CN112111608A - Macrobrachium rosenbergii dyskinesia nodavirus rapid detection primer group and kit - Google Patents
Macrobrachium rosenbergii dyskinesia nodavirus rapid detection primer group and kit Download PDFInfo
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Abstract
The invention relates to a rapid detection primer group and a kit for Macrobrachium rosenbergii dyskinesia nodavirus (MMDNV). The primer group of the invention comprises 5 primers, and the kit of the invention comprises: a sample collection tube, a nucleic acid washing tube filled with double distilled water, a template denaturation tube filled with TE buffer solution, an amplification color development tube filled with amplification reaction liquid and dye, a negative control tube, a positive control tube and an FTA membrane. The primer group can specifically detect the target nucleic acid of the low-copy MMDNV, and the detection method of the kit has higher convenience, sensitivity and specificity than a conventional PCR detection method, a histopathology detection method, an electron microscope detection method and the like, is convenient to use, is more accurate and rapid in detection, and is low in use cost. The primer group and the kit can be used in a conventional detection laboratory and can also be used in a field production field.
Description
Technical Field
The invention belongs to the technical field of marine organism pathogen detection, and particularly relates to a Macrobrachium rosenbergii movement disorder nodavirus (MMDNV) on-site rapid detection primer group and a kit.
Background
In recent years, the macrobrachium rosenbergii industry in China encounters a series of problems, wherein the prominent problem is that iron shrimp diseases are serious. The normal macrobrachium rosenbergii has a per mu yield of 300-400 kg generally, but the per mu yield of the shrimp pond with the macrobrachium rosenbergii is only 100-200 kg, and the macrobrachium rosenbergii adults have a much lower selling price than normal commodity-specification macrobrachium rosenbergii due to small size and light weight in the harvest period, so that the income of farmers is seriously influenced, and the farmers suffering from the macrobrachium rosenbergii are basically in loss states. In 2014, population events caused by the problem of 'iron shrimps' of thousands of farmers also occur in Gaoyou, Jiangsu and the like. 2018, the general benefit of the seedling raising enterprises in the Jiangsu Gaoyou area in 2019 is reduced due to the influence of the problem of the iron shrimps, and the proportion of the iron shrimps in the breeding process of the breeding households is over 50 percent.
In the work of the applicant, it was found that a new type of nodavirus infection was present in the aforementioned macrobrachium rosenbergii individuals presenting the "siderite" symptoms, which was tentatively designated macrobrachium rosenbergii dyskinesia nodavirus (MMDNV).
From the result of histopathological analysis, after the MMDNV is infected with the macrobrachium rosenbergii, obvious pathological changes are shown in compound eyes of the eyestalk part, such as damage of omentum cells, damage of onion organs and vacuolation of tissues. The artificial infection recovery experiment shows that the MMDNV can cause a certain proportion of macrobrachium rosenbergii to have the symptom of slow-growing 'iron shrimp', so that the MMDNV is considered as a pathogen closely related to the 'iron shrimp' disease. At present, the macrobrachium rosenbergii iron shrimp phenomenon still seriously damages macrobrachium rosenbergii culture areas such as Jiangsu, Zhejiang, Guangdong and Guangxi in China, so a rapid detection and early warning technology of pathogenic microorganisms of the disease is researched and established, the monitoring of MMDNV in parent macrobrachium rosenbergii, offspring seeds, culture process input products and culture environment is enhanced, the introduction of MMDNV in the breeding and culture process of the offspring seeds of the parent macrobrachium rosenbergii is avoided, effective prevention and control measures are actively taken to cut off the transmission of the MMDNV, the infection rate of the MMDNV in the macrobrachium rosenbergii culture area in China is reduced, and the high-quality development of the macrobrachium rosenbergii culture industry in China is ensured.
The early detection of the nodavirus in the dyskinesia of the macrobrachium rosenbergii is carried out, and preventive measures are taken to prevent the macrobrachium rosenbergii diseases from appearing on a large scale, still an effective means for reducing the epidemic risk of MMDNV in the macrobrachium rosenbergii cultivation at present and reducing the serious economic loss caused by the large-area epidemic of the macrobrachium rosenbergii is provided, so that the design of MMDNV detection primers, the establishment of a field-available, simple and feasible detection method aiming at the virus and the development of a corresponding detection kit become problems to be solved at present.
