CN108950067A - Detect LAMP primer group, kit and its method of prawn irido virus - Google Patents
Detect LAMP primer group, kit and its method of prawn irido virus Download PDFInfo
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Abstract
The invention discloses LAMP primer group, kit and its method of detection prawn irido virus, which includes a pair of of outer primer, a pair of of inner primer and a pair of of ring primer, and nucleotide sequence is as shown in NO.1~6 SEQ ID.High sensitivity, the specificity of primer sets and detection kit provided by the invention are high and stability is good, and the lowest detection limit can reach 1fg/ μ L.Detection method provided by the invention have the characteristics that rapidly and efficiently, easy to operate and identification it is easy, only need 30~60min that entire amplification procedure can be completed, expand yield up to 109~1010A copy, the early monitoring suitable for prawn irido virus subclinical infection.
Description
Technical field
The present invention relates to LAMP primer group, the examinations of technical field of molecular biology, more particularly to detection prawn irido virus
Agent box and its method.
Background technique
Prawn haemocyte irido virus (Shrimp hemocyte iridescent virus, SHIV), earliest by
Lightner and Redman is in 1993 in the rough prawn (Protrachypene of original in Ecuadorian prawn culturing field
Precipua Burkenroad) in be found.
Pathological study discovery, the epidermis epithelial cell of former rough prawn are carried out to the rough prawn of original of infection irido virus
Typical irido virus infection is presented in (gill, stomach internal layer are even throughout whole body)；Hematopoietic tissue, antennal gland epithelial tissue and the heart
Dirty, muscle, subcutaneous tissue, the gill, the phoirocyte in hepatopancrease and phagocyte are also infected by irido virus.
For the control method of the disease, domestic and foreign scholars are it is believed that comprehensive prevention is the method for relative efficiency, i.e., as early as possible
It was found that disease and many measures is taken to stop virus transmission.Therefore, sensitive, accurate, quick and easy SHIV Pathogen test is established
Method is the important channel for reducing iris disease and occurring and endangering.
Currently, the inspection whether prawn infects irido virus generallys use the micro- sem observation of pathology, biochemical measurement, is immunized
Learn test and cell culture etc., it is these method time-consuming, effort, complicated for operation, it is difficult to adapt to the current quick, need that accurately detect
It wants.
In recent years, there is a kind of new amplification method-ring mediated isothermal amplification (loop-mediated in molecular biology
Isothermal amplification, LAMP) technology, this method can rely on drawing for 6 specific regions on identification target sequence
Object and a kind of archaeal dna polymerase with strand displacement characteristic efficient, quick, high can specifically expand target sequence under isothermal conditions.
LAMP is a kind of novel nucleic acids amplification technique of the foundation such as Notomi, under 60~65 DEG C of constant temperature, it is only necessary to 30~60min
Nucleic acid amplification reaction can be completed.It can directly be sentenced by the variation of fluorescence signal by the way that fluorescent dye is added in LAMP reaction
Determine reaction result.The technology has the following characteristics that (1) high specificity: LAMP can be from the gene sample for differing only by 1 nucleotide
In, it finds out corresponding target sequence and is expanded；(2) isothermal efficiency: LAMP is expanded under isothermal conditions, will not be due to temperature changes
Leeway is caused, and is influenced by non-target sequences small, compared with PCR, detectable limit is smaller, only several copies；
(3) time-consuming short: can be target sequence amplification to 10 in 1h9Times；(4) product easily detects.LAMP is because having high specific, high efficiency
And the characteristics of fast reaction, the detection and communicable disease of aquatic products virus can be used for the target sequence needed for massive amplification
Diagnosis.
Application publication number is that the application for a patent for invention document of CN108034768A discloses a kind of prawn irido virus shell type
PCR detection kit, the kit include two pairs of specific primers: outer primer: upstream IVF145:
GGAGCATTGAAGTTGGATAC；Downstream IVR961:GAAGGATGGATACAACAACA；Inner primer: upstream IVF300:
AGTAATCGGCAGTCATCAC；Downstream IVR646:GGTGAATGGTGGTCTTATCC.
To disclose a kind of prawn irido virus real-time for the application for a patent for invention document that application publication number is CN108018379A
Fluorescent quantificationally PCR detecting kit, the kit includes specific primer: IVQF:
CAATCATGTTGTTGTATCCATCCTTCT；IVQR:GGTTTCATTCAACGTAAACGATCTCG.
