CN104911276A - Primers, kit and method for rapidly detecting Aleutian mink disease virus - Google Patents

Primers, kit and method for rapidly detecting Aleutian mink disease virus Download PDF

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Publication number
CN104911276A
CN104911276A CN201410084204.8A CN201410084204A CN104911276A CN 104911276 A CN104911276 A CN 104911276A CN 201410084204 A CN201410084204 A CN 201410084204A CN 104911276 A CN104911276 A CN 104911276A
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primer
test kit
pcr
sample
master mix
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赵家平
徐超
李光玉
杜锐
张秀丽
宋兴超
刘晗璐
朱洪伟
陈秀敏
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Institute Special Animal and Plant Sciences CAAS
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Abstract

The present invention discloses primers, a kit and a method for rapidly detecting Aleutian mink disease virus (AMDV), wherein the upstream primer sequence is represented by SEQIDNO.1, the downstream primer sequence is represented by SEQIDNO.2, the kit comprises the upstream primer, the downstream primer, PCRMastermix of a SYBRGreen dye, a virus lysate, a negative control, a positive control and the like, and the detection method comprises extraction of DNA of a sample to be detected, fluorescence PCR detection, and result determination. According to the present invention, the primers, the kit and the method can be used for AMDV identifying, have characteristics of simple and rapid operation, reliable result, strong specificity and high sensitivity, are suitable for on-site detection, and provide important effect for early stage detection and diagnosis, epidemiological epidemic situation investigation and import and export port detection.

