CN108411031A - A kind of detection kit and detection method of grass carp hemorrhage virus - Google Patents
A kind of detection kit and detection method of grass carp hemorrhage virus Download PDFInfo
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Abstract
The invention discloses a kind of primer pairs for the amplification of grass carp hemorrhage virus VP5 gene orders.The invention also discloses a kind of amplification methods of grass carp hemorrhage virus VP5 gene orders.The invention also discloses the detection kits and detection method of a kind of grass carp hemorrhage virus.The one-step method fluorescent quantitation RT PCR methods that the present invention establishes are compared with the method reported, not only simply, quickly, but also it is accurate, sensitive, detection limit is low.This method can virus and content quick, in Accurate Determining illness Fish tissue.
Description
Technical field
The present invention relates to the detection field of fish Causative virus, it is related to a kind of fish Causative virus detection kit and inspection
The detection kit and detection method of survey method more particularly to a kind of grass carp hemorrhage virus.
Background technology
(grass carp hemorrhage virus, GCHV, International Commission on Virus Classification are referred to as grass carp hemorrhage virus
For reovirus of grass carp, GCRV) it is the first fishes virus that China detaches, it is subordinate to Reoviridae, water
Lively object reovirus category, 70~80nm of diameter, 20 face body spheric granules, the double-stranded RNA containing 11 segments.Different regions
There are different strains.10 separation strains are had reported at present, which mainly causes CHINESE FRESHWATER to cultivate principal item grass carp
Hemorrhage occurs in the fingerling stage, the death rate is up to 90% or more, brings about great losses to culture fishery.
Grass carp viral hemorrhagic is often happened at the annual 4-10 months, when water temperature fades away in 15 DEG C or less the state of an illness.It is main
Symptom is congested, and the organ hemorrhages phenomenon such as sick lymphogranuloma inguinale chamber, the gill cover, fin base, enteron aisle, liver, spleen, whole-body muscle is in scarlet when serious
Color, it is in pale asphyxia that the gill, which loses scarlet, but enteron aisle, muscle occur without rotten, oedema phenomenon.
Occur to control cultured fishes fish disease in time, there is an urgent need to a kind of can quickly detect whether there is the cause in fish body
The technology of virus.Fluorescence quantitative polymerase chain reaction (Real-time Fluorescent Quantitative
Polymerase Chain Reaction, RtFQ-PCR) because its have it is easy, quickly, it is sensitive, special, be suitable for early stage and a large amount of
The advantages of sample detection, is widely applied in Pathogen test field.
At present for the detection of the grass carp hemorrhage virus country still without the research report in terms of relevant quantitative PCR detection technique
It accuses.Grass carp hemorrhage virus is a kind of RNA virus, and common detection process is complicated, and RNA is degradable, and false negative is caused to occur.
Invention content
Goal of the invention：The present invention the technical problem to be solved is that provide quickly to examine for fluorescent quantitation gene amplification method
VP5 gene orders (AF239175.1) primer and its application for surveying 873 strains of GCHV-strain, go out grass carp from molecular level
Blood virus is used for quickly detecting, and has the characteristics that easy, quick, high specific and sensitivity.
There is provided a kind of detections of grass carp hemorrhage virus fluorescent quantitation one-step method to try for the present invention also technical problems to be solved
Agent box, according to the VP5 gene orders (AF239175.1) of Genbank 873 strains of GCHV-strain announced, design specificity
Primer expands the specific region of target gene using fluorescence quantitative RT-RCR, is carried out to grass carp hemorrhage virus from molecular level fast
Speed detection has the characteristics that easy, quick, high specific and sensitivity.
There is provided a kind of one-step method fluorescence quantitative detection kits to detect grass carp for the present invention also technical problems to be solved
The method of bleeding virus can detect sick fish tissues and cell of GCHV infection etc..
Technical solution：Of the existing technology in order to solve the problems, such as, the present invention uses following technical scheme：One kind is for grass
The primer pair of fish bleeding virus VP 5 gene order amplification, the primer pair includes II-F of sense primer VP5- and downstream primer
The nucleotide sequence such as SEQ ID NO of VP5- II-R, the II-F of sense primer VP5-：Shown in 3, the downstream primer VP5-
The nucleotide sequence of II-R such as SEQ ID NO：Shown in 4.
II-F of sense primer VP5-：5’-CTCCGTGTTGACCCTGGATGTG-3'；
II-R of downstream primer VP5-：5’-ATGTTAGCAGCGGTAGTGACTTGTTG-3'.
A kind of amplification method of grass carp hemorrhage virus VP5 gene orders, includes the following steps：
1) bioinformatics method design primer group and progress primer screening obtain the primer pair；
2) fluorescence quantitative PCR detection of grass carp hemorrhage virus VP5 genes：It is extracted with viral RNA extracts kit and obtains grass
Fish bleeding virus genome RNA sample；
3) using the grass carp hemorrhage viral gene group RNA samples of step 2) as template, the primer pair is subjected to one-step method
Fluorescence quantitative RT-RCR obtains pcr amplification product；
4) pcr amplification product that step 3) obtains, which is analyzed and is sequenced, obtains grass carp hemorrhage virus VP5 gene orders.
Wherein, above-mentioned steps 1) it is that the detection of primer PCR amplification efficiency is carried out to which screening obtains institute by quantitative fluorescent PCR
The primer pair stated.
The detection kit of a kind of detection kit of grass carp hemorrhage virus, the grass carp hemorrhage virus includes described draws
Object pair.
