CN110218817A - Detect the kit and its method of inspection of infectious hematopoietic necrosis - Google Patents
Detect the kit and its method of inspection of infectious hematopoietic necrosis Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The present invention provides a kind of kit for detecting infectious hematopoietic necrosis, and it is suitable for polymerase chain reaction, kit includes primer pair, and primer pair includes reverse primer shown in the forward primer as shown in sequence mark number 1 and sequence mark number 2.The present invention separately provides a kind of method of inspection using mentioned reagent box detection infectious hematopoietic necrosis.Thus, it is possible to quick and specific detect infectious hematopoietic necrosis 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 infectious hematopoietic necrosis should be detected.
Background technique
Infectious hematopoietic necrosis is a kind of viral disease for infecting cold water fish.Host is mainly salmon fishes,
Such as rainbow trout and Atlantic salmon, when infectious hematopoietic necrosis's outburst, the death rate for occurring postlarva and juvenile fish first is prominent
So increase.The fish encroached on generally occurs within lethargic sleep symptom, is reluctant activity and avoids water flow, but also have fish performance it is violent run helter-skelter,
Spin equal abnormal phenomenas.Opaque or sepia vacation in tow at illness fish exophthalmos and blackening, abdominal distention and anus
Cast mucous stool is more typical feature, but not disease institute is exclusive.The fish gill is pale, fin base portion bleeding.In addition, usually existing
Subcutaneous hemorrhage is shown above side line behind head.
The cause of disease of infectious hematopoietic necrosis is infectious hematopoietic necrosis's virus (Infectious
Haematopoietic Necrosis Virus, IHNV), it is under the jurisdiction of outside Rhabdoviridae (Rhabdoviridae) grain and plays shape disease
Poison belongs to (Novirhabdovirus), is a sub-thread negative sense RNA virus, Genome Size 11130bp.Its form is in bullet shaped,
Length is about 160-180nm, and diameter is about 70-90nm, there is the cyst membrane of 15nm thickness.The base of infectious hematopoietic necrosis's virus
It is held from 3 ' to 5 ' because of group and holds successively encoding nuclear proteins (nucleoprotein, N), phosphoprotein (phosphoprotein, P), matrix
Albumen (matrix protein, M), glycoprotein (glycoprotein, G), non-structural protein (non-virion protein,
) and polymerase protein (polymerase, L) NV.
The dead situation of the fish of infectious hematopoietic necrosis's infection only has suitable close relationship with the fish age, generally
Juvenile fish is quite sensitive to virus, and biggish fish has the resistance of certain degree.Good environment can reduce the harm of this disease.In nature
Under environment, between the water temperature that infectious hematopoietic necrosis mainly occurs is 8-15 DEG C, the death rate and environment temperature, virus
Strain (virus strain) etc. is all related, and usually its cumulative mortality (cumulative mortality) may be up to when such as serious
90-95%.The usual various courses of disease can be all observed if becoming chronic (chronic cases), disease infection in such as followed by
The usual death of the bacterium infection for sending out Secondary is can be quite serious.
Therefore the kit and its method of inspection of quickly, conveniently and accurately detection infectious hematopoietic necrosis are developed,
To facilitate scene or the laboratory testing of infectious hematopoietic necrosis most crucial.
Summary of the invention
In view of this, a purpose of the invention be to provide it is a kind of detect infectious hematopoietic necrosis kit and its
Detection method, can be quick and specific detects infectious hematopoietic necrosis from sample to be tested.
A scheme of the invention is to provide a kind of kit for detecting infectious hematopoietic necrosis, and it includes a primers
Right, the primer pair includes reversely to draw shown in forward primer shown in a sequence mark number 1 and a sequence mark number 2
Object.
According to the kit of detection infectious hematopoietic necrosis above-mentioned, a reverse transcription polymerase can further include
Reagent needed for chain reaction.
According to the kit of detection infectious hematopoietic necrosis above-mentioned, it can further include and compiled just like sequence mark
Fluorescent probes shown in numbers 3.
The invention adopt another scheme that provide it is a kind of detect infectious hematopoietic necrosis the method for inspection, comprising with
Lower step.A sample to be tested is provided as a template.The template is subjected to a reverse transcription polymerase chain reaction, to obtain one
CDNA template.CDNA template is subjected to polymerase chain with the kit for detecting infectious hematopoietic necrosis described in leading portion
Reaction, to obtain a polymerase chain reaction product, and the kit of the detection infectious hematopoietic necrosis includes one
Primer pair, the primer pair include reversely to draw shown in forward primer shown in sequence mark number 1 and sequence mark number 2
Object.Whether the molecular size range for finally detecting the polymerase chain reaction product is 98bp.
