Method for detecting expression of TLR21 gene of Paralichthys olivaceus by fluorescent reverse transcription-polymerase chain reaction (RT-PCR) technology
A technology of RT-PCR and technical detection, which is applied in the field of detection of TLR21 gene expression of flounder by fluorescent RT-PCR technology, monitoring of early pathogenic infection of flounder, and can solve the problems such as the lack of immune prevention and control technology and treatment methods
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Embodiment 1
[0068] Flounder infection experiment:
[0069] Vibrio anguillarum (provided by Tianjin Aquatic Disease Control Center) was inoculated in Luriai-Bertani liquid medium with a pH value of 7.5 and supplemented with 2% NaCl, and cultured at 28°C for 24 h with constant temperature shaking (150 r / min). The OD value was measured at 550 nm, and the bacterial concentration and the total number of cells were calculated. Collect the thalline by centrifugation, wash the thalline three times with PBS centrifugation, and prepare 10 6 Individual / mL concentration of Vibrio eel bacteria liquid.
[0070] Fifty healthy flounder (body length 8-10 cm) that had not been vaccinated recently were cultured in an aquarium, and each fish was intraperitoneally injected with live bacteria 0.1 mL (10 5 Vibrio anguillaris), or 200 μg of Poly(I:C) simulated RNA virus was injected intraperitoneally into each tail, and the culture was continued, and the head and kidney tissues were dissected and separated fro...
Embodiment 2
[0072] Extraction and purification of total RNA:
[0073] (1) Take 100 mg of head kidney tissue, chop it up with scissors, add 1 mL of Trizol reagent (Invitrogen, USA) and 10 μL of heparin, grind the homogenate thoroughly in a homogenizer, then transfer to a 1.5 mL RNase-free centrifuge tube for shaking Mix well and place at room temperature for 5 min to fully lyse.
[0074] (2) Centrifuge at 4°C and 12,000 rpm for 5 minutes, and transfer the supernatant to a new RNase-free centrifuge tube.
[0075] (3) Add 0.1 mL of 5 mol / L NaCl to each tube and mix well, then add 0.3 mL of chloroform to each tube, shake vigorously for 15 s, place at room temperature for 3 min, and centrifuge at 12,000 r / min at 4°C After 15 min, the supernatant was aspirated and transferred to another clean 1.5 mL centrifuge tube without aspirating the middle layer as much as possible.
[0076] (4) Add isopropanol of equal volume to the obtained supernatant to each tube, mix gently, place at room temperatur...
Embodiment 3
[0080] Real-time fluorescent RT-PCR detection of TLR21 gene:
[0081] (1) Primer design:
[0082] According to the full-length sequence of TLR21 described in SEQ ID NO 1, use Primer 5 software to design specific primers suitable for real-time fluorescent PCR detection. The primer sequences are as follows:
[0083] TLR21-q-F: 5'-TAAACTTTGCCTACATCACA-3'
[0084] TLR21-q-R: 5'-AACACGAGCAGAAGAACAT-3'
[0085] The expected length of the PCR product of TLR21 is 198 bp. The results of agarose gel electrophoresis showed that the length of the amplified product was consistent with the expected length of the product, and it was a single band, indicating that the designed primers had strong specificity and were suitable for use in Real-time fluorescent RT-PCR detection; agarose gel electrophoresis results such as figure 1 shown.
[0086] At the same time, according to the cds sequence of flounder β-actin (EU090804) provided by NCBI, primers for real-time fluorescent PCR internal con...
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