Wherein, the molar concentration rate of above-mentioned II-F of sense primer VP5- and II-R of downstream primer VP5- is 1.5：1.
Wherein, above-mentioned detection kit includes：
1) grass carp hemorrhage virus reaction solution, Taq archaeal dna polymerases and reverse transcriptase (Quant Reverse
Transcriptase)：Grass carp hemorrhage virus reaction solution includes containing dNTPs, 10 × buffer, MgC12、SYBR green I、
The mixed liquor of DEPC water and the primer pair；
2) positive control：
3) critical positive control；
4) negative control object.
Wherein, above-mentioned positive control is the total serum IgE extracted containing high-purity grass carp hemorrhage virus.
The application of context of detection of the detection kit of above-mentioned grass carp hemorrhage virus in fish bacterial pathogens.
Wherein, above-mentioned detection method be by RNA reverse transcriptions and quantitative fluorescent PCR reaction in the same reaction tube continuously into
Row.
Wherein, above-mentioned detection method specifically includes following steps：
1) RNA of extraction fish sample tissue obtains sample RNA；
2) one-step method fluorescence quantitative RT-RCR reaction system is established；
3) determine whether to infect grass carp hemorrhage virus by comparing standard items real-time fluorescence quantitative RT-PCR amplification curve.
The detection method is one-step method real-time fluorescent quantitative RT-PCR detecting method, and reaction system is：
5 μ L, the 2.5U/ μ L of downstream primer 2.0 μ L, sample RNA of 3.0 μ L, the 10pmol/ μ L of sense primer of 10pmol/ μ L
2.5 μ L, 5U/ μ L reverse transcriptase (Quant Reverse Transcriptase) of Taq archaeal dna polymerases 0.5 μ L, 12 μ L DEPC
Water, 2 × import real-time fluorescence PCR buffer solution, 25 μ L；2 × import real-time fluorescence PCR buffer solution of the 25 μ L is 5 μ L
10 × buffer of ThermoFisher companies, 10mM dNTP of 4 μ L, 0.005 μ L 10000 × SYBR green I and
The mixture of 16 μ L DEPC water；
PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 75 DEG C
5sec, read plate are recycled for 45 totally.
Advantageous effect：Compared with prior art, it is an advantage of the invention that：
1, the present invention provides the primer special in the detection of grass carp hemorrhage virus VP5 gene fluorescence quantitative RT-PCRs for the first time,
Make it possible to detect grass carp hemorrhage virus VP5 genes using fluorescence quantitative RT-RCR.
2, the detection kit of grass carp hemorrhage virus of the invention further includes positive control；The positive control is to contain
There are the RNA extracts of high-purity grass carp hemorrhage virus.Without live virus, the bio-safety of this kit has been ensured to greatest extent
Property.
3, other viral common detection methods of detection method and grass carp hemorrhage, such as virus be separately cultured identification,
Enzyme-linked Immunosorbent Assay ELISA method is compared with regular-PCR, and sensitivity and specificity are high, and detection efficiency is greatly improved.
4, detection method is detected using one-step method fluorescence quantitative RT-RCR, i.e., by RNA reverse transcriptions and fluorescent quantitation
PCR reactions are carried out continuously in the same reaction tube.The step of reducing reverse transcription, with common fluorescence quantitative detecting method phase
Than the characteristics of having operation sequence easy to use, and can effectively prevent pollution.This reaction system due to can to amplified production into
Row detection in real time, substantially increases detection sensitivity, and the electrophoresis step after PCR reactions is omitted, and is highly suitable for micro RNA
Detection.
5, other detection methods of detection method and grass carp hemorrhage virus, such as virus are separately cultured identification, enzyme
Connection immuno absorbence ELISA method is compared with regular-PCR, has the characteristics that operation sequence is easy to use, this kit can be used for operating
Sequencing is suited large area to popularize and is applied.
In conclusion the present invention can quickly and accurately to detect that grass carp hemorrhage virus provides guarantee, for prevention disease,
Scientific Usage of Drugs, and ensure that healthy fish provides guarantee.The detection method and kit of primer special can be used for according to the present invention
The detection of nucleic acids of the main illing tissue of fish (gill, enteron aisle, liver, spleen) VP5 genes.
Description of the drawings
Fig. 1 is the electrophoretogram after being expanded respectively for the target target gene of grass carp hemorrhage virus using three pairs of primers；
DNA marker sizes are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp successively from top to bottom in figure；
Fig. 2 is the electrophoresis after being expanded using the target target gene of three pairs of non-target fish Causative virus of four kinds of primer pair
Figure；DNA marker sizes are 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp successively from top to bottom in figure；(N1-
Rainbow Medaka fish arc reovirus virus, N2- spring darter reoviruses, N3- Micropterus salmonoides reoviruses, N4- dog salmons exhale intestines lonely
Virus)
Fig. 3 is the standard items real-time fluorescence quantitative RT-PCR amplification curve carried out using second couple of II-F/R of primer VP5-
Figure；
Fig. 4 is to carry out real time fluorescent quantitative RT- to 10 grass carp total serum IgE samples using second couple of II-F/R of primer VP5-
PCR amplification curve graph.
Specific implementation mode
The present invention is described in further details with reference to specific embodiment.
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited
Invention.Following embodiment is in order to which invention is further described in detail, not to the limitation of invention.The experiment side of the present invention
The equal reference of method《Fine works molecular biology experiment guide》Proposed by (chief editors such as F.M. Ao Sibai, Science Press publish for 2005)
Experiment condition.