Above two method is based respectively on the operating method and real time fluorescence quantifying PCR method of Standard PCR, can increase pair
Cause of disease recall rate when shrimp irido virus subclinical infection, the early monitoring suitable for prawn irido virus subclinical infection；But often
It is not high to advise PCR primer specificity, is easy to appear false positive, and regular-PCR needs to be differentiated by electrophoresis result, only can only
As Qualitative Identification, operating process is complicated, and be easy to cause pollution；And quantitative fluorescent PCR solves false sun to a certain extent
The problem of property, it is also able to carry out quantitative detection, but its probe cost is excessively high, and needs to use fluorescence quantitative PCR instrument, it need to be in reality
Room completion is tested, the detection to prawn irido virus of field quickly, easy is unfavorable for.
LAMP detection kit of the present invention is due to being 4 species-specific primers for the design of 6 regions of target sequence, 6 areas
Any region and primer mismatch not can be carried out nucleic acid amplification in domain, increase the specificity of primer, effectively reduce false positive
Generation, and do not need initial denaturation and temperature cycles, can complete to test under the conditions of a temperature, shorten Check-Out Time.
Summary of the invention
The first object of the present invention is to provide a kind of for detecting the LAMP primer group of prawn irido virus, the primer sets
High sensitivity, specificity is high and stability is good, the early monitoring suitable for prawn irido virus subclinical infection.
The second object of the present invention is to provide a kind of for detecting the kit of prawn irido virus, and the kit is same
Also have the characteristics that highly sensitive, high specific and high stability, the early monitoring suitable for prawn irido virus subclinical infection.
The third object of the present invention is to provide a kind of method for detecting prawn irido virus, and this method can be quick
Efficiently expanded, it is not only easy to operate, but also identify easy to be also more easy.
Specific technical solution is as follows:
The present invention provides a kind of LAMP primer group for detecting prawn irido virus, including a pair of of outer primer, draw in one pair
Object and a pair of of ring primer, nucleotide sequence are as follows:
SHIV-F3:CAATCCAGAATTTTACAACTTGT；
SHIV-B3:TCAATGTTGGGGAATACCTT；
SHIV-FIP:GACTTCCATCCCTGAAAACATCATTTTTTAATAAAAATCCCAATTTGATCGC；
SHIV-BIP:AACTCCCAATCGATTACATTGACTTTTTGAAAAAGTTGTCTACTGCGA；
SHIV-LF:CGAAATCGTATACACCGTTCTTGAA；
SHIV-LB:GGCACGATTGATCATCCCGAAGT。
The present invention also provides a kind of kits for detecting prawn irido virus, including LAMP primer group, the LAMP to draw
The LAMP primer group of object group detection prawn irido virus as described above.
Preferably, the volume of outer primer is 0.5~2 μ L in the LAMP primer group, the volume of inner primer is 0.1~1 μ
L, the volume of ring primer is 0.2~1 μ L.
Further, the kit further include: LAMP reaction solution, archaeal dna polymerase, nucleotide fluorescent dye, positive control
And negative control.
Wherein, preferably, the nucleotide fluorescent dye is Eva Green.
Preferably, the archaeal dna polymerase is Bst archaeal dna polymerase；
Preferably, LAMP reaction solution by 2~5 μ L of 10mM dNTP, 10 × ThermoPol reaction buffer, 2~5 μ L,
150mM MgSO41~3 μ L of aqueous solution composition；
Preferably, the positive control is the carrier T containing prawn irido virus ATPase gene of MP segment；
Preferably, the negative control is ultrapure water.
The present invention also provides a kind of methods for detecting prawn irido virus, comprising the following steps:
(1) DNA of measuring samples is extracted；
(2) isothermal expansion is carried out to the DNA using the LAMP primer group for detecting prawn irido virus described in claim 1
Increase, and amplification is judged according to the change in fluorescence of amplified production.
Preferably, the DNA of the measuring samples is extracted using polishing.
In the above method, the viral nucleic acid in polishing extraction supernatant is can be used in the extraction of SHIV viral DNA, without using
The method of protease digestion tissue.Sample then mainly acquires glandula digestive, front foot etc., shrimp seedling is then directly ground if adult shrimp.It is right
In the detection of peeled shrimp, first several peeled shrimps are homogenized with refiner, then a small amount of shrimp is taken to carry out milling and extracting.It saves in this way
The time of albumen enzyme effect, it is often more important that eliminate a large amount of impurity and pigment, reduce the inhibition to subsequent LAMP reaction.
