This kit is suitable for the extraction of pathogen nucleic acid from aquatic animal samples. Using the purification method of magnetic nanoparticles with high binding force, the sample is lysed and digested under the action of the lysate, and the RNA/DNA is released into the lysate. Magnetic particles adsorb RNA/DNA, but proteins are not adsorbed and removed. The RNA/DNA-adsorbed nanoparticles are washed with a washing solution to remove proteins and other impurities, washed with an ethanol solution to remove salts, and finally treated with an eluent to obtain high-purity RNA/DNA.

The rapid separation and purification process eliminates the need for toxic phenol-chloroform extraction and time-consuming alcohol precipitation. Suitable for automated nucleic acid extraction equipment, the entire extraction process only takes 30 minutes. The obtained nucleic acid can be directly used in experiments such as PCR, fluorescence quantitative PCR, and virus detection.

【Product Components】

【 Storage conditions and expiration date】

This product is transported and stored at room temperature (15°C~25°C). The validity period is 12 months.

【 Preparations】

1. Instruments, consumables and tools that should be prepared before the experiment: automated nucleic acid extractor, matching single T-well plate, pipette and matching pipette tips, centrifuge, vortexer, scissors, tweezers, homogenization bag, centrifuge tube, Centrifuge tube rack, metal scoop (for scooping up shrimp or fish fry), alcohol lamp (for sterilizing scissors, tweezers, metal scoop), labeling pen, etc.

2. Before using the prepackaged 6-well plate nucleic acid extraction reagent, gently tap the 6-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. Put the matching single T-well plate in the nucleic acid extractor. Insert the disposable magnetic sleeve into the magnetic sleeve socket of the nucleic acid extraction instrument, and make sure that the disposable magnetic sleeve has been inserted to the end of the magnetic sleeve socket (see Note 4).

Note 4: A disposable magnetic sleeve must be installed in the nucleic acid extraction instrument to prevent the magnetic rod from directly touching the magnetic beads in the extraction reagent plate.

2. Tear off the sealing film of the nucleic acid extraction reagent from the prepackaged 6-well plate (see Note 5), add 200 μl of the sample liquid and 20 μl of Proteinase K prepared in the sample pretreatment in the first well (equilibrate to room temperature) in sequence.

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. Insert the prepackaged 6-well plate nucleic acid extraction reagent into the matching single T-well plate (see Figure 1), and check the correspondence between the disposable magnetic sleeve and the 6-well plate nucleic acid extraction reagent (see Note 6).
4. 

Note 6: The correct orientation of the nucleic acid extraction reagent for the 6-well plate with the parafilm removed is with the label facing outward. The initial position of the disposable magnetic sleeve is vertically above the first hole of the 6-well plate.

4. In the nucleic acid extraction instrument, set the nucleic acid extraction parameters of aquatic animal pathogens according to Table 1. (See Note 7)