Disclosure of Invention
The invention aims to provide a rapid and sensitive macrobrachium rosenbergii dyskinesia nodavirus detection method suitable for production field use, and concentrates detection reagents in a unified matched kit in a standard way, so that the defect that the early detection and early warning cannot be carried out on the macrobrachium rosenbergii pathogen in the industry at present is overcome, and the detection of MMDNV is more convenient, rapid and sensitive.
The invention firstly provides a primer group for detecting the macrobrachium rosenbergii dyskinesia nodavirus, and the sequence information of the primer group is as follows:
upstream primers 1-3(SEQ ID NO: 1):
5′-atcgcaacattgcacggcaatttttcgaaggttatctcgctacgg-3′；
downstream primers 1-3(SEQ ID NO: 2):
5′-ctctccttctgctggaaccgctttttgcagtcgaccaatagggtt-3′；
upstream primer 2-3(SEQ ID NO: 3): 5'-cagacgcttgctgtgacc-3', respectively;
downstream primers 2-3(SEQ ID NO: 4): 5'-tttgaggccaggaaccatc-3', respectively;
melting primers 2-3(SEQ ID NO: 5): 5'-gctagaacggtccagacgg-3' are provided.
The invention also provides a kit for detecting the macrobrachium rosenbergii dyskinesia nodavirus, which comprises the following components:
1) the sample collecting tube is used for collecting and homogenizing a sample to be detected;
2) a nucleic acid washing pipe filled with double distilled water;
3) a template denaturation tube filled with TE buffer solution;
4) an amplification color development tube, which is filled with amplification reaction liquid and nucleic acid dye, and the concentration and the dosage of each component are as follows: 1-3 muM of each of the upstream primer 1-3 and the downstream primer 1-3 of the amplification primer, 0.5-1.5 muM of each of the upstream primer 2-3 and the 2-3 of the amplification primer, 0.1-0.5 muM of each of the melting primer 3-3 of the amplification primer, 0.8-2.2 mM of each of dATP, dTTP, dGTP and dCTP, and22-8 mM, Betaine (0.6-1.5M), Tris-HCl 10-40 mM, KCl (10-20 mM), MgSO41～6mM，(NH4)2SO46-12 mM, Triton X-1000.05% -1.0%, 1-20U of reverse transcriptase and 2-20U of Bst DNA polymerase;
the nucleic acid dye may be anthocyanin nucleic acid dye, or complex formed by manganese ion and calcein. The anthocyanin-type nucleic acid dye has low toxicity and better color development effect, and the types of the anthocyanin-type nucleic acid dye include but are not limited to SYBR Green, GelRed, GelGreen and GoldViewTM、GeneFinderTMAnd the like. The anthocyanin nucleic acid dye is adhered to the upper part of the inner side of the tube wall of the amplification and color development tube or the inner side of the cover of the amplification and color development tube in advance; the complex formed by the manganese ions and the calcein needs to be premixed in the amplification reaction solution.
5) A negative control tube, wherein an FTA membrane adsorbing the nucleic acid of the healthy macrobrachium rosenbergii is arranged in the negative control tube;
6) a positive control tube, which is internally provided with an FTA membrane adsorbing MMDNV nucleic acid;
7) FTA membrane, grinding rod, toothpick and straw which are respectively packaged in sterile bags.
The kit is used for detecting the nucleic acid of the MMDNV according to the following steps, toothpicks are used for transferring a template in the detection process, and a new toothpick is used each time, so that the cross contamination of samples is avoided:
1) putting 0.01-1.5 g of a sample into a sample collection tube, and grinding the sample into a slurry state by using a grinding rod;
2) placing the FTA membrane in a sample collection tube to sufficiently adsorb sample nucleic acid;
3) drying the membrane at room temperature for 1-20 min;
4) transferring the membrane adsorbed with the nucleic acid into a nucleic acid washing tube to wash for 3min, and then placing the membrane into a template denaturation tube to keep the temperature at 70 ℃ for 3 min;
5) transferring the membrane which adsorbs the nucleic acid and completes template denaturation into an amplification developing tube, and incubating for 30-50 min at the temperature of 58-65 ℃;
6) repeatedly shaking the amplification color development tube up and down for 1-3min to repeatedly mix the dye with the amplification reaction solution;
7) centrifuging or shaking the amplification color development tube downwards by hands to enable the amplification reaction solution mixed with the dye in the tube to be gathered at the bottom of the amplification color development tube, and directly observing the color of the reaction solution by naked eyes, wherein if the color is green, the detection result of the MMDNV of the sample is positive, and if the color is orange, the detection result of the MMDNV of the sample is negative.
