Optimization Of The Detection Of AHPND Caused By Vibrio Parahaemolyticus On Shrimp Using Direct PCR Assay

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.