CN110218814A - Detect the kit and its method of inspection of huichun viremia virus - Google Patents
Detect the kit and its method of inspection of huichun viremia virus Download PDFInfo
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Abstract
The present invention provides a kind of kit for detecting huichun viremia virus, is suitable for polymerase chain reaction, and kit includes primer pair, and primer pair includes reverse primer shown in the forward primer as shown in sequence identification number 1 and sequence identification number 2.The present invention separately provides a kind of method of inspection using mentioned reagent box detection huichun viremia virus.Thus, it is possible to quickly and specifically detect huichun viremia virus from sample to be tested.
Description
Technical field
The present invention is measurement and the method for inspection in relation to a kind of nucleic acid, in particular to a kind of anti-using polymerase chain
The kit and its method of inspection of huichun viremia virus should be detected.
Background technique
Spring viremia (Spring viraemia of carp, SVC) is also known as carp infectiousness ascites symptoms, is by carp
Acute, hemorrhagic caused by spring viremia virusemia viral (Spring viremia of carp virus, SVCV) and with height
The epidemic disease of infection can seriously endanger fish production and can cause crushing blow.
Huichun viremia virus is classified in rhabdoviridae (Rhabdoviridae), blister Tobamovirus
(Vesiculovirus).It is made of single-stranded, negative stock RNA, genome includes 11019 nucleotide, can translate out 5 structure eggs
White matter, respectively nucleoprotein (nucleoprotein, N), phosphoprotein (phosphoprotein, P), stromatin (matrix
Protein, M), glycoprotein (glycoprotein, G) and ribonucleic acid associated polymerase (RNA-dependent RNA
polymerase,L).There is one layer of cyst membrane, viral size is about 180 × 70nm, and the buoyant density in CsCl is 1.195-
1.200g/mL.Huichun viremia virus can be included in vesicular stomatitis class according to its structural proteins component, and research card at present
Bright huichun viremia virus only has One serotype.
Huichun viremia virus usually infects cyprinid fish, in the fishes such as carp, fancy carp, grass carp, silver carp, flathead, black crucian carp and crucian carp
Apparent symptom can occur, but carp is wherein most sensitive host.Spring viremia is with systemic bleeding and ascites, morbidity is anxious,
Death rate height is characterized, and usually breaks out and cause juvenile fish and adult fish dead in spring.In acute infection, the liver for fish body of catching an illness,
Kidney, spleen, the gill and brain are that there are organs for high-tensile strength valence virus.Spring viremia is popular in Europe, the Middle East and Russia earliest, closely
America and Asia are traveled to over year.World Organization for Animal Health (OIE) is classified as the important epidemic disease that must be reported.Also for 2008 I
A kind of animal epidemic is classified as in the animal epidemic register that state newly promulgates.Therefore quickly, the conveniently and accurately detection carp spring is developed
The kit and its method of inspection of viremia virusemia virus, to facilitate scene or the laboratory testing of spring viremia most to close
Key.
Summary of the invention
In view of this, a purpose of the invention be provide it is a kind of detect huichun viremia virus kit and its inspection
Survey method, can quickly and huichun viremia virus is specifically detected from sample to be tested.
A mode of the invention is to provide a kind of kit for detecting huichun viremia virus, includes a primer pair,
The primer pair includes reverse primer shown in forward primer shown in sequence identification number 1 and sequence identification number 2.
According to the kit of detection huichun viremia virus above-mentioned, can further include just like shown in sequence identification number 3
Fluorescence probe.
According to the kit of detection huichun viremia virus above-mentioned, a reverse transcription polymerase chain reaction can be further included
Required reagent.
Another way of the invention is to provide a kind of method of inspection for detecting huichun viremia virus, includes following step
Suddenly.A sample to be tested is provided as a template.The template is subjected to a reverse transcription polymerase chain reaction, to obtain a cDNA
Template.CDNA template is subjected to polymerase chain reaction with the kit of detection huichun viremia virus described in leading portion, with
A polymerase chain reaction product is obtained, and the kit of the detection huichun viremia virus includes a primer pair, it is described
Primer pair includes reverse primer shown in forward primer shown in sequence identification number 1 and sequence identification number 2.Finally detect
Whether the molecular size range of the polymerase chain reaction product is 98bp.
According to the method for inspection of detection huichun viremia virus above-mentioned, wherein the polymerase chain reaction product can
With the sequence as shown in sequence identification number 4.
According to the method for inspection of detection huichun viremia virus above-mentioned, produced wherein detecting the polymerase chain reaction
Object can be carried out with colloid electrophoresis system.