Description

A kind of primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and method
Technical field
The present invention relates to Animal diseases checkout and diagnosis method and technology field, particularly relate to a kind of primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and method, qualitative and detection by quantitative is carried out to Aleutian Mink Disease Parvovirus in the scene of being highly suitable for, and detects have vital role for the diagnosis of feedlot early detection, the epidemiology investigation of epidemic situation and turnover port.
Background technology
Since the 1950's finds Aleutian disease (ADM), because ADM pathogenesis is more special, so not yet develop the conventional vaccine of effectively this disease of prevention so far, main to put prevention first in production, find that positive eliminates clean group control ADM mainly through detecting aleutian disease virus (AMDV) at present.So it is significant to set up the control of a kind of effective diagnostic method to ADM, traditional AMDV detection method comprises: the separation of cause of disease and the etiological diagnosis such as biological experiment, electron microscopy method, the Serology tests such as iodine agglutination test (IAT) and counterimmunoelectrophoresis (CIEP), complement fixation test (CFT), immunocomplex detection method (CIC); Biochip technology, polymerase chain reaction (PCR), nucleic acid hybridization technique equimolecular biometric diagnostic method.These method ubiquities, as shortcomings such as cycle length, complex operation, specificity, susceptibility and poor repeatability, are not suitable for Aulomatizeted Detect in enormous quantities, bring certain difficulty to the prevention and control of ADM.
Quantitative fluorescent PCR detects owing to have employed complete stopped pipe, do not need the PCR aftertreatments such as gel electrophoresis, avoid crossed contamination, whole testing process can realize automatization, and react quick, reproducible, highly sensitive, high specificity, result are clear, labour intensity is little, be easy to mass, stdn application.Along with the reduction of quantitative real time PCR Instrument and corresponding consumptive material price, the appearance of especially small-sized real-time fluorescence quantitative PCR and supporting automated operation equipment, significantly reduces the cost of quantitative fluorescent PCR, simplifies the process of detection by quantitative, improves working efficiency.Can predict soon in the future, declining further along with quantitative fluorescent PCR relevant cost and can carrying full-automatic quantitative real time PCR Instrument occurs, adopting this technology to carry out Site Detection to AMDV will become a kind of effective ways of control ADM.
Summary of the invention
The object of the present invention is to provide a kind of primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and method, for the detection to AMDV.
In order to realize rapid detection AMDV, the present invention is achieved by the following technical solutions:
A kind of primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and method, this primer comprises upstream primer and downstream primer, and upstream primer sequence is SEQ ID N0.1 5'-CCCTCCAGGTCAACTCTTTGTTAAA-3' and downstream primer sequence is SEQ ID N0.2 5'-GCCTCTCCAAGTAAAGTAACCATAGG-3'.
A kind of primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and method, this test kit comprises 1) PCR Master mix containing above-mentioned upstream primer and downstream primer and SYBR Green dyestuff, upstream and downstream primer final concentration is 0.25 μm of ol/L; 2) employing virus cracking liquid; 3) negative control-aseptic ultrapure water; 4) positive control-10 2copies/ μ L is connected with viral aliquots sequence amdv-f1's pMD19-T-amdv-f1 plasmid, amdv-f1 sequence is as shown in SEQ ID N0.3; 5) Virahol; 6) 75% ethanol.
pMDpreparation method is as follows for 19-T-amdv-f1 positive plasmid: utilize upstream primer SEQ ID N0.1 and downstream primer SEQ ID N0.2 with mink AMDV-G strain for template, carry out at EasyCycler gene-amplificative instrament (Analytikjena).Reaction system is 20 μ L, comprising: 10 μ L Premix Taq, 8 μ L ddH2O, 1 μ L template, upstream and downstream primer (each 5 μm of ol/L) mixture 1 μ L.Amplification is: 94 DEG C of denaturation 10s; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, totally 30 circulations; 7 min are extended after 72 DEG C.