Preferably, it is 25 μ L that the reaction system of the isothermal duplication, which includes: total volume,；Wherein, 3.5 μ of 10mM dNTP
2.5 μ L, 150mM MgSO of L, 10 × ThermoPol reaction buffer41.5 μ L, Bst archaeal dna polymerase of aqueous solution 0.5 μ l, 10 μM
SHIV-F3 0.2 μ L, 10 μM of SHIV-B30.2 μ L, 10 μM of SHIV-FIP0.8 μ L, 10 μM of SHIV-BIP 0.8 μ L, 10 μM
SHIV-LF0.4 μ L, 10 μM of 0.4 μ L of SHIV-LB, DNA profiling 2 μ L, surplus ddH2O。
Preferably, the reaction condition of the isothermal duplication are as follows: 63 DEG C of 30~60min of reaction.
Currently, the fluorescent dye that isothermal duplication uses is SYBR Green I；Through experiments, it was found that SYBRGreen I is to this
Invention Lamp reaction has certain inhibiting effect, and will appear non-specific amplification in amplification procedure, therefore preferably, institute
Stating the nucleotide fluorescent dye added in the reaction system of isothermal duplication is Eva Green.
Further, the method for the judgement amplification are as follows: fluorescence signal is collected, if there is " S " type amplification curve
Then it is determined as the positive；If being determined as feminine gender without " S " type amplification curve.
Compared with prior art, the invention has the following advantages:
(1) LAMP primer group provided by the invention is six specific primers, can expand 6 regions of target sequence, 6
Any region and primer mismatch not can be carried out nucleic acid amplification in region, therefore its specificity is high, and highly stable, and formation is drawn
The probability of object dimer is low, ensure that going on smoothly for reaction；The lowest detection limit can reach 1fg/ μ L.
(2) detection method rapidly and efficiently, only needs 30~60min that entire amplification procedure can be completed, and expands yield
Up to 109~1010A copy.
(3) detection method is easy to operate, does not need complicated instrument, does not need special reagent, does not need in advance
Carry out the tedious steps such as the denaturation of double-stranded DNA, it is only necessary to which controlling reaction temperature can react and detect for 63 DEG C, condition temperature
With.
(4) detection method identification is easy, by the way that Eva Green is added, is believed according to the fluorescence that instrument is read in real time
Number judge amplification, without other any analytical procedures such as electrophoresis, is suitble to on-site test.
Detailed description of the invention
Fig. 1 is the fluorescence detection result after the reaction of 1 isothermal amplification of embodiment.
Fig. 2 is the result of primer specificity experiment in embodiment 1.
Fig. 3 is the sensitivity experiment of distinct methods in embodiment 1；
Wherein, A:LAMP is detected；B nesting PCR detection.
Specific embodiment
The invention will be further described combined with specific embodiments below, and what is be exemplified below is only specific implementation of the invention
Example, but protection scope of the present invention is not limited only to this.
Embodiment 1
1, experimental material
Sample 1: in the adult prawn that 2017.06.16 is obtained in Pinghu.
Sample 2: in the shrimp seedling that 2018.04.02 is obtained in Hangzhou.
Positive quality control product: nucleotide sequence is as shown in SEQ ID NO.7.
2, the extraction of prawn irido virus DNA
(1) experiment sample (sample 1 takes the parotid gland, sample 2 to be rounded a shrimp seedling) for weighing 30mg, is added the physiology salt of 500 μ L
Water removes liquid after being mixed by inversion, it is primary to repeat the step；
(2) clean shrimp seedling is placed in grinding pipe, a grinding bead (diameter 8mm) is added, the cracking of 100 μ L is added
Liquid is put into homogeneous instrument, is homogenized 20s with 6m/s；
(3) lysate of 100 μ L is added, and the Proteinase K of 20 μ L, 56 DEG C of warm bath 1h are added；
(4) the combination liquid of 200 μ L is added, places 10min at 70 DEG C；
(5) dehydrated alcohol of 200 μ L is added, is added in adsorption column after being mixed by inversion, 12000rpm is centrifuged 30s；
(6) waste liquid is outwelled, the salt washing lotion of 500 μ L is added into adsorption column, 12000rpm is centrifuged 30s；
(7) waste liquid is outwelled, the rinsing liquid of 600 μ L is added into adsorption column, 12000rpm is centrifuged 30s；
(8) step 7 is repeated；
(9) rinsing liquid is thoroughly dried, 100 μ LTE Buffer are added, centrifugation obtains prawn irido virus DNA.