Another mode of the invention is to provide a kind of method of inspection for detecting huichun viremia virus, includes following step
Suddenly.A sample to be tested is provided as a template.The template is subjected to a reverse transcription polymerase chain reaction, to obtain a cDNA
Template.CDNA template is subjected to a fluorescence probe formula polymerase with the kit of detection huichun viremia virus described in leading portion
Chain reaction, and the kit of the detection huichun viremia virus includes a primer pair and a fluorescence probe, the primer
To including that forward primer shown in sequence identification number 1 and sequence recognize reverse primer shown in number 2, the fluorescence probe has
Just like sequence shown in sequence identification number 3.Finally detect whether that there is a fluorescence signal value.
According to the method for inspection of detection huichun viremia virus above-mentioned, wherein the fluorescence probe formula polymerase chain
Reaction can be the chain reaction of real time aggregation enzyme (Real-time PCR) or isolation thermostatic type polymerase chain reaction (insulated
isothermal PCR,iiPCR)。
Still another way of the invention is to provide a kind of method of inspection for detecting huichun viremia virus, includes following step
Suddenly.A sample to be tested is provided as a template.By the template with the reagent of detection huichun viremia virus described in leading portion
Box sequentially carries out a reverse transcription polymerase chain reaction and a DNA amplification reaction.The examination of the detection huichun viremia virus
Agent box is premixed form, and it includes reagents needed for a primer pair, a reverse transcription polymerase chain reaction and/or a fluorescence to visit
Needle, the primer pair include reverse primer shown in forward primer shown in sequence identification number 1 and sequence identification number 2, institute
Stating fluorescence probe has the sequence as shown in sequence identification number 3.And the DNA amplification reaction be a polymerase chain reaction or
One fluorescence probe formula polymerase chain reaction.Whether finally detect has an expected polymerase chain reaction product.DNA cloning is anti-
When should be polymerase chain reaction, when the molecular size range of a polymerase chain reaction product is 98bp, for expected poly-
The characterization of synthase chain reaction product.When DNA amplification reaction is fluorescence probe formula polymerase chain reaction, a fluorescence signal value is
Characterization with expected polymerase chain reaction product.
According to the method for inspection of detection huichun viremia virus above-mentioned, wherein the fluorescence probe formula polymerase chain
Reaction can be the chain reaction of real time aggregation enzyme or isolation thermostatic type polymerase chain reaction.
The kit and its method of inspection of detection huichun viremia virus of the invention as a result, can quick, special, side
Just it and accurately detects and whether there is huichun viremia virus in sample to be tested, display sample to be tested has infected spring viremia of carp virus blood
Syndrome virus.The testing result of the kit of detection huichun viremia virus of the invention and its method of inspection is in addition to can be via one
As routinely run glue confirmation result outside, can also carry out fluorescence polymerase chain reaction and isolation thermostatic type polymerase chain reaction confirmation
As a result, the testing result for wherein especially completely cutting off thermostatic type polymerase chain reaction is not required to complicated data interpretation, even if not received
The personnel of professional training can also be carried out the detection of huichun viremia virus using kit of the invention and the method for inspection, be taken
The detection that can be used for aquaculture fishery between field with portable fluorescence nucleic acids instrument, can fast and accurately diagnose cause of disease
Infection is for prevention and treatment ahead of time.
Foregoing invention content is intended to provide simplifying for this disclosure and makes a summary, so that reader has base to this disclosure
This understanding.The invention content is not the complete overview of this disclosure, and it is not intended to pointing out the embodiment of the present invention
Key/critical component defines the scope of the present invention.
Detailed description of the invention
For above and other purpose, feature, advantage and embodiment of the invention can be clearer and more comprehensible, institute's accompanying drawings are said
It is bright as follows:
Fig. 1 shows the structural representations of the kit of the detection huichun viremia virus according to one embodiment of the present invention
Figure.
Fig. 2 indicates the step process of the method for inspection of the detection huichun viremia virus of another embodiment of the present invention
Figure.
Fig. 3 indicates the step process of the method for inspection of the detection huichun viremia virus of another embodiment of the invention
Figure.
Fig. 4 indicates the step process of the method for inspection of the detection huichun viremia virus of another embodiment of the invention
Figure.
Fig. 5 is that the detection specificity of the kit of detection huichun viremia virus of the invention analyzes result figure.
Fig. 6 is that the detection sensitivity of the kit of detection huichun viremia virus of the invention analyzes result figure.
Fig. 7 is that the kit of detection huichun viremia virus of the invention carries out isolation thermostatic type polymerase chain reaction
Gel electrophoresis figure.
