CN107988427A - Prawn hepatopancreatic parvovirus（HPV）RAA constant temperature fluorescence detection method and reagent - Google Patents
Prawn hepatopancreatic parvovirus（HPV）RAA constant temperature fluorescence detection method and reagent Download PDFInfo
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Abstract
The invention discloses a kind of prawn hepatopancreatic parvovirus（HPV）RAA constant temperature fluorescence detection method and detection kit.Detection kit includes forward primer SEQ ID NO.1, reverse primer SEQ ID NO.2, specificity fluorescent probe SEQ ID NO.3, reaction solution, restructuring polymerase and reference substance.The kit high specificity of the present invention；Detection sensitivity is high, can reach 2fg/ μ L；Accuracy is high, reliable；It is simple and efficient to handle, it is adapted to Site Detection, has a wide range of applications scene.
Description
Technical field
The invention belongs to technical field of molecular biology, it is related to the detection method of marine aquaculture industry, and in particular to one
The RAA constant temperature fluorescence detection method and kit of kind prawn hepatopancreatic parvovirus.
Background technology
The rear molding larva of hepatopancreatic parvovirus (Hepatopancreatic Parvovirus, HPV) main infection prawn
(postlarvae) and young shrimp, general disease shrimp appearance is without obvious specific symptoms, and take action after being infected torpescence, appetite of young shrimp subtracts
Move back, is slow-growing, seldom husking, body surface often has many commensalism biologies or debris attachment；The juvenile prawn of cultivation phase or into shrimp, shrimp body
It is thin and weak, body colour is deeper, there are a large amount of black splotches on some shrimp body crust surfaces of falling ill, sometimes crust soften, abdominal muscles bleach, resist
Inverse performance force difference, the Secondary bacterium infections of Chang Bingfa are most common with vibrios (Vibrio spp.), and shrimp body of falling ill sometimes is except secondary
Outside property bacterium infection infected grass shrimp (P.monodon) can often detect grass shrimp baculoviral (Monodon baculovirus,
MBV=Pm SNPV) and white spot syndrome time group's virus (white spot syndrome virus, WSSV).(Umesha RK
Et al, 2003, R), very big death is caused, up to 50~90%, and as shrimp body increases, the state of an illness mitigates its death rate；Seed shrimp
More in subclinical infection with virus.The organ of the target of shrimp hepatopancreatic parvovirus infection is the remote cecum epithelium E cells of hepatopancrease pipe
And promesenteron back segment epithelial cell, and replicated in nucleus, and destroy histocyte.
Hepatopancreatic parvovirus is Single-stranded DNA virus (Single-Strand DNA), in nucleus internal breeding and forms circle
Shape or oval inclusion body (inclusion body), are positive, virion size is with infection through Feulgen Albert'stain Alberts
Shrimp species and it is variant；Grass shrimp hepatopancrease virion is such as infected to take for 22-24nm (Lightner＆Redman, 1985), infection bar
Horse prawn (banana shrimp, Penaeus=Fenneropenaeus merguiensis), hepatopancrease virion is 21-
22nm (Roubal et al.1989), infection spot section shrimp (kuruma shrimp, P.=Marsupenaeus japonicus),
Hepatopancrease virion is 17-20nm (Spann et al, 1997), and average virus particle is 22-24 μm.Existing frequently-used inspection
Survey method is PCR- electrophoresis, complicated.In addition, PCR detection method need expensive instrument and equipment, compared with high detection expense with
And it is set not to be suitable for the promotion and popularization of prawn scene to the higher technical requirements of testing staff.
Recombinase-mediated amplification (Recombinase-aid Amplification, RAA) technology be also it is a kind of at a constant temperature
It can make the method for nucleic acid rapid amplifying.Unlike RPA, RAA amplification uses what is obtained from bacterium or fungi
Recombinase, under 37 DEG C of constant temperature, which can combine closely with primed DNA, the condensate of enzyme and primer be formed, when primer exists
When the sequence of complete complementary therewith is searched on template DNA, in single-stranded DNA binding protein (single-strandedDNA
Binding, SSB) with the help of, template DNA is unwind, and under the action of archaeal dna polymerase, form new DNA complementary strands, instead
It is also to be increased with exponential to answer product, can obtain that the amplification piece that agarose gel electrophoresis detects can be used usually in 1h
Section.Fluorophor is added in RAA reaction systems, whole RAA amplification procedures are monitored in real time using the accumulation of fluorescence signal, 20 points
, it can be achieved that quantitative and qualitative analysis to starting template in clock.Entirely react simple and quick, because high temperature circulation is not required, institute
To be particularly suitable for using in the non-test in laboratory place for having a large amount of samples, suitable for field of rapid food detection.