3, the design of LAMP primer
Design of primers is carried out by the website http://loopamp.eiken.co.jp/e/lamp/primer.html,
Obtain one group of LAMP primer group, including a pair of of outer primer, a pair of of inner primer and a pair of of ring primer, nucleotide sequence are as follows
(NO.1~6 SEQ ID):
SHIV-F3:CAATCCAGAATTTTACAACTTGT；
SHIV-B3:TCAATGTTGGGGAATACCTT；
SHIV-FIP:GACTTCCATCCCTGAAAACATCATTTTTTAATAAAAATCCCAATTTGATCGC；
SHIV-BIP:AACTCCCAATCGATTACATTGACTTTTTGAAAAAGTTGTCTACTGCGA；
SHIV-LF:CGAAATCGTATACACCGTTCTTGAA；
SHIV-LB:GGCACGATTGATCATCCCGAAGT。
4, isothermal amplification
Reaction system is (25 μ L of total volume) as shown in table 1:
Table 1LAMP reaction system
After mentioned reagent is mixed, 20 μ L paraffin oil sealing fluids are added, mixes centrifugation, covers tightly pipe lid.
5, the method and result of fluorescence detection
Detection method:
Prepared reaction tube is centrifuged, is placed in ABI Step One instrument, program is set, program is as follows:
Testing result:
According to experimental result, " S " type curve occur is the positive, and what it is without " S " type curve is then feminine gender, two actual samples
All occur " S " type curve with positive quality control product, and negative control does not occur expanding (as shown in Figure 1).
The nucleotide sequence of above method China and foreign countries primer extension product is as shown in SEQ ID NO.8；Inner primer amplified production
Nucleotide sequence as shown in SEQ ID NO.9.
6, primer specificity is tested
Detection method:
For the primer specificity for detecting this kit, using above-mentioned LAMP detection method, respectively to prawn white spot syndrome
Viral (WSSV), prawn infectious subcutaneous and haematopoietic necrosis virus (IHHNV), prawn intestines born of the same parents parasitosis (EHP), SHIV are carried out
Detection analyzes this kit to the detection case of other common virus of SHIV virus and prawn.
Testing result: testing result shows that only " S " type amplification curve occurs in virus SHIV sample, and negative control is (ultrapure
Water) and virus WSSV, IHHNV, EHP sample do not occur expand (as shown in Figure 2).It is above-mentioned the experiment results show that this LAMP examine
Test agent box energy specific amplification goes out the target sequence in virus SHIV, without cross reaction occurs with other viral nucleic acids.Explanation
The method of the present invention and kit specificity are good, do not occur false positive.
7, sensitivity experiment
Detection method:
It extracts positive plasmid (carrier T containing target sequence segment), positive plasmid is determined by NanoDrop One
Amount, and it is diluted to respectively 10pg/ μ L, 1pg/ μ L, 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, 100ag/ μ L, 10ag/ μ L,
1ag/μL.Be respectively adopted above-mentioned LAMP detection method and nested PCR detection method (Liang Qiu, Meng-Meng Chen,
Xiao-Yuan Wan, et al.Scientific Reports.2017,7:11834), to each concentration positive plasmid after dilution
Carry out augmentation detection.
Nested PCR primer and amplification program (Scientific Reports.2017,7:11834)
Testing result: LAMP testing result is as shown in Figure 3A, curve from left to right be followed successively by 10pg/ μ L, 1pg/ μ L,
The amplification of the positive criteria product of 100fg/ μ L, 10fg/ μ L, 1fg/ μ L, 100ag/ μ L, it can be seen that of the invention
The sensitivity of LAMP kit detection is up to 1fg/ μ L, and the experimental result of sleeve type PCR such as Fig. 3 B, left side are external primer amplification production
Object, target fragment 457bp, right side are inner primer amplified production, target fragment 129bp, analysis shows sleeve type PCR
Detection sensitivity is 10fg/ μ L.