The performance evaluation analysis is carried out on the designed detection primer group and the kit, and the result shows that the detection technology can specifically detect the MMDNV isolate, has no cross reaction with nucleic acids of other common viruses, and has good specificity. The detection sensitivity of the primer of the invention is tested by real-time fluorescence quantitative equipment, and the primer designed by the invention can detect as low as 2.07 x 102A copied virus. According to the invention, reagent consumables such as FTA membrane, TE buffer solution, isothermal amplification reaction solution and dye which can complete MMDNV on-site rapid detection are uniformly prepared in the kit, detection operation is specified in the specification, and the sequencing and standardization of the detection of the kit are realized. The detection kit is suitable for use in the quarantine and detection laboratories of aquatic products, and can also be used for field crude condition detection of the environmental samples of the macrobrachium rosenbergii culture field, so that the detection kit can be used for improving the MMDNV detection level and preventing and treating the large-scale popularization of MMDNVCan play a good technical supporting role.
Drawings
FIG. 1: a specific detection result graph of the primer combination 3, wherein 3S-shaped curves in the graph are the amplification results of the MMDNV nucleic acid; the remaining lines represent CMNV, EHP, IHHNV, WSSV, Vp, respectivelyAHPADTSV and YHV (straight line with partial overlap because no amplification occurred).
FIG. 2: the sensitivity detection result graph of the primer combination 3 is shown in the figure, wherein an S-shaped curve is the amplification result of the artificial plasmid pMD19-T-MMDNV with different copy numbers.
Detailed Description
The invention firstly clones RNA-dependent polymerase (RdRp) genes of MMDNV from macrobrachium rosenbergii with diseases of 'iron shrimp', analyzes all the RdRp conserved sequences of the kindred nodavirus, and designs 3 primer combinations which can be used for detecting the MMDNV on the basis; after the primer combination is subjected to amplification optimization and test analysis on detection characteristic parameters including detection sensitivity and detection specificity, 1 group of 5 specific primers are screened from 3 groups of primer groups, so that the MMDNV virus can be quickly, specifically and sensitively detected. On the basis of screening the primer group, the invention also provides a kit capable of realizing reverse transcription and cDNA (or DNA) amplification in one step, the kit does not need to independently carry out reverse transcription on target nucleic acid (RNA), and can synchronously finish the reverse transcription and amplification of the target nucleic acid by keeping the temperature for a period of time at constant temperature, thereby realizing the rapid and high-sensitive detection of MMDNV.
The present invention is further illustrated by the following examples.
Example 1 design and screening of Macrobrachium rosenbergii dyskinesia nodavirus on-site Rapid detection primers
Firstly, aiming at RNA-dependent RNA polymerase (RdRp) genes of the MMDNV of the Macrobrachium rosenbergii which is cloned from the diseases of the Macrobrachium rosenbergii such as Jiangsu, Zhejiang, Guangdong and Guangxi, the variation condition of the sequences is compared by using an NCBI online program Blastn and molecular biology software BioEitd 7.0, a conservative region sequence of the RdRp gene of the MMDNV is selected, and amplification primers are designed through the software Lamp design 4.0, so that 3 sets of primers are designed in total (Table 1).
