Turbot reddish body iridovirus virus polymerase chain reaction detection method
An iridescent virus and turbot technology, which is applied in the field of molecular biology, can solve the problem that the turbot erythroid iridovirus cannot be detected, etc., and achieves enhanced virus detection and fish quarantine, sensitive detection, and high sensitivity. Effect
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Embodiment 1
[0071] Example 1: Extraction and purification of sample tissue DNA using Roche Diagnostics’ high-purity PCR template preparation kit
[0072] The specific operation steps for extracting and purifying sample tissue DNA using Roche Diagnostics’ High Pure PCR Template Preparation Kit are as follows:
[0073] (1) Take 25-50 mg of the sample tissue to be tested (such as 50 mg of turbot spleen tissue infected with TRBIV), put it in a 1.5 ml sterile centrifuge tube, cut it into pieces with ophthalmic scissors, and add Tissue Lysis Buffer to each 200 μl and 40 μl of proteinase K at 20 mg / ml were digested in a water bath at 55°C for more than 1 hour until the tissue was completely digested.
[0074] (2) Add 200 μl of binding buffer, mix thoroughly, and keep warm in a water bath at 72° C. for 10 minutes.
[0075] (3) Add 100 μl of isopropanol, mix thoroughly, and then absorb the insoluble matter in the centrifuge tube; place the high purity filter tube (High Pure Filter Tube) on the co...
Embodiment 2
[0085] Embodiment 2: Separation and purification of turbot red body disease iridescent virus
[0086] The following centrifugation was performed at 4°C. Get 1.7g of the spleen tissue of turbot diseased fish infected by TRBIV, and use phosphate buffered solution (abbreviated as PBS, which consists of: 2.68mM KCl, 1.47mM KH 2 PO 4 , 0.137M NaCl, 8.06mM Na 2 HPO 4, pH7.4) and washed 3 times, placed in a glass homogenizer, added 15ml of TEN solution (0.1mol / L NaCl, 10mmol / L Tris-HCl, 1mmol / L EDTA, pH8.0), and ground thoroughly on ice. Centrifuge the homogenate at 1500×g for 15min, discard the precipitate; take the supernatant and centrifuge at 5800×g for 30min (HITACHI P50AT2-721 angle rotor), discard the precipitate; take the supernatant obtained by the second centrifugation, and pass through 36400× Centrifuge at g for 2 hours, discard the supernatant; completely suspend the pellet in TEN solution, spread on the pre-prepared 20-60% (w / w) sucrose concentration gradient, and ul...
Embodiment 3
[0088] Example 3: Preparation of TRBIV main capsid protein gene and its nearby nucleotide sequence
[0089] 1. Extraction of nucleic acid from spleen tissue of turbot diseased fish containing TRBIV DNA
[0090] Take 50 mg of the spleen tissue of turbot diseased fish infected with TRBIV, and use the high-purity PCR template preparation kit from Roche Diagnostics to extract and purify nucleic acid from the spleen tissue of diseased fish containing viral DNA. The specific operation steps are the same as in Example 1.
[0091] 2. PCR amplification of TRBIV DNA
[0092] The extracted TRBIV DNA samples were amplified using primers iridoF and iridoR. Take a 200 μl sterile centrifuge tube, operate it on an ice bath, and add the following PCR reaction components: 5 μl of PCR reaction template solution, 2.5 U of Taq DNA polymerase, 250 μM of each of the four dNTPs, 10 mM of Tris-HCl (pH9.0), KCl 40mM, MgCl 2 1.5 mM, 50 pmol each of primers iridoF and iridoR, and the total volume of ...
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