The content of the invention
In view of this, the object of the present invention is to provide the RAA constant temperature fluorescence nucleic acid inspection of prawn hepatopancreatic parvovirus (HPV)
Test agent box and detection method.
In order to achieve the above object, the present invention uses following technical scheme：
A kind of detection kit of prawn hepatopancreatic parvovirus (HPV) nucleic acid, including：Prawn hepatopancreatic parvovirus is just
To primer, reverse primer and specificity fluorescent probe, wherein the prawn hepatopancreatic parvovirus forward primer nucleotides sequence
Row are as shown in SEQ ID NO.1, the prawn hepatopancreatic parvovirus reverse primer nucleotide sequence such as SEQ ID NO.2 institutes
Show, the nucleotide sequence such as SEQ ID NO.3 of the specificity fluorescent probe, its 5 ' end is marked with fluorescent reporter group, 3 ' ends
It is marked with fluorescent quenching group.
In some embodiments, the fluorescent reporter group of the specificity fluorescent probe be selected from FAM, VIC, JOE, TET,
One kind in CY3, CY5, ROX, Texas Red or LC RED460, fluorescent quenching gene be selected from BHQ1, BHQ2, BHQ3,
One kind in Dabcy1 or Tamra.
In some embodiments, the kit for detecting nucleic acid, further includes primer mixed liquor, specificity fluorescent is visited
Pin, A Buffer, B Buffer, RAA powdered reagents, prawn hepatopancreatic parvovirus standard items and ddH2At least one of O.
In some embodiments, the kit, wherein, the ABuffer is 20%PEG；B Buffer are
280mM MgAc。
In some embodiments, the kit, wherein, the component of the RAA powdered reagents is as follows：1mmol/
L dNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA restructuring zymoproteins (SC-recA/BS-recA) or 30ng/ μ L
Rad51,30ng/ μ LBsu archaeal dna polymerases, 100mmol/L Tricine, 20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/
μ L creatine kinases, Exo exonucleases.
In some embodiments, the kit for detecting nucleic acid, prawn hepatopancreatic parvovirus standard items be containing
The positive plasmid of prawn hepatopancreatic parvovirus conserved region gene partial sequence.
In some embodiments, the kit, it is described to contain prawn hepatopancreatic parvovirus conserved region gene portion
The sequence of the positive plasmid of sub-sequence is as shown in SEQ ID NO.4.
Present invention also offers a kind of RAA constant temperature fluorescence detection methods of prawn hepatopancreatic parvovirus, test sample is treated in extraction
The DNA of product, using the DNA of sample to be tested as template, in the forward primer, reverse primer, specificity of prawn hepatopancreatic parvovirus
Fluorescence probe and RAA powdered reagents, ABuffer, B Buffer and ddH2Real-time fluorescence RAA reactions are carried out in the presence of O, according to reality
When fluorescence RAA amplification curves analysis sample to be tested；Wherein described prawn hepatopancreatic parvovirus forward primer nucleotide sequence is such as
Shown in SEQ ID NO.1, the prawn hepatopancreatic parvovirus reverse primer nucleotide sequence as shown in SEQ ID NO.2, institute
The nucleotide sequence such as SEQ ID No.3 of specificity fluorescent probe are stated, its 5 ' end is marked with fluorescent reporter group, and 3 ' ends are marked with
Fluorescent quenching group.
In some embodiments, the implementation fluorescence RAA response procedures are：37 DEG C, 40s；37 DEG C, 20min, amount to 40
A circulation；
Detection method of the present invention needs real-time fluorescence RAA after reaction, is analyzed using real-time fluorescence RAA instrument soft
Part, sample to be tested is analyzed according to the amplification curve of real-time fluorescence RAA.Preferably, the analysis sample to be tested is sample to be tested FAM
Channel fluorescence curve is in " S " type and CT value≤35, is judged as prawn hepatopancreatic parvovirus positive findings；When sample to be tested curve
It is not in " S " type or CT values ＞ 35, is judged as prawn hepatopancreatic parvovirus negative findings.
