Application of isothermal nucleic acid amplification on shrimp viral disease diagnosis

• Main Authors: Teng Ping-Hua, 鄧秉華
• Other Authors: Ou Bor-Rung
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/39938024179183265321

博士 === 東海大學 === 畜產與生物科技學系 === 97 === Shrimp viral diseases are important issues in aquaculture industries. Until now, there have been not efficient vaccines or therapeutic strategies against these viral diseases. To face the threats, farmers tried to decrease cultivation risks with more efficient methods. However, they had no tools to early detect and prevent viruses into cultivation environment. For the purpose of pathogen detection before disease outbreak, a diagnostic platform with efficiency and accuracy plays an important role for health management of shrimp culture. Traditional strategies, such as histochemistry or immune-related techniques, only could be used as disease determination because of their insufficient sensitivities. To pathogen prevention, however, applications of molecular diagnostic technologies are feasible strategies to conquer the defects of sensitivities and complexities of histochemistry and immuo-related techniques. Polymerase chain reaction (PCR), for example, is a powerful diagnostic tool that can detect even 10 molecules in the reaction. PCR related techniques had been used to establish the culture system of specific pathogen free (SPF) and daily diagnosis for large-scale or integrated farms. However, the needs of equipments and technicians for a PCR laboratory operating are too expensive for medium or small-sized farms. Although some diagnostic centers may provide services of PCR diagnosis, the time to get results is always too long. With so many difficults to get efficient diagnostic tools, the pathogenic threats are accomplished with these smaller farms. In the study, some isothermal amplification techniques will be applied to develop a simple, economic and accurate molecular diagnostic platform to match farmers’ needs. Isothermal amplification techniques, including ramification amplification (RAM), nucleic acid sequence based amplification (NASBA) and loop-mediated isothermal amplification (LAMP), will be evaluated the feasibilities. In addition, real-time detection of these products amplified by isothermal amplifications is also tested to avoid post-amplification procedures and simplify data interpretation. The results showed that the sensitivity of RAM in detecting IHHNV was 100 copies per reaction, and the sensitivity of NASBA and reverse transcription LAMP in detecting TSV was 50 and 100 copies per reaction, respectively. Three kinds of isothermal amplification methods could be achieved high sensitivities comparable to that of PCR at 3 hours. The specificities and simplicities of these methods are also verified under different conditions to match the purpose of on-site use. For the purposes of real-time detection and quantification, LAMP coupled with fluorescence resonance energy transfer (FRET) techniques is studied and the results are available for follow-up developments. For further utilities, a novel real-time isothermal amplification detection machine should be developed to fit the purpose of diagnosis with simple, economic and accurate.