The method of inspection according to detection infectious hematopoietic necrosis above-mentioned, wherein the polymerase chain reaction
Product can have the sequence as shown in sequence mark number 4.
The method of inspection according to detection infectious hematopoietic necrosis above-mentioned, wherein detect the polymerase chain
Reaction product can be carried out with colloid electrophoresis system.
Yet another aspect of the invention be providing it is a kind of detect infectious hematopoietic necrosis the method for inspection, comprising with
Lower step.A sample to be tested is provided as a template.The template is subjected to a reverse transcription polymerase chain reaction, to obtain one
CDNA template.CDNA template is subjected to a fluorescent probes with the kit for detecting infectious hematopoietic necrosis described in leading portion
Formula polymerase chain reaction, and the kit of the detection infectious hematopoietic necrosis includes that a primer pair and a fluorescent are visited
Needle, the primer pair include reverse primer shown in forward primer shown in sequence mark number 1 and sequence mark number 2, institute
Fluorescent probes are stated with the sequence as shown in sequence mark number 3.Finally detect whether that there is a Fluorescent signal value.
According to the method for inspection of detection infectious hematopoietic necrosis above-mentioned, wherein the fluorescent probes formula polymerase
Chain reaction can be instant polymerase chain reaction (Real-time PCR) or isolation thermostatic type polymerase chain reaction
(insulated isothermal PCR,iiPCR)。
Yet another aspect of the invention is to provide a kind of method of inspection for detecting infectious hematopoietic necrosis, comprising following
Step.A sample to be tested is provided as a template.By the template with detection infectious hematopoietic necrosis described in leading portion
Kit sequentially carry out a reverse transcription polymerase chain reaction and a DNA amplification reaction.The detection infectious hematopoietic organ
The kit of downright bad disease is premixed form, it includes reagent needed for a primer pair, a reverse transcription polymerase chain reaction and/or
One fluorescent probes, the primer pair include reversed shown in forward primer shown in sequence mark number 1 and sequence mark number 2
Primer, the fluorescent probes have the sequence as shown in sequence mark number 3.And the DNA amplification reaction is polymerase company
Lock reactor or a fluorescent probes formula polymerase chain reaction.Finally detect whether that there is an expected polymerase chain reaction product.
When DNA amplification reaction is polymerase chain reaction, when the molecular size range of a polymerase chain reaction product is 98bp, for tool
There is the characterization of expected polymerase chain reaction product.When DNA amplification reaction is fluorescent probes formula polymerase chain reaction, a fluorescent
Signal value is the characterization with expected polymerase chain reaction product.
The method of inspection according to detection infectious hematopoietic necrosis above-mentioned, wherein the fluorescent probes formula polymerization
Enzyme chain reaction can be instant polymerase chain reaction or isolation thermostatic type polymerase chain reaction.
The kit and its method of inspection of detection infectious hematopoietic necrosis of the invention as a result, can be quick, special
It is different, conveniently and accurately detect sample to be tested in the presence or absence of infectious hematopoietic necrosis virus, display sample to be tested
Infect infectious hematopoietic necrosis.The kit and its method of inspection of detection infectious hematopoietic necrosis of the invention
Testing result other than it can confirm result via generally conventional race glue, fluorescent polymerase chain reaction can also be carried out and isolation is permanent
Warm formula polymerase chain reaction confirmation is as a result, the testing result for wherein especially completely cutting off thermostatic type polymerase chain reaction is not required to complexity
Data interpretation can also be infected using kit and the method for inspection of the invention even if not received the personnel of professional training
Property Hematopoietic Necrosis's disease detection, and the detection for aquaculture fishery between field 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 present disclosure and makes a summary, so that reader has base to present disclosure
This understanding.The invention content is not the complete overview of present disclosure, and it is not intended to pointing out the embodiment of the present invention
Key/critical element 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, the description of the drawings
It is as follows.
Fig. 1 shows the structures of the kit of the detection infectious hematopoietic necrosis according to one embodiment of the present invention
Schematic diagram.