Wherein, reference numeral is described as follows:
100: detecting the kit of huichun viremia virus；
210: box body；
220: box cover；
300: liner；
310: premix buffer solution module；
320: positive control module；
330: nucleic acid buffer solution module；
400: connecing sample ring assemblies；
410: connecing sample ring；
500: premix reagent component；
600: detecting the method for inspection of huichun viremia virus；
610,620,630,640: step；
700: detecting the method for inspection of huichun viremia virus；
710,720,730,740: step；
800: detecting the method for inspection of huichun viremia virus；
810,820,830: step.
Specific embodiment
This specification disclosure provides a kind of kit for quickly detecting huichun viremia virus and its inspection party
Method.It designs specific primer pair with the height reserved area of huichun viremia virus, with the kit comprising primer pair with to
Test sample this progress polymerase chain reaction is that there are carp spring diseases in sample to be tested when there is expected polymerase chain reaction product
The characterization of toxaemia virus, display sample to be tested have infected huichun viremia virus.And it can further design and carp spring disease
Toxaemia virus has the fluorescence probe of specificity, with the kit comprising primer pair and fluorescence probe, carries out with sample to be tested
Fluorescence probe formula polymerase chain reaction is that there are the tables of huichun viremia virus in sample to be tested when there is fluorescent signal
Sign, display sample to be tested have infected huichun viremia virus.
Aforementioned so-called " fluorescence probe " of the invention refers to that one includes the molecule of continuous at least eight nucleotide, to choose PCR
One section of sequence between upstream and downstream primer marks upper fluorophor (reporter), 3`- as probe, and at the end 5`- of probe
End marks upper corresponding Control of Fluorescence stain (quencher), since two groups are in close proximity, constitutes fluorescent energy transmitting
Relationship is generated without fluorescence signal, and during annealing (annealing) of each circulation, which can be with template phase
In conjunction in subsequent extension, when primer is blended into probe and when template junction, the 5`- 5 prime excision enzyme activity of Taq enzyme can be with
The end 5`- of degradation probe, and separate fluorophor with the quenching group on Control of Fluorescence stain, allow fluorophor to discharge
Fluorescence out.Such as FAM (520nm) or JOE, VIC (550nm) etc. can be used in the end the 5' fluorophor of fluorescence probe, and fluorescence is visited
The end the 3' Control of Fluorescence stain of needle can be used black hole quencher (black hole quencher) (BHQ1) or non-fluorescence be quenched
Agent (non fluorescent quencher) (NFQ) etc., so its is merely illustrative not as the limitation of the invention.
The present invention it is aforementioned it is so-called " isolation thermostatic type polymerase chain reaction (insulated isothermal PCR,
IiPCR) " it is a new detection technique of fluorescence, the principle of thermal convection is controlled and utilized with single point temperature, it is anti-completes polymerase chain
The reaction (denaturing) of dissociation DNA double stock structure, primer annealing it should react (annealing) in the process and extend reaction
(extension) circulation of three steps such as.Because only needing the single temperature control in bottom, and replaced mechanically with natural temperature gradient
Heating and cooling, machine more lightweight can be made, avoid machine because of mechanical breakdown caused by heating and cooling repeatedly, and saved needed for heating and cooling
Time.Because testing result is not required to data interpretation, even if not received the personnel of professional training, detection carp of the invention can be also utilized
The kit and detection method of spring viremia virusemia virus carry out the detection of huichun viremia virus.
Following experiments example illustrates the present invention for demonstrating, to be conducive to the usual skill of the technical field of the invention,
The present invention can be completely utilized and practice in the case of being not required to and excessively interpreting, without these experimental examples should be considered as to model of the present invention
The limitation enclosed, but for how to implement material and method of the invention.
[kit of detection huichun viremia virus]
The kit of detection huichun viremia virus of the invention, includes a primer pair, and the primer pair includes a sequence
Reverse primer shown in forward primer shown in column identification number 1 and sequence identification number 2.Preferably, inspection of the invention
The kit for surveying huichun viremia virus can further include the fluorescence probe as shown in sequence identification number 3.More preferably, of the invention
Detection huichun viremia virus kit can further include a reverse transcription polymerase chain reaction needed for reagent, to will
Template in sample to be tested is cDNA template via reverse transcription polymerase chain reaction reverse transcription, so that the detection carp spring of the invention
The kit of viremia virusemia virus be premixed form, can sequentially be carried out in same developmental tube reverse transcription polymerase chain reaction and
DNA amplification reaction.
Fig. 1 is please referred to, indicates the kit 100 of the detection huichun viremia virus according to one embodiment of the present invention
Structural schematic diagram.The kit 100 of detection huichun viremia virus includes box body 210, box cover 220, liner 300, connects sample
Ring assemblies 400 and premix reagent component 500.
