| dc.description.abstract | In 2009, a new threat, Early Mortality Syndrome (EMS) or Acute Hepatopancreatic
Necrosis Disease (AHPND) presented itself with the potential to devastate the global shrimp
farming market. So far, Vibrio parahaemolyticus is known to be the very reason behind any
AHPND outbreaks. With a death rate of over 80% in 3-5 days of infection, it brings losses
and disaster to any farm it occurs. In Vietnam, since its occurrence, the disease reduced 50%
of total shrimp product and is estimated to be 1 million USD damaged in 2013. PCR
appeared to be a reliable method that helps in producing precise results for detecting
AHPND in shrimp. However, the need for a faster and more cost-effective method is
questioning researchers caused there is still potential in developing a better PCR version.
Direct PCR is a DNA amplification technique that basically is a short PCR version
following the principles of conventional PCR method. This method does not require the
DNA isolation step but it is a straight one-step PCR from raw samples. The condition,
temperatures, protocols, chemicals are nearly the same except for some differences in
techniques and additives with a considerably shorter time requirement. Initially, the direct
PCR is optimized and compared with the conventional PCR for highlighting the efficiency,
time requirement. However, because of the policies of the laboratory, raw samples of
AHPND are not allowed in the lab so the obtained isolated DNA samples of AHPND are
mixed together with lysate samples of normal shrimp with different ratios to test out the
range of AHPND detection. A direct multiplex PCR is carried out following by gel
electrophoresis to evaluate the DNA amplification. In the scope of this study, the
denaturation and extension temperature is 95oC and 72oC while the annealing temperature
is tested with different primer pairs, all duration is 30s. With this condition after
optimization, the result is high efficiency with a less time requirement and lower cost per
PCR run. | en_US |