Table 1: amplification primer designed according to MMDNV protein A gene
Using the 3 sets of primer combinations designed and synthesized above, a reaction system was prepared and an amplification reaction (25. mu.L/reaction, referred to as method 1) was performed: 1.6. mu.M each of the forward primer 1 and the reverse primer 1 of the amplification primer, 0.8. mu.M each of the forward primer 2 and the reverse primer 2 of the amplification primer, 0.2. mu.M each of the melting primer 3 of the amplification primer, 1.4mM each of dATP, dTTP, dGTP and dCTP, and MgCl28mM, Betaine 1.2M, Tris-HCl20mM, KCl 10mM, MgSO4 1mM，(NH4)2SO410mM, Triton X-1000.1%, reverse transcriptase 5U, Bst DNA polymerase 8U; the dye is a complex formed by 50 mu M of calcein and manganese chloride; the template is 50ng of MMDNV positive prawn nucleic acid, and the blank control is 2 mu L of sterile water; then placing the prepared reaction reagent into a fluorescent quantitative PCR instrument (A)
RealPlex 2), detecting a fluorescence signal once per minute (1 cycle), and comparing the amplification conditions of different primer combinations according to an amplification curve recorded by a fluorescent quantitative PCR instrument. The experimental result shows that the amplification efficiency of the primer combination 3 is highest. Thus primer set 3 was used for the next step of specificity and sensitivity analysis.
Respectively using MMDNV, Cover Metal Nodal Virus (CMNV), (enterocytozon hepapenaei) EHP, infection hypoderm and proteomic virus (IHHNV), and Acute hepa detailed disease (Vp)AHPAD) The nucleic acids of Yellow Head Virus (YHV), Taura Syndrome Virus (TSV) and White Spot Syndrome Virus (WSSV) areThe template was amplified according to the reaction system and reaction procedure described in method 1 above, and the specificity of MMDNV was determined using primer set 3. The amplification results showed that the amplification reaction was positive only when MMDNV nucleic acid was used as a template. This indicates that primer combination 3 is capable of specifically amplifying MMDNV nucleic acids without cross-amplification reactions with nucleic acids of currently common prawn viruses (fig. 1).
Preparing 20 mu L of PCR reaction system: 2 μ L of cDNA for MMDNV, 2.0 μ L of 10 XPCR master mix (Life Technologies),1 μ L of 10 μ M forward primer 2-3,1 μ L of 10 μ M reverse primer 2-3, 14 μ L H2O, and amplification was performed according to the following procedure: incubation at 94 ℃ for 5min, followed by 35 cycles (94 ℃ for 30s, 50 ℃ for 30s, 72 ℃ for 50s), and finally extension at 72 ℃ for 7 min. The amplified product was electrophoresed, a desired band was excised with a scalpel and recovered with a gel, and the gel-recovered product fragment was inserted into plasmid pMD19 to construct an artificial plasmid pMD19-T-MMDNV containing an MMDNV fragment (see the construction methods of CMNV plasmids: Qinlgi Zhang et al, Journal of viral methods,2013, 187:384-9, for related methods). The plasmid containing the MMDNV target fragment (2.07X 10)8copies/. mu.l) was diluted in 10-fold gradient and then used as a template for amplification according to the reaction system and reaction procedure described above in method 1, and the detection sensitivity of primer set 3 was analyzed. The amplification result shows that the lowest detectable level of the primer combination 3 is 2.07 multiplied by 102Copied viral plasmid (fig. 2).
Example 2 the test kit of the present invention consists of the following components (package capable of testing 8 samples):
(1) 8 sample collecting tubes for containing and grinding samples to be detected;
(2) 10 nucleic acid washing tubes, wherein each tube is filled with 1mL of double distilled water;
(3) template denaturation tubes, 10, each filled with 20. mu.L of TE buffer (containing 10mM Tris-HCl and 1mM EDTA, pH 8.0);
(4) 10 amplification and color development tubes, wherein each tube contains 24 mu L of amplification reaction liquid and 1 mu L of dye, and the amplification reaction liquid comprises the following components: the upstream primer 1 and the downstream primer 1 of the amplification primer are respectively 1.6 mu M, the melting primer 1 and the 2 position of the amplification primer are 0.8 mu M, and the amplification primerThe upstream primer 2 of (2) was 0.2. mu.M, 1.4mM each of dATP, dTTP, dGTP and dCTP, and MgCl22mM, Betaine 1.2M, Tris-HCl20mM, KCl 10mM, MgSO4 6mM，(NH4)2SO410mM, Triton X-1000.1%, reverse transcriptase 5U, Bst DNA polymerase 8U; the dye is a complex formed by 50 mu M of calcein and manganese chloride;
(5) negative control tubes, 1, containing FTA membrane without Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid;
(6) 1 positive control tube, which is internally provided with FTA membranes adsorbing the nucleic acid of the Macrobrachium rosenbergii dyskinesia nodavirus;
(7) the number of the FTA membranes and the number of the grinding rods are respectively 8; 80 toothpicks are arranged; 1 straw is arranged; packaging the above materials in sterilized sterile bag;
(8) the packaging box comprises 1 packaging box, a pipe frame of EPPDORF pipes formed by folding a piece of hard board is arranged in the packaging box, and the paper board is provided with 8 multiplied by 10 small holes for containing various pipes and consumables;
(9) instruction book, 1 part.