Reaction product utilizes 2% agarose gel electrophoresis, utilize gel to reclaim test kit to reclaim PCR primer purifying, the PCR primer of recovery pMD19-T Vector is connected, in the PCR pipe of 0.2 mL, get PCR and reclaim product 4 μ L and pMD19-T Vector 1 μ L, add 5 μ L and connect damping fluid, 16 DEG C of connections of spending the night.Connecting fluid full dose (10 μ L) is added to 100 μ L and dissolves in ice in Trans5 α competent cell, places 30 min in ice.After 42 DEG C of heating 45 s, then in ice, place 1 min.Add 890 μ L SOC substratum, 37 DEG C of shaking culture 60min.L-Agar Plating containing X-Gal, IPTG, Amp is cultivated, forms single bacterium colony.Select white colony, use PCR method to confirm the length scale of Insert Fragment in carrier.From flat board, picking 4 clones are inoculated in the LB substratum of 5 mL 100 μ g/mL ammonia benzyls, cultivate 14h for 37 DEG C.Get 4mL incubated overnight bacterium liquid, with plasmid extraction kit extracting plasmid, send plasmid to check order.By plasmid called after correct for sequencing result pMD19-T-amdv-f1 and at BioPhotometer(eppendorf) on measure concentration, according to recombinant plasmid extract concentrations calculate copy number.
Preparation 1 × 10 10the solution of copies/ μ L as standard substance stoste ,-80 DEG C of preservations.Standard substance stoste 10 times of gradient dilutions are become 10 10-10 0copies/ μ L standard substance carry out real-time fluorescence quantitative PCR detection, with ddH on quantitative real time PCR Instrument 2o is template is negative control, occurs that the minimum concentration gradient of standard " S " type amplification curve is as the sensitivity detected using threshold cycle number (Ct)≤30.Reaction system is as follows: 2 × PCR mix(Power SYBR Green PCR Master Mix, Applied Biosystems) 10 μ L, template 1 μ L, upstream and downstream primer mixture (5 μm of ol/L) 1 μ L, ddH 2o 8 μ L.Amplification program is as follows: 95 DEG C of denaturation 10 min; 95 DEG C of sex change 15 s, 60 DEG C of annealing 60 s, 40 circulations.The susceptibility that present method result shows the method reaches 10 copies/ μ L, 10 2copies/ μ L and above time there is good specificity and repeatability, at negative control without amplification, show that the method specificity is good.
Standard substance stoste 10 times of gradient dilutions are got 10 9, 10 8, 10 7, 10 6, 10 5, 10 4, 10 3, 10 2copies/ μ L, as standard substance, quantitative real time PCR Instrument carries out real-time fluorescence quantitative PCR detection, and each sample 3 times repeats, reaction conditions as above, according to Ct value and template starting copy number production standard curve and standard equation.As can be seen from amplification curve (Fig. 1): totally see that all knee point are clear, exponential phase, is obvious, and the overall collimation of amplification curve is good, and baseline is flat without raising up phenomenon, and low concentration sample amplification curve exponential phase is obvious; As can be seen from Fig. 2 melting curve, template gradient melting curve only has 1 obvious peak, illustrates that design of primers does not rationally produce non-specific amplification and dimer.Present method sets up AMDV real-time fluorescence PCR quantitation curves (Fig. 3) equation: Y=-3.082X+36.762, r 2=0.995, wherein Y represents Ct value, and X represents the logarithmic value of template amount.Its linearity range can reach 8 orders of magnitude.
The test kit production and assembly of rapid detection Aleutian Mink Disease Parvovirus and application: the specification of rapid detection Aleutian Mink Disease Parvovirus test kit is 96 reaction/boxes.Therefore be prepared and packing according to each component of amount detection to reagent.
Test kit production and assembly:
(1) reference substance and water
Positive control: 1 × 10 2copies/ μ L's pMD19-T-amdv plasmid is packed as 25 μ L/ and manages ,-20 DEG C of preservations.
Negative control and dissolving DNA water: after ultrapure water autoclaving, be packed as 2.5mL/ pipe ,-20 DEG C of preservations.
(2) reagent
Employing virus cracking liquid: preparation contains the employing virus cracking liquid of 3mol/L guanidinium isothiocyanate, 0.4% sarcosyl, 0.12 mmol/L beta-mercaptoethanol, 20mmol/L Trisodium Citrate, the packing of 40mL/ bottle ,-4 DEG C of preservations.