Fig. 2 indicates the step of method of inspection of detection infectious hematopoietic necrosis of another embodiment of the present invention
Flow chart.
Fig. 3 indicates the step of method of inspection of detection infectious hematopoietic necrosis of another embodiment of the invention
Flow chart.
Fig. 4 indicates the step of method of inspection of detection infectious hematopoietic necrosis of another embodiment of the invention
Flow chart.
Fig. 5 is that the detection specificity of the kit of detection infectious hematopoietic necrosis of the invention analyzes result figure.
Fig. 6 is that the detection sensitivity of the kit of detection infectious hematopoietic necrosis of the invention analyzes result figure.
Fig. 7 is that the kit of detection infectious hematopoietic necrosis of the invention carries out isolation thermostatic type polymerase chain
The gel electrophoresis figure of reaction.
Wherein, the reference numerals are as follows.
100: detecting the kit of infectious hematopoietic necrosis.
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 infectious hematopoietic necrosis.
610,620,630,640: step.
700: detecting the method for inspection of infectious hematopoietic necrosis.
710,720,730,740: step.
800: detecting the method for inspection of infectious hematopoietic necrosis.
810,820,830: step.
Specific embodiment
The content of this disclosure provide a kind of quickly detection infectious hematopoietic necrosis kit and its
The method of inspection.It is to design specific primer pair with the height reserved area of infectious hematopoietic necrosis's virus, comprising drawing
The kit and sample to be tested of object pair carry out polymerase chain reaction, are to be measured when there is expected polymerase chain reaction product
There are the characterization of infectious hematopoietic necrosis's virus in sample, show that sample to be tested has infected infectious hematopoietic organ necrosis
Disease.And the fluorescent probes that there is specificity with infectious hematopoietic necrosis's virus can be further designed, to include primer
Pair and fluorescent probes kit, with sample to be tested carry out fluorescent probes formula polymerase chain reaction be when there is fluorescent signal
There are the characterization of infectious hematopoietic necrosis's virus in sample to be tested, show that sample to be tested has infected infectious hematopoietic organ
Downright bad disease.
Aforementioned so-called " fluorescent probes " of the invention refer to that one includes the molecule of continuous at least eight nucleotide, to choose
One section of sequence between PCR upstream and downstream primer marks upper fluorescent group (reporter) as probe, and at the end 5`- of probe, and 3
The end `- marks upper corresponding fluorescent quencher (quencher), since two groups are in close proximity, constitute fluorescent energy transmission
Relationship is generated without Fluorescent 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 fluorescent group with the quenching group on fluorescent quencher, allow fluorescent group to release
Fluorescent.Such as the end 5' fluorescent group usable FAM (520nm) or JOE, VIC (550nm) etc. of fluorescent probes, and fluorescent probes
The end 3' fluorescent quencher can be used black hole quencher (black hole quencher) (BHQ1) or non-fluorescent quencher
(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 fluorescent detection technique, 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, kit of the invention can be also utilized
And detection method carries out the detection of infectious hematopoietic necrosis.
Following experiments example illustrates the present invention for demonstrating, and is to be conducive to the usual knowledge of the technical field of the invention
Person can completely utilize and practice the present invention in the case of being not required to and excessively interpreting, without these experimental examples should be considered as to this hair
The limitation of bright range is only for how implementing material and method of the invention.
[kit of detection infectious hematopoietic necrosis]
The kit of detection infectious hematopoietic necrosis of the invention, includes a primer pair, the primer pair includes
Reverse primer shown in forward primer shown in one sequence mark number 1 and a sequence mark number 2.Preferably, of the invention
The kit of detection infectious hematopoietic necrosis can further include the fluorescent probes as shown in sequence mark number 3.
It is highly preferred that the kit of detection infectious hematopoietic necrosis of the invention can further include reverse transcription polymerase company
Reagent needed for lock reactor, to by the template in sample to be tested via reverse transcription polymerase chain reaction reverse transcription be cDNA mould
Plate so that the kit of detection infectious hematopoietic necrosis of the invention is premixed form, can in same developmental tube according to
Sequence carries out reverse transcription polymerase chain reaction and DNA amplification reaction.
Fig. 1 is please referred to, indicates the reagent of the detection infectious hematopoietic necrosis according to one embodiment of the present invention
The structural schematic diagram of box 100.The kit 100 for detecting infectious hematopoietic necrosis includes box body 210, box cover 220, liner
300, sample ring assemblies 400 and premix reagent component 500 are connect.