Box cover 220 closes box body 210 for covering, and the inner surface of box cover 220 is provided with sponge buffer.Liner 300 connects sample ring
Component 400 and premix reagent component 500 are sequentially arranged in box body 210.
Liner 300 includes premix buffer solution module 310, positive control module 320 and nucleic acid buffer solution module 330.
It premixes buffer solution module 310 and is equipped with corresponding hole location, to place the first buffer solution of dissolution mix primer.Positive control
Module 320 is to place positive nucleic acid dry powder.Nucleic acid buffer solution module 330 dissolves the second of positive nucleic acid dry powder to place
Buffer solution.
Connecing sample ring assemblies 400 includes that at least one bag of independent packaging connects sample ring packet, every bag connect be placed in sample ring packet it is several
It is a to connect sample ring 410.
The premix reagent packet that reagent component 500 includes at least one bag independent packaging is premixed, if having in every bag of premix reagent packet
Main pipe premixes Reagent Tube, places freeze-dried powder in premix Reagent Tube, and freeze-dried powder includes dNTPs, primer pair, fluorescence probe, anti-
Transcriptase and archaeal dna polymerase.Wherein primer pair includes that forward primer shown in sequence identification number 1 and sequence recognize number 2
Shown in reverse primer.The sequence of fluorescence probe is as shown in sequence identification number 3, in this embodiment, 3 ' end tools of fluorescence probe
There is NFQ Control of Fluorescence stain, 5 ' ends have the modification of FAM fluorophor.
Detecting premix Reagent Tube in the kit 100 of huichun viremia virus is premixed form mixed in advance, is not added
Add ROX reference dye, when use need to only take out the premix Reagent Tube of corresponding amount according to number of awaiting test sample, will premix the jelly of Reagent Tube
The first buffer solution mixing in dry particl, premix buffer solution module 310, and be transferred to sample to be tested with connecing sample ring 410
It premixes in Reagent Tube, can start to react.Such as need to be added fluorescent dye correction Real-time PCR instrument, it can be according to each reality
ROX reference dye is added in the standard test procedure for testing room.The kit 100 for detecting huichun viremia virus is simple, square
Portable belt, can room temperature carrying and preservation.Also, if carrying out Aluminium Foil Package to the kit 100 of detection huichun viremia virus
Dress sealing, it is constant that freezen protective in -20 DEG C can then store 1 year or more efficiency.
[method of inspection of detection huichun viremia virus]
Referring to figure 2., the method for inspection 600 of the detection huichun viremia virus of another embodiment of the present invention is indicated
Step flow chart.The method of inspection 600 for detecting huichun viremia virus includes step 610, step 620, step 630 and step
Rapid 640.
Step 610 is to provide a sample to be tested as a template.The sample to be tested can be fish gill sample, the spleen sample of fish
Sheet and digestive organ's sample.And it can be further using in business extraction agent box or other prior art methods extraction sample to be tested
Nucleic acid, step and principle are had usually intellectual and are known in related fields, thus are no longer described in detail herein.
Step 620 is that the template is carried out reverse transcription polymerase chain reaction, to obtain a cDNA template.
Step 630 is by the cDNA template to recognize forward primer shown in number 1 and sequence identification comprising sequence
The primer pair of reverse primer shown in number 2 carries out polymerase chain reaction, to obtain a polymerase chain reaction product.
Step 640 is to detect whether the molecular size range of the polymerase chain reaction product is 98bp.If polymerase connects
The molecular size range of lock reactor product is 98bp, then it represents that there are huichun viremia virus in sample to be tested, show to test sample
This has infected spring viremia.Preferably, polymerase chain reaction product has the sequence as shown in sequence identification number 4.
And polymerase chain reaction product can be detected with colloid electrophoresis system.
Referring to figure 3., the method for inspection 700 of the detection huichun viremia virus of another embodiment of the present invention is indicated
Step flow chart.The method of inspection 700 for detecting huichun viremia virus includes step 710, step 720, step 730 and step
Rapid 740.
Step 710 is to provide a sample to be tested as a template.The sample to be tested can be fish gill sample, the spleen sample of fish
Sheet and digestive organ's sample, and can be further using in business extraction agent box or other prior art methods extraction sample to be tested
Nucleic acid.
Step 720 is that the template is carried out reverse transcription polymerase chain reaction, to obtain a cDNA template.
Step 730 for by the cDNA template with sequence recognize number 1 shown in forward primer, sequence identification 2 institute of number
Fluorescence probe shown in reverse primer and sequence the identification number 3 shown carries out a fluorescence probe formula polymerase chain reaction.Compared with
Goodly, fluorescence probe formula polymerase chain reaction can be the chain reaction of real time aggregation enzyme (Real-time PCR) or isolation constant temperature
Formula polymerase chain reaction (insulated isothermal PCR, iiPCR).
