Vibrio harveyi nucleic acid detection kit (one-tube PCR-fluorescent probe method) instruction manual

Vibrio harveyi nucleic acid detection kit (one-tube PCR-fluorescence probe method)

â—† Product Description

The animal disease detection series can amplify specific nucleic acid fragments of pathogens in samples such as foods and animal tissues, and judge the results by real-time amplification curves. This product is used for the detection of Vibrio harveyi with a detection limit of 10 ³ copies/μL parasite genomic DNA .

• Product composition (96 test)

• Applicable instrument

Real-time fluorescence PCR instrument such as ABI 7500, CFX 96, Mx 3005P, LineGene9600.

â—† Self-supplied supplies and instruments

1 ice box; 2 pipettes (0.5-10μL, 10-100μL, 100-1000μL) and matching sterilization tips; 3 centrifuges; 4 vortex mixers; 5 metal baths; 6 homogenizers, mixers or Grinding tools such as mortar; 7 electronic balance.

â—† Notes

1. This reagent has high detection sensitivity. In order to prevent pollution, the experiment is to be partitioned.

1) First zone: sample preparation zone.

2) The second zone: the template addition zone.

3) Zone 3: Amplification and product analysis zone.

★ It is best to physically isolate the partitions to avoid contamination caused by human factors.

2. Work clothes and latex gloves are worn during the experiment, and tools are used independently in different areas. Gloves and lab coats need to be replaced.

3. Strictly follow the operation steps, reagent preparation and sample loading steps, please operate in strict accordance with the instructions on the ice box.

4. The components in the reaction solution are sensitive to light and should be stored away from light . The reagent should be completely thawed before use, but repeated freezing and thawing should be avoided. It is recommended to centrifuge for 30 seconds before use, and store the reaction solution in an appropriate volume according to the frequency of detection.

5. After the reaction is completed, the expansion tube should be placed in a sealed bag and discarded. On the same day, the lid is opened and the aerosol is easily contaminated. It is forbidden to open the lid.

6. Do not mix different batches of reagents used within the validity period.

â—† Sample processing

The sample was processed as described below.

1. Direct detection

Lesion tissue

(1) If the sample is not directly inoculated, the lesion tissue of the diseased animal is taken for homogenization.

2. Water body

(1) taking 10 to 100 mL of culture water, filtering with a sieve, and taking the filtrate through a 0.22 um nitrocellulose membrane for filtration;

(2) The filter was chopped and shaken and resuspended in 500 μL of 0.9% NaCl aqueous solution or sterile physiological saline, centrifuged at 12000 rpm for 5 min, and the supernatant was discarded as much as possible.

2. Enrichment detection

Take 25g (mL) of the sample to be tested, add 225mL of TYE liquid medium, homogenize for 1 min at 8000r/min with a rotary blade homogenizer, or slap for 2 min with a tapping homogenizer to prepare 1:10 Uniform dilution. If there is no homogenizer, the sample is ground in a sterile mortar or homogenized bag, and then added with 10 mL of TYE liquid medium and cultured on a shaker for 24 hours.

For detailed steps, please follow the standard operation or check the food safety software.

• Experimental operation

1. Template preparation (sample preparation area)

Please refer to the extraction procedure of the instructions for rapid extraction of genomic DNA from aquatic animal diseases.

2. Add a template (template addition area, placed on the ice box)

Cut the PCR tube containing the reaction number, and place it at room temperature to be thawed. After centrifugation for 30 seconds, uncover the sealing film. Add 5 μL of template to each reaction solution in the order of NG, sample template to be tested. , PG-VH-P. After the matching PCR tube cap was covered, the mixture was vortexed for 30 s, centrifuged for 1 min, and the PCR amplification reaction was immediately performed.

3. Amplification reaction (amplification and product analysis area)

Using a real-time PCR instrument, the fluorophore was selected for FAM and the quencher group was selected for TAMRA.

Set up the amplification reaction according to the following conditions:

• Result determination

The test sample has no Ct value or ≥40, the curve is straight or slightly oblique, and there is no “S” type amplification curve. The sample can be reported negative, does not contain Vibrio harveyi or the content is lower than the detection limit;

The test sample Ct ≤ 36, the curve shows an "S" type amplification curve, which can directly report the sample positive, containing Vibrio harveyi;

Test sample 36

• The NG reaction is a smooth straight line, and the PG reaction is an "S" type amplification curve. The test result is valid, otherwise it is invalid. If the duplicate test results are still invalid, please contact technical support.
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