Embodiment 1 carries out design of primers and the screening of fluorescence quantitative RT-RCR detection for grass carp hemorrhage virus VP5 genes
1, bioinformatics method design primer and progress primer screening
Download the VP5 sequences (GeneBank for the grass carp hemorrhage virus that GenBank is included：AF239175.1), download simultaneously
Negative control virus genome, rainbow Medaka fish arc reovirus virus, spring darter reovirus, Micropterus salmonoides reovirus, big horse
Fish reovirus is breathed out, see the following table 1.After being compared by Clustal X, appropriate area design primer, the region are selected
It is expressed in grass carp hemorrhage virus internal specific, but at least there is the difference of 10%-30% relative to negative control, after selection area
Using 3.0 real-time fluorescence quantitative RT-PCR primer-design softwares of ABI Primer Express, synthetic primer is designed.It will be carried
The alternative primer taken is screened as claimed below：
1) it is designed in primer application nucleic acid series conserved region and with specificity；
2) product cannot form secondary structure (free energy is less than 58.61KJ/mol)；
3) for primer length generally between 17-25 bases, upstream and downstream primer cannot differ too big；
4) G+C contents are between 40%~60%；
5) base wants random distribution, as possible uniformly；
6) primer itself cannot have the complementation for continuing to exceed 4 bases；
7) there cannot be the complementation for continuing to exceed 4 bases between primer；
8) end of primer 5 ' can be modified；
9) 3 ' ends can not be modified, and avoid AT, the region (2-3) of GC rich；
10) 5 ' end of primer global design free energy distribution is more than 3 ' ends, and 3 ' end free energys are more preferably less than 9KJ/mol；
11) design of primers avoids DNA pollution, preferably across exon connector area；
12) homology of primer and non-specific amplification sequence is more preferably less than 70% or has 8 complementary bases homologous；
13) presence whether there is or not pseudogene is checked.Pseudogene is exactly non-functional DNA sequence dna, the purpose piece expanded with needs
Segment length is similar；
14) Tm values are between 55-65 DEG C.
PCR primer used is synthesized by Shanghai Sangon Biotech Company, and primer requires PAGE purifying, is primer dry powder when arrival, with nothing
It is spare after primer to 100pmol/ μ L storage liquid to dry powder progress assay after bacterium water redissolves.
For grass carp hemorrhage virus VP5 genes, 3 groups of upstream and downstream primers are devised altogether, and sequence is as follows：
VP5- I (PCR product length is 122bp)
I-F of sense primer VP5-：5’-CGACTCACTTGCCATCGTTA-3'；
I-R of downstream primer VP5-：5’-CGCTTGGAATAAAGGTTGCC-3'；
VP5- II (PCR product length is 135bp)
II-F of sense primer VP5-：5’-CTCCGTGTTGACCCTGGATGTG-3'；
II-R of downstream primer VP5-：5’-ATGTTAGCAGCGGTAGTGACTTGTTG-3'；
VP5- III (PCR product length is 116bp)
III-F of sense primer VP5-：5’-GGTATGCTTTCCGCCCTAAC-3'；
III-R of downstream primer VP5-：5’-CTCTCTGTGCTCTTGCCC-3'.
Table 1
| Negative control species name | Negative control species name | GenBank |
| Rainbow Medaka fish arc reovirus virus | Guppy reovirus | AAP72181 |
| Spring darter reovirus | Etheostoma fonticola aquareovirus | NC_030406.1 |
| Micropterus salmonoides reovirus | Micropterus salmoides reovirus | KJ740730.1 |
| Dog salmon reovirus | Chum salmon reovirus | NC_007592.1 |
2, molecular biology experiment carries out primer detection
2.1 experiment materials and reagent
Material：873 strains of grass carp hemorrhage virus GCHV-strain are (referring to document Qiu T, Lu R H, Zhang J, et
al.Molecular characterization and expression of the M6gene of grass carp
hemorrhage virus(GCHV),an aquareovirus.[J].Archives of Virology,2001,146(7):
1391.), rainbow Medaka fish arc reovirus virus, spring darter reovirus, Micropterus salmonoides reovirus, dog salmon exhale the lonely disease of intestines
Poison.Five kinds of viruses are all from Wuhan aquatile research institute of the Chinese Academy of Sciences.It is activated with corresponding culture medium using preceding, by activation
Virus infects healthy grass carp respectively, takes the gill of illness fish, enteron aisle, liver, spleen tissue as sample to be tested respectively after illness embodiment.
The grass carp gill, enteron aisle, liver, spleen tissue and the grass of infection different virus sample are extracted with viral RNA extracts kit (Tiangen)
873 strain total serum IgEs of fish bleeding virus GCHV-strain, with RT-PCR kit (ThermoFisher) reverse transcription.With
ThermoFisher PCR kits evaluate specificity and the sensitivity of PCR system.
RNA reagents：Lysate RL, protein liquid removal RW1, rinsing liquid RW, Proteinase K, RNase-Free ddH2O, RNase-
Free adsorption columns CR3,1500U DNase I, save backup in -20 DEG C.
ThermoFisher RT-PCR reagents：(1μg)Positive control RNA、5U/μL AMV reverse
transcriptase、RNase inhibitor、10mM dNTP、RNase free H2O、10×RT buffer、25mM
MgCl2, 10 μM of Random primer, saved backup in -20 DEG C.