Beneficial effect
1st, rapidly and efficiently：Whole amplification only needs 20-30min to complete, and amplification yield can reach 109-1010It is a to copy
Shellfish；
2nd, it is easy to operate：Special reagent is not required, it is not necessary to carry out the tedious steps such as the deformation of double-stranded DNA in advance, only need
The luminoscope of constant temperature is wanted, condition is gentleer；
3rd, high specific：The present invention is to prawn others illness prawn infectious hypodennal and haematopoietic necrosis virus
(IHHNV), prawn Acute Hepatic pancreatic necrosis (AHPND), White Spot Syndrome Virus (WSSV), prawn irido virus
(SIV), the DNA of prawn liver sausage born of the same parents worm (EHP) is not expanded.
4th, high sensitivity：The detectable limit of the present invention can reach 2fg/ reactions
5th, identification is simple：According to real-time fluorescence data, amplification is directly judged, without electrophoresis detection, be adapted to scene inspection
Survey.
Brief description of the drawings
Fig. 1 is 4 pairs of primer RAA amplification curve diagrams involved in the present invention.
Fig. 2 is RAA detection methods to the sensitivity experiment figure of HPV, be from left to right followed successively by 2pg/ μ L, 200fg/ μ L,
20fg/ μ L, 2fg/ μ L, 0.2fg/ μ L positive criteria product amplification.
Fig. 3 is specificity experiments figure of the RAA detection methods to HPV.
Specific implementation method
Below by way of specific embodiment, the present invention is further described, but is not limited thereto.
Embodiment 1：
The present invention searches for prawn hepatopancreatic parvovirus strain to prawn hepatopancreatic parvovirus in Genebank databases
Gene order, is compared multisequencing using 6.0 softwares of DNAMAN, finds out conservative section.4 are devised in conservative region
Group primer and probe, and BLAST comparisons are carried out in ncbi database, the sequence of primer and probe is as shown in table 1.Positive sample
Amplification curve is as shown in Figure 1.
1 primer and probe sequence of table:
By Fig. 1 results as it can be seen that the amplification curve of the 4th group of primer and probe is the most typical, there are obvious exponential phase and platform
Phase, has compared with high fluorescent (ordinate value), and CT values smaller (abscissa corresponding to the crosspoint of curve and threshold line) are tied
Fruit analysis in table 2.Other primed probe curve lifting heights are relatively low, and CT values are larger, plateau unobvious；Or without expanding
Increase, missing inspection occur.Illustrate the reproduction speed of the 4th group of primer and probe purpose product faster, more, amplified reaction efficiency
Higher.
2 primed probe the selection result of table is analyzed
| Group result | CT values | Fluorescence intensity |
| First group | 11.95 | 180,000 |
| Second group | 8.21 | 310,000 |
| 3rd group | 12.03 | 160,000 |
| 4th group | 4.13 | 680,000 |
Real-time example 2：The kit prawn hepatopancreatic parvovirus
Kit for detecting nucleic acid of the present invention, further includes primer mixed liquor, specificity fluorescent probe, A Buffer, B
Buffer, RAA powdered reagent, prawn hepatopancreatic parvovirus standard items and ddH2O。
Kit of the present invention, wherein, the ABuffer is 20%PEG；B Buffer are 280mM MgAc.
Kit of the present invention, wherein, the component of the RAA powdered reagents is as follows：1mmol/L dNTP、
90ng/ μ L SSB albumen, 120ng/ μ L recA restructuring zymoproteins (SC-recA/BS-recA) or 30ng/ μ L Rad51,30ng/
μ L Bsu archaeal dna polymerases, 100mmol/LTricine, 20%PEG, 5mmol/L dithiothreitol (DTT), 100ng/ μ L creatine kinases,
Exo exonucleases.
In primer mixed liquor of the present invention, the forward primer base sequence is described as shown in SEQ ID NO.1
Shown in the base sequence SEQ ID NO.2 of reverse primer, the mol ratio of forward primer and reverse primer is SEQ ID NO.1：
SEQ ID NO.2 are 1:1.
The specific probe base sequence of prawn hepatopancreatic parvovirus provided by the invention as shown in SEQ ID NO.3,
5 ' ends of probe are marked with FAM fluorescent reporter groups, and 3 ' ends are marked with BHQ1 fluorescent quenching groups.
