2. Before using the prepackaged deep-well plate nucleic acid extraction reagent, gently tap the deep-well plate vertically so that no reagents or magnetic beads remain on the parafilm.

【Sample type】

Shrimp fry, fish fry, animal tissues (such as caterpillars, artemia, shelled artemia eggs, prawn tissue, fish tissue, mollusk tissue, etc.), unshelled artemia eggs, and aquaculture water.

【 Sample pretreatment】

1. Shrimp fry, fish fry, animal tissues (such as caterpillars, Artemia, shelled Artemia eggs, prawn tissue, fish tissue, mollusk tissue, etc.), unshelled Artemia eggs (see Note 1):

Note 1: Unshelled samples of Artemia eggs should be thoroughly ground with a mortar after shelling by appropriate methods.

Put the sample into the homogenization bag, and grind it thoroughly (with a blunt instrument, such as a plastic bottle) to repeatedly scrape the surface of the homogenization bag containing the sample, so that the sample is fully ground. Take 30 mg (about the size of mung bean, the center of the sample slope is about 0.5cm high after centrifugation. See Note 2) of the milled sample into a centrifuge tube, centrifuge for 5s, centrifuge the sample to the bottom of the tube, and observe the size of the sample. 30mg, excess sample can be removed with a pipette.

Add 500 μl of Buffer TL to the centrifuge tube containing the sample, and use a vortexer to mix for 15 s and set aside.

Note 2: The sample size will affect the final test results. If the sampling amount is too small, the pathogen content will be insufficient; if the sampling amount is too large, the qPCR detection may be inhibited due to the excessive content of inhibitor components.

2. Aquaculture water

Use a water-based filter membrane with a pore size of 0.22 μm (the water-based membrane is not easy to block) for filtration (the filter membrane can retain bacterial pathogens such as EHP, Vibrio (including EMS), etc.), after filtering more than 500mL of culture water, take out the filter membrane and cut it into small pieces Then put it into a centrifuge tube.

Add 500 μl of Buffer TL to the centrifuge tube containing the sample, mix well with a vortexer for 15 s, centrifuge at 10,000 rpm for 1 min, and use the supernatant for later use. (See Note 3)

Note 3: During the process of transferring the supernatant, be careful not to suck the solid. If the solid is accidentally adsorbed into the pipette tip, the liquid should be pumped back into the centrifuge tube, centrifuged again for 1 min, and the supernatant should be carefully aspirated again.

【 Experimental steps】

1. Insert the disposable magnetic sleeve into the magnetic sleeve socket of the nucleic acid extraction instrument, and make sure that the disposable magnetic sleeve is inserted to the end of the magnetic sleeve socket (see Note 4).

Note 4: Disposable magnetic sleeves must be installed in the nucleic acid extraction instrument to avoid direct contact of the magnetic rods with the magnetic beads in the extraction reagent plate.

2. Tear off the sealing film of the nucleic acid extraction reagent from the deep-well plate (see Note 5), and add 200 μl of the sample liquid to be used in the sample pretreatment and 20 μl of Proteinase K (equilibrate to room temperature) into the wells in the 1/7 column.

Note 5: When tearing the parafilm, if there is glue residue or burr on the mouth of the tube, please clean it with sterilized tweezers.

3. In the nucleic acid extractor, set the parameters for nucleic acid extraction of aquatic animal pathogens according to Table 1. (See Note 6)

 Table 1: Nucleic acid extraction parameters for aquatic animal pathogens

Note 6: The parameters of "Nucleic Acid Extraction of Aquatic Animal Pathogens" have been preset in the FD-E32 Nucleic Acid Extractor, and the program can be directly run.

4. The nucleic acid extraction process is complete after the nucleic acid extractor runs for about 30 minutes. Take out the deep-well plate and transfer the liquid nucleic acid product extracted from the 6/12th column for use. (See Note 7)

Note 7: The extracted liquid nucleic acid product should be used for qPCR detection as soon as possible. If it cannot be used for detection immediately, if the nucleic acid type of the pathogen to be tested is DNA, it should be stored at -20°C as soon as possible; if the nucleic acid type of the pathogen to be tested is RNA, it should be stored at -80°C as soon as possible, or at -20°C. more than 24 hours.

5. Discard waste liquid and consumables according to standard procedures.

V1.1 20211222