DETEKSI AGEN PENYAKIT IKAN DENGAN POLYMERASE CHAIN REACTION (PCR) - part I

Summary

This video demonstrates a practical experiment aimed at detecting shrimp pathogens using molecular techniques like Polymerase Chain Reaction (PCR). The process begins with DNA extraction from shrimp tissues, including the carapace, gills, and muscles. Key steps involve tissue homogenization, proteinase-K treatment, centrifugation, and multiple washings with commercial kits. The extracted DNA is then prepared for PCR amplification to identify viruses. The video offers a step-by-step guide to this molecular method, emphasizing its precision and speed for diagnosing fish diseases. The experiment showcases both the technical procedures and the tools required for successful pathogen detection.

Takeaways

• 😀 The objective of the practical session is to detect fish disease agents using molecular techniques like PCR.
• 😀 Students are expected to diagnose fish diseases accurately and quickly after completing the practical.
• 😀 Shrimp is used as the test animal in this procedure.
• 😀 The specific organ to be sampled in shrimp depends on the type of pathogen being detected, including organs like the carapace, swimming legs, gills, hepatopancreas, muscles, and tail.
• 😀 DNA extraction is the first step in detecting the presence of viruses in shrimp.
• 😀 The process starts by homogenizing shrimp tissue, followed by DNA extraction using a commercial kit.
• 😀 30 mg of tissue is used for DNA extraction, and the tissue is placed in an Eppendorf tube for homogenization.
• 😀 After homogenization, proteinase-K is added, and the mixture is vortexed and centrifuged.
• 😀 The mixture is incubated at 56°C for 3 to 4 hours or overnight, depending on the tissue type.
• 😀 Following incubation, the solution is centrifuged, and the supernatant is collected for further purification with ethanol and buffers.
• 😀 Final DNA purification steps involve washing with buffer solutions and incubating the DNA to extract a clean sample, which is then stored at -20°C for later use.

Q & A

What is the main objective of this practical session?

The main objective is to detect fish pathogens using molecular techniques, specifically through PCR (Polymerase Chain Reaction).

What organism is used for testing in this experiment?

Shrimp is used as the test organism in this practical session.

Which organs of the shrimp are analyzed for virus detection?

The organs analyzed include the carapace, swimming legs, gills, hepatopancreas, muscles, and tail, depending on the pathogen being targeted.

What is the first step in the DNA extraction process from the shrimp?

The first step is to homogenize the shrimp tissues to break them down into a fine consistency.

What is the role of Proteinase K in the DNA extraction process?

Proteinase K is used to break down proteins, aiding in the release of DNA from the cells during the extraction process.

At what temperature should the samples be incubated during the DNA extraction process?

The samples should be incubated at 56°C for 3 to 4 hours or overnight, depending on the type of tissue.

What is the purpose of adding ethanol during DNA extraction?

Ethanol is added to precipitate the DNA, allowing it to be separated from other substances in the solution.

How is the DNA purified during this process?

DNA is purified using a column-based purification system, where the sample is washed with buffers and then centrifuged to remove impurities.

What speed and duration should the centrifugation be at during the DNA extraction?

Centrifugation should be performed at speeds of 8,000 to 14,000 RPM, with durations ranging from 1 to 5 minutes, depending on the step.

How is the final DNA stored before it is used in PCR?

The final DNA is stored at -20°C before being used in PCR.