Prawn hepatopancreatic parvovirus standard items provided by the invention include prawn hepatopancreatic parvovirus conserved region gene
The positive plasmid of sequence, the base sequence of the plasmid is as shown in SEQ ID NO.4.
The base sequence (SEQ ID NO.4) of plasmid:
GGTAGTAAGAGCAGCAAACGAAGCAATAAGAAGTGGTGGAGATAGATTAGCAGAATTAGTACAAGCATA
CGCATCAGGATTTTCAGACAGCACAGAAATAGTAGAAGTAAGACAAGAAGATAGAGTACAGAGAGACATATTCCAAG
AAGAAGGACAGAATTTATTGGCTATTGAGATTGCATTACAAGAACCAAGCAGTGTAGCGCAACAGTTAGACCAGGAG
AGAACTCCAGCAATCAAGAGAGCTCTAGAACTAACAGCAGAAGAAGAACGGATAGAACGCATAGAAAACGCTAAGAA
ATATATTGAAGAAGTCATAGAAGAGACAAATCAAGAACTACAAGAACAAGAGAGACAAGAAGTAAGTGCGGCGGCGG
AAGATACGATGAACACTGAAGCACCCGTCCCGATGGAAACTTCTGAATCCGGAGCCACCGCCGCACCGCAGCAGCGA
GCTGCGGCGGGCGGCGGCGGTAGCGGAGGTGGAGGAGAATCTGCAGGGTACGGAAAAAACCCTAGCGATTCATTCCA
GCGCCACCGCAATAAGCCAGTTGATCTCAAACACATCGGAGACAACATATATGTGGCTCAGCGAGTTTATAAAGTAG
AGGCTGAATGTAAGCTGGTAGGCGACAAGTTATCATGGAATAACACAACAAACAGTAAATATCT
Example 3：Kit prawn hepatopancreatic parvovirus of the present invention
1st, the extraction of positive nucleic acid
1.1st, nucleic acid extraction：DNA extractions are carried out using marine animal tissue DNA extracts kit.
2nd, the configuration of RAA reaction systems：Each detection sample corresponds to a RAA reaction dry powder pipe, and each RAA reacts dry powder
Each reactive component and the volume added are as shown in table 3 in pipe.
Table 3:
| RAA reaction system components | Volume (μ L) |
| A Buffer | 12.5μL |
| B Buffer | 2.5μL |
| Primer mixed liquor | 4μL |
| Specificity fluorescent probe | 0.6μL |
| DNA profiling | 2μL |
| ddH2O | 28.4μL |
| Cumulative volume | 50μL |
A Buffer are 20%PEG；B Buffer are 280mM MgAc
3rd, the RAA reaction tubes for having configured reaction system are positioned in ABI7500 amplification instruments, are carried out according to following procedure
RAA is expanded：37 DEG C, 40s；37 DEG C, 20min, amount to 40 circulations.The fluorescence of each circulating collection FAM passages.
4th, judged after expanding according to fluorescence curve and CT values judge prawn hepatopancreatic parvovirus positive or negative knot
Fruit.
Judge result：FAM channel fluorescences curve is in " S " type and CT value≤35, is judged as prawn hepatopancreatic parvovirus sun
Property result；When sample to be tested curve is not in " S " type or CT values ＞ 35, it is judged as prawn hepatopancreatic parvovirus negative findings.
Embodiment 4：Assessment of the RAA detection kits of the present invention in clinical practice application
The experiment of clinical blind sample is carried out using kit of the present invention, detects 500 portions of prawns；Test result indicates that the present invention
The 4th primer pair can distinguish shrimp liver born of the same parents worm, it is very high with nest-type PRC positive coincidence rate.In 500 parts, nest-type PRC, has
308 parts are positive findings, and 192 parts are negative findings, and it is positive that the result detected by RAA methods, which is 309 parts, there is 191 parts
It is different there are a positive findings for negative findings, PCR amplification is carried out to this sample DNA and is sequenced, sequencing result shows this
Sample is the positive, illustrates the RAA detection reagents of the present invention and has the accuracy rate of higher.