Step 740 is to detect whether there is a fluorescence signal value, if having fluorescence signal value, then it represents that deposit in sample to be tested
In huichun viremia virus, show that sample to be tested has infected spring viremia.
Referring to figure 4., the method for inspection 800 of the detection huichun viremia virus of another embodiment of the invention is indicated
Step flow chart.The method of inspection 800 for detecting huichun viremia virus includes step 810, step 820 and step 830.
Step 810 is to provide a sample to be tested as a template.The sample to be tested can be fish gill sample, the spleen sample of fish
Sheet and digestive organ's sample, and can be further using business extraction agent box or the extraction of other prior art methods in test sample sheet
Nucleic acid.
Step 820 be by the template in same developmental tube with the reagent of detection huichun viremia virus of the invention
Box sequentially carries out a reverse transcription polymerase chain reaction and a DNA amplification reaction.The examination of the detection huichun viremia virus
Agent box includes reagent and/or a fluorescence probe needed for a primer pair, a reverse transcription polymerase chain reaction, the primer pair packet
Containing reverse primer shown in forward primer shown in sequence identification number 1 and sequence identification number 2, the fluorescence probe has such as
Sequence recognizes sequence shown in number 3.And the DNA amplification reaction is that a polymerase chain reaction or a fluorescence probe formula polymerize
Enzyme chain reaction.And fluorescence probe formula polymerase chain reaction can be the chain reaction of real time aggregation enzyme or isolation thermostatic type polymerase
Chain reaction.
Step 830 is whether detecting has an expected polymerase chain reaction product.DNA amplification reaction is polymerase chain
When reaction, when the molecular size range of a polymerase chain reaction product is 98bp, to be produced with expected polymerase chain reaction
The characterization of object.When DNA amplification reaction is fluorescence probe formula polymerase chain reaction, a fluorescence signal value is with expected polymerase
The characterization of chain reaction product.
[test example]
1. real time aggregation enzyme chain reaction
Fig. 1 and Fig. 4 are please referred to, in detail, if carrying out real time aggregation to detect the kit 100 of huichun viremia virus
Enzyme chain reaction is for detecting huichun viremia virus.
Positive controls nucleic acid dry powder is first dissolved with the second buffer solution of 100 μ L, to obtain concentration as 4 × 10-5ng/μL
Positive control, and by the positive control after back dissolving be stored in 4 DEG C it is spare.Premix reagent packet is opened again, and according to sample to be tested
Number takes out the premix Reagent Tube of corresponding amount.
And premix freeze-drying particle back dissolving mode such as the following steps in Reagent Tube.If being not required to addition ROX reference dye, pipe is opened
Particle is lyophilized in lid, the first buffer solution back dissolving that 50 μ L are added in each pipe premix Reagent Tube, i.e., acquisition real time aggregation enzyme is chain anti-
Answer mixed liquor.In real time aggregation enzyme chain reaction mixed liquor, the concentration of dNTPs is 0.5mM, sequence recognizes shown in number 1 just
Concentration to reverse primer shown in primer acid and sequence identification number 2 is respectively 0.5 μM, the concentration of fluorescence probe is 0.05 μ
M, the concentration of reverse transcriptase is 16U and the concentration of archaeal dna polymerase is 13U.If ROX reference dye correction Real- need to be added
The ROX reference dye back dissolving freeze-drying of the first buffer solution and 1 μ L of 49 μ L is added in time PCR instrument, each pipe premix Reagent Tube
Particle.
In the dedicated reaction tube of fluorescent PCR for taking 23 μ L to be dispensed into 0.2mL the good every pipe of freeze-drying particle solution of back dissolving.Take 2 μ
The dedicated reaction tube of fluorescent PCR containing 23 μ L freeze-drying particle solution is added in sample to be tested, positive control or the negative control of L.Its
In, negative control does not contain the nucleic acid of target gene.
It is carried out amplification reaction again according to reaction condition.Probe in detecting mode setting are as follows: Reporter Dye-FAM,
Quencher Dye-MGB or NONE, depending on type.Reaction condition is set as 42 DEG C and reacts 30 minutes, follows subsequently into 40 times
Ring, each cycling condition are reacted 15 seconds, 60 DEG C for 93 DEG C and are reacted 60 seconds.