ThermoFisher PCR reagents：5U/ μ L Taq DNA polymerase (containing 10 × reaction buffer and
25mM Mg2+)、10mM dNTP、DNA marker I；Millipore H2It dispenses after O high pressure sterilizations, is saved backup in -20 DEG C.
Reverse transcriptase (Quant Reverse Transcriptase are purchased from Tiangen Biotech (Beijing) Co.,
LTD, article No. ER103-03).
2.2 primers, virus test
With the designed primer Test Virus of Standard PCR test, PCR reaction systems are prepared according to the concentration of each component, are expanded
Increase program to be write according to the Tm values of primer and the size of product.Standard PCR amplification is in Bio-Rad Mycycler gradient amplification instruments
Upper progress, agarose gel electrophoresis attached gel imaging system detect obtained PCR product.
RNA reverse transcription systems are：1 μ g, 5U/ μ L AMV reverse of Positive control RNA
2 μ L, 10 × RT buffer of transcriptase 1 μ L, RNase inhibitor, 1 μ L, 10mM dNTP 2 μ L, 25mM
MgCl22 μ L, 10 μM of 2 μ L, RNase free H of Random primer2O supplies 20 μ L；
RNA reverse transcription conditions are：37℃30min；
The PCR system of routine PCR reaction is：The downstream primer of 2.0 μ L, the 10pmol/ μ L of sense primer of 10pmol/ μ L
1 μ L, 5U/ μ L Taq DNA polymerase of product, 1 μ L, 10 × reaction buffer, 2 μ after 2.0 μ L, RNA reverse transcriptions
L、25mM Mg2+1μL、10mM dNTP1μL、Millipore H2O supplies 20 μ L；
Routine PCR reaction condition is：95℃3min；95 DEG C of 20sec, 58 DEG C of 20sec, 72 DEG C of 20sec, totally 35 recycle；
72℃5min。
2.3 result
2.3.1 grass carp hemorrhage virus
A. the primer of Standard PCR test grass carp hemorrhage virus and design
Three pairs of primer sequences of design are as follows：
VP5-Ⅰ
I-F of sense primer VP5-：5’-CGACTCACTTGCCATCGTTA-3'；
I-R of downstream primer VP5-：5’-CGCTTGGAATAAAGGTTGCC-3'；
VP5-Ⅱ
II-F of sense primer VP5-：5’-CTCCGTGTTGACCCTGGATGTG-3'；
II-R of downstream primer VP5-：5’-ATGTTAGCAGCGGTAGTGACTTGTTG-3'；
VP5-Ⅲ
III-F of sense primer VP5-：5’-GGTATGCTTTCCGCCCTAAC-3'；
III-R of downstream primer VP5-：5’-CTCTCTGTGCTCTTGCCC-3'.
It will be seen from figure 1 that for grass carp hemorrhage virus, designed three pairs of primers it is amplifiable go out target band, table
The primer of grass carp hemorrhage virus, designed, designed and the PCR amplification system used are used equally for subsequent experiment used in bright.
B. the specificity of Standard PCR detection architecture
For four kinds of common non-target fish Causative virus, the Standard PCR detection architecture of foundation, I Hes of VP5- are used
II primer detection results of VP5- are feminine gender, and III primer pairs of VP5- weak non-spy occur to rainbow Medaka fish arc reovirus virus testing result
Anisotropic band.The designed non-target fish Causative virus of four kinds of three pairs of primer pairs is detected as feminine gender, is examined to grass carp hemorrhage virus
It surveys to be positive, this shows that established Standard PCR detection architecture has preferable specificity, can be used for grass carp hemorrhage virus
Quickly detection (as shown in Figure 2).
3, primer PCR amplification efficiency detects
The screening principle of primer is：The higher primer of PCR amplification efficiency is selected to draw as candidate in target gene region
Object.
The preparation of 3.1 positive controls and standard form
After cultivating grass carp hemorrhage virus GCHV-strain 873, disease is isolated and purified by sucrose density gradient method
Poison is obtained more than 1 × 108The virion of PFU/ml, 873 samples of grass carp hemorrhage virus GCHV-strain after extraction purification
Total serum IgE measures the concentration of the RNA of extraction, is diluted to certain multiple and is detected for PCR as positive control.
3.2 primer PCR amplification efficiencies detect
The screening principle of primer is：Select the higher primer of PCR amplification efficiency as candidate drugs in target fragment area.
3.2.1 gradient template prepares
By positive control RNA (above-mentioned 3.1 gained), with DEPC water with 10 times of gradient dilutions, as fluorescence quantitative RT-RCR
Reaction system optimization template.Take 10-4、10-5、10-6、10-7、10-8Dilution, number be corresponding in turn to for L1, L2, L3, L4,
L5.It is saved backup in -80 DEG C after packing.
3.2.2 fluorescence quantitative RT-RCR buffer solution and PCR programs
PCR reaction systems are：The 2.0 μ L of downstream primer of 2.0 μ L, the 10pmol/ μ L of sense primer of 10pmol/ μ L are positive
Compare 5 μ L, 2.5U/ μ L Taq archaeal dna polymerases of RNA, 2.5 μ L, 5U/ μ L reverse transcriptase (Quant Reverse
Transcriptase is purchased from Tiangen Biotech (Beijing) Co., LTD, article No. ER103-03) 0.5 μ L, 13 μ L
DEPC water, 2 × import real-time fluorescence PCR buffer solution, 25 μ L (are purchased from ThermoFisher companies, 5 μ L10 × buffer, 4 μ
L10mM dNTP, 0.005 μ L10000 × SYBR green I and 16 μ L DEPC water)；
The PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 75
DEG C 5sec, read plate totally 45 cycles.The results are shown in Table 2.