Test example 5：The sensitivity test of kit of the present invention
The prawn hepatopancreatic parvovirus standard items plasmid that kit described in the embodiment of the present invention 2 provides, extracts positive matter
Grain, and with the concentration of NanoDrop measurement positive plasmids, and it is diluted to respectively 2pg/ μ L, 200fg/ μ L, 20fg/ μ L,
5 2fg/ μ L, 0.2fg/ μ L concentration gradients carry out sensitivity test.
Testing result is as shown in Fig. 2, be from left to right followed successively by 2pg/ μ L, 200fg/ μ L, 20fg/ μ L, 2fg/ μ L, 0.2fg/
The amplification of the positive criteria product of μ L, it can be seen that the sensitivity of the RAA amplified fluorescences reagent of the present invention and detection can
Up to 100fg/ μ L, accuracy is better than regular-PCR detection method, shows RAA constant temperature fluorescence detection reagent kit and the detection of the present invention
Diagnosis sensitivity with height of the method to HPV.
Test example 6：The specific test of kit of the present invention
In order to detect the specificity of kit of the present invention, using the detection method in example 3, respectively to viral WSSV,
IHHNV, EHP, AHPND, SIV sample are detected, and analyze detection feelings of this kit to other common virus of HPV and prawn
Condition.
Testing result shows：Only there is normal amplification, negative control (ddH in HPV samples2O) and IHHNV, WSSV, AHPND,
SIV, EHP sample do not occur expanding (as shown in Figure 3).The above results explanation, RAA constant temperature fluorescence detection reagent kit energy of the present invention
Specific amplification goes out the target sequence in HPV, without cross reaction occurs with other viral nucleic acids.Illustrate the method for the present invention and reagent
Box specificity is good, does not occur false negative.
Meanwhile 1-3 designed by the invention carries out primer same specificity experiments, it is found that these primers cannot be very
Good specifically distinguishes different samples, and specificity is not fine (summary of specific experiment data).
In the case where lacking any element specifically disclosed herein, limitation, it is possible to achieve illustrated and described herein
Invention.Used terms and expressions method is used as the term of explanation and unrestricted, and is not intended in these terms and table
Any equivalent shown in being excluded up in the use of method with the feature or part thereof, and should be realized that various remodeling exist
All it is feasible in the scope of the present invention.It is therefore to be understood that although specifically disclosed by various embodiments and optional feature
The present invention, but the modifications and variations of concept as described herein can use by those of ordinary skill in the art, and recognize
Fallen into for these modifications and variations within the scope of the present invention of the appended claims restriction.
It is specifically described herein or record article, patent, patent application and every other document and can electronically obtain
The content of information include in full to a certain extent herein by reference, just as each individually publication by specific and single
Solely point out by reference.Applicant retains from any of any this article, patent, patent application or other documents
And all material and information are incorporated into the right in the application.
Sequence table
<110>Hangzhou Zhong Ce bio tech ltd
<120>Prawn hepatopancreatic parvovirus（HPV）RAA constant temperature fluorescence detection method and reagent
<130> 17-100070-00002920
<141> 2017-11-10
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<210> 3
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<210> 4
<211> 672
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<213>Artificial sequence (Artificial Sequence)
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ggtagtaaga gcagcaaacg aagcaataag aagtggtgga gatagattag cagaattagt 60
acaagcatac gcatcaggat tttcagacag cacagaaata gtagaagtaa gacaagaaga 120
tagagtacag agagacatat tccaagaaga aggacagaat ttattggcta ttgagattgc 180
attacaagaa ccaagcagtg tagcgcaaca gttagaccag gagagaactc cagcaatcaa 240
gagagctcta gaactaacag cagaagaaga acggatagaa cgcatagaaa acgctaagaa 300
atatattgaa gaagtcatag aagagacaaa tcaagaacta caagaacaag agagacaaga 360
agtaagtgcg gcggcggaag atacgatgaa cactgaagca cccgtcccga tggaaacttc 420
tgaatccgga gccaccgccg caccgcagca gcgagctgcg gcgggcggcg gcggtagcgg 480
aggtggagga gaatctgcag ggtacggaaa aaaccctagc gattcattcc agcgccaccg 540
caataagcca gttgatctca aacacatcgg agacaacata tatgtggctc agcgagttta 600
taaagtagag gctgaatgta agctggtagg cgacaagtta tcatggaata acacaacaaa 660
cagtaaatat ct 672
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<213>Artificial sequence (Artificial Sequence)
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<210> 8
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<213>Artificial sequence (Artificial Sequence)
<400> 8
tagaagtaag acaagaagat agagtacag 29
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<213>Artificial sequence (Artificial Sequence)
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<213>Artificial sequence (Artificial Sequence)
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<210> 11
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caagaagata gagtacagag agacatattc c 31
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cttgtagttc ttgatttgtc tcttctatga c 31
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Claims (8)
- A kind of 1. prawn hepatopancreatic parvovirus（HPV）The detection kit of nucleic acid, including：Prawn hepatopancreatic parvovirus is positive Primer, reverse primer and specificity fluorescent probe, wherein the prawn hepatopancreatic parvovirus forward primer nucleotide sequence As shown in SEQ ID NO.1, the prawn hepatopancreatic parvovirus reverse primer nucleotide sequence as shown in SEQ ID NO.2, The nucleotide sequence of the specificity fluorescent probe such as SEQ ID NO.3, its 5 ' end are marked with fluorescent reporter group, 3 ' end marks There is fluorescent quenching group.