Detection data file, and further progress result judgement are saved after reaction.Analysis condition is set as first setting
The amount of positive criteria product in standard curve.The function that automatically analyzes of program is clicked again, obtains analysis result.Wherein positive criteria product
Value < 32 Ct, i.e. expression whole group reagent efficiency and reaction is normal.If thering is obvious logarithm to increase in FAM channel amplification curve, for
Positive findings indicate to show that sample to be tested has infected spring viremia disease there are huichun viremia virus in sample to be tested
Poison.If increasing in FAM channel amplification curve without logarithm, for negative findings, indicate that spring viremia of carp virus blood is not present in sample to be tested
Syndrome virus, or there is the virus quantity for being lower than detection sensitivity.
Firstly, this test example examines the kit and its method of inspection of detection huichun viremia virus of the invention
Specificity analysis is surveyed, used sample to be tested includes huichun viremia virus (SVCV), Viral Hemorrhagic septicaemia disease
Poison (VHSV), infectious hematopoietic necrosis's virus (IHNV), infectivity pancreatic necrosis virus (IPNV), salmon infectious anemia
The nucleic acid of viral (ISAV), salmon Alphavirus (SAV).
Referring to figure 5., result is analyzed for the detection specificity of the kit of detection huichun viremia virus of the invention
Figure.The results show that the kit and its method of inspection of detection huichun viremia virus of the invention are only to spring viremia
Virus has specific amplification, and it is other then have no amplification curve for prelibation strain, show detection spring viremia disease of the invention
The kit and its method of inspection of poison have good specificity.
This test example further the kit to detection huichun viremia virus of the invention and its method of inspection into
The analysis of row detection sensitivity.The plastid for the target gene that one includes huichun viremia virus is constructed, then first with this plastid benefit
Reverse transcription RNA in test tube is used to utilize ultraviolet spectrometry luminance meter measurement reverse transcription RNA template later as the template of positive control group
Concentration and purity, according to the concentration calculation copy number of measurement.It is dilute that reverse transcription RNA template is carried out by 10 times of sequences with DEPC water again
It releases, is diluted to 3.2 × 107Duplicate is to 3.2 × 100 duplicates, as standard items with measurement sensitivity, and after foregoing sequences are diluted
Standard items detected with the kit and its method of inspection of detection huichun viremia virus of the invention, detailed detection
Step is as previously mentioned, details are not described herein.
Fig. 6 is please referred to, is that the detection sensitivity of the kit of detection huichun viremia virus of the invention analyzes result
Figure, wherein curve from left to right is sequentially 3.2 × 107Duplicate, 3.2 × 106Duplicate, 3.2 × 105Duplicate, 3.2 × 104It is multiple
Originally, 3.2 × 103Duplicate, 3.2 × 102Duplicate, 3.2 × 101The detection curve of duplicate, and 3.2 × 100Duplicate does not expand song
Line.The results show that the inspection of the kit and its method of inspection of detection huichun viremia virus of the invention to carp edema virus
High sensitivity is surveyed up to 32 duplicates.
2. completely cutting off thermostatic type polymerase chain reaction
If carrying out isolation thermostatic type polymerase chain reaction to detect the kit 100 of huichun viremia virus to detect
For huichun viremia virus, positive controls nucleic acid dry powder is first dissolved with the second buffer solution of 100 μ L, to obtain concentration
It is 4 × 10-5The positive control of ng/ μ L, and by the positive control after back dissolving be stored in 4 DEG C it is spare.Premix reagent packet is opened again, and
The premix Reagent Tube of corresponding amount is taken out according to number of awaiting test sample, and premixes the freeze-drying for example following step of particle back dissolving mode in Reagent Tube
Suddenly.Pipe lid is opened, the first buffer solution back dissolving that 50 μ L are added in each pipe premix Reagent Tube is lyophilized particle, that is, obtains isolation constant temperature
Solution is mixed and is moved back into isolation thermostatic type polymerase chain reaction pipe, to completely cut off perseverance by formula polymerase chain reaction mixed liquor
Warm formula polymerase chain reaction device carries out isothermal duplication.Reaction condition is 42 DEG C of reverse transcription reactions 10 minutes, bottom-heated 95
DEG C 30 minutes i.e. available reaction result.
In order to understand the kit of detection huichun viremia virus of the invention, to be thermally shielded equality of temperature polymerase chain anti-
The stability for answering the polymerase chain reaction product of amplification 98bp, reverse transcription RNA in test tube, which is sequentially diluted to number of copies, is
100 be used as positive controls, then with to detection huichun viremia virus of the invention kit and its method of inspection into
Row tests and analyzes, and isolation thermostatic type polymerase chain reaction product is separately separated with colloid electrophoresis, to ensure accuracy of the invention.
Fig. 7 and table one are please referred to, Fig. 7 is that the kit of detection huichun viremia virus of the invention carries out isolation constant temperature
The gel electrophoresis figure of formula polymerase chain reaction, wherein M is the molecular weight marker of nucleic acid, and N is without containing target gene core
The negative control group of acid.Table one is that the kit of detection huichun viremia virus of the invention carries out isolation thermostatic type polymerase
The fluorescent signal of chain reaction analyzes result.