Example 3 the test kit of the invention may also consist of the following components (package capable of testing 8 samples):
(1) 8 sample collecting tubes for containing and grinding samples to be detected;
(2) 10 nucleic acid washing tubes, wherein each tube is filled with 1mL of double distilled water;
(3) template denaturation tubes, 10, each filled with 20. mu.L of TE buffer (containing 10mM Tris-HCl and 1mM EDTA, pH 8.0);
(4) 10 amplification and color development tubes, wherein each tube is filled with 25 mu L of amplification reaction liquid and 1 mu L of nucleic acid dye, and the amplification reaction liquid comprises the following components: 1.6. mu.M each of the forward primer 1 and the reverse primer 1 of the amplification primer, 10.8. mu.M each of the melting primer of the amplification primer, 0.2. mu.M each of the forward primer 2 and the reverse primer 2 of the amplification primer, 1.4mM each of dATP, dTTP, dGTP and dCTP, and MgCl28mM, Betaine 1.2M, Tris-HCl20mM, KCl 10mM, MgSO42mM，(NH4)2SO410mM, Triton X-1000.1%, reverse transcriptase 5U, Bst DNA polymerase 8U; nucleic acid staining1 μ L of the feed, the ingredient of which is 10 times diluted GeneFinderTMAnd the nucleic acid dye is adhered and fixed on the inner side of the tube cover of the amplification color development tube.
(5) Negative control tubes, 1, containing FTA membrane without Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid;
(6) 1 positive control tube, which is internally provided with FTA membranes adsorbing the nucleic acid of the Macrobrachium rosenbergii dyskinesia nodavirus;
(7) the number of the FTA membranes and the number of the grinding rods are respectively 8; 80 toothpicks are arranged; 1 straw is arranged; packaging the above materials in sterilized sterile bag;
(8) the packaging box comprises 1 packaging box, a pipe frame of EPPDORF pipes formed by folding a piece of hard board is arranged in the packaging box, and the paper board is provided with 8 multiplied by 10 small holes for containing various pipes and consumables;
(9) instruction book, 1 part.
Example 4 method for detecting Macrobrachium rosenbergii dyskinesia nodavirus Using the detection kit of the present invention
(1) Preparing 4 macrobrachium rosenbergii samples suffering from the iron shrimp diseases, wherein the number of the macrobrachium rosenbergii samples is 1, 2, 3 and 4; preparing 4 healthy macrobrachium rosenbergii samples which are respectively numbered as 5, 6, 7 and 8; about 0.1g of tissue from each sample was placed in a sample collection tube and quickly homogenized with a grinding rod;
(2) fully wetting the No. 1-8 membrane with the ground tissue fluid;
(3) standing the FTA membrane for 5min at room temperature;
(4) respectively transferring the FTA membrane into a No. 1-8 nucleic acid washing tube by using a toothpick, rinsing for 3min, and then placing the membrane in a membrane denaturation tube, and incubating for 5min at 70 ℃;
(5) transferring the modified FTA membrane to No. 1-8 tube amplification and color development tubes respectively, and incubating at 63 deg.C for 50 min;
(6) repeatedly shaking the amplification and color development tube up and down for 2 min;
(7) then the amplification developing tube is swung downwards by force, and the amplification reaction solution is observed by eyes; the result shows that the reaction liquid in the amplification and color development tubes No. 1-4 corresponding to the Macrobrachium rosenbergii samples No. 1-4 with the iron shrimp disease is green, which indicates that the MMDNV detection result of the samples No. 1-4 is positive; the reaction liquid in the amplification color development tubes No. 5-8 corresponding to No. 5-8 of the healthy Macrobrachium rosenbergii sample is orange yellow, which indicates that the MMDNV detection result of the No. 5-8 sample is negative.