2 different primers amplification efficiency of table
Note：It is Ct values that value is indicated in table.
From the point of view of amplification efficiency, II primer pairs VP5-II-F/VP5-II-R is selected to continue subsequent experimental.Subsequent experimental
The reverse transcriptase of use is reverse transcriptase (Quant Reverse Transcriptase).
The optimization of 2 fluorescence quantitative RT-PCR primer dosage of embodiment
1, the first suboptimization of fluorescence quantitative RT-PCR primer dosage
With the L1 (10 diluted-4) and L2 (10-5) template that optimizes as primer amount of positive control RNA, respectively to primer
The dosage of upstream and downstream carry out gradient optimizing, primer working solution concentration 10pmol/ μ L, PCR reaction systems are：Upstream and downstream primer amount
3 are shown in Table, the reverse transcriptase (Quant of 5 μ L, 2.5U/ μ L Taq archaeal dna polymerases of positive control RNA, 2.5 μ L, 5U/ μ L
Reverse Transcriptase) 0.5 μ L, 2 × import real-time fluorescence PCR buffer solution, 25 μ L are (purchased from ThermoFisher companies
5 μ L10 × buffer, 4 μ L10mM dNTP, 0.005 μ L10000 × SYBR green I and 16 μ L DEPC water), supplement
Sterile water is to 50 μ L.PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec,
75 DEG C of 5sec, read plate are recycled for 45 totally.The results are shown in Table 3, can be analyzed from result, PCR primer is with descending stair
Degree combination can work normally, wherein primer combination (3.0 μ L × 10pmol/ μ L, 2.0 μ L × 10pmol/ μ L), (4.0 μ L ×
10pmol/ μ L, 3.0 μ L × 10pmol/ μ L), (2.5 μ L × 10pmol/ μ L, 2.0 μ L × 10pmol/ μ L) reaction efficiency highest,
It is carried out sense primer amount 2.5-4 μ L × 10pmol/ μ L, downstream primer amount 2.0-3.0 μ L × 10pmol/ μ L as primer dosage
Programmed screening.
The first time optimum results of 3 real-time fluorescence quantitative RT-PCR primer dosage of table
Note：It is Ct values that value is indicated in table, and " 2.0,2.5,3.0,4.0 " is (a concentration of for primer in 50 μ L PCR systems
10pmol/ μ L) dosage (μ L).
2, the second suboptimization of fluorescence quantitative RT-PCR primer dosage
The influence for reagent sensitivity is combined for test different primers dosage, the lower RNA templates of concentration is selected to carry out
Test.Gradient dilution, which is carried out, using positive control RNA (positive control RNA in embodiment l) takes L3 (10-6)、L4(10-7), L5
(10-8) as second of primer amount optimization template, each group primer is further optimized on the basis of the first suboptimization.
PCR reaction systems are：Upstream and downstream primer amount is shown in Table 3,5 μ L, 2.5U/ μ L Taq archaeal dna polymerases of positive control RNA, 2.5 μ L,
5U/ μ L reverse transcriptase (Quant Reverse Transcriptase) 0.5 μ L, 2 × import real-time fluorescence PCR buffer solution, 25 μ L
(it is purchased from 5 μ L10 × buffer, 4 μ L10mM dNTP, the 0.005 μ L10000 × SYBR green I of ThermoFisher companies
And 16 μ L DEPC water), supplement sterile water to 50 μ L.PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec,
60 DEG C of 20sec, 72 DEG C of 20sec, 75 DEG C of 5sec, read plate are recycled for 45 totally.The results are shown in Table 4, this suboptimization upstream
For primer in 3 μ L, downstream primer testing result in 2 μ L is preferable, this volume is selected to draw for real-time fluorescence quantitative RT-PCR of the present invention
The use volume of object.
Second of optimum results of 4 real-time fluorescence quantitative RT-PCR primer dosage of table
Note：It is Ct values, " 2.5,3.0,4.0 " that value is indicated in table；" 2.0,2.5,3.0 " are primer in 50 μ L PCR systems
The dosage (μ L) of (a concentration of 10pmol/ μ L)
The above optimum results show basic group of grass carp hemorrhage virus real-time fluorescence quantitative RT-PCR detection reagent of the present invention
At substantially as shown in table 5 with each component content.
The basic composition and each component content of 5 grass carp hemorrhage virus real-time fluorescence quantitative RT-PCR detection reagent of table
3 grass carp hemorrhage virus VP5 gene fluorescence quantitative RT-PCRs of embodiment detect
1, the foundation of standard curve
Fluorescence quantitative RT-RCR detection is carried out using positive control RNA as template, establishes standard curve.Concrete operations are such as
Under：Positive control RNA progress is serially diluted into I for 10 times：1.0×105pg/μL；II：1.0×104pg/μL；III：1.0×
103pg/μL；Ⅳ：1.0×102pg/μL；V：10pg/μL.Standard items detect PCR reaction systems：Draw the upstream of 10pmol/ μ L
The 2.0 μ L of downstream primer of 3.0 μ L, 10pmol/ μ L of object, 5 μ L, 2.5U/ μ L Taq archaeal dna polymerases of positive control RNA, 2.5 μ L,
0.5 μ L of 5U/ μ L reverse transcriptase (Quant Reverse Transcriptase), 12 μ L DEPC water, 2 × import real-time fluorescence
25 μ L of PCR buffer solutions (purchased from 5 μ L10 × buffer of ThermoFisher companies, 4 μ L10mM dNTP, 0.005 μ L10000 ×
SYBR green I and 16 μ L DEPC water configure).PCR reaction conditions are：50℃30min；95℃3min；95℃
10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 75 DEG C of 5sec, read plate are recycled for 45 totally.Standard items real time fluorescent quantitative RT-
(Fig. 3, abscissa are Ct values to PCR amplification curve, and ordinate is fluorescent value, I as shown in Figure 3：1.0×105pg/μL；II：1.0×
104pg/μL；III：1.0×103pg/μL；Ⅳ：1.0×102pg/μL；V：10pg/μL.)
