-II viruses, choose the DNA of CyHV-II, IHHNV, WSSV, GCRV as template,
Products therefrom carries out lateral flow immunochromatography technique (LFD) with gel electrophoresis and differentiates, as a result as shown in attached drawing 3 and Fig. 4.
5 RPA kit agarose gel electrophoresis detection sensitivity of embodiment is evaluated
The extraction of CyHV-II viruses takes the DNA of CyHV-II viruses to carry out multiple dilution, each dilution factor with embodiment 2
RPA amplifications are carried out, product is detected into row agarose gel electrophoresis after 15min is expanded at 37 DEG C, as shown in Figure 5, Ke Yijian
Measure 10-3Copy.
Embodiment 6RPA test strips lateral flow immunochromatographies detection sensitivity is evaluated
The extraction of CyHV-II viruses takes the DNA of CyHV-II to carry out multiple dilution, each dilution factor carries out with embodiment 2
RPA is expanded, and 15min is cultivated in 38 DEG C of Metal constant temperature casees, treats to carry out lateral flow immunochromatography technique to product after reaction
(LFD) detect, as shown in Figure 6, can detect 10-4Copy (about 5pg).
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>For detecting the RPA kits of II type carp herpesvirals and its primer special and probe
<130> 20180201
<141> 2018-02-01
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 249
<212> DNA
<213> Artificial Sequence
<220>
Claims (7)
1. for detecting the RPA primer specials of II type carp herpesvirals, designed according to II type carp herpesviral target gene,
And the II types carp herpesviral objective gene sequence is：
caatcagggtcagtggacgagactggcgttgtgcggtctggccaacacggccaactaccccatcgatctgtac
gaccagaaggacaccaagtacagaatcatcaacaccggggccgagcctctgcactctggcgacgcgtttgtggttga
accgccatcggtggaggcttcaaaggcgcaggtggacagcatgaaaaacaacaccatcagagccgagggttccttca
gaccgggtaacatggctctggg。
2. it is used to detect the RPA primer specials of II type carp herpesvirals as described in claim 1, which is characterized in that the RPA is special
It is with the forward primer sequence of primer
CAATCAGGGTCAGTGGACGAGACTGGCGTTGT；
The reverse primer sequences of the RPA primer specials are
CCTCCCAGAGCCATGTTACCCGGTCTGAAGGA。
3. for detecting the probe of II type carp herpesvirals, designed according to II type carp herpesviral target gene, and it is described
II type carp herpesviral objective gene sequences are：
caatcagggtcagtggacgagactggcgttgtgcggtctggccaacacggccaactaccccatcgatctgtac
gaccagaaggacaccaagtacagaatcatcaacaccggggccgagcctctgcactctggcgacgcgtttgtggttga
accgccatcggtggaggcttcaaaggcgcaggtggacagcatgaaaaacaacaccatcagagccgagggttccttca
gaccgggtaacatggctctggg。
4. as claimed in claim 3 for detecting the probes of II type carp herpesvirals, which is characterized in that the probe sequence is：
(FAM)cactctggcgacgcgtttgtggttgaaccgccaTc(THF)gTggaggcttcaaaggc(C3spacer)。
5. for detecting the RPA kits of II type carp herpesvirals, which is characterized in that special including the RPA of claim 1 or 2
With any one of primer and claim 3~5 probe.
6. as claimed in claim 5 for detecting the RPA kits of II type carp herpesvirals, which is characterized in that further include containing
The positive control of ORF72 plasmids.
7. as claimed in claim 5 for detecting the RPA kits of II type carp herpesvirals, which is characterized in that further include containing
Sterile ddH2The negative control of O.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624718A (en) * | 2018-06-20 | 2018-10-09 | 上海海洋大学 | Constant temperature detects the RPA kits and its primer special and probe of Tilapia mossambica lake virus in real time |
| CN109628640A (en) * | 2018-12-29 | 2019-04-16 | 中国水产科学研究院珠江水产研究所 | A kind of RPA-LFD primer, method and the kit of quick detection huichun viremia virus |
| 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 |
| CN110964848A (en) * | 2019-10-29 | 2020-04-07 | 北京市水产技术推广站 | RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method |
| CN116356080A (en) * | 2023-04-14 | 2023-06-30 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | RPA-LFD primer, probe and kit for detecting koi herpesvirus |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624718A (en) * | 2018-06-20 | 2018-10-09 | 上海海洋大学 | Constant temperature detects the RPA kits and its primer special and probe of Tilapia mossambica lake virus in real time |
| CN109628640A (en) * | 2018-12-29 | 2019-04-16 | 中国水产科学研究院珠江水产研究所 | A kind of RPA-LFD primer, method and the kit of quick detection huichun viremia virus |
| 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 |
| 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 |
| CN110964848A (en) * | 2019-10-29 | 2020-04-07 | 北京市水产技术推广站 | RAA amplification primer and probe for rapidly detecting carp herpesvirus II, detection kit and use method |
| CN116356080A (en) * | 2023-04-14 | 2023-06-30 | 岭南现代农业科学与技术广东省实验室肇庆分中心 | RPA-LFD primer, probe and kit for detecting koi herpesvirus |
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