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 reagent of dissolution mix primer.Positive control
Module 320 is to include infectious hematopoietic organ necrosis to place positive controls nucleic acid dry powder, the positive controls nucleic acid
The plastid of the full genome of virus.Nucleic acid buffer solution module 330 is to place the second of dissolution positive controls nucleic acid dry powder
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, fluorescent probes, anti-
Transcriptase and archaeal dna polymerase.Wherein primer pair includes forward primer and sequence mark number 2 shown in sequence mark number 1
Shown in reverse primer.The sequence of fluorescent probes is as shown in sequence mark number 3, in this embodiment, 3 ' end tools of fluorescent probes
There is NFQ fluorescent quencher, 5 ' ends have the modification of FAM fluorescent group.
Detecting premix Reagent Tube in the kit 100 of infectious hematopoietic necrosis is premixed form mixed in advance,
It is not added with 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 Reagent Tube
Freeze-drying particle, premix buffer solution module 310 in the first buffer reagent mixing, and with connect sample ring 410 by sample to be tested turn
It moves in premix Reagent Tube, can start to react.It such as needs to be added fluorescent dye and corrects (Real-time) PCR instrument immediately, it can
ROX reference dye is added according to the standard test procedure in each laboratory.Detect the reagent of infectious hematopoietic necrosis
Box 100 is simple, is convenient for carrying, can room temperature carrying and preservation.Also, if to the reagent of detection infectious hematopoietic necrosis
Box 100 carries out In Aluminium Foil Packing sealing, and it is constant that freezen protective in -20 DEG C can then store 1 year or more efficiency.
[method of inspection of detection infectious hematopoietic necrosis]
Referring to figure 2., the inspection party of the detection infectious hematopoietic necrosis of another embodiment of the present invention is indicated
The step flow chart of method 600.The method of inspection 600 for detecting infectious hematopoietic necrosis includes step 610, step 620, step
Rapid 630 and step 640.
Step 610 is to provide a sample to be tested as a template.The sample to be tested can for the kidney sample of fish, spleen sample,
Brain sample and digestive organ's sample.And it can be further using business extraction agent set group or the extraction of other known methods for test sample
Nucleic acid in this, step and principle are that have usually intellectual in related fields to be known, thus no longer go to live in the household of one's in-laws on getting married in detail herein
It states.
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 include forward primer shown in sequence mark number 1 and sequence mark
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 infectious hematopoietic necrosis's virus in sample to be tested, is shown
Show that sample to be tested has infected infectious hematopoietic necrosis.Preferably, polymerase chain reaction product has such as sequence mark
Sequence shown in number 4.And polymerase chain reaction product can be detected with colloid electrophoresis system.
Referring to figure 3., the inspection party of the detection infectious hematopoietic necrosis of another embodiment of the present invention is indicated
The step flow chart of method 700.The method of inspection 700 for detecting infectious hematopoietic necrosis includes step 710, step 720, step
Rapid 730 and step 740.
Step 710 is to provide a sample to be tested as a template.The sample to be tested can for the kidney sample of fish, spleen sample,
Brain sample and digestive organ's sample, and can be further using business extraction agent set group or the extraction of other prior art methods to test sample
Nucleic acid in this.
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 forward primer shown in sequence mark number 1,2 institute of sequence mark number
Fluorescent probes shown in the reverse primer and sequence mark number 3 shown carry out a fluorescent probes formula polymerase chain reaction.It is excellent
Selection of land, fluorescent probes formula polymerase chain reaction can be instant polymerase chain reaction (Real-time PCR) or isolation constant temperature
Formula polymerase chain reaction (insulated isothermal PCR, iiPCR).
Step 740 is to detect whether there is a Fluorescent signal value, if having Fluorescent signal value, then it represents that deposit in sample to be tested
In infectious hematopoietic necrosis's virus, display sample to be tested has infected infectious hematopoietic necrosis.
Referring to figure 4., the inspection party of the detection infectious hematopoietic necrosis of another embodiment of the invention is indicated
The step flow chart of method 800.Detect infectious hematopoietic necrosis the method for inspection 800 include 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 for the kidney sample of fish, spleen sample,
Brain sample and digestive organ's sample, and can be further using business extraction agent set group or the extraction of other prior art methods to test sample
Nucleic acid in this.