- 2. kit for detecting nucleic acid according to claim 1, the fluorescent reporter group of the specificity fluorescent probe is selected from One kind in FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460, fluorescent quenching gene are selected from One kind in BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.
- 3. according to the kit for detecting nucleic acid described in claim 1 and 2, primer mixed liquor, specificity fluorescent probe, A are further included Buffer, B Buffer, RAA powdered reagents, prawn hepatopancreatic parvovirus standard items and ddH2At least one of O.
- 4. kit according to claim 3, wherein, the A Buffer are 20% PEG；B Buffer are 280mM MgAc。
- 5. kit according to claim 5, wherein, the component of the RAA powdered reagents is as follows：1mmol/L DNTP, 90ng/ μ L SSB albumen, 120ng/ μ L recA restructuring zymoproteins（SC-recA/BS-recA）Or 30ng/ μ L Rad51, 30ng/ μ L Bsu archaeal dna polymerases, 100mmol/L Tricine, 20% PEG, 5mmol/L dithiothreitol (DTT), 100ng/ μ L fleshes Acid kinase, Exo exonucleases.
- 6. according to the kit for detecting nucleic acid described in claim 1-5 any one, prawn hepatopancreatic parvovirus standard items are Positive plasmid containing prawn hepatopancreatic parvovirus conserved region gene partial sequence.
- 7. kit according to claim 6, described to contain prawn hepatopancreatic parvovirus conserved region gene partial sequence Positive plasmid sequence as shown in SEQ ID NO.4.
- 8. the RAA constant temperature fluorescence detection methods of prawn hepatopancreatic parvovirus, extract the DNA of sample to be tested, with sample to be tested DNA is template, is tried in the forward primer of prawn hepatopancreatic parvovirus, reverse primer, specificity fluorescent probe and RAA dry powder Agent, A Buffer, B Buffer and ddH2Real-time fluorescence RAA reactions are carried out in the presence of O, according to real-time fluorescence RAA amplification curves Analyze sample to be tested；Wherein described prawn hepatopancreatic parvovirus forward primer nucleotide sequence as shown in SEQ ID NO.1, The prawn hepatopancreatic parvovirus reverse primer nucleotide sequence is as shown in SEQ ID NO.2, the specificity fluorescent probe Nucleotide sequence such as SEQ ID No.3, its 5 ' end is marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group.
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| CN110592275A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Infectious Spleen and Kidney Necrosis Virus (ISKNV) of mandarin fish |
| CN112251432A (en) * | 2020-10-22 | 2021-01-22 | 中国水产科学研究院黄海水产研究所 | Liver pancreas parvovirus and nucleocapsid protein affinity screening method and application thereof |
| CN116814848A (en) * | 2023-04-12 | 2023-09-29 | 中国医学科学院医学实验动物研究所 | Primers, probes and methods for detecting mouse parvovirus based on fluorescent RAA |
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| CN110592275A (en) * | 2018-06-13 | 2019-12-20 | 杭州众测生物科技有限公司 | RAA constant temperature fluorescence detection method and reagent for Infectious Spleen and Kidney Necrosis Virus (ISKNV) of mandarin fish |
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| CN116814848A (en) * | 2023-04-12 | 2023-09-29 | 中国医学科学院医学实验动物研究所 | Primers, probes and methods for detecting mouse parvovirus based on fluorescent RAA |
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