In the above steps, from the 4 th step, the FTA membrane without the Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid, the FTA membrane with the Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid and the FTA membrane with the sample nucleic acid are processed together until the detection reaction is finished, and the FTA membrane is respectively used as a negative control and a positive control of the detection; the reaction liquid in the amplification color development tube corresponding to the negative control diaphragm is orange yellow, and the reaction liquid in the amplification color development tube corresponding to the positive control diaphragm is green.
Example 5 detection of the Presence of Virus in various samples Using the kit of the invention
Samples such as bottom sediment, fresh bait and the like in the culture environment of macrobrachium rosenbergii are collected from part of provinces and cities, and the detection kit in the embodiment 2 or the embodiment 3 is used for carrying out detection analysis of MMDNV on the samples by the method in the embodiment 4. The method comprises the following steps:
(1) the sediment sample is named as sample 1, and the bait sample is named as sample 2; a part of fresh bait of macrobrachium rosenbergii prepared in a laboratory is taken out and named as a No. 3 sample (the sample does not contain MMDNV through PCR detection); about 0.2g of the 0.3g samples 1, 2 and 3 were placed in a sample collection tube and the samples were ground to a slurry with a grinding rod;
(2) fully wetting FTA membranes numbered as No. 1, No. 2 and No. 3 with 3 pasty samples respectively;
(3) placing FTA membranes numbered as No. 1, No. 2 and No. 3 at room temperature for drying for 3 min;
(4) transferring the membrane adsorbed with the nucleic acid into a nucleic acid washing tube to wash for 3min, and then placing the membrane into a template denaturation tube to keep the temperature at 70 ℃ for 3 min;
(5) transferring the membrane which adsorbs the nucleic acid and completes template denaturation into an amplification developing tube, and incubating for 30-50 min at the temperature of 58-65 ℃;
(6) repeatedly shaking the amplification color development tube up and down for 1-3min to repeatedly mix the dye with the amplification reaction solution;
(7) centrifuging or swinging the amplification and color development tube downwards by hands to enable the amplification reaction liquid mixed with the dye in the tube to be gathered at the bottom of the amplification and color development tube, and directly observing the color of the reaction liquid by naked eyes, wherein the results show that the reaction liquid in the No. 1 and No. 2 amplification and color development tubes corresponding to the No. 1 sample and the No. 2 sample is green, which indicates that the No. 1 sample and the No. 2 sample are MMDNV positive; the reaction solution in the No. 3 amplification color development tube corresponding to the No. 3 sample is orange yellow, which indicates that the No. 3 sample is MMDNV negative.
In the above steps, from step 4, the FTA membrane free of the Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid, the FTA membrane having adsorbed the Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid and the FTA membrane having adsorbed the sample nucleic acid are treated together until the detection reaction is completed, and are used as negative and positive controls for detection, respectively. The detection result shows that the reaction liquid in the amplification color development tube corresponding to the negative control diaphragm is orange yellow, and the reaction liquid in the amplification color development tube corresponding to the positive control diaphragm is green; thus proving that the kit of the invention can effectively detect the nodavirus in the wild field without the dyskinesia of the macrobrachium rosenbergii.
In conclusion, the detection kit and the detection method thereof can be applied to detection of MMDNV in the sediment sample and detection of MMDNV in the bait.
The FTA patch of the present invention is an FTA card used by Whatman, uk, for the preparation of nucleic acids; the FTA card is cut into paper sheets with the dimensions of length multiplied by width multiplied by 1 millimeter or punched into paper sheets with the diameter of 1 millimeter, and then the FTA membrane can be manufactured.
Reagents and materials used in the kit of the invention: the primer is synthesized by optisco biotechnology limited; disodium ethylenediaminetetraacetate, tris (hydroxymethyl) aminomethane, dNTP, Betaine (Betaine) Sigma Co; dATP, dGTP, dCTP and dTTP, KCl, MgSO4、(NH4)2SO4、MgCl2Triton X-100 and reverse transcriptase were purchased from TAKARA; calcein, manganese chloride, etc. are purchased from Beijing GmbH, chemical reagents of national drug group; bst DNA polymerase was purchased from NEB; nucleic acid dyes were purchased from xiamen baiweixin biotechnology limited. The above reagents and materials may be selected from those commonly used in the art, andand is not limited to the specific limitations of the embodiments of the present invention.