2, the drafting of standard curve
Standard curve, real-time fluorescence quantitative RT-PCR inspection are drawn according to the logarithm of the corresponding standard items of gained Ct values
It is 1 × 10 to survey the positive control RNA range of linearity5~10pg copies/μ L reaction systems, the coefficient R square value of standard curve
Amplification efficiency for 0.998 (y=-0.3075x+13.273), fluorescence quantitative RT-RCR reaction is 102.7%.Standard curve is aobvious
Show：The areas the grass carp hemorrhage virus VP5 gene real-time fluorescence quantitative RT-PCR detection method that the present invention establishes has the line of 5 orders of magnitude
Property detection range, further illustrate the detection method have very high sensitivity.
3, real-time fluorescence quantitative RT-PCR method detects 10 parts of fish samples
The RNA of 3.1 extraction fish sampling tissues
Acquire 4 parts of illness grass carp and 6 parts of healthy grass carp samples, use viral RNA extracts kit to extract hepatic tissue
Total serum IgE.It is detected with the Standard PCR detection architecture of foundation.Remaining template is preserved in -20 DEG C in case reinspection.
3.2 detect sick fish RNA samples with the method established in embodiment 1,2
Sick fish detects PCR reaction systems：The downstream primer 2.0 of 3.0 μ L, the 10pmol/ μ L of sense primer of 10pmol/ μ L
μ L, 5 μ L, 2.5U/ μ L Taq archaeal dna polymerases of sick fish hepatic tissue total serum IgE, 2.5 μ L, 5U/ μ L reverse transcriptase (Quant Reverse
Transcriptase) 0.5 μ L, 12 μ L DEPC water, 2 × import real-time fluorescence PCR buffer solution, 25 μ L (are purchased from ThermoFisher
5 μ L10 × buffer, 4 μ L10mM dNTP, 0.005 μ L10000 × SYBR green I and the 16 μ L DEPC water of company are matched
It sets).PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 75 DEG C
5sec, read plate are recycled for 45 totally.Sick fish RNA sample real-time fluorescence quantitative RT-PCRs amplification curve as shown in Figure 4 (Fig. 4,
Abscissa is Ct values, and ordinate is fluorescent value), as a result show：There is purpose when using 4 parts of illness grass carp total tissue RNAs template
Fluorescent amplification curve, without amplification curve when using remaining 6 parts of healthy grass carp sample RNA as template.
4 grass carp hemorrhage virus VP5 gene fluorescence quantitative RT-PCR detection kits of embodiment
The areas grass carp hemorrhage virus VP5 gene fluorescence quantitative RT-PCR detection kit includes the 5 pipes grass of respective independent packaging
250 μ L/ pipes of fish bleeding virus reaction solution 1.25mL/ pipes and 1 pipe Taq archaeal dna polymerases (2.5U/ μ L, each to react 2.5 μ L), 1
50 μ L/ pipes of pipe reverse transcriptase (Quant Reverse Transcriptase) (5U/ μ L, each to react 0.5 μ L), 7 Reagent Tubes
It is commonly assembled at again in an external packing box.Wherein, grass carp hemorrhage virus reaction solution contains dNTPs, MgC12、SYBR green I
And primer mixed liquor, by 2 × buffer of 25 μ L, (use is purchased from 10 × buffer5 μ L of ThermoFisher companies, 4 μ L's
10mM dNTP and 10000 × SYBR green I, 0.005 μ L and the mixtures of 16 μ L DEPC water be formulated), implement
2 determined combination of example, 3 μ L sense primers, 2 μ L downstream primers and 12 μ L's mixes in proportion without RNase water, and when preparation will be each
Component is multiplied by a coefficient (such as 10000, according to output fixed), after mixing, packing, and every 1.25mL totally 5.For convenience of inspection
It surveys, kit includes the positive control RNA (10 of in addition respective independent packaging-5The positive control RNA L2 of dilution), the critical positive
Compare RNA (10-8The positive control RNA L5 of dilution), negative controls (sterile water), and with grass carp hemorrhage virus reaction solution and
Taq polymerase Reagent Tube is commonly assembled in external packing box.PCR reaction systems：Grass carp hemorrhage virus reaction solution 42 μ L, it is to be detected
RNA (L2 or L5) 5 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 2.5 μ L, reverse transcriptase (Quant Reverse
Transcriptase) (5U/ μ L) 0.5 μ L compositions.PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60
DEG C 20sec, 72 DEG C of 20sec, 75 DEG C of 5sec, read plate totally 45 cycles.The result shows that (table 6), by 5 replications
Results contrast is stablized, and determines that L2 is the positive control of kit of the present invention, L5 is the critical positive control of kit of the present invention.