By the comparison of the two detection mode, show the accuracy of LAMP detection method better than regular-PCR detection method, card
Bright LAMP kit and detection method of the invention have the sensitivity of height to the diagnosis of viral SHIV.
Sequence table
<110>Hangzhou Ao Sheng Instrument Ltd.
Scientific and Technological Institutes Of Zhejiang
Zhejiang Fisheries Technology Extension Station
<120>LAMP primer group, kit and its method of prawn irido virus are detected
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
caatccagaa ttttacaact tgt 23
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tcaatgttgg ggaatacctt 20
<210> 3
<211> 52
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gacttccatc cctgaaaaca tcatttttta ataaaaatcc caatttgatc gc 52
<210> 4
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aactcccaat cgattacatt gactttttga aaaagttgtc tactgcga 48
<210> 5
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cgaaatcgta tacaccgttc ttgaa 25
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ggcacgattg atcatcccga agt 23
<210> 7
<211> 1200
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atggtagaat ctcaggaagt gttttacaat ccagaatttt acaacttgtt aaataaaaat 60
cccaatttga tcgcattcaa gaacggtgta tacgatttcg aaaatgatgt tttcagggat 120
ggaagtccag aagattatct ttcagttaaa ctcccaatcg attacattga ctacggcacg 180
attgatcatc ccgaagttat cgcagtagac aactttttcc aaaaggtatt ccccaacatt 240
gaaatcagag attattttct agatcaggcc agttttgtat tcgttggtgg aaatcacaac 300
aaggtcattc tattttggac aggagaggga aataacggga aaacggtaac tcaaacgtta 360
tttgagaaaa tgttgggaaa gtttgcaatt aaattcaaca catctctgat tacgggtaaa 420
aaggcaaaca tgggagctgc aagtcccgaa ttggccaggg cgggagatgg tgttagatgg 480
gcagtcatgg atgaaccaaa tgctgacgaa atcatcagtt cgggaacgtt aaagggtctc 540
acgggaaacg attcgtattg ggctcgagat ttgttccaac gaggaaagga aacgaaagaa 600
attataccct ttttcaaatt acacatgatt tgcaacaagc ttccagcaat caaggatgcc 660
gatcaagcaa cgtggaatcg aatcagggtt attccattcg aaagtacatt caaacatgaa 720
aacgattgcc ccgttgaatt tgaagaacaa atgaaacaga aaacattccc catggataaa 780
aatttcacag aaaagattcc cgaaatggta aaacccctgg cttggtatct tattcagaga 840
tggaagacta tcaggaagtg tgaaattgta gagccagaga ttgtaacggt agctacatct 900
tcgtaccgaa acgaaaacga tatttacaag caattcgaag atcagtgtat ccatcaagag 960
aaaaatggaa atctcagctt taccgtttta tattcagtat tcaaggattg gttcaaagaa 1020
gagtatccta atatgaccat cccaatcaga caaacgatca gaaaacattt catttccaaa 1080
tttggacaac ttgagagagg tagatggaag aactttatat gcaagaagga cgaagacgac 1140
tttggtcggg atagtgatga agatggtgac gatgttgtga atcccgctct tttagtttaa 1200
<210> 8
<211> 457
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<213>artificial sequence (Artificial Sequence)
<400> 8
gggcgggaga tggtgttaga tgggcagtca tggatgaacc aaatgctgac gaaatcatca 60
gttcgggaac gttaaagggt ctcacgggaa acgattcgta ttgggctcga gatttgttcc 120
aacgaggaaa ggaaacgaaa gaaattatac cctttttcaa attacacatg atttgcaaca 180
agcttccagc aatcaaggat gccgatcaag caacgtggaa tcgaatcagg gttattccat 240
tcgaaagtac attcaaacat gaaaacgatt gccccgttga atttgaagaa caaatgaaac 300
agaaaacatt ccccatggat aaaaatttca cagaaaagat tcccgaaatg gtaaaacccc 360
tggcttggta tcttattcag agatggaaga ctatcaggaa gtgtgaaatt gtagagccag 420
agattgtaac ggtagctaca tcttcgtacc gaaacga 457
<210> 9
<211> 129
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgggaaacga ttcgtattgg gctcgagatt tgttccaacg aggaaaggaa acgaaagaaa 60
ttataccctt tttcaaatta cacatgattt gcaacaagct tccagcaatc aaggatgccg 120
atcaagcaa 129
Claims (10)
1. detecting the LAMP primer group of prawn irido virus, which is characterized in that including a pair of of outer primer, a pair of of inner primer and a pair
Ring primer, nucleotide sequence are as follows:
SHIV-F3:CAATCCAGAATTTTACAACTTGT；
SHIV-B3:TCAATGTTGGGGAATACCTT；
SHIV-FIP:GACTTCCATCCCTGAAAACATCATTTTTTAATAAAAATCCCAATTTGATCGC；
SHIV-BIP:AACTCCCAATCGATTACATTGACTTTTTGAAAAAGTTGTCTACTGCGA；
SHIV-LF:CGAAATCGTATACACCGTTCTTGAA；
SHIV-LB:GGCACGATTGATCATCCCGAAGT。
2. detecting the kit of prawn irido virus, including LAMP primer group, which is characterized in that the LAMP primer group such as right
It is required that shown in 1.