Virahol: 60 mL/ bottle packing, normal temperature is preserved.
75% ethanol: prepare 75% ethanol after ultrapure water autoclaving, the packing of 120mL/ bottle, normal temperature is preserved.
PCR Master mix: get commercialization 2 × SYBR Green PCR Master mix, aseptic ultrapure water, 5 μm of ol/L upstream and downstream primer mixtures in the mixing of 10:7:1 ratio, 18 μ L/ pipes are sub-packed in 8 connecting legs or 96 orifice plates, and-20 DEG C keep in Dark Place.
(3) test kit assembling
Each 1 pipe of positive control, negative control and dissolving DNA water, each 1 bottle of employing virus cracking liquid, Virahol, 75% ethanol, 1.5mL centrifuge tube 96, containing 18 μ L/ pipe PCR Master mix 8 PCR reaction tubess 12 groups or containing 18 μ L/ pipe PCR Master mix 96 orifice plate 1 piece.
Present invention also offers and a kind ofly adopt above-mentioned test kit to detect Aleutian Mink Disease Parvovirus method, comprise the following steps:
(1) DNA extraction of sample to be checked; 200 μ L testing liquid samples (serum, tissue juice, urine etc.) are added in each 1.5mL plastic centrifuge tube.Add 400 μ L employing virus cracking liquids, after vibration 40s, room temperature places 10min.600 μ L Virahols are added after vibration 30s mixing, after vibration 45s mixing, the centrifugal 16min of 15000g room temperature.Move and abandon supernatant, add 1.2mL75% ethanol, vibrate the centrifugal 5min of 15000g room temperature after 15 seconds, abandons supernatant, and centrifugal 15s abandons residual, and uncap standing 30s, adds 20 μ L ultrapure waters, and vibration is dissolved stand-by.
(2) fluorescent PCR detects; Get above-mentioned solution 2 μ L respectively to get yin and yang attribute simultaneously and contrast 2 μ L and add containing 18 μ L/ pipe PCR Master mix 8 PCR reaction tubess or as follows containing carrying out amplification amplification program after mixing in 18 μ L/ pipe PCR Master mix 96 orifice plates on quantitative real time PCR Instrument: 95 DEG C of denaturation 10 min; 95 DEG C of sex change 15 s, 60 DEG C of annealing 60 s, 40 circulations.
(3) result judges: reaction solution measure Ct value be all greater than 35 or without the sample of numerical value as feminine gender; The sample of Ct≤30 is positive sample; The sample measuring 30 < Ct < 35 needs repetition measurement, and repetition measurement result Ct < 35 is positive, and repetition measurement result Ct >35's is negative.
The present invention and prior art contrast and are characterized in:
1, detect easy, quick, efficient: utilize this test kit only need prepare whizzer, quantitative real time PCR Instrument and pipettor and supporting rifle head, other consumptive materials without the need to prepare, easy to use, be applicable to onsite application.This detection method is after quantitative PCR reaction terminates, result judgement can be carried out immediately by instrument software, do not need agarose gel electrophoresis, EB dyeing observations, decrease environmental pollution and sample cross contamination, judge only to need 2 hours from nucleic acid extraction to result; Once can carry out the detection of 96 samples, there is high efficiency.
2, accurate quantitative analysis can be realized: by preparation standard product and drawing standard curve, then according to the Ct value of testing sample, can carry out quantitatively the Aleutian Mink Disease Parvovirus in testing sample, accuracy is high, has accomplished detection by quantitative truly.
3, sensing range is wide, highly sensitive, is 10 at standard substance 2-10 8have fabulous linear relationship within the scope of copy, sensing range can reach 8 orders of magnitude.
Accompanying drawing explanation
Figure 1A MDV gene amplification curve.
Fig. 2 AMDV gene melting curve.
Fig. 3 AMDV gene recombination plasmid typical curve.
Embodiment
The primer (the raw work in Shanghai) of AMDV is designed with reference to Aleutian Mink Disease Parvovirus G strain sequence (NC_001662.1) of having announced in NCBI, and by BLAST Preliminary detection primer specificity.Best primer sequence is: upstream primer SEQ ID N0.1 5'-CCCTCCAGGTCAACTCTTTGTTAAA-3' and downstream primer SEQ ID N0.2 5'-GCCTCTCCAAGTAAAGTAACCATAGG-3', expected product amplification length 109 bp.