Sequence listing
<110> Huanghai aquatic product research institute of China aquatic product science research institute of Jiangsu county high-yield water breeding limited company
<120> Macrobrachium rosenbergii dyskinesia nodavirus rapid detection primer group and kit
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<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctctccttct gctggaaccg ctttttgcag tcgaccaata gggtt 45
<210> 3
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cagacgcttg ctgtgacc 18
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tttgaggcca ggaaccatc 19
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<213> Artificial Sequence (Artificial Sequence)
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gctagaacgg tccagacgg 19
Claims (9)
1. A primer group for rapidly detecting macrobrachium rosenbergii dyskinesia nodavirus has a nucleotide sequence of SEQ ID NO. 1-5.
2. Use of the primer set according to claim 1 for the preparation of a product for detecting macrobrachium rosenbergii dyskinesia nodavirus.
3. The use of claim 2, wherein the article of manufacture is a nucleic acid detection kit.
4. A kit for detecting macrobrachium rosenbergii dyskinesia nodavirus, comprising the primer set of claim 1.
5. The kit of claim 4, further comprising the following components:
1) the amplification color development tube is filled with amplification reaction liquid and dye, and the amplification reaction liquid comprises the following components: 1-3 muM of each of the upstream primer 1-3 and the downstream primer 1-3 of the amplification primer, 0.5-1.5 muM of each of the upstream primer 2-3 and the downstream primer 2-3 of the amplification primer, 0.1-0.5 muM of each of the melting primer 3-3 of the amplification primer, 0.8-2.2 mM of each of dATP, dTTP, dGTP and dCTP, MgCl2 2～8mM，Betaine 0.6～1.5M，Tris-HCl10～40mM，KCl 10～20mM，MgSO4 1～6mM，(NH4)2SO46-12 mM, Triton X-1000.05% -1.0%, 1-20U of reverse transcriptase and 2-20U of Bst DNA polymerase;
2) negative control, TE buffer solution without Macrobrachium rosenbergii dyskinesia nodavirus nucleic acid is filled in;
3) and a positive control, wherein positive nucleic acid of the macrobrachium rosenbergii dyskinesia nodavirus is filled in the kit.
6. The kit according to claim 4, wherein the dye is a cyanine-based nucleic acid dye or a complex of manganese ions and calcein.
7. The kit of claim 4, wherein the anthocyanidin-based nucleic acid dye is SYBR Green, GelRed, GelGreen, GoldView, or GeneFinder.
8. The kit of claim 4, further comprising the following components:
1) the sample collecting tube is used for collecting and homogenizing a sample to be detected;
2) a nucleic acid washing pipe filled with double distilled water;
3) a template denaturation tube filled with TE buffer solution;
4) FTA membrane, grinding rod, toothpick and straw which are respectively packaged in sterile bags.
9. Use of a kit according to any one of claims 4 to 8 for the detection of macrobrachium rosenbergii dyskinesia nodavirus for non-disease diagnostic and therapeutic purposes.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212915A (en) * | 2014-09-10 | 2014-12-17 | 中国水产科学研究院黄海水产研究所 | Primer group and kit for carrying out spot quick detection on shrimp covert mortality nodavirus |
| CN105331739A (en) * | 2015-11-09 | 2016-02-17 | 中国水产科学研究院黄海水产研究所 | Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212915A (en) * | 2014-09-10 | 2014-12-17 | 中国水产科学研究院黄海水产研究所 | Primer group and kit for carrying out spot quick detection on shrimp covert mortality nodavirus |
| CN105331739A (en) * | 2015-11-09 | 2016-02-17 | 中国水产科学研究院黄海水产研究所 | Prawn covert mortality nodavirus fluorescent quantitative RT-PCR detection method |
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| Title |
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| 林峰: "罗氏沼虾野田村病毒致病性及其快速检测技术研究", CNKI, no. 10 * |
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