3. kit as claimed in claim 2, which is characterized in that the volume of outer primer is 0.5~2 in the LAMP primer group
μ L, inner primer volume be 0.1~1 μ L, the volume of ring primer is 0.2~1 μ L.
4. kit as claimed in claim 2, which is characterized in that further include: LAMP reaction solution, archaeal dna polymerase, nucleic acid fluorescent
Dyestuff, positive control and negative control；
The nucleotide fluorescent dye is Eva Green.
5. kit as claimed in claim 2, which is characterized in that the archaeal dna polymerase is Bst archaeal dna polymerase；
The LAMP reaction solution is by 2~5 μ L of 10mM dNTP, 10 × ThermoPol reaction buffer 2~5 μ L and 150mM
MgSO41~3 μ L of aqueous solution composition；
The positive control is the carrier T containing prawn irido virus ATPase gene of MP segment；
The negative control is ultrapure water.
6. a kind of method for detecting prawn irido virus, comprising the following steps:
(1) DNA of measuring samples is extracted；
(2) isothermal duplication is carried out to the DNA using the LAMP primer group for detecting prawn irido virus described in claim 1, and
Amplification is judged according to the change in fluorescence of amplified production.
7. method as claimed in claim 6, which is characterized in that the reaction system of the isothermal duplication includes: that total volume is 25 μ
l；
Wherein, 3.5 μ L, 10 × ThermoPol reaction buffer of 10mM dNTP, 2.5 μ L, 150mM MgSO41.5 μ L of aqueous solution,
Bst archaeal dna polymerase 0.5 μ l, 10 μM of SHIV-F3 0.2 μ L, 10 μM of SHIV-B3 0.2 μ L, 10 μM of SHIV-FIP0.8 μ L,
10 μM of SHIV-BIP 0.8 μ L, 10 μM of SHIV-LF 0.4 μ L, 10 μM of 0.4 μ L of SHIV-LB, 2 μ L of DNA profiling, surplus are
ddH2O。
8. method as claimed in claim 6, which is characterized in that the reaction condition of the isothermal duplication are as follows: 63 DEG C of reactions 30~
60min。
9. method as claimed in claim 6, which is characterized in that the nucleic acid fluorescent added in the reaction system of the isothermal duplication
Dyestuff is Eva Green.
10. method as claimed in claim 6, which is characterized in that the method for the judgement amplification are as follows: collect fluorescence letter
Number, then it is determined as the positive if there is " S " type amplification curve；If being determined as feminine gender without " S " type amplification curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810717869.6A CN108950067A (en) | 2018-07-03 | 2018-07-03 | Detect LAMP primer group, kit and its method of prawn irido virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810717869.6A CN108950067A (en) | 2018-07-03 | 2018-07-03 | Detect LAMP primer group, kit and its method of prawn irido virus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
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| CN112795702A (en) * | 2021-03-07 | 2021-05-14 | 珠海市迪奇孚瑞生物科技有限公司 | LAMP (loop-mediated isothermal amplification) detection primer group, kit and method for shrimp hemocyte iridovirus |
| CN115976272A (en) * | 2022-09-13 | 2023-04-18 | 青岛嘉智生物技术有限公司 | TaqMan fluorescent quantitative PCR method for rapidly detecting prawn hemangiocyte iridovirus |
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