Step 820 is that the template is detected infectious hematopoietic necrosis's in same developmental tube with of the invention
Kit sequentially carries out a reverse transcription polymerase chain reaction and a DNA amplification reaction.The detection infectious hematopoietic organ is bad
The kit of dead disease includes reagent and/or a fluorescent probes needed for a primer pair, a reverse transcription polymerase chain reaction, described
Primer pair includes reverse primer shown in forward primer shown in sequence mark number 1 and sequence mark number 2, and the fluorescent is visited
Needle set is just like sequence shown in sequence mark number 3.And the DNA amplification reaction is that a polymerase chain reaction or a fluorescent are visited
Pin type polymerase chain reaction.And fluorescent probes formula polymerase chain reaction can be instant polymerase chain reaction or isolation constant temperature
Formula polymerase chain reaction.
Step 830 is to detect whether there is 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 fluorescent probes formula polymerase chain reaction, a Fluorescent signal value is with expected polymerase
The characterization of chain reaction product.
[test example]
1. instant polymerase chain reaction
Please with reference to Fig. 1 and Fig. 4, in detail, if to detect the progress of the kit 100 of infectious hematopoietic necrosis
Instant polymerase chain reaction is first dissolved for detecting infectious hematopoietic necrosis with the second buffer solution of 100 μ L
Positive controls nucleic acid dry powder, to obtain concentration as 4 × 10-5The positive control of ng/ μ L, and the positive control after back dissolving is saved
It is spare in 4 DEG C.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 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
Lid, the first buffer solution back dissolving that 50 μ L are added in each pipe premix Reagent Tube are lyophilized particle, that is, it is anti-to obtain instant polymerase chain
Answer mixed liquor.In instant polymerase chain reaction mixed liquor, the concentration of dNTPs is 0.5mM, shown in sequence mark number 1 just
Concentration to reverse primer shown in primer and sequence mark number 2 is respectively 0.5 μM, the concentration of fluorescent probes is 0.05 μM,
The concentration of reverse transcriptase is 16U and the concentration of archaeal dna polymerase is 13U.If ROX reference dye correction Real-time need to be added
Particle is lyophilized in the ROX reference dye back dissolving of PCR instrument, 49 μ L are added in each pipe premix Reagent Tube the first buffer solution and 1 μ L.
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
Sample to be tested, positive controls or the negative control group of L is added the fluorescent PCR Special reverse containing 23 μ L freeze-drying particle solution and answers
Pipe.Wherein, 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 profile, 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 formula 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 amplification curve has obvious logarithm to increase in the channel FAM, for
Positive findings indicate that, there are infectious hematopoietic necrosis's virus in sample to be tested, display sample to be tested has infected infectiousness
Hematopoietic Necrosis's disease virus.If increasing in the channel FAM amplification curve without logarithm, for negative findings, indicate in sample to be tested
There is no infectious hematopoietic necrosis's viruses, or there is the virus quantity for being lower than detection sensitivity.
First to the kit and its method of inspection of detection infectious hematopoietic necrosis of the invention in this test example
Detect specific analysis, used sample to be tested includes infectious hematopoietic necrosis viral (IHNV), viral
Hemorrhagic septicemia virus (VHSV), huichun viremia virus (SVCV), infectivity pancreatic necrosis virus (PNV), salmon are infected
The nucleic acid of property anemia virus (ISAV), salmon Alphavirus (SAV).
It referring to figure 5., is the detection specificity analysis of the kit of detection infectious hematopoietic necrosis of the invention
Result figure.The results show that the kit and its method of inspection of detection infectious hematopoietic necrosis of the invention are only to infection
Property Hematopoietic Necrosis's disease virus has specific amplification, and it is other then have no amplification curve for prelibation strain, show inspection of the invention
The kit and its method of inspection for surveying infectious hematopoietic necrosis have good specificity.
This test example is further to the kit for detecting infectious hematopoietic necrosis of the invention and its inspection party
Method carries out detection sensitivity analysis.The plastid for the target gene that one includes infectious hematopoietic necrosis's virus is constructed first,
Again using this plastid using reverse transcription RNA in test tube as the template of positive control group, it is rear measured using ultraviolet specrophotometer it is anti-
The concentration and purity for transcribing RNA template, according to the concentration calculation copy number of measurement.Again with DEPC water by reverse transcription RNA template into
10 times of serial dilutions of row, are diluted to 3.5 × 108Be copied to 3.5 × 100 copy, as standard items with measurement sensitivity, and will before
Standard items after stating serial dilution with the kit of detection infectious hematopoietic necrosis of the invention and its method of inspection into
Row detection, detailed detecting step is as previously mentioned, details are not described herein.