The test result of 6 positive control of table
| It repeats | 1 | 2 | 3 | 4 | 5 |
| L2 | 20.8 | 20.5 | 20.7 | 20.5 | 20.9 |
| L5 | 30.1 | 30.5 | 30.4 | 30.8 | 30.3 |
Embodiment 5：The Performance Evaluation of grass carp hemorrhage virus VP5 gene fluorescence quantitative RT-PCR detection kits
It is assessed for the performance to kit of the present invention, product sample is repeatedly made according to the technique after products perfection
To the sensitivity of kit, specificity, accuracy, stability and with trial production product carries out clinical test, with examine or check produce
The performance of product.
1, the range of linearity of kit detection and sensitivity test
Grass carp hemorrhage virus RNA positive controls are subjected to 10 times of serial dilutions to 10 with DEPC water diluents-9, i.e., clever
Sensitivity quality-control product.Take L1 (10-4Dilution), L2 (10-5Dilution), L3 (10-6Dilution), L4 (10-7Dilution), L5 (10-8
Dilution), L6 (10-9Dilution) quality-control product as kits for evaluation sensitivity, number L1-L6, in -80 DEG C of preservations after packing
It is spare.It is detected using kit of the present invention.PCR reaction systems：42 μ L of grass carp hemorrhage virus reaction solution, positive control RNA
5 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 2.5 μ L, reverse transcriptase (Quant Reverse Transcriptase) (5U/ μ L)
0.5 μ L compositions.PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 75 DEG C
5sec, read plate are recycled for 45 totally.As a result (table 7) is shown, 10-8Kit is in most cases when dilution (L5)
Detection.To the product samples of different batches into line sensitivity and linear analysis, as a result shows that primer can be stablized and measure 10-8Dilution
The positive control RNA sample of degree, for 10-9Dilution sample, kit of the present invention cannot detect, so kit of the present invention is most
It is low to detect containing 10-8The positive control RNA of dilution has higher sensitivity.Therefore set 10-8Dilution is minimum detection value.
The test result of the 7 kit range of linearity of the present invention of table
| Dilution gradient | 10-4 | 10-5 | 10-6 | 10-7 | 10-8 | 10-9 |
| Ct values | 17.4 | 20.3 | 23.7 | 26.9 | 30.3 | No Ct |
2, the specificity analysis of kit detection
Using the non-target fish Causative virus of four kinds of product sample pair (rainbow Medaka fish arc reovirus virus N1, the spring of different batches
Darter reovirus N2, Micropterus salmonoides reovirus N3, dog salmon reovirus N4) it is detected.PCR reactants
System：Grass carp hemorrhage virus reaction solution 42 μ L, sample RNA 5 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 2.5 μ L, reverse transcriptase
(Quant Reverse Transcriptase) (5U/ μ L) 0.5 μ L compositions.PCR reaction conditions are：50℃30min；95℃
3min；95 DEG C of 10sec, 60 DEG C of 20sec, 72 DEG C of 20sec, 75 DEG C of 5sec, read plate are recycled for 45 totally.As a result (table 8) N1-
Ct values are not detected in N4, it was demonstrated that kit of the invention has good specificity.
The test result of 8 kit of the present invention specificity of table
| Sample | N1 | N2 | N3 | N4 |
| Ct values | No Ct | No Ct | No Ct | No Ct |
3, the accuracy detected in kit batch
(positive control RNA is diluted to 1.0 × 10 using accuracy quality-control product-5As accuracy quality-control product) for detecting
The accuracy of kit.It carries out 10 repetitions respectively to reaction system to detect, PCR reaction systems：Grass carp hemorrhage virus reaction solution
42 μ L, positive control RNA 5 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 2.5 μ L, reverse transcriptase (Quant Reverse
Transcriptase) (5U/ μ L) 0.5 μ L compositions.PCR reaction conditions are：50℃30min；95℃3min；95 DEG C of 10sec, 60
DEG C 20sec, 72 DEG C of 20sec, 75 DEG C of 5sec, read plate totally 45 cycles.The CV% of 10 precision quality-control product Ct values<
8% (table 9), it was demonstrated that kit of the invention has good accuracy.
The precision test result of 9 kit of the present invention of table
| Sample | 1 | 2 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | CV% |
| Ct values | 19.8 | 20.7 | 21.1 | 20.4 | 20.5 | 21.4 | 20.7 | 20.8 | 19.5 | 2.8 |
4, the accuracy of kit measures
Using sequencing approach confirm the accuracy of kit of the present invention.Amplified production is sequenced, sequence with it is pre-
Phase result is completely the same, it was demonstrated that the testing result of kit of the present invention is accurate.
5, the Stability Determination of kit
The stability of 5.1 kits
The stability of product depends on the stability of each composition.The reaction of grass carp hemorrhage virus is prepared in the present invention
Liquid, Taq archaeal dna polymerases, reverse transcriptase, positive control are preserved at -20 DEG C, are preserved always after taking-up to 4 DEG C of refrigerators, longest is protected
It deposits and has not yet to see within one week performance decline.
In the Product transport prepared for clinical test, a full set of product (including grass carp hemorrhage virus reaction solution, Taq DNA
Polymerase, reverse transcriptase, positive control), formerly after through go through -20 DEG C of 3 days by a definite date freezings, 4 DEG C of long-distance transports, -20 DEG C of freezings,
It is multiple a series of two-way process such as melt after, detected using quality-control product, testing result has no that there were significant differences.Show kit of the present invention
Each component quite stable.