AMDV gene fragment pcr amplification reaction carries out at EASYCycler gene-amplificative instrament, and reaction system is 20 μ L, comprising: 10 μ L Premix Taq (TaKaRa), 8 μ L ddH 2o, 1 μ L Aleutian Mink Disease Parvovirus G strain template, upstream and downstream primer (5 μm of ol/L) mixture 1 μ L.Amplification is: 94 DEG C of denaturation 10s, 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, and 72 DEG C extend 30s, totally 30 circulations; 7min is extended after 72 DEG C.
Reaction product 2% agarose gel electrophoresis, reclaims test kit (TaKaRa) and reclaims by target stripe gel, the PCR primer of recovery is used pMD19-T Vector(TaKaRa) connect, in the PCR pipe of 0.2 mL, get PCR reclaim product 4 μ L and pMd19-T Vector 1 μ L, adds 5 μ L and connects damping fluid, 16 DEG C of connections of spending the night.Connecting fluid full dose (10 μ l) is added to 100 μ l and dissolves in ice in Trans5 α (the full formula gold in Beijing) competent cell, places 30 min in ice.After 42 DEG C of 45 seconds of heating, then place 1min in ice.Add 890 μ L SOC substratum, 37 DEG C of shaking culture 60min.L-Agar Plating containing X-Gal, IPTG, Amp is cultivated, forms single bacterium colony.Select white colony, use PCR method to confirm the length scale of Insert Fragment in carrier.
From flat board, picking 4 clone inoculates in the LB substratum of 5mL 100 μ g/mL ammonia benzyl, cultivates 14h for 37 DEG C.Get 4mL incubated overnight bacterium liquid, with the little extraction reagent kit of plasmid (TaKaRa) extracting plasmid.Plasmid is sent to check order (the raw work in Shanghai).
By plasmid correct for sequencing result, with BioPhotometer(eppendorf) measure concentration, calculate purified product copy number.Preparation 1 × 10 10the solution of copies/ μ L as standard substance stoste ,-80 DEG C of preservations.
Standard substance stoste 10 times of gradient dilutions are become 10 10-10 0copies/ μ L standard substance utilize this test kit to detect on quantitative real time PCR Instrument, occur that the minimum concentration gradient of standard " S " type amplification curve is as the sensitivity detected using threshold cycle number (Ct)≤30.Standard substance stoste 10 times of gradient dilutions are got 10 9, 10 8, 10 7, 10 6, 10 5, 10 4, 10 3, 10 2copies/ μ L, as standard substance, quantitative real time PCR Instrument carries out real-time fluorescence quantitative PCR detection, and each sample 3 times repeats, reaction conditions as above, according to C tand template starting copy number production standard curve and standard equation.
As can be seen from amplification curve (Fig. 1): totally see that all knee point are clear, exponential phase, is obvious, and the overall collimation of amplification curve is good, and baseline is flat without raising up phenomenon, and low concentration sample amplification curve exponential phase is obvious; As can be seen from Fig. 2 melting curve, template gradient melting curve only has 1 obvious peak, illustrates that design of primers does not rationally produce non-specific amplification and dimer.Present method sets up AMDV real-time fluorescence PCR quantitation curves (Fig. 3) equation; Y=-3.082X+36.762; r 2=0.995, wherein Y represents Ct value, and X represents the logarithmic value of template amount.Its linearity range can reach 8 orders of magnitude.
Get 8 parts of mink serum, in each 1.5mL plastic centrifuge tube, add 200 μ L test serums.Add 400 μ L employing virus cracking liquids, after vibration 40s, room temperature places 10min.600 μ L Virahols are added after vibration 30s mixing, after vibration 45s mixing, the centrifugal 16min of 15000g room temperature.Move and abandon supernatant, add 1.2mL75% ethanol, vibrate the centrifugal 5min of 15000g room temperature after 15 seconds, abandons supernatant, and centrifugal 15s abandons residual, and uncap standing 30s, adds 20 μ L ultrapure waters, and vibration is dissolved stand-by.
Get above-mentioned solution 2 μ L respectively to get yin and yang attribute simultaneously and contrast 2 μ L and add containing carrying out amplification amplification program after mixing in 18 μ L/ pipe PCR Master mix 8 PCR reaction tubess on quantitative real time PCR Instrument as follows: 95 DEG C of denaturation 10 min; 95 DEG C of sex change 15 s, 60 DEG C of annealing 60 s, 40 circulations.6 parts of aobvious positives as a result, positive rate 75%.
SEQUENCE LISTING
 