Fig. 6 is please referred to, for the detection sensitivity analysis of the kit of detection infectious hematopoietic necrosis of the invention
Result figure, wherein curve from left to right is sequentially 3.5 × 107Copy, 3.5 × 106Copy, 3.5 × 105Copy, 3.5 × 104
Copy, 3.5 × 103Copy, 3.5 × 102Copy, 3.5 × 101The detection curve of copy, and 3.5 × 100 copies are bent without amplification
Line.The results show that the kit and its method of inspection of detection infectious hematopoietic necrosis of the invention are to carp edema virus
Detection sensitivity it is high, detectable limit be 35 copy.
2. completely cutting off thermostatic type polymerase chain reaction
If with detect the kit 100 of infectious hematopoietic necrosis carry out isolation thermostatic type polymerase chain reaction with
For detecting infectious hematopoietic necrosis, positive controls nucleic acid dry powder is first dissolved with the second buffer solution of 100 μ L, with
Obtaining concentration 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 is opened again
Reagent packet, and according to the premix Reagent Tube of number of awaiting test sample taking-up corresponding amount, and premix freeze-drying particle back dissolving mode in Reagent Tube
Such as the following steps.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
Completely cut off thermostatic type polymerase chain reaction mixed liquor, solution mixed and is moved back into isolation thermostatic type polymerase chain reaction pipe,
Isothermal duplication is carried out to completely cut off thermostatic type polymerase chain reaction device.Reaction condition is 42 DEG C of reverse transcription reactions 10 minutes, bottom
Heat 95 DEG C, 30 minutes i.e. available reaction result in portion.
Equality of temperature polymerase company is thermally shielded in order to understand the kit of detection infectious hematopoietic necrosis of the invention
Lock reactor expands the stability of the polymerase chain reaction product of 98bp, and reverse transcription RNA in test tube is sequentially diluted to copy number
Mesh is 100 as positive controls, then to the kit for detecting infectious hematopoietic necrosis of the invention and its inspection
Proved recipe method is tested and analyzed, and isolation thermostatic type polymerase chain reaction product is separately separated with colloid electrophoresis, to ensure the present invention
Accuracy.
Fig. 7 and table one are please referred to, Fig. 7 is that the kit of detection infectious hematopoietic necrosis of the invention completely cuts off
The gel electrophoresis figure of thermostatic type polymerase chain reaction, wherein M is the molecular weight marker of nucleic acid, and N is without containing target base
Because of the negative control group of nucleic acid.Table one is that the kit of detection infectious hematopoietic necrosis of the invention carries out isolation constant temperature
The fluorescent signal of formula polymerase chain reaction analyzes result.
Table one, the florescence analysis 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 copy number, are made using detection infectiousness of the invention
It is the kit of blood organ necrosis disease, i.e. forward primer shown in sequence mark number 1, reversed as shown in sequence mark number 2
Fluorescent probes shown in primer and sequence mark number 3 can all be stablized in 4 independent experiment pipes and amplify size and be
The polymerase chain reaction product of 98bp.In the fluorescent signal analysis of table one, the 520nm signal value after progress iiPCR removes
A signal-to-noise ratio (signal-to-noise ratio) can be obtained to carry out the 520nm signal value before iiPCR, when signal-to-noise ratio is more than anti-
When answering the preset threshold value of device, interpretation be with fluorescent signal (with+indicate).Positive controls all can be detected in wavelength 520nm
Fluorescent signal, and do not add the negative group of template then without fluorescent signal.The results show that no matter with fluorescent detection or colloid electrophoresis point
Reaction result is analysed, the kit of detection infectious hematopoietic necrosis of the invention can all be examined in sample to be tested and whether have
Be infectious Hematopoietic Necrosis's disease virus.