The stability of 5.2 reference substances
The stability of reference substance has a significant impact to the analytical judgment of test result, and the reference substance of this kit is mainly pair
Reaction system carries out quality control.This kit has used a positive control, a critical positive and a negative control, right
It carries out freeze thawing detection.Experimental result is as shown in table 10, the results showed that the reference substance in kit of the present invention also has good
Stability.
The freezing-thawing test result of 10 positive reference substance of table
| Number of freezing and thawing | The CT values of positive control | The CT values of critical positive control | The CT values of negative control |
| 1 | 19.52 | 29.8 | No Ct |
| 2 | 19.54 | 30.7 | No Ct |
| 3 | 20.14 | 30.16 | No Ct |
| 4 | 20.42 | 30.4 | No Ct |
| 5 | 20.96 | 30.5 | No Ct |
| 6 | 21.15 | 31.4 | No Ct |
| 7 | 20.53 | 30.7 | No Ct |
| 8 | 20.75 | 30.8 | No Ct |
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should manage
Solution, technical scheme of the present invention can be modified or replaced equivalently, without departing from technical solution of the present invention essence and
Range.
SEQUENCE LISTING
<110>Transduction progresses greatly（Wuhan）Bioisystech Co., Ltd
<120>A kind of detection kit and detection method of grass carp hemorrhage virus
<130> 20170113001
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>Sense primer VP5-I-F
<400> 1
cgactcactt gccatcgtta 20
<210> 2
<211> 20
<212> DNA
<213>Downstream primer VP5-I-R
<400> 2
cgcttggaat aaaggttgcc 20
<210> 3
<211> 22
<212> DNA
<213>Sense primer VP5-II-F
<400> 3
ctccgtgttg accctggatg tg 22
<210> 4
<211> 26
<212> DNA
<213>Downstream primer VP5-II-R
<400> 4
atgttagcag cggtagtgac ttgttg 26
<210> 5
<211> 20
<212> DNA
<213>Sense primer VP5-III-F
<400> 5
ggtatgcttt ccgccctaac 20
<210> 6
<211> 18
<212> DNA
<213>Downstream primer VP5-III-R
<400> 6
ctctctgtgc tcttgccc 18
Claims (10)
1. a kind of primer pair for the amplification of grass carp hemorrhage virus VP5 gene orders, which is characterized in that the primer pair includes upper
Swim the nucleotide sequence such as SEQ ID NO of II-F and downstream primer VP5- II-R, the II-F of sense primer VP5- of primer VP5-：
Shown in 3, the nucleotide sequence such as SEQ ID NO of the II-R of downstream primer VP5-：Shown in 4.
2. a kind of amplification method of grass carp hemorrhage virus VP5 gene orders, which is characterized in that include the following steps：
1）Bioinformatics method design primer group and progress primer screening obtain primer pair as described in claim 1；
2）The fluorescence quantitative PCR detection of grass carp hemorrhage virus VP5 genes：Hemorrhagic disease of grass carp is extracted with viral RNA extracts kit
Virus gene group RNA；
3）With step 2）Grass carp hemorrhage viral gene group RNA samples be template, by primer pair described in claim 1 carry out one
Footwork fluorescence quantitative RT-RCR obtains pcr amplification product；
4）Step 3）Obtained pcr amplification product, which is analyzed and is sequenced, obtains grass carp hemorrhage virus VP5 gene orders.
3. a kind of amplification method of grass carp hemorrhage virus VP5 gene orders according to claim 2, which is characterized in that institute
State step 1）It is that the detection of primer PCR amplification efficiency is carried out to which screening obtains described in claim 1 by fluorescence quantitative RT-RCR
Primer pair.
4. a kind of detection kit of grass carp hemorrhage virus, which is characterized in that the detection kit packet of the grass carp hemorrhage virus
Include primer pair described in claim 1.
5. the detection kit of grass carp hemorrhage virus according to claim 4, which is characterized in that the sense primer VP5-
The molar concentration rate of II-R of II-F and downstream primer VP5- is 1.5：1.
6. the detection kit of grass carp hemorrhage virus according to claim 4, which is characterized in that the detection kit
Including：
1）Grass carp hemorrhage virus reaction solution, Taq archaeal dna polymerases and reverse transcriptase：Grass carp hemorrhage virus reaction solution includes containing
dNTPs、10×buffer、MgC12, SYBR green I, DEPC water and primer pair described in claim 1 mixed liquor；
2）Positive control：
3）Critical positive control；
4）Negative control object.
7. the detection kit of grass carp hemorrhage virus according to claim 4, which is characterized in that the positive control is
Total serum IgE containing the extraction of high-purity grass carp hemorrhage virus.
8. the detection kit of claim 4 ~ 7 any one of them grass carp hemorrhage virus is in the context of detection of fish bacterial pathogens
Using.
9. the detection method of claim 4 ~ 7 any one of them grass carp hemorrhage virus, which is characterized in that the detection method is
RNA reverse transcriptions and quantitative fluorescent PCR reaction are carried out continuously in the same reaction tube.
10. detection method according to claim 9, which is characterized in that the detection method specifically includes following steps：
1）The RNA of extraction fish sample tissue obtains sample RNA；
2）Establish one-step method fluorescence quantitative RT-RCR reaction system；
3）Determine whether to infect grass carp hemorrhage virus by comparing standard items real-time fluorescence quantitative RT-PCR amplification curve.
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| CN112048575A (en) * | 2020-10-26 | 2020-12-08 | 深圳市莱孚生物科技有限公司 | Primer, probe and detection method for identifying grass carp hemorrhagic fever virus |
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