Table one, the fluorometric result for completely cutting off thermostatic type polymerase chain reaction
By Fig. 7's the results show that the positive controls for being 100 in number of copies, utilize detection spring viremia of carp virus of the invention
The kit of mass formed by blood stasis virus, i.e. sequence recognize forward primer shown in number 1, the reverse primer as shown in sequence identification number 2,
And fluorescence probe shown in sequence identification number 3, can all stablize amplification in 3 independent experiment pipes to go out size is the poly- of 98bp
Synthase chain reaction product.In the fluorescent signal analysis of table one, by the 520nm signal value after progress iiPCR, divided by progress
520nm signal value before iiPCR can obtain a signal-to-noise ratio (signal-to-noise ratio), when signal-to-noise ratio is more than that reactor is pre-
If threshold value when, interpretation be with fluorescent signal (with+indicate).Positive controls all may detect that fluorescence is interrogated in wavelength 520nm
Number, and the negative of template is not added to organize then unstressed configuration signal.The results show that no matter with fluorescence detecting or gel electrophoresis reaction
Whether as a result, the kit of detection huichun viremia virus of the invention, can all examine in sample to be tested has spring viremia of carp virus
Mass formed by blood stasis virus.
In conclusion the kit and its method of inspection of detection huichun viremia virus of the invention can be sensitive, special
It is anisotropic good, quick and simple to operate huichun viremia virus is detected.Detect huichun viremia virus
Kit special, sensitively can not only detect huichun viremia virus, and detect huichun viremia virus
If kit arrange in pairs or groups light, portable, the fluorescence nucleic acids instrument of combined type portable, such as the intelligent portable core of POCKIT
Acid analysis instrument (the auspicious letter of gold is prompt) can carry out on-site test to huichun viremia virus on pond side, be spring viremia of carp virus blood
The quick detection and prevention and control of syndrome virus provide effective scientific method and important evidence, the disengaging to China's cyprinid fish is ensured
Mouth trade and domestic production cultivation safety are of great significance.
The present invention is disclosed above with embodiment, and however, it is not to limit the invention, any to be familiar with this those skilled in the art,
It does not depart from the spirit and scope of the present invention, when can be used for a variety of modifications and variations, therefore protection scope of the present invention is after
Subject to attached those as defined in claim.
Sequence table (SEQUENCE LISTING)
< 110> Food Inspection & Quarantine Technology Center of Shenzhen Entry-Exit Inspection
Prompt (Xiamen) Biotechnology Co., Ltd of the auspicious letter of gold
< the kit and its method of inspection of 120> detection huichun viremia virus
< 160> 4
< 170> PatentIn version 3.3
< 210> 1
< 211> 23
< 212> DNA
< 213> artificial sequences (Artificial sequence)
<220>
<221>
<222>
<223>primer (primer), amplification huichun viremia virus Zheng to Yin Wu
< 400> 1
gtagtacccc attctgttca ttt 23
< 210> 2
< 211> 23
< 212> DNA
< 213> artificial sequences (Artificial sequence)
<220>
<221>
<222>
<223>primer (primer), the Fan of amplification huichun viremia virus are to Yin Wu
< 400> 2
ttcatttcgc acactttttc tct 23
< 210> 3
< 211> 20
< 212> DNA
< 213> artificial sequences (Artificial sequence)
<220>
<221>
<222>
< 223> primers (primer), fluorescence probe
< 400> 3
tgatcgatcc agtgtcctcc 20
< 210> 4
< 211> 98
< 212> DNA
< 213> spring viremias (Spring viraemia of carp)
< 220>
<221>
<222>
< 223> CDS, the huichun viremia virus deoxyribonucleic acid fragment of amplification
< 400> 4
gtagtacccc attctgttca tttagagcca tatggaggac attggatcga tcacgaattt 60
aatgggggcg aatgcagaga aaaagtgtgc gaaatgaa 98
Claims (10)
1. it is a kind of detect huichun viremia virus kit, characterized by comprising:
One primer pair includes:
One sequence recognizes forward primer shown in number 1；And
One sequence recognizes reverse primer shown in number 2.
2. the kit of detection huichun viremia virus as described in claim 1, wherein further include to recognize just like sequence and compile
Fluorescence probe shown in numbers 3.
3. the kit of detection huichun viremia virus as claimed in claim 1 or 2, wherein it is poly- to further include a reverse transcription
Reagent needed for synthase chain reaction.