In conclusion the kit and its method of inspection of detection infectious hematopoietic necrosis of the invention being capable of spirits
It is quick, specificity is good, quick and simple to operate detects to infectious hematopoietic necrosis.Detect infectious hematopoietic
The kit of organ necrosis disease special, sensitively can not only detect infectious hematopoietic necrosis, and detect
If the kit collocation of infectious hematopoietic necrosis is light, portable, in conjunction with portable fluorescent nucleic acids instrument, such as
The intelligent portable nucleic acids instrument of POCKIT (the auspicious letter of gold is prompt), pond side can to infectious hematopoietic necrosis into
Row on-site test, for infectious hematopoietic necrosis it is quick detection and prevention and control provide effective scientific method and it is important according to
According to foreign trade and domestic production cultivation safety to guarantee China's salmon fishes are of great significance.
The present invention has passed through the embodiment and has carried out disclosed above, but it is not intended to limit the invention, Ren Heben
Field technical staff can carry out various change and modification, therefore the present invention without departing from the spirit and scope of the present invention
Protection scope should be subject to defined by appended claims.
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 infectious hematopoietic necrosis
< 160> 4
< 170> PatentIn version 3.3
< 210> 1
< 211> 24
< 212> DNA
< 213> artificial sequences (artificial sequence)
<220>
<221>
<222>
< 223> primers (primer)
< 400> 1
gtacgataac cctccctcta tttt 24
< 210> 2
< 211> 21
< 212> DNA
< 213> artificial sequences (artificial sequence)
<220>
<221>
<222>
< 223> primers (primer)
< 400> 2
ctttctcgtt ccctctcctc c 21
< 210> 3
< 211> 15
< 212> DNA
< 213> artificial sequences (artificial sequence)
<220>
<221>
<222>
< 223> primers (primer), fluorescent probes
< 400> 3
actcaccgcc cgctt 15
< 210> 4
< 211> 98
< 212> DNA
< 213> infectious hematopoietic necrosis are viral (Infectious Haematopoietic Necrosis Virus)
<220>
<221>
<222>
< 223> CDS, infectious hematopoietic necrosis's virus nuclifort segment of amplification
< 400> 4
gtacgataac cctccctcta tttttctcca agacagactt agacctagag atgatcaagc 60
gggcggtgag tcaagtcgga ggagagggaa cgagaaag 98
Claims (10)
1. a kind of kit for detecting infectious hematopoietic necrosis, which is characterized in that it includes a primer pair, the primer pairs
Include reverse primer shown in forward primer shown in a sequence mark number 1 and a sequence mark number 2.
2. the kit of detection infectious hematopoietic necrosis as described in claim 1, wherein it further includes a sequence
Fluorescent probes shown in column marker number 3.
3. the kit of detection infectious hematopoietic necrosis as claimed in claim 1 or 2, wherein it is further included
Reagent needed for one reverse transcription polymerase chain reaction.
4. a kind of method of inspection for detecting infectious hematopoietic necrosis, which is characterized in that it includes:
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 infectious hematopoietic necrosis as claimed in claim 4, wherein the polymerase chain is anti-
Answer product that there is sequence shown in sequence mark number 4.
6. detecting the method for inspection of infectious hematopoietic necrosis described in claim 4, wherein detect polymerase company
Lock reactor product is carried out by colloid electrophoresis system.
7. a kind of method of inspection for detecting infectious hematopoietic necrosis, which is characterized in that it includes:
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 fluorescent probes formula polymerase chain reaction with kit as claimed in claim 2；And
Detect whether that there is a Fluorescent signal value.
8. the method for inspection of the detection infectious hematopoietic necrosis as described in claim 7, wherein the fluorescent probes formula
Polymerase chain reaction is an instant polymerase chain reaction or an isolation thermostatic type polymerase chain reaction.
9. a kind of method of inspection for detecting infectious hematopoietic necrosis, which is characterized in that it includes:
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, the DNA amplification reaction are a polymerase chain reaction or a fluorescent probes formula polymerase chain reaction；And
Detect whether that there is 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 is
When 98bp, for the characterization with the expection polymerase chain reaction product；
When the DNA amplification reaction is the fluorescent probes formula polymerase chain reaction, a Fluorescent signal value is to polymerize with the expection
The characterization of enzyme chain reaction product.
10. the method for inspection of detection infectious hematopoietic necrosis as claimed in claim 9, wherein the fluorescent probes formula
Polymerase chain reaction is an instant polymerase chain reaction or an isolation thermostatic type polymerase chain reaction.
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Cited By (1)
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| CN112795705A (en) * | 2021-03-12 | 2021-05-14 | 长沙海关技术中心 | Primer and kit for efficient triple detection of SVCV, IHNV and CEV |
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