<110> Gao Yun
 
The primer of a <120> rapid detection Aleutian Mink Disease Parvovirus, test kit and method
 
<130> 1
 
<160> 3
 
<170> PatentIn version 3.3
 
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ccctccaggt caactctttg ttaaa 25
 
 
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gcctctccaa gtaaagtaac catagg 26
 
 
<210> 3
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<212> DNA
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ccctccaggt caactctttg ttaaactaac agaaaacctc actgatacat ttaactatga 60
 
tgaaaatcca gacagaataa aaacctatgg ttactttact tggagaggc 109
 
 

Claims (6)

1. the primer of a rapid detection Aleutian Mink Disease Parvovirus, test kit and method, it is characterized in that, its primer nucleotide sequences is: upstream primer sequence is SEQ ID N0.1 5'-CCCTCCAGGTCAACTCTTTGTTAAA-3' and downstream primer sequence is SEQ ID N0.2 5'-GCCTCTCCAAGTAAAGTAACCATAGG-3'.
2. the test kit containing rapid detection Aleutian Mink Disease Parvovirus amplimer described in claim 1.
3. the primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and a method, this test kit comprises: positive control: 1 × 10 2copies/ μ L contains aleutian disease virus partial sequence pMD19-T-amdv-f1 plasmid (amdv-f1 plasmid sequence is as shown in SEQ ID N0.3) is packed as 25 μ L/ and manages ,-20 DEG C of preservations; Negative control and dissolving DNA water: after ultrapure water autoclaving, be packed as 2.5mL/ pipe ,-20 DEG C of preservations; Employing virus cracking liquid: containing the employing virus cracking liquid of 3mol/L guanidinium isothiocyanate, 0.4% sarcosyl, 0.12 mmol/L beta-mercaptoethanol, 20mmol/L Trisodium Citrate, the packing of 40mL/ bottle ,-4 DEG C of preservations; Virahol: 60 mL/ bottle packing, normal temperature is preserved; 75% ethanol: prepare 75% ethanol after ultrapure water autoclaving, the packing of 120mL/ bottle, normal temperature is preserved; PCR Master mix: get commercialization 2 × SYBR Green PCR Master mix, aseptic ultrapure water, 5 μm of ol/L upstream and downstream primer mixtures in the mixing of 10:7:1 ratio, 18 μ L/ pipes are sub-packed in 8 connecting legs or 96 orifice plates, and-20 DEG C keep in Dark Place.
4. the primer of a rapid detection Aleutian Mink Disease Parvovirus, test kit and method, this test kit comprises: each 1 pipe of positive control, negative control and dissolving DNA water, each 1 bottle of employing virus cracking liquid, Virahol, 75% ethanol, 1.5mL centrifuge tube 96, containing 18 μ L/ pipe PCR Master mix 8 PCR reaction tubess 12 groups or containing 18 μ L/ pipe PCR Master mix 96 orifice plate 1 piece.
5. the primer of rapid detection Aleutian Mink Disease Parvovirus, test kit and a method, the method comprises the following steps: the DNA extraction of (1) sample to be checked; Add 200 μ L testing liquid samples in 1.5mL plastic centrifuge tube, add 400 μ L employing virus cracking liquids, after vibration 40s, room temperature places 10min, adds 600 μ L Virahols after vibration 30s mixing, after vibration 45s mixing, and the centrifugal 16min of 15000 g room temperature; Abandon supernatant, add 1.2mL 75% ethanol, the centrifugal 5min of 15000 g room temperature after vibration 15s, abandon supernatant, centrifugal 15s abandons residual supernatant, and uncap standing 30s, adds 20 μ L ultrapure waters, and vibration is dissolved stand-by; (2) fluorescent PCR detects: get above-mentioned solution 2 μ L and respectively get yin and yang attribute simultaneously and contrast 2 μ L and add and increase on quantitative real time PCR Instrument after mixing containing 18 μ L/ pipe PCR Master mix 8 PCR reaction tubess or containing in 18 μ L/ pipe PCR Master mix 96 orifice plates.
6. amplification program is as follows: 95 DEG C of denaturation 10 min; 95 DEG C of sex change 15 s, 60 DEG C of annealing 60 s, 40 circulations; (3) result judges: reaction solution measure Ct value be all greater than 35 or without the sample of numerical value as feminine gender; The sample that FAM passage measures Ct≤30 is positive sample; Measure the sample repetition measurement of 30 < Ct < 35, repetition measurement result Ct < 35 is positive, and repetition measurement result Ct >35's is negative.
CN201410084204.8A 2014-03-10 2014-03-10 Primers, kit and method for rapidly detecting Aleutian mink disease virus Pending CN104911276A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652594A (en) * 2019-01-09 2019-04-19 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） One kind is for detecting the PCR kit for fluorescence quantitative and application thereof of Aleutian Mink Disease Parvovirus (AMDV)
CN114250322A (en) * 2021-12-24 2022-03-29 吉林农业大学 Dual fluorescent quantitative PCR kit for detecting mink circovirus and mink Aleutian virus

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110086339A1 (en) * 2009-08-21 2011-04-14 Mark Stahl Elimination of pathogenic infection in farmed animal populations
CN102154447A (en) * 2010-12-06 2011-08-17 中国农业科学院特产研究所 Method for screening potential Aleutian disease resistant minks
CN102181581A (en) * 2011-04-21 2011-09-14 中国农业科学院特产研究所 Ternary PCR (polymerase chain reaction) kit for canine distemper virus, enteritis parvovirus and Aleutian disease virus

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110086339A1 (en) * 2009-08-21 2011-04-14 Mark Stahl Elimination of pathogenic infection in farmed animal populations
CN102154447A (en) * 2010-12-06 2011-08-17 中国农业科学院特产研究所 Method for screening potential Aleutian disease resistant minks
CN102181581A (en) * 2011-04-21 2011-09-14 中国农业科学院特产研究所 Ternary PCR (polymerase chain reaction) kit for canine distemper virus, enteritis parvovirus and Aleutian disease virus

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652594A (en) * 2019-01-09 2019-04-19 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） One kind is for detecting the PCR kit for fluorescence quantitative and application thereof of Aleutian Mink Disease Parvovirus (AMDV)
CN114250322A (en) * 2021-12-24 2022-03-29 吉林农业大学 Dual fluorescent quantitative PCR kit for detecting mink circovirus and mink Aleutian virus
CN114250322B (en) * 2021-12-24 2024-04-30 吉林农业大学 Dual-fluorescence quantitative PCR (polymerase chain reaction) kit for detecting mink circovirus and mink Albikino virus

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