4. it is a kind of detect huichun viremia virus the method for inspection, characterized by comprising:
A sample to be tested is provided as a template；
The template is subjected to a reverse transcription polymerase chain reaction, to obtain a cDNA template；
The cDNA template is subjected to a polymerase chain reaction with kit described in claim 1, is connected with obtaining a polymerase
Lock reactor product；And
Whether the molecular size range for detecting the polymerase chain reaction product is 98bp.
5. the method for inspection of detection huichun viremia virus as claimed in claim 4, wherein the polymerase chain reaction
Product has the sequence as shown in sequence identification number 4.
6. the method for inspection of detection huichun viremia virus as claimed in claim 4, wherein detect the polymerase chain
Reaction product is with the progress of colloid electrophoresis system.
7. it is a kind of detect huichun viremia virus the method for inspection, characterized by comprising:
A sample to be tested is provided as a template；
The template is subjected to a reverse transcription polymerase chain reaction, to obtain a cDNA template；
The cDNA template is subjected to a fluorescence probe formula polymerase chain reaction with kit as claimed in claim 2；And
Detect whether that there is a fluorescence signal value.
8. the method for inspection of detection huichun viremia virus as claimed in claim 7, wherein the fluorescence probe formula polymerization
Enzyme chain reaction is a real time aggregation enzyme chain reaction or an isolation thermostatic type polymerase chain reaction.
9. it is a kind of detect huichun viremia virus the method for inspection, characterized by comprising:
A sample to be tested is provided as a template；
The template is sequentially subjected to a reverse transcription polymerase chain reaction and a DNA cloning with kit as claimed in claim 3
Reaction, wherein the DNA amplification reaction is a polymerase chain reaction or a fluorescence probe formula polymerase chain reaction；And
Whether detecting has an expected polymerase chain reaction product；
When the DNA amplification reaction is the polymerase chain reaction, when the molecular size range of a polymerase chain reaction product
When for 98bp, for the characterization with the expection polymerase chain reaction product；
When the DNA amplification reaction is the fluorescence probe formula polymerase chain reaction, a fluorescence signal value is with the expection
The characterization of polymerase chain reaction product.
10. the method for inspection of detection huichun viremia virus as claimed in claim 9, wherein the fluorescence probe formula is poly-
Synthase chain reaction is a real time aggregation enzyme chain reaction or an isolation thermostatic type polymerase chain reaction.
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| CN201820294877 | 2018-03-02 | ||
| CN201820294877X | 2018-03-02 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876810A (en) * | 2012-10-19 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method for spring viraemia of carp virus |
| US20130244221A1 (en) * | 2012-03-15 | 2013-09-19 | Industry Foundation Of Chonnam National University | Method for isolating hepatitis a virus or spring viremia of carp virus |
| CN105002298A (en) * | 2015-05-21 | 2015-10-28 | 上海市水产研究所 | Fluorescent quantitative PCR detection method of spring viraemia of carp virus |
| CN105112568A (en) * | 2015-09-14 | 2015-12-02 | 山东出入境检验检疫局检验检疫技术中心 | Carp spring viremia virus detection kit based on pyrosequencing |
-
2019
- 2019-02-27 CN CN201910147246.4A patent/CN110218814B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130244221A1 (en) * | 2012-03-15 | 2013-09-19 | Industry Foundation Of Chonnam National University | Method for isolating hepatitis a virus or spring viremia of carp virus |
| CN102876810A (en) * | 2012-10-19 | 2013-01-16 | 中国水产科学研究院长江水产研究所 | RT-PCR (reverse transcription-polymerase chain reaction) detection kit and detection method for spring viraemia of carp virus |
| CN105002298A (en) * | 2015-05-21 | 2015-10-28 | 上海市水产研究所 | Fluorescent quantitative PCR detection method of spring viraemia of carp virus |
| CN105112568A (en) * | 2015-09-14 | 2015-12-02 | 山东出入境检验检疫局检验检疫技术中心 | Carp spring viremia virus detection kit based on pyrosequencing |
Non-Patent Citations (6)
| Title |
|---|
| ZHIQIN YUE等: ""Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR"", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| ZONGXIAO LIU等: ""Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay"", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| 中华人民共和国国家质量监督检验检疫总局: ""鲤春病毒血症检疫技术规范"", 《中华人民共和国出入境检验检疫行业标准》 * |
| 吴斌等: ""数字RT-PCR 检测鲤春病毒血症的方法建立与应用"", 《中国动物检疫》 * |
| 王娟等: ""恒温热隔绝式PCR技术的原理与应用"", 《动物医学进展》 * |
| 高隆英等: ""用RT-PCR法快速检测鲤春病毒血症病毒基因"", 《水生生物学报》 * |
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