Grass carp hemorrhagic disease type III virus (GCRV III) nucleic acid detection kit (one tube constant temperature fluorescence method) instruction manual

Grass carp hemorrhagic disease type III virus (GCRV III) nucleic acid detection kit (one-tube thermostat fluorescence method)

â—† Product Description

The animal disease detection series is based on a unique thermostated fluorescence detection technology that can amplify specific nucleic acid fragments of viruses in samples such as food and animal tissues, and judge the results by real-time amplification curves. This product is used for the detection of grass carp hemorrhagic disease type III virus (GCRV III) with a detection limit of 10 ³ copies/μl genomic RNA .

â—† Product composition ( 96 test)

â—† Applicable instruments

Dhelix-C, ESE Tube Scanner, Genie II, Deaou-308C and other constant temperature fluorescence detectors, ABI 7500, LightCycler480, CFX 96 and other fluorescent PCR instruments.

â—† Self-supplied supplies and instruments

1 Sterilize 1.5mL or 2.0mL centrifuge tube; 2 ice box; 3 pipette (0.1-2.5μL, 0.5-10μL, 10-100μL, 100-1000μL) and matching sterilization tips; 4 centrifuge; 5 vortex Rotary mixer; 6 metal bath

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 pollution 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.

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

Samples were processed according to the SN/T 3584-2013 Technical Specifications for Grass Carp Bleeding Diseases or other standards, and the prepared samples were kept for use.

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

â—† Experimental operation

1. Template preparation (sample preparation area)

It is recommended to use the aquatic animal virus genomic DNA/RNA extraction kit series. The specific process is detailed in the product manual.

2. Add a template (template add area, placed in the ice box)

The required test number of the PCR tube containing the reaction solution was taken out, the reagent was completely thawed, and after thawing, the tube cover was opened and 0.8 μL of BI and 0.2 μL of RI were added to the bottom of each tube, and the tube lid was centrifuged for 1 min. Open the tube cover and add 2 μL of template along each tube wall in the order of NG-I, sample template to be tested, and PG-GCRV III -I. After the PCR tube cover was capped, it was centrifuged for 3 min, and the PCR amplification reaction was immediately performed.

3. Amplification reaction (amplification and product analysis area)

1 The constant temperature instrument was reacted at 63 ° C for 45 min.

2 If a real-time PCR instrument is used, the fluorescent group is selected as FAM, the quenching group is selected as None, 63 ° C for 15 s, 63 ° C for 45 s as a cycle, and fluorescence signal is collected at 63 ° C for 45 s, 45 cycles.

For other instruments, please refer to the instrument manual for setting.

â—† Result judgment

1 The instrument automatically determines the result. If “positive” is displayed, the sample contains grass carp hemorrhagic disease type III virus (GCRV III); if “negative” is displayed, the sample does not contain grass carp hemorrhagic disease type III virus (GCRV III) or The content is below the detection limit. For example, if the curve rises between 40min and 45min and the automatic interpretation result is negative, it may be due to the low sample content. It is recommended to enrich the sample or prolong the reaction time to 60min for review.

2 On the fluorescence quantitative PCR machine, the results were determined based on the presence or absence of the "S" type amplification curve. If there is an "S" type amplification curve, the sample contains grass carp hemorrhagic disease type III virus (GCRV III); if there is no "S" type amplification curve, the sample does not contain grass carp hemorrhagic disease type III virus (GCRV III) Or the content is below the detection limit.

• The result of the NG reaction tube showed "negative", and the result of the PG reaction tube showed "positive". The test result was valid, otherwise it was invalid. If the duplicate test results are still invalid, please contact technical support.
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