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A. Safety operation in a PCR lab:

  • Hazardous chemicals.
  • Safety rules for machines operation.
  • Reagent preservation and lab security

B. Basic theory of PCR:

  • DNA structure.
  • The function of DNA Polymerase.
  • The principle of polymerase chain reaction (PCR).
  • Basic requirements and design of PCR lab.
  • Potential problems of PCR.

C. Introduction of PCR related reagents:

  • DNA extraction kit
  • IQ2000TM WSSV Kit.
  • Reagents for electrophoresis.

D. Introduction of PCR related equipment

E. Review of some major shrimp viral diseases:

  • White spot syndrome virus (WSSV).
  • Yellow-head virus/Gill-associated virus (YHV/GAV).
  • Infectious hypodermal and haematopoietic necrosis virus (IHHNV).
  • Taura syndrome virus (TSV).

F. Basic theory of viral disease prevention:

  • Avoid vertical transmission.
  • Avoid horizontal transmission.
A. Practice of DNA extraction

B. Practice of PCR operation procedure:

  • Reaction condition.
  • Reagents preparation.
  • Reaction Procedure.

C. Practice of gel electrophoresis:

  • Agarose gel preparation.
  • Electrophoresis.
  • Gel staining and data assay.

D. Sampling procedure and sample preservation:

  • Preservation of shrimp (tissue) samples.
  • Preservation of DNA samples.
  • Introduction of sample dissecting tools.
  • Disinfection procedure of the sample dissecting tools.
  • Sampling strategy for broodstocks.
  • Sampling strategy for eggs and nauplii.
  • Sampling strategy for PLs.
  • Sampling strategy for juveniles in the grow-out pond.
  • Sampling strategy for environmental factors.
A. Perform the assay by using real samples:
  • To compare the sensitivity, reproducibility, and to practice more, it is necessary to go through the whole procedure by real samples again.
  • Positive standards and negative control are required.
  • Requirements of sample.

B. Detail description of PCR diagnostic procedure:

  • Sampling, sensitivity, and statistical issue.
  • False positive or false negative result.
  • Reproducibility.
  • Lab management.

C. How to integrate the diagnostic results into practical shrimp farming industry:

  • Environmental screening.
  • Choosing the source of brooders and PLs.
  • Viral risk management for the grow-out pond.
A. Real samples operation: to confirm the operation and process the other diagnostic systems.

B. Discussion.


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