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Specific primers and method used for detecting paralichthys olivaceus infected with beta nodavirus
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A nodavirus, specific technology, applied in the field of applied biology, to achieve the effect of simple reaction instrument, simple and fast operation, and improved detection sensitivity
Inactive Publication Date: 2012-11-28
TIANJIN NORMAL UNIVERSITY
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After searching, the present invention utilizes specially designed primers, and adopts the combined method of reverse transcription and strand displacement amplification to detect the infection of flounder by β-noda virus, but there is no literature report yet
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Embodiment 1
[0042] Detection of Brain RNA Samples of Juvenile Flounder Infected with β-Nodavirus
[0043] (1) Primer synthesis: Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0044] (2) Preparation of brain RNA samples from juvenile fish infected with β-nodavirus:
[0045] ① Place 10 juvenile flounder fish on a pre-cooled dissecting plate, dissect them with scissors and tweezers that have been baked at 180°C for 8 hours, and quickly transfer about 0.1g of brain tissue to a 1.5mL centrifuge tube without RNase Add 1mL Trizol, fully homogenize in an ice bath, and let stand at room temperature for 5min. Centrifuge at 4°C (12000rpm, 10min);
[0046] ②Take the supernatant from ① above into a 1.5mL clean centrifuge tube, add 0.5mL chloroform to shake for 15s, and let stand for 2min;
[0047] ③Centrifuge the above ② at 4°C, 12000rpm for 15min and take the colorless supernatant;
[0048] ④ Add 0.5 mL of isopropanol to the above ③, mix the liquid in the tube gently, and let stand a...
Embodiment 2
[0058] Detection of Crude Brain Extracts from Juvenile Flounder Infected with β-Nodavirus
[0059] The basic operation is the same as that in Example 1, except that the test sample is the crude extract of the brain tissue of juvenile flounder, specifically, place the juvenile flounder on a pre-cooled dissecting tray, and use scissors that have been baked at 180°C for 8 hours Dissect with tweezers, weigh about 1 g of its brain tissue, add 1 mL of DEPC water to homogenate. Centrifuge the homogenate (4000rpm, 1min), draw the supernatant into a new 1.5mL centrifuge tube, and then take 5μL of the supernatant as a template. After the reaction, add 2 μL of SYBR GREEN I dye and observe the results. The result judged the green fluorescence as positive (see Figure 5 ), that is, the test sample contains the nucleic acid sequence of beta nodavirus.
Embodiment 3
[0060] Example 3 Application example
[0061] Detection by conventional methods:
[0062] ① RNA was extracted from the brain tissue of animals infected with β-noda virus;
[0063] ② Use the RNA obtained in ① as a template to perform reverse transcription to prepare cDNA;
[0064] ③ Design specific primers, and use the cDNA prepared in ② as a template for PCR reaction;
[0065] ④ Nucleic acid electrophoresis to observe the band size and perform gel recovery;
[0066] ⑤ Ligate the recovered fragments to the sequencing carrier (usually PMD18-T carrier);
[0067] ⑥ transformation;
[0068] ⑦ Pick a single clone and send it for sequencing;
[0069] ⑧The sequencing results were compared with the published β nodavirus genome for homologous determination.
[0070] The example that the present invention actually detects (cultured fish):
[0071] ① Dissect and weigh 1 g of the brain tissue of the diseased animal, add 1 mL of DEPC water to homogenate;
[0072] ② Centrifuge the ho...
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Abstract
The invention relates to specific primers and a method used for detecting paralichthys olivaceus infected with beta nodavirus. According to the invention, 4 specific primers are designed aiming at 4 interrelated regions of a target gene sequence, such that the specificity and accuracy of the detection are ensured. A scheme with reverse transcription efficacy is adopted, such that the whole detection process can be finished in one PCR tube. Only one time of sample application is required, such that human operation disturbance is reduced. The detection can be finished in 90 minutes, and no expensive equipment is needed. When a reaction is finished, a SYBRGREENI dye is added, and positive results can be rapidly identified by naked eyes. With ultraviolet auxiliary observation, the detection sensitivity can be further improved. The detection limit of the method is similar to that of PT-PCT with complicated operations. The detection samples can be crude extraction liquid of disease focus tissues, or extracted RNA thereof, or cDNA obtained by reverse transcription. Therefore, the primers and the method are suitable to be used in on-site qualitative and quantitative detections. The primers and the method have high values to be popularized in aquaculture disease controlling.
Description
[0001] This patent application was completed by Tianjin Normal University School of Life Sciences / Tianjin Key Laboratory of Cell Genetics and Molecular Regulation, and was supported by National Natural Science Foundation of China (30901094), Tianjin Science and Technology Plan (09JCYBJC15000), Tianjin Education Commission Fund (20080618) and National Science and Technology Funding from the Support Program (2011BAD13B07, 2011BAD13B04). technical field [0002] The invention belongs to the field of applied biotechnology, and relates to the detection of infection of flounder by beta noda virus by using reverse transcription and strand displacement amplification combined method. More specifically: it is a specific primer for detecting beta nodavirus gene and its detection method. Background technique [0003] Beta nodavirus belongs to the Nodaviridae family. Its genome consists of two positive strands of RNA. It can infect more than 40 species of farmed fish and cause viral neur...
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